Detection of biological threats. A challenge for directed molecular evolution

Journal of Microbiological Methods 58 (2004) 147 – 168 www.elsevier.com/locate/jmicmeth

Review

Detection of biological threats. A challenge for directed molecular evolution Valery A. Petrenko *, Iryna B. Sorokulova Department of Pathobiology, College of Veterinary Medicine, Auburn University, 253 Greene Hall, Auburn, AL 36849, USA Received 12 January 2004; received in revised form 3 April 2004; accepted 6 April 2004 Available online 14 May 2004

Abstract The probe technique originated from early attempts of Anton van Leeuwenhoek to contrast microorganisms under the microscope using plant juices, successful staining of tubercle bacilli with synthetic dyes by Paul Ehrlich and discovery of a stain for differentiation of gram-positive and gram-negative bacteria by Hans Christian Gram. The technique relies on the principle that pathogens have unique structural features, which can be recognized by specifically labeled organic molecules. A hundred years of extensive screening efforts led to discovery of a limited assortment of organic probes that are used for identification and differentiation of bacteria. A new challenge—continuous monitoring of biological threats—requires long lasting molecular probes capable of tight specific binding of pathogens in unfavorable conditions. To respond to the challenge, probe technology is being revolutionized by utilizing methods of combinatorial chemistry, phage display and directed molecular evolution. This review describes how molecular evolution methods are applied for development of peptide, antibody and phage probes, and summarizes the author’s own data on development of landscape phage probes against Salmonella typhimurium. The performance of the probes in detection of Salmonella is illustrated by a precipitation test, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) and fluorescent, optical and electron microscopy. D 2004 Elsevier B.V. All rights reserved. Keywords: Phage display; Detection; Monitoring; Threat agents; Landscape library; Phage antibodies; Salmonella typhimurium; Fluorescenceactivated cell sorting; Electron microscopy; Food safety; Food security

1. Introduction Development of systems for the routine monitoring of the environment and food for biological threat agents is a challenge because it requires large financial investments and extensive collaborative efforts of specialists in different areas of science and technology. New * Corresponding author. Tel.: +1-334-844-2897; fax: +1-334844-2652. E-mail address: [email protected] (V.A. Petrenko). 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.04.004

criteria for mass biological monitoring—fast, sensitive, accurate and inexpensive—depreciate the significance of traditional microbiological procedures and call for new ideas, concepts and methods. In our recent review (Petrenko and Vodyanoy, 2003), we identified phage display (Smith and Petrenko, 1997) as a new technique for development of diagnostic probes which may meet the strong criteria for biological monitoring. Since our previous review was published, we have observed an exponential rise in publications that support the high potential of new molecular selection and evolution

148

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

techniques for detection of biological agents. The appearance of new supportive published data and our own encouraging results lead us to take a closer look at the challenges of continuous environmental monitoring and the ways in which molecular evolution methods can be used for development of environmentally stable, long lasting, sensitive and specific diagnostic probes.

2. In real time monitoring versus postmortem tests Today, a bioterrorist attack with pathogenic bacteria or viruses may be recognized only based on clinical manifestations of a widespread infectious disease outbreak, followed by cultural tests and biochemical reactions (Dennis et al., 2001; Henderson et al., 1999; Inglesby et al., 1999, 2000). An attack with biological toxins may be identified using a bioassay, in which toxin-specific antibodies protect mice against the toxin present in the sample (reviewed in (Arnon et al., 2001). These microbiological, biochemical and animal tests dominate in all clinical and biomonitoring laboratories as ‘‘gold standards’’ because they are sensitive, specific and accurate (Deisingh and Thompson, 2002). Their intrinsic drawback, however, is that they are exceedingly time-consuming: detection takes at least 2 –7 days (and up to 2 – 3 weeks) to complete, thus making the analysis often postmortem. The only systems in place today for detecting a covert biological release, such as the Laboratory Response Network (Nulens and Voss, 2002), exploit standard public health surveillance procedures, such as those for tracking influenza outbreaks. Because these systems rely on the onset of an observation of unusual or ‘‘suspicious’’ cases, many days could elapse between the biological attack and its recognition. By that time, it would be very late to save the lives of many of the exposed individuals. The fatal effect of many biowarfare agents, such as anthrax, may be minimized if treatment is initiated immediately after exposure to the agent (Inglesby et al., 1999; O’Brien et al., 2003; Varkey et al., 2002). To respond effectively to a bioterrorist attack, food poisoning or an environmental biocontamination, methods of detection and identification are needed which provide information on a biological agent in real time, without any delay for sample harvesting and preparation.

Current biomonitoring systems, such as Biological Aerosol Sentry and Information System (BASIS), designed by Lawrence Livermore and Los Alamos national laboratories (Livermore, CA; Los Alamos, NM), consist of a network of sampling units deployed in and around potential target sites (Science and Technology, http://www.llnl.gov/str/October03/ October03.html). Each unit continuously collects, registers and stores samples. The air samplers suck air through filters and collect microbes onto the filter’s surface. The filters with samples are retrieved 3 –4 times per day and brought to the mobile field laboratory where they are analyzed via the polymerase chain reaction (PCR). Thus, even the most advanced monitoring systems, like BASIS, require expansive bulky equipment and a very skilled crew. Such systems also require 8 – 10 h from collecting samples to identifying a threat organism, leading to at least 1 day delay of response to the attack. Another approach to biological monitoring may be grounded on using automatic continuous in real time detectors coupled to an air-to-liquid concentrator (McBride et al., 2003; Barnes et al., 2000; Cage et al., 1996). The essential element of a real time detector is a probe that binds bacterium, spore, virus or toxin and, as a part of an analytical platform, generates a measurable signal. The platform measures changes in mass, frequency of oscillation, capacitance, resistance, surface plasmon resonance, reflectometric interference, fluorescence and other effects caused by interaction of the probe with the biological threat agent (reviewed in (Gizeli and Lowe, 2002; Ivnitski et al., 1999; Luppa et al., 2001). More sophisticated platforms, such as electrochemiluminescence (ECL), enzyme-linked imunosorbent assay (ELISA) and other immuno-sandwich platforms, use two different probes: capture probe (immobilized) and detector or reporter, tracer probe (in solution; McBride et al., 2003; Hayhurst and Georgiou, 2001; Higgins et al., 1999; King et al., 2000; Poli et al., 2002; Rowe-Taitt et al., 2000; Szilagyi et al., 2000). The choice of the detection probes is dictated by their specificity, selectivity, performance, storage, operational and environmental stability. In most platforms, the probes are polyclonal or monoclonal antibodies (Luppa et al., 2001; Ziegler and Gopel, 1998). However, polyclonal antibodies (pAb) obtained from immunized animals are not selective because

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

they recognize all antigens to which the animal has been exposed in the past. Furthermore, because of the limitations of immunization procedures in which bacteria, viruses or toxins must be inactivated before immunizing an animal and in which spore germination processes occur in vivo, the antibodies obtained often lack affinity and specificity against the desired native target. Therefore, it is difficult to standardize and certify pAb for routine applications. To be used for detection of a biological agent, they should be affinity purified on columns with the immobilized agent or its isolated antigens. Affinity purification of pAb dramatically increases their cost. Monoclonal antibodies (mAb) are more selective, but their application is also hindered by high production cost and inherent sensitivity to unfavorable environmental conditions (Pancrazio et al., 1999; Shone et al., 1985). Furthermore, obtaining a mAb against nondominant epitopes (that may be selective markers of a biological agent) is a challenge because the limited number of clones generated by hybridoma technique preferably produce antibodies against major nonunique epitopes (Emanuel et al., 1996). Both pAb and mAb are often directed against epitopes that are shared by different microorganisms, which causes cross-reactions and ambiguous results in their detection (Bettelheim et al., 1993; Lior and Borczyk, 1987; Marsden et al., 1994; Perry and Bundle, 1990; Vinogradov et al., 2000). Thus, there is need for a new type of probe – substitute antibodies –antigen binding molecules having engineered immunoglobulin or nonimmunoglobulin scaffolds that might be selected and evolutionary improved in vitro, bypassing the biased immune system (Emanuel et al., 2000; Petrenko and Smith, 2000; Skerra, 2000; reviewed in Smith and Petrenko, 1997). It was shown recently that substitute antibodies can be recruited from huge multibillion clone libraries of peptides or mutagenized functional domains of various proteins (including single-chain Fv and Fab fragments of antibodies) displayed on the surface of bacteriophages. This powerful technology of molecular selection and evolution was named ‘‘phage display’’ (reviewed in (Barbas et al., 2001; Hoogenboom, 2002; Smith and Petrenko, 1997). In contrast to hybridoma technique, relying on a screening of limited number of clones, display technologies operate with billions of potential binders and allow exploration of genetic

149

manipulations and molecular evolution to control specificity (Short et al., 2001) and selectivity of the probes (Ditzel, 2002). Such techniques can also be used to enhance probe affinity (Barbas et al., 1994; Chowdhury, 2002; Maynard et al., 2002; Pini et al., 1997; Worn and Pluckthun, 2001) and stability (Jermutus et al., 2001; Reiter et al., 1994).

3. Diagnostic probes: hundred years of screening efforts Probe technology relies on the principle that every biological agent exposes surface receptors or ligands that bind other specific molecules. This idea probably visited first the Dutch microscopist Anton van Leeuwenhoek, who in 1719 attempted differentiation of bacteria by using naturally colored agents, such as beet juice, intuitively assuming that these agents may stain bacteria in the same manner as they stain fabrics (Wainwright and Lederberg, 1992). Enormous growth and expansion of the I.G. Farben Industries in Germany at the end of 19th Century highly stimulated and encouraged investigations of the effect of synthetic dyes on live and dead cells. The scientific articles published in Germany during the first two decades of the 20th Century contain hundreds of papers describing applications of various dyes and stains to experimental cytology and medicine (Sokoloff, 1949). The theoretical basis of these works was formulated by Paul Ehrlich. At the Seventeenth International Congress of Medicine in 1913 – 1914, he wrote (translated by Himmelweit, 1960, and cited by Gale et al., 1972): ‘‘Now, if we are to look for specific medicaments, the first condition is that they must possess a definite group which is chemically allied to one of the chemoreceptors of the parasites. . . The organotropism of medicaments can, of course, be explained. . .by supposing that, in the different cells of the body and its organs, there are present a great variety of chemoreceptors. . . The chemical differences of the organs become clearly evident after vital staining. I mention here the methylene blue staining of nerve trunks, the neutral red staining of cell granules, and the distribution of isamine blue in the pirrole cells. . .these phenomena can be explained only by the fact that, at the sites concerned, certain chemical combinations of a specific nature must take place

150

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

and are to be attributed to the presence of certain chemoreceptors. . . Parasites possess a whole series of chemoreceptors which differ specifically from each other. Now, if we were to succeed in discovering amongst these a receptor which was not represented in the organs of the host, we would have the possibility of constructing an ideal medicament by selecting a haptophore group which fits exclusively this particular receptor of the parasite.’’ (Reprinted with permission from The Collected Papers of Paule Ehrich, (Ed. F. Himmelweit), 1960, Pergamon Press, pp. 505– 510). Thus, by 1882, Paul Ehrlich and Robert Koch first succeeded in staining the tubercle bacillus with an aniline dye. Using the aniline water gentian violet, Ehrlich prepared slides of bacteria, which appeared a brilliant shade of violet under the microscope. In these experiments, the idea that there must be an affinity of the cell of the bacterium for some specific dye found convincing proof. Two years later, Hans Christian Gram, a Danish pathologist, developed the Gram stain in an attempt to differentiate bacterial cells from infected tissue. The stain is still commonly used in the differentiation of gram-positive and gram-negative bacteria (Donnelly, 1962; Wainwright and Lederberg, 1992). The original method of staining involved several steps, including heat fixation of the bacteria, a two-step staining protocol, alcohol extraction and counterstaining. Over the years, several improved gram-staining techniques have been developed, but most still involve a cell-fixation or cell-permabilization step that kills bacteria being tested. A new generation of stains allows researchers to rapidly classify bacteria as either gram-negative or grampositive in minutes. For example, the LIVE BacLight Bacterial Gram Stain Kit (Molecular Probes) contains green Fluorescent SYTO 9 and red-fluorescent hexidium iodine nucleic acid stains. These two dyes differ in both their spectral characteristics and in their ability to label live gram-negative and gram-positive bacteria. When a mixed population of live gram-negative and gram-positive bacteria is simultaneously stained with the membrane penetrating SYTO 9 dye in combination with hexidium iodide, gram-negative bacteria fluoresce green, and gram-positive bacteria fluoresce red – orange (Barker et al., 1997). Another stain, acridine orange, was first described by Strugger and Hilbrich (1942). It is still widely used for detecting microorganisms in direct smears by the

fluorescent staining technique. Acridine orange possesses differential staining properties with regard to clinical materials: certain bacteria stain bright orange and are differentiated from human cells and tissue debris, which stain pale green to yellow (Kronvall and Myhre, 1977). Microscopic staining techniques, the earliest methods devised for detecting bacterial cells, still remain a standard procedure in clinical microbiology. For example, a microscopic acid-fast staining procedure is the dominant technique used in analysis of the tubercle bacillus because of its speed and simplicity, the ease of examining the slide and the reliability of the method (Smithwick et al., 1995; Taylor, 1966). Stain and fluorescent probes have been developed by screening—a process when various candidates are tested one by one for required function, such as ability to mark target biological agents. The new era of genomics revealed another approach—rational design of nucleic acid probes targeting unique sequences of bacterial and viral genomes (Almadidy et al., 2003; reviewed by Pillai, 1997). ‘‘Gene probes’’ bind to their complementary sequences like a ‘‘zipper’’ and can generate a measurable signal when they are coupled to fluorescent or enzyme labels (Cathers and Waring, 2002). Higher sensitivity of DNA tests is achieved when probes serve as primers in the polymerase chain reaction (Pillai, 1997; Saiki et al., 1988). Although diagnostic methods based on hybridization and amplification of nucleic acids have already found numerous applications for identification, typing and quantitative analysis of microorganisms (Almadidy et al., 2003; Meng et al., 2001), they are not in focus of this review because they are not suitable for in real time continuous monitoring.

4. Molecular evolution versus screening in the development of diagnostic probes All bacterial stains and fluorescent probes have been discovered during the last century using screening of available chemical compounds. Further development of the probe technology relies on using new methods of selection and directed molecular evolution evolved during the last decade of the century (reviewed by Smith and Petrenko, 1997; Szostak, 1997). Directed molecular evolution imitates the strat-

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

egy of natural evolution in the test tube iterating between diversification, selection and amplification. Diversification of potential candidates can be achieved using methods of combinatorial chemistry, such as random oligonucleotide or peptide synthesis (reviewed in Lam et al., 1997; Osborne and Ellington, 1997), or their combination with random mutagenesis (Sidhu et al., 2000). The major feature of combinatorial chemistry is that it uses a finite number of building blocks, such as nucleotides or amino acids, and a finite number of chemically addressable positions in a linear sequence or a scaffold compound, as illustrated in Fig. 1. Therefore, the diversity of structures obtained by combinatorial approaches lies in the number of combinations, not in chemical complexity. For example, a library of relatively short segments of random DNA—30-mer composed of four building blocks—consists of f 1018 various structures that encode f 1013 unique 10-mer peptides—a reservoir considerably exceeding the number of chemical compounds synthesized during the history of organic chemistry, and the diversity of natural antibodies in a human body. The remarkable feature of combinatorial chemistry is that this tremendous amount of

151

various chemical structures can be synthesized at once, in one test tube, during one working day. Another necessary component of molecular evolution is selection. Selection allows choosing from the library one or more individuals that fit to a new requirement, for example—ability to bind a target receptor. Best binders may be revealed in one experiment using affinity selection protocols. Thus, selection contrasts to traditional screening, when the members of the library are tested separately, one by one. Purely chemical libraries can be used in only one round of affinity selection, followed by analysis and identification of the bound entities. Biologically based libraries, such as phage display and nucleic acid libraries (reviewed by Osborne and Ellington, 1997; Smith and Petrenko, 1997), have a strong advantage compared with chemical libraries because they can be amplified. Amplification of primary candidates isolated in the first round of selection allows multiple rounds of selection that may be combined with generation of new diversity using mutagenesis. Multiple rounds of selection coupled with mutagenic amplification mimic Darwinian evolution, a process that automatically optimizes properties subjected to selective pressure. At the molecular level, this process of evolution helps to optimize the properties required in the probe technology such as specificity, selectivity and binding affinity of the probe towards the target receptor or cell.

5. Phage display—amplification system for directed molecular evolution

Fig. 1. Creation of probe diversity by combinatorial approach. Probes are generated by occupying N positions of a scaffold (exemplified by three positions) by K different ligands (exemplified by four different ligands) having an equal chances to be placed in any position. Indefinitely large number of copies of the scaffold, which are used as a matrix for the synthesis (such as polyfunctional cyclic compounds), or which may be generated during the synthesis (peptides or oligonucleotides backbones) create as many as KN various structures (exemplified by 4 from 64 possible structures).

To provide a background for the discussion of phage-derived probes, we will give a brief outline of the phage display technology, which is sufficient for our purposes. The reader can find more detailed information on this subject in the recent review (Smith and Petrenko, 1997). Although useful display systems based on bacteriophages T4, T7 and E have been introduced (Ansuini et al., 2002; Houshmand et al., 1999; Malys et al., 2002), the technology is most fully developed in the Ff class of filamentous phage, which includes three wild-type strains: f1, first isolated in New York City (Loeb, 1960); M13, isolated in Munich, Germany (Hofschneider, 1963); and fd, iso-

152

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

lated in Heidelberg, Germany (Marvin and HoffmanBerling, 1963). These phages are flexible, thread-like particles approximately 1 Am long and 6 nm in diameter. The bulk of their protective tubular capsid (outer shell) consists of 2700 identical subunits of the 50-residue major coat protein pVIII arranged in a helical array with a fivefold rotational axis and a coincident twofold screw axis with a pitch of 3.2 nm. The major coat protein constitutes 87% of total virion mass (Berkowitz and Day, 1976). Each pVIII subunit is largely ahelical and rod-shaped; its axis lies at a shallow angle to the long axis of the virion. About half of its 50 amino acids are exposed to the solvent, the other half being buried in the capsid (for review of phage structure, see Marvin et al., 1994). At the leading tip of the particle—the end that emerges first from the cell during phage assembly—the outer tube is capped with five copies each of minor coat proteins pVII and pIX (encoded by genes VII and IX); five copies each of minor coat proteins pIII and pVI (encoded by genes III and VI) cap the trailing end; it is assumed, but not proven, that the minor proteins form rings that match the fivefold rotational symmetry of the pVIII array. The capsid encloses a single-stranded DNA (ssDNA)—the viral or plus strand, whose length is 6407– 6408 nucleotides in wild-type strains but is not constrained by the geometry of the helical capsid. Longer or shorter plus strands—including recombinant genomes with foreign DNA inserts— can be accommodated in a capsid whose length matches the length of the enclosed DNA by including proportionally fewer or more pVIII subunits. In 1985, recombinant DNA techniques were applied to phage to fashion a new type of molecular chimera that underlies today’s phage display technology (Smith, 1985). To create one of these chimeras, a foreign coding sequence is spliced in-frame into a phage coat protein gene, so that the ‘‘guest’’ peptide encoded by that sequence is fused to a coat protein, and thereby displayed on the exposed surface of the virion. A phage display library is an ensemble of up to about 10 billion such phage clones, each harboring a different foreign coding sequence, and therefore displaying a different guest peptide on the virion surface. The foreign coding sequence can derive from a natural source, or it can be deliberately designed and synthesized chemically. For instance, phage libraries dis-

playing billions of random peptides can be readily constructed by splicing degenerate synthetic oligonucleotides, obtained by combinatorial approach (see previous section), into the coat protein gene. Surface exposure of guest peptides underlies affinity selection, a defining aspect of phage display technology. A target binding molecule, which we’ll call generically the ‘‘selector,’’ is immobilized on a solid support of some sort (e.g., on a magnetic bead or on the polystyrene surface of an ELISA well) and exposed to a phage display library. Phage particles whose displayed peptides bind the selector are captured on the support and can remain there while all other phage are washed away. The captured phage—generally a minuscule fraction of the initial phage population—can then be eluted from the support without destroying phage infectivity and propagated or cloned by infecting fresh bacterial host cells. A single round of affinity selection is able to enrich for selector-binding clones by many orders of magnitude; a few rounds suffice to survey a library with billions or even trillions of initial clones for exceedingly rare guest peptides with particularly high affinity for the selector. After several rounds of affinity selection, individual phage clones are propagated and their ability to bind the selector confirmed. Affinity selection represents a sort of in vitro evolution: the phage library is analogous to a natural population of ‘‘organisms’’; affinity for the selector is an artificial analogue to the ‘‘fitness’’ that governs an individual’s survival in the next generation. By introducing ongoing mutation into the ‘‘evolving’’ phage population, the analogy with natural selection can be made even closer. Because selection parameters can be designed and controlled, the phage display is an ideal instrument for development and directed molecular evolution of diagnostic probes. Phage antibodies are a special type of phage display construct in which the displayed peptide is an antibody molecule—or, more exactly, a domain of the antibody molecule that includes the site that binds antigen (see Fig. 2 and legend for explanation). A phage antibody library includes billions of clones, displaying billions of antibodies with different antigen specificities (reviewed in Hoogenboom, 2002). After selection of phage binders, the antibody gene(s) can be transferred to a high-level expression system in

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

Fig. 2. Display formats of antibodies. Immunoglobulin G (IgG) molecule consists of four chains: two identical heavy (H) and two identical light (L) chains, which are held together by both noncovalent interactions and disulfide – S – S – bonds. The H and L chains are made up of a number of domains of about 110 amino acids each, which fold independently and provide a modular structure to the antibody molecule. Antigen biding domains Fv ( f 25 kDa) can be stabilized by introducing disulfide bonds (reviewed in Hoogenboom, 2002). Single-chain Fv fragments (scFv) are contiguous polypeptides consisting of the heavy-(VH) and light (VL) chain variable domains of an immunoglobulin linked together by a 15- to 20-amino-acid flexible linker (Huston et al., 1991). They combine the specificity and affinity of the parental antibody but are smaller than intact IgG (30 kDa as opposed to 150 kDa). Fab fragments ( f 50 kDa) are cleaved from native IgG by papain and consist of the light chain coupled with a fragment of the heavy chain. Phage display libraries may be assembled from the variable (V) regions expressed by B-lymphocytes from an individual with a particular immune response (immune libraries), or a nonimmunized individual (native libraries). An alternative approach to create diverse libraries exploits the large collections of cloned V genes to which randomized antigen binding regions CD3 are fused in vitro by PCR (synthetic libraries).

order to produce the antibody in usable quantities in a recombinant DNA host. Selected antibodies can also be fused to reporter molecules (Casey et al., 2000; Kerschbaumer et al., 1997; Morino et al., 2001; Muller et al., 1999; Pearce et al., 1997) or subjected to molecular evolution procedures to improve their affinity, specificity (Chen et al., 1999; Short et al., 2001), stability (Reiter et al., 1994) and avidity (Kortt et al., 2001).

153

A single phage antibody library can be distributed to thousands of users and serve as the source of cloned antibodies against an unlimited array of antigens. Because selection is based solely on affinity, many toxic and biological threat agents that could not be used to immunize animals without their prior inactivation can nevertheless serve as ‘‘native antigens’’ in this artificial immune system. Furthermore, phage display allows selecting of antibodies recognizing unique epitopes on biological agents that may be missed in hybridoma screening (Emanuel et al., 1996). Another advantage of phage display contrasting it to the hybridoma technique is that the quantity of antigens required for selection of phage antibodies may be astonishingly small (1 – 10 ng in Liu and Marks, 2000), and the properties of selected probes can be further improved by affinity maturation and molecular evolution (Chowdhury, 2002; Deng et al., 1994; Worn and Pluckthun, 2001). Thus, for many purposes, this system may well come to replace natural immunity in animals (Liu and Marks, 2000). General principles and numerous applications of phage display technology are summarized in recent review (Smith and Petrenko, 1997) and manuals (Barbas et al., 2001; Kay et al., 1996; O’Brien and Aitken, 2002). Here, we will focus specifically on the application of phage display for development of diagnostic probes.

6. Phage-borne peptides and antibodies as probes for biological detection 6.1. Peptides Peptides are short chains of amino acids connected sequentially by peptide bonds. It is accepted that chains with 2 – 50 units are classified as peptides, while longer chains are classified as proteins, although the boundary between these two classes of polymers is blurry. Because peptides can be synthesized chemically in large scale as pure standard products or can be fused to carrier proteins and expressed at high levels, they are considered potentially valuable diagnostic probes (Turnbough, 2003). Usually, primarily selected peptides demonstrate modest affinity to target receptors, but their performance may be enhanced by maturation using iterative mutagenesis and selection

154

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

(Li et al., 1995). It has also been shown that high affinity ligands can be more likely selected from disulfide constrained peptide libraries rather than from linear peptide libraries (O’Neil et al., 1992; reviewed in Smith and Petrenko, 1997). Diagnostic peptide probe technology is still in the infant stages of development, and its prospects may be evaluated after accumulation of a sufficient pool of data. Nevertheless, the promise of this technology may be confirmed by several examples of its successful use, listed in Table 1. Detection of viral particles by phage-borne peptide probes can be exemplified by the work of Gough et al. (1999). The authors selected several proline-rich peptides that bind specifically to the coat protein of cucumber mosaic virus (CuMV) from a pVIII-fused random 9-mer library. Some of the selected peptides were produced in Escherichia coli cells as fusions to the N-terminus of thioredoxin. Both the phage-displayed and thioredoxin-fusion versions of the peptides could detect purified CuMV and CuMV present in crude leaf extracts from infected plants. The fused peptide probes detected as little as 5 ng of CuMV in dot blots and did not bind to other plant viruses. Because peptides isolated by phage display can be fused to carrier proteins and expressed at high levels, this approach may provide a cheap, adaptable and convenient source of diagnostic protein. Recently, peptide probes were derived against spores of Bacillus species in nonbiased selection experiments (without depletion of libraries or blocking undesirable binding sites) from 7- and 12-mer pIII-fused libraries (Knurr et al., 2003; Steichen et al., 2003; Turnbough, 2003). It was demonstrated by fluorescence-activated cell sorting (FACS) that the probes bind spores (but not vegetative cells) in a highly selective manner, binding only to the target spores and spores of phylogenetically similar species. For example, the peptide probe ATYPLPIRGGGC coupled with a highly fluorescent protein phycoerythrin (PE) was shown in FACS to bind well to spores of multiple strains of Bacillus anthracis, while binding poorly or not at all to spores of phylogenetically similar species, allowing one to distinguish between spores of B. anthracis and the other Bacillus species. Detection of Mycobacterium paratuberculosis with phage-derived peptides in artificially contaminated

milk was demonstrated recently by Stratmann et al. (2002). Peptides were selected from a commercial 12mer pIII-fused M13 phage display library in nonbiased selection with whole bacterial cells, and these peptides were immobilized to magnetic beads through biotin – streptavidin linkage. The beads were used for detection of M. paratuberculosis by capture PCR with primers specific for the insertion element ISMav2. The method competes well with previously invented immunomagnetic PCR (Grant et al., 2000) but solves the problem of standardization inherent for polyclonal antibodies. A selected phage itself can be used as a probe in a detection device, without chemical synthesis of the displayed peptide or fusion to a carrier protein. For example, to be used in an automated fluorescencebased sensing assay (Goldman et al., 2000) or FACS (Turnbough, 2003), phage, exposing thousands of reactive amino-groups, can be conjugated with Cys5, Alexa or other fluorescent labels. In this format, phage can successfully compete with antibody-derived probes. Thus, a phage –probe binding staphylococcal enterotoxin B (SEB), selected from a pIII-fused random 12-mer library, and control anti-SEB antibody showed comparable sensitivity of detection of SEB (1.4 ng/well) in the plate-based fluoroimmunoassay. However, in SEB coated fibers, the antibodies generated much higher and more specific signal than phage. Fusion of a peptide to the pIII minor coat protein, located on the tip of the phage capsid, is probably not optimal for obtaining phage probes because this expression format does not take advantage of the avidity effect gained when the binding peptides are displayed multivalently on the major coat protein pVIII (Petrenko and Smith, 2000; reviewed in Mammen et al., 1998; Smith and Petrenko, 1997). Binding peptides were selected against botulinum neurotoxins (Zdanovsky et al., 2001) and many other molecular targets (reviewed in Smith and Petrenko, 1997), but their potential in detection of the target molecules was not evaluated. 6.2. Antibodies The display of antibody domains on the surface of filamentous phage particles and the selection of recombinant phages by binding to a target antigen provide a powerful means of isolating antibody with

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

155

Table 1 Diagnostic and therapeutic probes against biological threats developed using phage display technology Probe nature//library

Target agent

Analytical test

Sensitivity

Reference

Toxins Staphylococcal enterotoxin B (SEB), Botulinum neurotoxins

fluoroimmunoassay on the plates n.d.

1.4 ng/well n.d.

(Goldman et al., 2000) (Zdanovsky et al., 2001)

dot blot analysis

5 ng of CaMV

(Gough et al., 1999)

FACS with PEcoupled peptides

107 spores

(Knurr et al., 2003)

coprecipitation

n.d.

FACS with PEcoupled peptides FACS with PEcoupled peptides or Alexa-labeled phage; fluorescence microscopy

107 spores

(Bishop-Hurley et al., 2002) (Williams et al., 2003) (Turnbough, 2003)

paramagnetic beads capture PCR ELISA, Flow cytometry, optical and fluorescence microscopy, transmission and scanning electron microscopy, microbalance

10 – 100 cell/ml

Peptides Phage//pIII-fused 12-mer peptide library Peptide//pIII-fused 12-mer peptide library Phage and peptide fused with thioredoxin //pVIIIfused 9-mer peptide library Peptides//7- and 12-mer pIII-fused peptide libraries Phage//landscape pVIII-fused 8-mer library Peptides//7- and 12-mer pIII-fused peptide libraries Peptides, phage//7- and 12-mer pIII-fused peptide libraries

Peptide, phage//12-mer pIII-fused peptide library Landscape phage//8-mer pVIII-fused peptide library

Viruses cucumber mosaic virus (CaMV) spores B. subtilis Spores Phytophthora capsici zoospores spores B. anthracis spores B. anthracis, B. subtilis, Bacillus cereus, Bacillus globigii Bacteria Mycobacterium avium subsp. paratuberculosis S. typhimurium

107 spores

(Stratmann et al., 2002) This work

n.d.

Antibodies scAb, expressed in E. coli//immune library

Purified scFv //nonimmune libraries Fab//human immune Fab-library

Fab//human immune Fab-library Fab//simian immune Fab-library

Toxins botulinum toxins

botulinum toxins

Kd = 2nM surface plasmon resonance, flow cytometry, enzyme linked immunosorbent assay (ELISA, and hand -held immunochromatographic assay SPR Kd = 26 – 72 nM

Viruses Human immunodeficiency surface plasmon virus (HIV) type 1 resonance, virus neutralization test

Kd = 7.7  10 IC50 = 10 9 10 10 M

Hepatitis B

n.d.

n.d.

Simian immunodeficiency n.d. virus (SIV)

n.d.

(Emanuel et al., 1996, 2000; Zwick et al., 2001)

(Sheets et al., 1998)

10

M;

(Barbas et al., 1994; Burton et al., 1991; Zwick et al., 2001) (Zebedee et al., 1992) (Glamann et al., 1998)

(continued on the next page)

156

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

Table 1 (continued ) Probe nature//library

Target agent

Analytical test

Sensitivity

Reference

Viruses respiratory syncytial virus

n.d.

n.d.

Puumala virus

immunoblot

n.d.

measles virus

n.d.

hepatitis C virus (HCV)

radioimmunoprecipitation assays ELISA

n.d.

Herpes simplex virus

n.d.

n.d.

Human cytomegalovirus (HCMV)

competition assay with surface Ka = 4.3  107 M plasmon resonance sensor

(Barbas et al., 1992; Crowe et al., 1998) (Salonen et al., 1998) (de Carvalho Nicacio et al., 2002) (Chan et al., 1996; Plaisant et al., 1997) (De Logu et al., 1998; Sanna et al., 1995) (Pini et al., 1997)

Human cytomegalovirus HCMV Rabies virus

n.d.

n.d.

n.d.

n.d.

Vaccinia virus

ELISA

n.d.

Epstein – Barr virus

n.d.

n.d.

Ebola virus

n.d.

n.d.

(Maruyama et al., 1999a,b; Meissner et al., 2002)

fluorescence microscopy, ELISA, competition ELISA

107 cfu/ml (in inhibition test)

(Zhou et al., 2002)

EIA, SPR

Ka = 4  107 M

ELISA

n.d.

(Deng et al., 1994, 1995) (Hayhurst et al., 2003)

M. catarrhalis

immunoblot, ELISA

n.d.

(Boel et al., 1998)

S. suis

n.d.

n.d.

(de Greeff et al., 2000)

L. monocytogenes

bioelectrochemical sensor

500 cell/ml

(Benhar et al., 2001)

C. trachomatis

ELISA, dot-blot, immunoblot and immunocytochemistry

n.d.

(Lindquist et al., 2002)

Antibodies Fab//human Fab-library Fab//human immune Fab-library Fab//human immune Fab-library scFv//human immune scFv-library Fab//human immune Fab-library scFv//human nonimmune scFv-library, affinity maturation Fab//human immune Fab-library scFv//human mAb-derived scFv-library Fab//human immune Fab-library Fab//human immune Fab-library Fab// human immune Fab-library

Phage-fused scFv// Naı¨ve human scFv scFv//mutagenized murine mAb-derived scFv-library scFv//mice immune scFv-library scFv//semisynthetic scFv-library scFv//semisynthetic scFv library scFv//’’Griffin 1’’ human synthetic library scFv//large human nonimmune scFv-library

Spores spores of the genus Bacillus Bacteria Salmonella serogroup B O-polysaccharide Brucella melitensis

1

(Williamson et al., 1997) (Muller et al., 1997) (Schmaljohn et al., 1999) (Bugli et al., 2001)

1

n.d.—Not defined; all antibody display libraries in the table are pIII-fused.

predetermined specificity. Effective display formats for antibodies are immunoglobulin variable fragments (Fv) with an engineered intermolecular disulfide bonds between the VH – VL pair, single-chain Fv with VH and VL domains connected through a peptide linker, and Fabs-light chains connected by disulfide bonds with VH1 – CH1 part of a heavy chain, as illustrated in Fig. 2 (reviewed in Hoogenboom, 2002). There are several examples where phage antibodies selected

against various biological threats have been used beneficially in various detection platforms (reviewed in Iqbal et al., 2000). Emanuel et al. (1996, 2000) constructed an immune phage antibody library using mRNA from mice immunized with botulinum toxin. The phage antibodies selected from the library using a biopanning protocol were expressed individually in E. coli cells and isolated in a highly purified form following metal

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

chelate affinity chromatography, at levels 0.5– 1.6 mg/ l LB broth. Recombinant antibodies revealed high affinity to neurotoxin (0.9 – 7 nM) and demonstrated higher performance than monoclonal antibody in a variety of assay formats including surface plasmon resonance, flow cytometry, enzyme-linked immunosorbent assay (ELISA) and hand-held immunochromatographic assay (Emanuel et al., 2000). Another group used similar methods to isolate antibotulinum toxin scFvs with affinities 26 –72 nM from very large nonimmune human phage antibody library (Sheets et al., 1998). In the example above, mRNA for the antibody phage library was isolated from animals immunized with toxoid of botulinum neurotoxin, and therefore contained species demonstrating high affinity for the intrinsic antigen. In an alternative approach, phage antibodies were selected from a universal ‘‘synthetic antibody library’’ that was constructed by appending highly diverse complementarity determining region 3 (CDR 3) gene segments to germ line VH-genes by polymerase chain reaction (PCR) and cloning the resulting repertoire, then pairing them to a single light chain in a phagemid vector (Nissim et al., 1994). Antibodies from this ‘‘universal’’ library typically have affinities in the 10 6 – 10 7 M 1 range. However, because the library consists of 5  108 potentially different heavy chains paired to a single-VL chain, it lends itself to combinatorial mutagenesis of the light chain by PCR with degenerate oligonucleotide primers. New sublibraries are rich reservoirs for selecting antibodies with higher affinity for the target antigen. This process of in vitro evolution (Barbas et al., 1994) mimics affinity maturation of antibodies in natural immune systems and allows adjustment of affinities of the primarily selected antibodies to the required level. For example, Pini et al. (1997) successfully used affinity maturation for development of antibodies against cytomegalovirus (HCMV) with affinity 4.3  107 M 1. Earlier, Barbas et al. (1994) used molecular evolution techniques to enhance affinity for gp120 protein of human immunodeficiency virus (HIV) 6– 8 times compared with a parental antibody. As a paradigm for production of human antibodies against spores of B. anthracis, Zhou et al. (2002) investigated selection of human antibodies that bind viable, native Bacillus spores from a

157

phage-displayed naı¨ve human antibody repertoire. In the direct nonbiased selection procedure, the authors demonstrated the possibility of discovering human antibodies specific for antigens on the live spore surface. For isolation of antispore antibodies, suspended spores of Bacillus subtilis were used for biopanning with a phage-displayed naı¨ve human antibody repertoire. Some of the selected antibodies were highly selective for B. subtilis versus other strains of the genus. Furthermore, phage-displayed antibodies, as well as phage-displayed peptides (Turnbough, 2003), were used for obtaining highly specific fluorescent probes that allow identification of Bacillus spores with high resolution and sensitivity by using fluorescence microscopy. A large, nonimmune scFv library was successfully used for selection of probes to surface components on elementary bodies (EB) of Chlamydia trachomatis (Lindquist et al., 2002). Sc Fv antibodies selected on the surface of purified EBs enabled the detection and identification of a variety of EB-associated antigens, some of which were chlamydial in origin whereas others were host-cell antigens. scFv antibody specificity and selectivity towards different serotypes and species of Chlamydia were assayed by ELISA, immunoblot and immunofluorescence testing. One important advantage that phage display technology offers over immunization and hybridoma techniques is that it allows the use of subtractive selection to obtain phage probes against differentially expressed structures on the surface of different cell types (Boel et al., 1998; Cai and Garen, 1995; Marks et al., 1993; Radosevic and van Ewijk, 2002; Samoylova et al., 2003). Boel et al. (1998) first reported that subtractive selection is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells. To select antibodies which can distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcus Moraxella (Branhamella) catarrhalis, the authors used competitive panning in which the target strain absorbed to MaxiSorp tubes was treated with the semisynthetic phage library (de Kruif et al., 1995) mixed and preincubated with the competing strain. It was shown by Western blotting analysis and inhibition ELISA that all phage antibodies were

158

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

directed against the outer membrane protein of M. catarrhalis, which earlier was found in multiple isolates of complement-resistant but not complement-sensitive strains. de Greeff et al. (2000) applied a subtractive selection procedure to isolate antibody fragments that can differentiate pathogenic and nonpathogenic strains of Streptococcus suis. The subtraction of the library was achieved by its preliminary treatment with the nonpathogenic strain followed by biopanning against the pathogenic strain. The selected phage antibody was found to be directed against extracellular factor (EF), which is differentially expressed by pathogenic and nonpathogenic strains. These results demonstrate a very high selectivity of the procedure. Phage antibodies were obtained against Brucella abortus, a gram-negative facultative intracellular pathogen responsible for zoonoses worldwide which is considered a potential offensive biological warfare weapon (Hayhurst et al., 2003). Detection of this bacterium by immunoassay methods is hampered by prevalence on its surface of O:9 epitope shared with another pathogen—Yersinia (Bundle et al., 1984). As a result, many existing anti-Brucellae sera exhibit cross-reactivity with the Yersinia. Phage-borne scFv against B. abortus were selected by panning an immune library against radiated bacterial cells absorbed onto plastic tubes at room temperature overnight—conditions favoring selection of strong nonpolysaccharide targeting binders. Indeed, the selected antibody allows distinguishing between Brucella and Yersinia, in contrast to a control monoclonal antibody demonstrating the reaction with both strains. In attempt to use phage displayed antibodies as probes for immunoelectrochemical detection of bacterial cells, Benhar et al. (2001) selected anti-Listeria monocytogenes scFv from ‘‘Greiffin 1’’ human synthetic library. Both purified scFv and phage-derived antibodies were studied as probes in the detector. The principal of the detection system used is ELIFA—enzyme-linked immunofiltration assay, where complexes of bacteria and labeled probe are separated from excess of probes by filtration, followed by electrochemical measurement of horseradish peroxidase (HRP) activity. While the work demonstrates the feasibility of using phage-derived

antibody probes in microbial detectors, the electrochemical platform itself is not suitable in its current format for continuous monitoring and requires essential improvements. Phage display has also been used to isolate recombinant antibodies against Hepatitis C (Chan et al., 1996; Plaisant et al., 1997), herpes simplex virus (Sanna et al., 1995), human cytomegalovirus (Williamson et al., 1997), rabies virus (Muller et al., 1997), vaccinia virus (Schmaljohn et al., 1999), Ebola virus (Maruyama et al., 1999a,b) and spores of the genus Bacillus (Zhou et al., 2002). Although these antiviral antibodies were developed primarily for prophylaxis and treatment of viral diseases, they may be considered also as prospective diagnostic probes. Antibodies selected from phage display libraries can be genetically fused to reporter proteins, such as fluorescent proteins (Casey et al., 2000; Morino et al., 2001), alkaline phosphatase (Kerschbaumer et al., 1997; Muller et al., 1999) or streptavidin (Pearce et al., 1997). These bifunctional proteins possessing both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for onestep immunodetection of biological agents. Despite its promise, however, phage antibody technology is not without difficulties. The last step in particular—expressing the selected antibody genes to make usable quantities of antibody—has proven troublesome, differing idiosyncratically from one antibody to another, as discussed in numerous reports (Brichta et al., 2003; Emanuel et al., 2000; Hayhurst, 2000; Hayhurst and Georgiou, 2001; Hayhurst et al., 2003). Besides, recombinant antibodies are sensitive to many stresses, so their engineered modification may be required to enhance their stability in nonphysiological conditions (Brichta et al., 2003; Dooley et al., 1998; Jermutus et al., 2001; Jung and Pluckthun, 1997; Reiter et al., 1994; Strachan et al., 2000; reviewed in Worn and Pluckthun, 2001). These concerns have led researchers to consider various nonimmunoglobulin scaffolds for artificial antibodies, such as minibodies, affibodies, etc. (Bianchi et al., 1995; Dennis et al., 1995; Koide et al., 1998; Ku and Schultz, 1995; Legendre et al., 2002; Martin et al., 1996; McConnell and Hoess, 1995; Nord et al., 2001; Sollazzo et al., 1997; references in Legendre et al., 2002; reviewed in Hoess, 2001).

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

7. Landscape phage probes against Salmonella 7.1. A challenge of food monitoring Salmonella is the leading cause of food-borne infections. Furthermore, Salmonella and several other organisms associated with food-borne illnesses are designated by the CDC as Category B threat agents—potential biological weapons that may be used in a bioterrorist attack. Salmonella has already been used by members of a religious sect to infect 751 individuals in Oregon through 10 salad bars in an attempt to sway local political elections (Torok et al., 1997). The effect of a deliberate Salmonella bioterrorism event may be estimated by considering actual epidemics, specifically, the 1985 contamination of pasteurized milk which caused 170,000 infections, and the 1994 contamination of ice cream that caused 224,000 illnesses (Sobel et al., 2002). Therefore, detecting pathogens in food products prior to consumption is becoming a critical part of a comprehensive strategic plan to ensure the safety of the public food supply. Monitoring of food for Salmonella differs from monitoring of air for biological aerosols. It does not require the bulky continuous monitoring systems, described in Section 2 of this review but rather needs portable high throughput devices able to inspect numerous samples in a short time. An alternative concept of food monitoring proposes enclosing small biosensors in food packages to accumulate information about package history, including storage and transportation conditions, and alert of contamination with Salmonella and other pathogens. While rapid detection of pathogens is recognized as essential for food safety, detection of Salmonella species still relies

159

on culture and plating methods (reviewed in Deisingh and Thompson, 2002). These methods usually require more than 48 h to yield results, even when the most advanced media, agars, automated microbial identification systems and standard identification kits are used (Cox et al., 2000; Davies et al., 2001; Hanai et al., 1997; Odumeru et al., 1999). Instrumental methods, like Enzyme ImmunoAssay (EIA), allow identification of Salmonella in overnight cultures but require a high concentration of bacterial cells (>106 cfu/g), which is difficult to reach because of competing microflora (Hanai et al., 1997). Anti-Salmonella monoclonal antibody probes can facilitate the analysis. Conjugated with superparamagnetic beads, they allow enrichment of Salmonella cells from overnight culture before PCR or ELISA testing (Fluit et al., 1993; Hanai et al., 1997). 7.2. Selection of the landscape probes against Salmonella To attack the problem of continuous food monitoring, we developed a new type of probe against Salmonella—landscape phage—phage display constructs with random peptides fused to all copies of major coat protein pVIII (4000 of them in the fd-tettype vectors; Petrenko and Smith, 2000; Petrenko et al., 1996; Petrenko and Vodyanoy, 2003). Landscape phage have been shown to serve as substitutes for antibodies against various soluble and cell-displayed antigens and receptors (Legendre and Fastrez, 2002; Petrenko and Smith, 2000; Petrenko et al., 2002; Romanov et al., 2001; Samoylova et al., 2003). To derive probes against Salmonella typhimurium, we explored three differing selection procedures outlined in Table 2. In procedure A, a portion of

Table 2 Procedures for selection of phage probes against S. typhimurium Procedure

Separation of bacteria/ phage complexes

Blockinga

Library depletion

Washingb

Elution of phagec

Rounds

A B

Immobilization Immobilization

0.1% BSA 0.1% BSA

0.1% Tween, TBS 0.5% Tween, TBS

(a) pH 2.2; (b) DOC (a) pH 2.2; (b) DOC

5 4

Precipitation

NO

NO Plastic, BSA-coated plastic Centrifugation

0.5% Tween, TBS

(a) pH 2.2; (b) DOC

3

C a

BSA: Bovine serum albumin. b TBS: (50 mM Tris – HCl, pH 7.5, 150 mM NaCl). c pH 2.2: 0.1 N HCl, 1 mg/ml BSA, pH adjusted to 2.2 with glycine; DOC: 2% sodium deoxycholate, 10 mM Tris, 2mM EDTA, pH 8.0.

160

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

f8-1/8-mer phage library (Petrenko et al., 1996; 2  109 clones, 1011 virions) in blocking buffer was added to 35 mm Petri dish coated with immobilized Salmonella cells. After incubation 1 h at room temperature, the unbound phages were washed away, and phages bound to Salmonella cells were eluted with mild acid. To recover residual cellassociated phages, Salmonella cells were lysed in deoxycholate buffer (Ivanenkov et al., 1999). Two phage fractions (eluate and lysate) were amplified separately in the presence of kanamycin that kills Salmonella and spares E. coli K91BK host strain. Sublibraries enriched for Salmonella-binders were used in subsequent rounds of selection. In procedure B, we used more stringent conditions for selection. To exclude selection of unrelated clones, the library was depleted against plastic and BSA and a higher concentration of Tween-20 was used for washing. Taking into account that SalmoFamily

I II III

nella cells can display different receptors under different conditions (Gawande and Bhagwat, 2002), we exploited also procedure C, elaborated for selection of phage interacting with suspended Salmonella cells. In this procedure, complexes of bacterial cells with absorbed phage were formed in solution and separated by centrifugation. Cell-bound virions were eluted with mild acid and deoxycholate, amplified separately in E. coli strain K91BK and used for next rounds of selection. The yield of Salmonellaassociated phages determined as a ratio of output to input phage increased from one round to another, indicated the successful selection of specific phage clones. Following the 5th round (A), the 4th round (B) or the 3rd round (C), individual phage clones were used for PCR and DNA sequencing and propagated for further screening. Selected peptides can be grouped into three families, shown below.

Position in peptide 1

2

3

4

5

6

7

8

V D D/E

P/S/T P R

P H/K/R P/S/T

N/P/Q G/L/P/S P/S/T

P/Q/S/T A/P P/S

A/N/Q/S A/G/H/L/Q A/N/S

H/P/S G/H/Q/S H/P/T

A/P/Q/S L/M/T T/V

Peptides from elution fractions isolated in all three procedures demonstrated striking similarities (family I) and differed remarkably from homologous families of peptides present in the lysis fractions (families II and III). 7.3. Binding of phage-derived probes to Salmonella Representative phage clones were assessed for their ability to bind Salmonella in phage capture and Salmonella capture ELISA (Fig. 3). Clones demonstrating higher signals than control wild-type phage were characterized in precipitation test to confirm their specificity to Salmonella cells. This test, used by other authors for analysis of phage binding to zoospores and bacterial spores (Bishop-Hurley et al., 2002; Knurr et al., 2003), was optimized in this work to avoid aggregation and self-precipitation of phage. Phage ( f 109 cfu/ml in TBS buffer) was heated 10 min at 70 jC and centrifuged at 13,000 rpm to precipitate aggregated phages. Phage from superna-

tant was incubated with Salmonella cells 1 h, centrifuged at 3500 rpm, washed and titered in parallel with input phage. Phage without Salmonella cells subjected to the same procedures served as a control. In the precipitation test, yields of Salmonella-bound phage were 12,000 – 22,000 times higher than the yield of the control wild-type phage, indicating very high specificity of the selection procedure. Binding of phage probes to Salmonella was also assayed by fluorescence-activated cell sorting (FACS), fluorescent microscopy and transmission electron microscopy (TEM). The phage was fluorescently labeled by Alexa Fluor 488 (Molecular Probes) with a density of 300 dye molecules per phage. For the FACS assay, fluorescently labeled phage (109 cfu) was incubated with Salmonella cells at room temperature for 1 h and centrifuged at 3500 rpm, as in the precipitation test. The pellet was washed and analyzed by FACS and fluorescence microscopy. Fig. 4 exemplifies results of FACS assay showing binding of selected phage to Salmonella

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

161

Fig. 5), demonstrating a multivalent character of the phage – bacteria binding. We also observed binding of Salmonella to immobilized phage directly, by high-power optical microscope (Signaton). In this test, one gold plate was coated with selected phage and another—with unrelated phage using chemisorptions and/or hydrophobic absorption. The plates were soaked in a suspension of Salmonella cells, washed and analyzed microscopically. Using this test, we observed specific binding of bacterial cells to immobilized phage in real time and in the liquid flow. 7.4. Landscape phage as diagnostic probes Data accumulated by studying Salmonella-binding landscape phage show good prospects of their use for monitoring of bacterial infections. Three major factors contribute to the high affinity and specificity of their binding to their targets: (a) constrained conformation of foreign peptides; (b) their multivalent display— thousands of binding sites/phage filament; and (c) extremely high local concentration of binding sites. The surface area density of the phage is 300 to 400

Fig. 3. Screening of Salmonella-binding phage clones by enzymelinked immunosorbent assay (ELISA). (A) Phage capture ELISA. Wells of 96-well ELISA plate were coated with Salmonella cells and blocked with 1% BSA. Phage clones were deposited into the wells, incubated for 1 h at room temperature; washed, reacted with biotinylated IgG against fd phage for 1 h at room temperature; washed, reacted for 1.5 h at room temperature with alkaline phosphatase conjugated with streptavidin, washed again and reacted with p-nitro-phenylphosphate (NPP) substrate. The kinetic of reaction was measured on a kinetic plate reader. The slope of yellow color development measured in terms of change in optical density per minute (mOD/min), was taken as the ELISA signal. (B) Salmonella capture ELISA. Wells of ELISA plate coated with phage and blocked with 1% BSA were incubated with Salmonella cells (108 cfu/ml in TBS/0.05%Tween), washed, reacted for 1 h with Salmonella O Coly-1 Antiserum (Becton – Dickinson), washed, reacted for 1 h at room temperature with alkaline phosphataseconjugated goat antirabbit antibodies (Jackson ImmunoResearch Laboratories), washed and developed with NPP substrate as described in (A) above.

cells. The complex of phage with bacteria was visualized directly by fluorescence microscopy (not shown) and transmission electron microscopy (TEM;

Fig. 4. FACS analysis of the binding of ALEXA-conjugated phage to S. typhimurium. The phage was fluorescently labeled by using Alexa Fluor 488 Protein Labeling Kit (Molecular Probes). The density of Alexa labeling was 300 dye molecules per phage. Fluorescently labeled phage (2  108 2  1011 cfu/ml) was incubated with suspension of Salmonella cells (109 cfu/ml) in TBS/0.5% Tween at room temperature for 1 h on rotor. The mixture was then centrifuged at 3500 rpm for 10 min. The pellet was washed twice with TBS/0.5% Tween, resuspended in 1 ml of PBS and analyzed by using fluorescence-activated cell sorting.

162

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

Fig. 5. Complex of S. typhimurium with the landscape phage probe. To distinguish the phage bound to Salmonella from bacterial fimbria, the phage was labeled with gold beads. For this aim, phage was first biotinylated by Sulfo-NHS-LC-LC-Biotin (Pierce) and mixed with Salmonella cells. Complex of phage with bacteria was absorbed onto Formvar-coated grid and treated with Immunogold conjugate EM streptavidin (BB International, Ted Pella). Arrows point to bound and free phage particles.

m2/g, corresponding to the best-known absorbents and catalysts. The randomized amino acids that form the ‘‘active receptors’’ of a landscape phage comprise up to 25% by weight of the phage and up to 50% of its surface area—an extraordinarily high fraction compared to natural proteins, including antibodies. When landscape phage serve as antibodies, one of the most troublesome steps of phage antibody technology is bypassed. No reengineering of selected antibody genes to express them at a high level is required: the phage themselves serve as the final product. Indeed, a culture of cells secreting filamentous phage is an efficient, convenient and discontinuous protein production system. The yield of wildtype particles regularly reaches 300 mg/l (20 mg/l for the engineered landscape phage). They are secreted from the cell nearly free of intracellular components; further purification is easily accomplished by simple, routinizable steps that do not differ from one clone to another. The phage structure is extraordinarily robust, being resistant to heat (Holliger et al., 1999), organic solvents (Olofsson et al., 2001), urea (up to 6 M), acid, alkali and other stresses. Purified phage can be stored indefinitely at moderate temperatures and retains functional probe activity at 37 jC during at least 6 months. These characteristics commend land-

scape phages very well as probes for continuous monitoring of biological threats.

8. Conclusion The probe technique was derived from early attempts of Anton van Leeuwenhoek to contrast microorganisms under the microscope using plant juices, a successful staining of tubercle bacillus with synthetic dyes by Paul Ehrlich and discovery of a staining procedure for differentiation of gram-positive and gram-negative bacteria by Hans Christian Gram. The technique relies on the principle that pathogens have unique structural features or distinctive receptors, which can be recognized by specifically labeled organic molecules. A hundred years of extensive screening efforts led to the discovery of a limited assortment of organic probes that are used for identification and differentiation of bacteria. A new challenge—continuous monitoring of biological threats— requires long lasting molecular probes capable of tight specific binding of pathogens in unfavorable conditions. To respond to the challenge, probe technology is being revolutionized by utilizing methods of combinatorial chemistry, phage display and directed mo-

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

lecular evolution. Although these methods are in an infant stage of development, they demonstrate a great promise for development of peptide, antibody and phage probes. Our experience with landscape phage shows that this format may be the most suitable for obtaining of long lasting specific probes against S. typhimurium and other pathogens. The performance of the probes in binding to Salmonella is illustrated by precipitation tests, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) and fluorescent, optical and electron microscopy. The next step towards continuous monitoring of biological threats requires integration of molecular probes into efficient analytical platforms. This sparely studied area requires considerable joint efforts of specialists in chemistry, biochemistry, material engineering and electronics to demonstrate performance and future prospects of the molecular probes obtained by combinatorial approaches. Acknowledgements This work was supported by U.S. Army grant DAAD19-01-10454 (to V.A.P.), NIH (NIAID) Grant # NIH-1 R21 AI05564501 (to V.A.P.), Animal Health Research (AHR) Grant, Auburn University ALAV353 (to V.A.P.) and Detection and Food Safety Center of Auburn University. We gratefully thank Dr. M.A. Toivio-Kinnucan for excellent electron microscopic support, Dr. R.C. Bird for discussion of FACS data, R.R. Young-White for technical expertise in FACS and Jennifer Brigati for reading the manuscript and making valuable notes. References Almadidy, A., Watterson, J., Piunno, P.A.E., Foulds, I.V., Horgen, P.A., Krull, U., 2003. A fibre-optic biosensor for detection of microbial contamination. Can. J. Chem. 81, 339 – 349. Ansuini, H., Cicchini, C., Nicosia, A., Tripodi, M., Cortese, R., Luzzago, A., 2002. Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands. Nucleic. Acids Res. 30, e78. Arnon, S.S., Schechter, R., Inglesby, T.V., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Hauer, J., Layton, M., Lillibridge, S., Osterholm, M.T., O’Toole, T., Parker, G., Perl, T.M., Russell, P.K., Swerdlow, D.L., Tonat, K. Working Group on Civilian B, 2001. Botulinum toxin as a

163

biological weapon: medical and public health management. JAMA 285, 1059 – 1070. Barbas III, C.F., Crowe Jr., J.E., Cababa, D., Jones, T.M., Zebedee, S.L., Murphy, B.R., Chanock, R.M., Burton, D.R., 1992. Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc. Natl. Acad. Sci. U. S. A. 89, 10164 – 10168. Barbas, C.F., Hu, D., Dunlop, N., Sawyer, L., Cababa, D., Hendry, R.M., Nara, P.L., Burton, D.R., 1994. In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity. Proc. Natl. Acad. Sci. U. S. A. 91, 3809 – 3813. Barbas III, C.F., Barton, D.R, Scott, J.K., Silverman, G.J. (Eds.), 2001. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Barker, L.P., George, K.M., Falkow, S., Small, P.L., 1997. Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. Infect. Immun. 65, 1497 – 1504. Barnes, C., Schreiber, K., Pacheco, F., Landuyt, J., Hu, F., Portnoy, J., 2000. Comparison of outdoor allergenic particles and allergen levels. Ann Allergy, Astma, Immunol 84, 47 – 54. Benhar, I., Eshkenazi, I., Neufeld, T., Opatowsky, J., Shaky, S., Rishpon, J., 2001. Recombinant single chain antibodies in bioelectrochemical sensors. Talanta 55, 899 – 907. Berkowitz, S.A., Day, L.A., 1976. Mass, length, composition and structure of the filamentous bacterial virus fd. J. Mol. Biol. 102, 531 – 547. Bettelheim, K.A., Evangelidis, H., Pearce, J.L., Sowers, E., Strockbine, N.A., 1993. Isolation of a Citrobacter freundii strain which carries the Escherichia coli O157 antigen. J. Clin. Microbiol. 31, 760 – 761. Bianchi, E., Folgori, A., Wallace, A., Nicotra, M., Acali, S., Phalipon, A., Barbato, G., Bazzo, R., Cortese, R., Felici, F., et al., 1995. A conformationally homogeneous combinatorial peptide library. J. Mol. Biol. 247, 154 – 160. Bishop-Hurley, S.L., Mounter, S.A., Laskey, J., Morris, R.O., Elder, J., Roop, P., Rouse, C., Schmidt, F.J., English, J.T., 2002. Phage-displayed peptides as developmental agonists for Phytophthora capsici zoospores. Appl. Environ. Microbiol. 68, 3315 – 3320. Boel, E., Bootsma, H., de Kruif, J., Jansze, M., Klingman, K.L., van Dijk, H., Logtenberg, T., 1998. Phage antibodies obtained by competitive selection on complement-resistant Moraxella (Branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein. Infect. Immun. 66, 83 – 88. Brichta, J., Vesela, H., Franek, M., 2003. Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naive phage library. Vet. Med. (Prague) 48, 237 – 247. Bugli, F., Bastidas, R., Burton, D.R., Williamson, R.A., Clementi, M., Burioni, R., 2001. Molecular profile of a human monoclonal antibody Fab fragment specific for Epstein – Barr virus gp350/ 220 antigen. Hum. Immunol. 62, 362 – 367. Bundle, D.R., Gidney, M.A., Perry, M.B., Duncan, J.R., Cherwonogrodzky, J.W., 1984. Serological confirmation of Brucella abortus and Yersinia enterocolitica O:9 O-antigens by monoclonal antibodies. Infect. Immun. 46, 389 – 393.

164

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

Burton, D.R., Barbas III, C.F., Persson, M.A., Koenig, S., Chanock, R.M., Lerner, R.A., 1991. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. U. S. A. 88, 10134 – 10137. Cage, B.R., Schreiber, K., Barnes, C., Portnoy, J., 1996. Evaluation of four bioaerosol samplers in the outdoor environment. Ann. Allergy, Astma, Immunol. 77, 401 – 406. Cai, X.H., Garen, A., 1995. Antimelanoma antibodies from melanoma patients immunized with genetically-modified autologous tumor-cells—selection of specific antibodies from single-chain Fv fusion phage libraries. Proc. Natl. Acad. Sci. U. S. A. 92, 6537 – 6541. Casey, J.L., Coley, A.M., Tilley, L.M., Foley, M., 2000. Green fluorescent antibodies: novel in vitro tools. Protein Eng. 13, 445 – 452. Cathers, B.E., Waring, M.J., 2002. DNA interactions. In: Gizeli, E., Lowe, C.R. (Eds.), Biomolecular Sensors. Taylor and Francis, London, pp. 33 – 46. Chan, S.W., Bye, J.M., Jackson, P., Allain, J.P., 1996. Human recombinant antibodies specific for hepatitis C virus core and envelope E2 peptides from an immune phage display library. J. Gen. Virol. 77, 2531 – 2539. Chen, G., Dubrawsky, I., Mendez, P., Georgiou, G., Iverson, B.L., 1999. In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site. Protein Eng. 12, 349 – 356. Chowdhury, P.S., 2002. Targeting random mutations to hotspots in antibody variable domains for affinity improvement. In: O’Brien, P.M., Aitken, R. (Eds.), Antibody Phage Display: Methods and Protocols. Humana Press, Totowa, NJ, pp. 269 – 286. Cox, N.A., Berrang, M.E., Dickens, J.A., 2000. Inadequacy of selective plating media in field detection of Salmonella efficacy of an herbal extract on the microbiological quality of broiler carcasses during a simulated chill. J. Appl. Poult. Res. 9, 403 – 406. Crowe, J.E., Firestone, C.Y., Crim, R., Beeler, J.A., Coelingh, K.L., Barbas, C.F., Burton, D.R., Chanock, R.M., Murphy, B.R., 1998. Monoclonal antibody-resistant mutants selected with a respiratory syncytial virus-neutralizing human antibody Fab fragment (Fab 19) define a unique epitope on the fusion (F) glycoprotein. Virology 252, 373 – 375. Davies, R.H., Bedford, S., Shankster, S., 2001. Enhanced culture techniques for the detection of Salmonella. Vet. Rec. 148, 539 – 540. de Carvalho Nicacio, C., Williamson, R.A., Parren, P.W., Lundkvist, A., Burton, D.R., Bjorling, E., 2002. Neutralizing human Fab fragments against measles virus recovered by phage display. J. Virol. 76, 251 – 258. de Greeff, A., van Alphen, L., Smith, H.E., 2000. Selection of recombinant antibodies specific for pathogenic Streptococcus suis by subtractive phage display. Infect. Immun. 68, 3949 – 3955. Deisingh, A.K., Thompson, M., 2002. Detection of infectious and toxigenic bacteria. Analyst 127, 567 – 581. de Kruif, J., Boel, E., Logtenberg, T., 1995. Selection and application of human single chain Fv antibody fragments from a semisynthetic phage antibody display library with designed CDR3 regions. J. Mol. Biol. 248, 97 – 105.

De Logu, A., Williamson, R.A., Rozenshteyn, R., Ramiro-Ibanez, F., Simpson, C.D., Burton, D.R., Sanna, P.P., 1998. Characterization of a type-common human recombinant monoclonal antibody to herpes simplex virus with high therapeutic potential. J. Clin. Microbiol. 36, 3198 – 3204. Deng, S.J., MacKenzie, C.R., Sadowska, J., Michniewicz, J., Young, N.M., Bundle, D.R., Narang, S.A., 1994. Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display. J. Biol. Chem. 269, 9533 – 9538. Deng, S.J., MacKenzie, C.R., Hirama, T., Brousseau, R., Lowary, T.L., Young, N.M., Bundle, D.R., Narang, S.A., 1995. Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries. Proc. Natl. Acad. Sci. U. S. A. 92, 4992 – 4996. Dennis, M.S., Herzka, A., Lazarus, R.A., 1995. Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display. J. Biol. Chem. 270, 25411 – 25417. Dennis, D.T., Inglesby, T.V., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Layton, M., Lillibridge, S.R., McDade, J.E., Osterholm, M.T., O’Toole, T., Parker, G., Perl, T.M., Russell, P.K., Tonat, K., 2001. Tularemia as a biological weapon: medical and public health management. JAMA 285, 2763 – 2773. Ditzel, H.J., 2002. Rescue of a broader range of antibody specificities using an epitope-masking strategy. In: O’Brien, P.M. (Ed.), Antibody Phage Display. Humana Press, Totowa, NJ, pp. 179 – 186. Donnelly, J.P., 1962. The secrets of Gram’s stain. Infect. Dis. Alert 15, 109 – 112. Dooley, H., Grant, S.D., Harris, W.J., Porter, A.J., Shelton, S.A., Graham, B.M., Strachan, G., 1998. Stabilization of antibody fragments in adverse environments immunomethods for detecting a broad range of polychlorinated biphenyls. Biotechnol. Appl. Biochem. 28, 77 – 83. Emanuel, P., O’Brien, T., Burans, J., DasGupta, B.R., Valdes, J.J., Eldefrawi, M., 1996. Directing antigen specificity towards botulinum neurotoxin with combinatorial phage display libraries. J. Immunol. Methods 193, 189 – 197. Emanuel, P.A., Dang, J., Gebhardt, J.S., Aldrich, J., Garber, E.A., Kulaga, H., Stopa, P., Valdes, J.J., Dion-Schultz, A., 2000. Recombinant antibodies: a new reagent for biological agent detection. Biosens. Bioelectron. 14, 751 – 759. Fluit, A.C., Widjojoatmodjo, M.N., Box, A.T., Torensma, R., Verhoef, J., 1993. Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay. Appl. Environ. Microbiol. 59, 1342 – 1346. Gale, E.F., Cundliffe, E., Reynolds, P.E., Richmond, M.H., Waring, M.J., 1972. The Molecular Basis of Antibiotic Action. Wiley, London. Gawande, P.V., Bhagwat, A.A., 2002. Inoculation onto solid surfaces protects Salmonella spp. during acid challenge: a model study using polyethersulfone membranes. Appl. Environ. Microbiol. 68, 86 – 92. Gizeli, E., Lowe, C.R., 2002. Biomolecular Sensors. Taylor and Francis, London. Glamann, J., Burton, D.R., Parren, P.W., Ditzel, H.J., Kent, K.A., Arnold, C., Montefiori, D., Hirsch, V.M., 1998. Simian immunodeficiency virus (SIV) envelope-specific Fabs with high-level

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168 homologous neutralizing activity: recovery from a long-termnonprogressor SIV-infected macaque. J. Virol. 72, 585 – 592. Goldman, E.R., Pazirandeh, M.P., Mauro, J.M., King, K.D., Frey, J.C., Anderson, G.P., 2000. Phage-displayed peptides as biosensor reagents. J. Mol. Recognit. 13, 382 – 387. Gough, K.C., Cockburn, W., Whitelam, G.C., 1999. Selection of phage-display peptides that bind to cucumber mosaic virus coat protein. J. Virol. Methods 79, 169 – 180. Grant, I.R., Pope, C.M., O’Riordan, L.M., Ball, H.J., Rowe, M.T., 2000. Improved detection of Mycobacterium avium subsp. paratuberculosis in milk by immunomagnetic PCR. Vet. Microbiol. 77, 369 – 378. Hanai, K., Satake, M., Nakanishi, H., Venkateswaran, K., 1997. Comparison of commercially available kits with standard methods for detection of Salmonella strains in foods. Appl. Environ. Microbiol. 63, 775 – 778. Hayhurst, A., 2000. Improved expression characteristics of singlechain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain. Protein Expr. Purif. 18, 1 – 10. Hayhurst, A., Georgiou, G., 2001. High-throughput antibody isolation. Curr. Opin. Chem. Biol. 5, 683 – 689. Hayhurst, A., Happe, S., Mabry, R., Koch, Z., Iverson, B.L., Georgiou, G., 2003. Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis. J. Immunol. Methods 276, 185 – 196. Henderson, D.A., Inglesby, T.V., Bartlett, J.G., Ascher, M.S., Eitzen, E., Jahrling, P.B., Hauer, J., Layton, M., McDade, J., Osterholm, M.T., O’Toole, T., Parker, G., Perl, T., Russell, P.K., Tonat, K., 1999. Smallpox as a biological weapon: medical and public health management. Working group on civilian biodefense. JAMA 281, 2127 – 2137. Higgins, J.A., Ibrahim, M.S., Knauert, F.K., Ludwig, G.V., Kijek, T.M., Ezzell, J.W., Courtney, B.C., Henchal, E.A., 1999. Sensitive and rapid identification of biological threat agents. Ann. N.Y. Acad. Sci. 894, 130 – 148. Himmelweit, F., 1960. The Collected Papers of Paqule Ehrich. Pergamon Press, London. Hoess, R.H., 2001. Protein design and phage display. Chem. Rev. 101, 3205 – 3218. Hofschneider, P.H., 1963. Untersuchungen uber ‘‘kleine’’ E. coli K12 bacteriophagen M12, M13, und M20. Z Naturforsch 18b, 203 – 205. Holliger, P., Riechmann, L., Williams, R.L., 1999. Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability. J. Mol. Biol. 288, 649 – 657. Hoogenboom, H.R., 2002. Overview of antibody phage-display technology and its applications. In: O’Brien, P.M., Aitken, R. (Eds.), Antibody Phage Display: Methods and Protocols. Humana Press, Totowa, NJ, pp. 1 – 39. Houshmand, H., Froman, G., Magnusson, G., 1999. Use of bacteriophage T7 displayed peptides for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction. Anal. Biochem. 268, 363 – 370. Huston, J.S., Mudgetthunter, M., Tai, M.S., Mccartney, J., Warren, F., Haber, E., Oppermann, H., 1991. Protein engineering of

165

single-chain Fv analogs and fusion proteins. Methods Enzymol. 203, 46 – 88. Inglesby, T.V., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Friedlander, A.M., Hauer, J., McDade, J., Osterholm, M.T., O’Toole, T., Parker, G., Perl, T.M., Russell, P.K., Tonat, K., 1999. Anthrax as a biological weapon: medical and public health management. Working group on civilian biodefense. JAMA 281, 1735 – 1745. Inglesby, T.V., Dennis, D.T., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Koerner, J.F., Layton, M., McDade, J., Osterholm, M.T., O’Toole, T., Parker, G., Perl, T.M., Russell, P.K., Schoch-Spana, M., Tonat, K., 2000. Plague as a biological weapon: medical and public health management. Working group on civilian biodefense. JAMA 283, 2281 – 2290. Iqbal, S.S., Mayo, M.W., Bruno, J.G., Bronk, B.V., Batt, C.A., Chambers, J.P., 2000. A review of molecular recognition technologies for detection of biological threat agents. Biosens. Bioelectron. 15, 549 – 578. Ivanenkov, V., Felici, F., Menon, A.G., 1999. Uptake and intracellular fate of phage display vectors in mammalian cells. Biochim. Biophys. Acta 1448, 450 – 462. Ivnitski, D., Abdel-Hamid, I., Atanasov, P., Wilkins, E., 1999. Biosensors for detection of pathogenic bacteria. Biosens. Bioelectron. 14, 599 – 624. Jermutus, L., Honegger, A., Schwesinger, F., Hanes, J., Pluckthun, A., 2001. Tailoring in vitro evolution for protein affinity or stability. Proc. Natl. Acad. Sci. U. S. A. 98, 75 – 80. Jung, S., Pluckthun, A., 1997. Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Protein Eng. 10, 959 – 966. Kay, B.K., Winter, J., McCafferty, J. (Eds.), 1996. Phage Display of Peptides and Proteins: A Laboratory Manual. Academic Press, New York. Kerschbaumer, R.J., Hirschl, S., Kaufmann, A., Ibl, M., Koenig, R., Himmler, G., 1997. Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. Anal. Biochem. 249, 219 – 227. King, K.D., Vanniere, J.M., Leblanc, J.L., Bullock, K.E., Anderson, G.P., 2000. Automated fiber optic biosensor for multiplexed immunoassays. Environ. Sci. Technol. 34, 2845 – 2850. Knurr, J., Benedek, O., Heslop, J., Vinson, R.B., Boydston, J.A., McAndrew, J., Kearney, J.F., Turnbough, C.L., 2003. Peptide ligands that bind selectively to spores of Bacillus subtilis and closely related species. Appl. Environ. Microbiol. 69, 6841 – 6847. Koide, A., Bailey, C.W., Huang, X., Koide, S., 1998. The fibronectin type III domain as a scaffold for novel binding proteins. J. Mol. Biol. 284, 1141 – 1151. Kortt, A.A., Dolezal, O., Power, B.E., Hudson, P.J., 2001. Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting. Biomol. Eng. 18, 95 – 108. Kronvall, G., Myhre, E., 1977. Differential staining of bacteria in clinical specimens using acridine orange buffered at low pH. Acta Pathol. Microbiol. Scand., Sect B 85, 249 – 254. Ku, J., Schultz, P.G., 1995. Alternate protein frameworks for molecular recognition. Proc. Natl. Acad. Sci. U. S. A. 92, 6552 – 6556.

166

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

Lam, K.S., Lebl, M., Krchnak, V., 1997. The one-bead – one-compound combinatorial library method. Chem. Rev. 97, 411 – 448. Legendre, D., Fastrez, J., 2002. Construction and exploitation in model experiments of functional selection of a landscape library expressed from a phagemid. Gene 290, 203 – 215. Legendre, D., Vucic, B., Hougardy, V., Girboux, A.L., Henrioul, C., Van Haute, J., Soumillion, P., Fastrez, J., 2002. TEM-1 betalactamase as a scaffold for protein recognition and assay. Protein Sci. 11, 1506 – 1518. Li, B., Tom, J.Y., Oare, D., Yen, R., Fairbrother, W.J., Wells, J.A., Cunningham, B.C., 1995. Minimization of a polypeptide hormone. Science 270, 1657 – 1660. Lindquist, E.A., Marks, J.D., Kleba, B.J., Stephens, R.S., 2002. Phage-display antibody detection of Chlamydia trachomatis-associated antigens. Microbiology 148, 443 – 451. Lior, H., Borczyk, A.A., 1987. False positive identifications of Escherichia coli O157. Lancet 1, 333. Liu, B., Marks, J.D., 2000. Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose. Anal. Biochem. 286, 119 – 128. Loeb, T., 1960. Isolation of bacteriophage specific for the F+ and Hfr mating types of Escherichia coli K12. Science 131, 932 – 933. Luppa, P.B., Sokoll, L.J., Chan, D.W., 2001. Immunosensors— principles and applications to clinical chemistry. Clin. Chim. Acta. 314, 1 – 26. Malys, N., Chang, D.Y., Baumann, R.G., Xie, D., Black, L.W., 2002. A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4 terminase (gp17) and late sigma factor (gp55) interaction. J. Mol. Biol. 319, 289 – 304. Mammen, M., Choi, S.K., Whitesides, G.M., 1998. Polyvalent interactions in biological systems—implications for design and use of multivalent ligands and inhibitors. Angew. Chem. 37, 2755 – 2794. Marks, J.D., Ouwehand, W.H., Bye, J.M., Finnern, R., Gorick, B.D., Voak, D., Thorpe, S.J., Hughes-Jones, N.C., Winter, G., 1993. Human antibody fragments specific for human blood group antigens from a phage display library. Bio/Technology 11, 1145 – 1149. Marsden, B.J., Bundle, D.R., Perry, M.B., 1994. Serological and structural relationships between Escherichia coli O:98 and Yersinia enterocolitica O:11,23 and O:11,24 lipopolysaccharide Oantigens. Biochem. Cell Biol. 72, 163 – 168. Martin, F., Toniatti, C., Salvati, A.L., Ciliberto, G., Cortese, R., Sollazzo, M., 1996. Coupling protein design and in vitro selection strategies: improving specificity and affinity of a designed beta-protein IL-6 antagonist. J. Mol. Biol. 255, 86 – 97. Maruyama, T., Parren, P.W., Sanchez, A., Rensink, I., Rodriguez, L.L., Khan, A.S., Peters, C.J., Burton, D.R., 1999a. Recombinant human monoclonal antibodies to Ebola virus. J. Infect. Dis. 179, S235 – 239. Maruyama, T., Rodriguez, L.L., Jahrling, P.B., Sanchez, A., Khan, A.S., Nichol, S.T., Peters, C.J., Parren, P.W., Burton, D.R., 1999b. Ebola virus can be effectively neutralized by antibody produced in natural human infection. J. Virol. 73, 6024 – 6030. Marvin, D.A., Hoffman-Berling, H., 1963. Physical and chemical

properties of two new small bacteriophages. Nature (Lond.) 197, 517 – 518. Marvin, D.A., Hale, R.D., Nave, C., Citterich, M.H., 1994. Molecular models and structural comparisons of native and mutant class I filamentous bacteriophages Ff (fd, f1, M13), If1 and IKe. J. Mol. Biol. 235, 260 – 286. Maynard, J.A., Chen, G., Georgiou, G., Iverson, B.L., 2002. In vitro scanning – saturation mutagenesis. Methods Mol. Biol. 182, 149 – 163. McBride, M.T., Masquelier, D., Hindson, B.J., Makarewicz, A.J., Brown, S., Burris, K., Metz, T., Langlois, R.G., Tsang, K.W., Bryan, R., Anderson, D.A., Venkateswaran, K.S., Milanovich, B.W., Colston Jr., B.W., 2003. Autonomous detection of aerosolized Bacillus anthracis and Yersinia pestis. Anal. Chem. 75, 5293 – 5299. McConnell, S.J., Hoess, R.H., 1995. Tendamistat as a scaffold for conformationally constrained phage peptide libraries. J. Mol. Biol. 250, 460 – 470. Meissner, F., Maruyama, T., Frentsch, M., Hessell, A.J., Rodriguez, L.L., Geisbert, T.W., Jahrling, P.B., Burton, D.R., Parren, P.W., 2002. Detection of antibodies against the four subtypes of Ebola virus in sera from any species using a novel antibody – phage indicator assay. Virology 300, 236 – 243. Meng, Q., Wong, C., Rangachari, A., Tamatsukuri, S., Sasaki, M., Fiss, E., Cheng, L., Ramankutty, T., Clarke, D., Yawata, H., Sakakura, Y., Hirose, T., Impraim, C., 2001. Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA. J. Clin. Microbiol. 39, 2937 – 2945. Morino, K., Katsumi, H., Akahori, Y., Iba, Y., Shinohara, M., Ukai, Y., Kohara, Y., Kurosawa, Y., 2001. Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression. J. Immunol. Methods 257, 175 – 184. Muller, B.H., Lafay, F., Demangel, C., Perrin, P., Tordo, N., Flamand, A., Lafaye, P., Guesdon, J.L., 1997. Phage-displayed and soluble mouse scFv fragments neutralize rabies virus. J. Virol. Methods 67, 221 – 233. Muller, B.H., Chevrier, D., Boulain, J.C., Guesdon, J.L., 1999. Recombinant single-chain Fv antibody fragment – alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization. J. Immunol. Methods 227, 177 – 185. Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D., Winter, G., 1994. Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents. EMBO J 13, 692 – 698. Nord, K., Nord, O., Uhlen, M., Kelley, B., Ljungqvist, C., Nygren, P.A., 2001. Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A. Eur. J. Biochem. 268, 4269 – 4277. Nulens, E., Voss, A., 2002. Laboratory diagnosis and biosafety issues of biological warfare agents. Clin. Microbiol. Infect. 8, 455 – 466. O’Brien, P.M., Aitken, R. (Eds.), 2002. Antibody Phage Display: Methods and Protocols. Humana Press, Totowa, NJ. O’Brien, K.K., Higdon, M.L., Halverson, J.J., 2003. Recognition and management of bioterrorism infections. Am. Fam. Phys. 67, 1927 – 1934.

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168 Odumeru, J.A., Steele, M., Fruhner, L., Larkin, C., Jiang, J., Mann, E., McNab, W.B., 1999. Evaluation of accuracy and repeatability of identification of food-borne pathogens by automated bacterial identification systems. J. Clin. Microbiol. 37, 944 – 949. Olofsson, L., Ankarloo, J., Andersson, P.O., Nicholls, I.A., 2001. Filamentous bacteriophage stability in non-aqueous media. Chem. Biol. 8, 661 – 671. O’Neil, K.T., Hoess, R.H., Jackson, S.A., Ramachandran, N.S., Mousa, S.A., DeGrado, W.F., 1992. Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library. Proteins 14, 509 – 515. Osborne, S.E., Ellington, A.D., 1997. Nucleic acid selection and the challenge of combinatorial chemistry. Chem. Rev. 97, 349 – 370. Pancrazio, J.J., Whelan, J.P., Borkholder, D.A., Ma, W., Stenger, D.A., 1999. Development and application of cell-based biosensors. Ann. Biomed. Eng. 27, 697 – 711. Pearce, L.A., Oddie, G.W., Coia, G., Kortt, A.A., Hudson, P.J., Lilley, G.G., 1997. Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents. Biochem. Mol. Biol. Int. 42, 1179 – 1188. Perry, M.B., Bundle, D.R., 1990. Antigenic relationships of the lipopolysaccharides of Escherichia hermannii strains with those of Escherichia coli O157:H7, Brucella melitensis, and Brucella abortus. Infect. Immun. 58, 1391 – 1395. Petrenko, V.A., Smith, G.P., 2000. Phages from landscape libraries as substitute antibodies. Protein Eng. 13, 589 – 592. Petrenko, V.A., Vodyanoy, V.J., 2003. Phage display for detection of biological threat agents. J. Microbiol. Methods 1768, 1 – 10. Petrenko, V.A., Smith, G.P., Gong, X., Quinn, T., 1996. A library of organic landscapes on filamentous phage. Protein Eng. 9, 797 – 801. Petrenko, V.A., Smith, G.P., Mazooji, M.M., Quinn, T., 2002. Alpha-helically constrained phage display library. Protein Eng. 15, 943 – 950. Pillai, S.D., 1997. Rapid molecular detection of microbial pathogens—breakthroughs and challenges. Arch. Virol., 67 – 82. Pini, A., Spreafico, A., Botti, R., Neri, D., Neri, P., 1997. Hierarchical affinity maturation of a phage library derived antibody for the selective removal of cytomegalovirus from plasma. J. Immunol. Methods 206, 171 – 182. Plaisant, P., Burioni, R., Manzin, A., Solforosi, L., Candela, M., Gabrielli, A., Fadda, G., Clementi, M., 1997. Human monoclonal recombinant Fabs specific for HCV antigens obtained by repertoire cloning in phage display combinatorial vectors. Res. Virol. 148, 165 – 169. Poli, M.A., Rivera, V.R., Neal, D., 2002. Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F. Toxicon 40, 797 – 802. Radosevic, K., van Ewijk, W., 2002. Subtractive isolation of singlechain antibodies using tissue fragments. Methods Mol. Biol. 178, 235 – 243. Reiter, Y., Brinkmann, U., Jung, S.H., Lee, B., Kasprzyk, P.G., King, C.R., Pastan, I., 1994. Improved binding and antitumor activity of a recombinant anti-erbB2 immunotoxin by disulfide stabilization of the Fv fragment. J. Biol. Chem. 269, 18327 – 18331.

167

Romanov, V.I., Durand, D.B., Petrenko, V.A., 2001. Phage display selection of peptides that affect prostate carcinoma cells attachment and invasion. Prostate 47, 239 – 251. Rowe-Taitt, C.A., Hazzard, J.W., Hoffman, K.E., Cras, J.J., Golden, J.P., Ligler, F.S., 2000. Simultaneous detection of six biohazardous agents using a planar waveguide array biosensor. Biosens. Bioelectron. 15, 579 – 589. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487 – 491. Salonen, E.M., Parren, P.W., Graus, Y.F., Lundkvist, A., Fisicaro, P., Vapalahti, O., Kallio-Kokko, H., Vaheri, A., Burton, D.R., 1998. Human recombinant Puumala virus antibodies: crossreaction with other Hantaviruses and use in diagnostics. J. Gen. Virol. 79, 659 – 665. Samoylova, T.I., Petrenko, V.A., Morrison, N.E., Globa, L.P., Bekker, H.J., Cox, N.R., 2003. Phage probes for molecular profiling of malignant glioma cells. Mol. Cancer Pharmacol. 2, 1 – 9. Sanna, P.P., Williamson, R.A., De Logu, A., Bloom, F.E., Burton, D.R., 1995. Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries. Proc. Natl. Acad. Sci. U. S. A. 92, 6439 – 6443. Schmaljohn, C., Cui, Y., Kerby, S., Pennock, D., Spik, K., 1999. Production and characterization of human monoclonal antibody Fab fragments to vaccinia virus from a phage-display combinatorial library. Virology 258, 189 – 200. Sheets, M.D., Amersdorfer, P., Finnern, R., Sargent, P., Lindquist, E., Schier, R., Hemingsen, G., Wong, C., Gerhart, J.C., Marks, J.D., Lindqvist, E., 1998. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci. U. S. A. 95, 6157 – 6162. Shone, C., Wilton-Smith, P., Appleton, N., Hambleton, P., Modi, N., Gatley, S., Melling, J., 1985. Monoclonal antibody-based immunoassay for type a Clostridium botulinum toxin is comparable to the mouse bioassay. Appl. Environ. Microbiol. 50, 63 – 67. Short, M.K., Jeffrey, P.D., Demirjian, A., Margolies, M.N., 2001. A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs. Protein Eng. 14, 287 – 296. Sidhu, S.S., Lowman, H.B., Cunningham, B.C., Wells, J.A., 2000. Phage display for selection of novel binding peptides. Methods Enzymol. 328, 333 – 363. Skerra, A., 2000. Engineered protein scaffolds for molecular recognition. J. Mol. Recognit. 13, 167 – 187. Smith, G.P., 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315 – 1317. Smith, G.P., Petrenko, V.A., 1997. Phage display. Chem. Rev. 97, 391 – 410. Smithwick, R.W., Bigbie Jr., M.R., Ferguson, R.B., Karlix, M.A., Wallis, C.K., 1995. Phenolic acridine orange fluorescent stain for mycobacteria. J. Clin. Microbiol. 33, 2763 – 2764. Sobel, J., Khan, A.S., Swerdlow, D.L., 2002. Threat of a biological terrorist attack on the US food supply: the CDC perspective. Lancet 359, 874 – 880.

168

V.A. Petrenko, I.B. Sorokulova / Journal of Microbiological Methods 58 (2004) 147–168

Sokoloff, B., 1949. The Miracle Drugs Ziff-Davies Publishing, Chicago, NY. Sollazzo, M., Venturini, S., Lorenzetti, S., Pinola, M., Martin, F., 1997. Engineering minibody-like ligands by design and selection. Chem. Immunol. 65, 1 – 17. Steichen, C., Chen, P., Kearney, J.F., Turnbough Jr., C.L., 2003. Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. J. Bacteriol. 185, 1903 – 1910. Strachan, G., Whyte, J.A., Molloy, P.M., Paton, G.I., Porter, A.J.R., 2000. Development of robust, environmental, immunoassay formats for the quantification of pesticides in soil. Environ. Sci. Technol. 34, 1603 – 1608. Stratmann, J., Strommenger, B., Stevenson, K., Gerlach, G.F., 2002. Development of a peptide-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk. J. Clin. Microbiol. 40, 4244 – 4250. Strugger, S., Hilbrich, P., 1942. Die fluoreszenzmikroskopische unterscheidung lebender und toten bakterienzeillen mit hilfe des akridinoragefarbung. Dtsch Teira¨ rztl Wochenschr 50, 121 – 130. Szilagyi, M., Rivera, V.R., Neal, D., Merrill, G.A., Poli, M.A., 2000. Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes A and B. Toxicon 38, 381 – 389. Szostak, J.W., 1997. In vitro selection and directed evolution. Harvey Lect. 93, 95 – 118. Taylor, R.D., 1966. Modification of the Brown and Brenn Gram Stain for the differential staining of gram-positive and gram-negative bacteria in tissue section. Am. J. Clin. Pathol. 46, 472 – 474. Torok, T.J., Tauxe, R.V., Wise, R.P., Livengood, J.R., Sokolow, R., Mauvais, S., Birkness, K.A., Skeels, M.R., Horan, J.M., Foster, L.R., 1997. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA 278, 389 – 395. Turnbough Jr., C.L., 2003. Discovery of phage display peptide ligands for species-specific detection of Bacillus spores. J. Microbiol. Methods 53, 263 – 271. Varkey, P., Poland, G.A., Cockerill III, F.R., Smith, T.F., Hagen,

P.T. 2002. Confronting bioterrorism: physicians on the front line. Mayo Clin. Proc. 77, 661 – 672. Vinogradov, E., Conlan, J.W., Perry, M.B., 2000. Serological crossreaction between the lipopolysaccharide O-polysaccharide antigens of Escherichia coli O157:H7 and strains of Citrobacter freundii and Citrobacter sedlakii. FEMS Microbiol. Lett. 190, 157 – 161. Wainwright, M., Lederberg, J., 1992. History of microbiology. In: Lederberg, J. (Ed.), Encyclopedia of Microbiology. Academic Press, New York, NY. Williams, D.D., Benedek, O., Tournbough, C.L., 2003. Speciesspecific peptide ligands for the detection of Bacillus anthracis spores. Appl. Environ. Microbiol. 69, 6288 – 6293. Williamson, R.A., Lazzarotto, T., Sanna, P.P., Bastidas, R.B., Dalla Casa, B., Campisi, G., Burioni, R., Landini, M.P., Burton, D.R., 1997. Use of recombinant human antibody fragments for detection of cytomegalovirus antigenemia. J. Clin. Microbiol. 35, 2047 – 2050. Worn, A., Pluckthun, A., 2001. Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305, 989 – 1010. Zdanovsky, A.G., Karassina, N.V., Simpson, D., Zdanovskaia, M.V., 2001. Peptide phage display library as source for inhibitors of clostridial neurotoxins. J. Protein Chem. 20, 73 – 80. Zebedee, S.L., Barbas III, C.F., Hom, Y.L., Caothien, R.H., Graff, R., DeGraw, J., Pyati, J., LaPolla, R., Burton, D.R., Lerner, R.A., et al., 1992. Human combinatorial antibody libraries to hepatitis B surface antigen. Proc. Natl. Acad. Sci. U. S. A. 89, 3175 – 3179. Zhou, B., Wirsching, P., Janda, K.D., 2002. Human antibodies against spores of the genus Bacillus: a model study for detection of and protection against anthrax and the bioterrorist threat. Proc. Natl. Acad. Sci. U. S. A. 99, 5241 – 5246. Ziegler, C., Gopel, W., 1998. Biosensor development. Curr. Opin. Chem. Biol. 2, 585 – 591. Zwick, M.B., Labrijn, A.F., Wang, M., Spenlehauer, C., Saphire, E.O., Binley, J.M., Moore, J.P., Stiegler, G., Katinger, H., Burton, D.R., Parren, P.W., 2001. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J. Virol. 75, 10892 – 10905.