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Immunoassay
Immunoassay That T. Ngo BioProbe International Incorporation, Tustin, California, USA Recent developments in immunoassay are reviewed with emphasis on the chemistry of label conjugation, new solid phases, enzyme substrates for enzyme-linked immunosorbent assays, new assay techniques and detection systems. Current Opinion in Biotechnology 1991, 2:102-109
Introduction Immunoassay is a quantitative analytical technique that is characterized by a very high degree of sensitivity and specificity. It is based on the interaction between an antibody and its complementary antigen or hapten. Immunoassays can be classified into six groups [1o]. Group one includes the classic 'competitive' immunoassays of antigens or haptens, using limiting amounts of both antibody and labeled analyte. Group two is similar to Group one in that all reagents are present in limiting amounts. The difference is that the labeled reagent is the antibody rather than the analyte, and the immobilized reagent is a derivative of the analyte instead of the antibody. Nephelometric, turibidimetric and gravimetric precipitation assays comprise Group three. These assays involve a quantification of the sizes of molecular complexes and aggregates formed through the antibody-antigen interactions. Group four includes assays in which all reagents are used in excess. One example is a two-site sandwich immunoassay in which both the immobilized antibody and labeled antibody are used in excess. Similarly, in the one-site immunoenzymometric assay for hapten, a calculated excess of labeled antibody is first incubated with the analyte. Any labeled antibody that is not occupied by analyte is removed by the addition of an excess of immobilized analyte to which it binds. The amount of labeled antibody with bound analyte is then quantified. Group five includes assays for specific antibodies using immobiliT.ed antigens and labeled secondary antibodies. Finally, group six consists of labeled reagent immunoassays that do not require separation of bound from unbound reagents before the intensity of the signal is measured. These assays are generally known as separation-free or homogeneous assays, and involve the signal from the labeled reagent being modulated by the immunochemical binding reaction [2,3].
All immunoassays that require a separation of either antibody or antigen-bound labeled reagents from the unbound labeled reagents will have, at some points during the assay, a heterogeneous phase to facilitate the separation process. Accordingly, such assays are termed heterogeneous assays. With the possible exception of assays in Group three, all immunoassays require, in addition to antibody, labeled reagents. Additionally, heterogeneous assays require a solid phase. This review will focus on recent developments in novel detection systems and new assay principles as well as methods for preparing labeled reagents and solid phases.
Label conjugation A direct covalent attachment of monomeric labels to the amino groups of either antibody or antigen can be achieved for labels carrying any of the following groups: aldehyde, carboxyiate, isocyanate, imidate, epoxide, aziridine, activated halogen, sulfonyi chloride or 2-alkoxy N-methylpyridiniu [4]. For example, labels which carry maleimide groups can be reacted with the thiol groups of a protein. Similarly, the histidine or tyrosine groups of a protein can be linked to labels carrying/>aminophenyl groups. The advent of hybridoma technology has enabled the production of large quantities of monoclonal antibodies with defined specificities. This has created an opportunity for using monoclonal antibodies in vivo for either radio-immunotherapeutic or tumor imaging applications [5]. For instance, monodonal antibodies reactive with human glioma were successfully labeled with the 0t-emitting nuclide astatine 211At [6]. Although At is a halogen, proteins that are labeled with 211At using the procedure commonly used for protein iodination do not give a stable product. A new procedure of labeling employs N-succinimidyt, 31211At] astatobenzoate to acyiate the protein to give a stable product of high specific activ-
Abbreviations AMPPD--3•(2••spir•adamantane)•4-meth•xy•4-(3•-ph•sph•ry••xy)pheny•-1•2-di•xetane; CEA-carcinoembryonic antigen; DAB---p-dimethylaminobenzene; DNP~dinitrophenyl; EA~nzyme-acceptor; ED enzyme-donor; ELISA--enzyme-linked immunosorbent assay; HRP--horseradish peroxidase;Ig--immunoglobulin; Q ~ u a r t z crystal microbalance; SERRS---surface-enhanced resonance Raman scattering; SMCC---succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate; TSH--thyroid-stimulating hormone. 102
~) Current Biology Ltd ISSN 0958-1669
ImmunoassayNgo 103 ity (4 mCi/mg protein), within the time-frame consistent with the half-life of the radionuclide. The label, N-succinimidyl 31211At]astatobenzoate, was synthesized by reacting N-succ'mimidyl 3-(trimethylstannyl)benzoate with 211At in the presence of t-butyl hydroperoxide [6]. The radio-labeling of antibody with metal ion can be carried out with the aid of a bifunctional reagent which reacts with the antibody at one end whilst allowing a metal ion to chelate to the other end. A series of heterobifunctional reagents have been prepared. The new cross-linking reagents are capable of reacting at one end of the reagent with thiol groups of a protein, whilst the thiol groups at the other end of the reagent are protected. Upon deprotection, metal ions such as technetium-99m can chelate to these thiol groups [plo]. The labeling of antibody or antigen with polymeric and poly-functional molecules, such as enzymes or phycobiliproteins, is mostly carried out by using suitable cross-linking reagents. The use of homobifunctional reagents such as glutaraldehyde or bisimidate tends to result in a heterogeneous mixture containing molecular species of different sizes and specific activities. Ishikawa [7] has developed a procedure that uses heterobifunctional cross-linking reagents to prepare enzyme-labeled reagents of high specific activity and with a narrow molecular size distribution. The use of heterobifunctional cross-linking reagents allows a reaction sequence to be more easily controlled. The most useful of these reagents are succinimidyt-4(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and its more water-soluble sulfo-derivative (sulfo-SMCC), N-succinimidyl (4-iodoacetyi) aminobenzoate, succinimidyl-m-maleimidobenzoate and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). In addition to chemical methods for preparing labeled immunochemical reagents, certain enzyme-labeled antigens can be prepared by using gene fusion methods. For example, Peterhans et al. [8] have carried out the in-frame fusion of the gene encoding the amino-terminal 461 bp of human interferon-0t2 with the gene for Escherichia coli J3-galactosidase. The expression of this fused protein in E. coli produced a conjugate that was enzymatically active as well as immunochemically reactive towards a monoclonal antibody directed against the amino-terminal region of human interferon
produced a fusion protein of Protein A and neomycin phosphotransferase II in E. coll. This conjugate serves as an enzyme-labeled second antibody reagent by virtue of the Protein A moiety, and it has been used to establish an ultrasensitive enzymatic radioimmunoassay with a detection level of 10 femtogram (1 pg ml-1). Procedures for preparing luminescent chimeric proteins have been described [P2"]. These consist of a photoprorein (aequorin) and a second protein such as a light or heavy chain immunoglobulin (Ig), an antigenic peptide, avidin, or Protein A. For example, Lindbladh, Mosbach and B/ilow have recently prepared a genetically fused Protein A and luciferase conjugate and demonstrated its use in a bioluminescent immunoassay (personal communication). The advantages of using gene fusion techniques for producing enzyme-labeled antigens are the ability to precisely control the stoichiometric ratio of enzyme and antigen, the ease with which the conjugate can be produced in large amounts, and the relatively low cost of such a production process. For further discussion on genetic fusion techniques, see the reviews by Brodelius (this issue, pp 23-29) and Scouten (this issue, pp 37-43).
Solid phases The solid phase on which the antibodies or antigens are immobilized is one of the most important elements in heterogeneous immunoassays because it contributes towards the reproducibility, accuracy, precision and sensitivity of an assay [12]. The most popular solid phases used for enzyme immunoassays are microwell plates or strips, which are usually made of polyvinylchloride or polystyrene. Antibodies or protein antigens are mostly immobilized by passive adsorption. The mechanism of protein adsorption to solid phases is not very well understood. It is postulated that the first stage involves redistribution of the functional groups in order to overcome potential repulsive forces between the protein molecules and the solid phase. The adsorption of a protein to the solid phase requires the exposure of the hydrophobic functional groups of the protein. The second event in the adsorption process is thought to involve van der Waals' interactions brought about by local dipoles that occur in the contact zone. These interactions result in the dehydration and subsequent binding of the protein to the solid phase. The release of water or other solvent molecules from the surface of a protein leads to a considerable gain in translational entropy which could be the driving force in the passive adsorption of protein to a solid phase [13]. Gamma-irradiation of polystyrene surface can increase protein adsorptivity. Polar and hydrophobic polystyrene surfaces, however, have been found to achieve the best protein adsorption [13]. The effect of the surface charge of a solid phase on the non-specific binding of rabbit IgG in solid phase immunoassays has been investigated [14]. The binding capacity was found to increase with increasing positive charges on the solid phase surface. Kinetic
104 Analyticalbiotechnology studies on the adsorption of human IgG to potyvinylchloride microwells demonstrate that binding to the solid phase is controlled by rate-limiting diffusion onto the surface, followed by a rapid and irreversible adsorption [ 15]. Although the use of passive adsorption as a technique to immobilize antibody onto a solid phase has been significantly developed since its first introduction by Catt and Tregear in 1967, the concern over the stability of the non-covalently bound antibody has continued. This concern has stimulated research aimed at improving the bond stability between the immobilized protein and the solid phase. For example, Nunc Inc. have introduced microwell dishes which are coated with a secondary amine, methyl amine. Any ligand carrying a carboxylate group can thus be linked covalently with the aid of a condensing reagent such as a water soluble carbodiimide. Bio-Products Ltd recently introduced microwells with a hydrophilic surface of reactive aldehyde groups that enable the binding of amino group-containing ligands. Covalent immobilization of a protein through its amino acid residues always runs the risk of inactivating the biological function of the protein. Whenever possible it is, therefore, desirable to attach the protein via its non-amino acid portion, such as carbohydrate groups in the case of a glycoprotein. BioProbe International Inc. have introduced microwell plates (Avidplate-HZ) with hydrophilic hydrazide surface groups capable of binding to the Fc region of oxidized Igs. Unlike other microwell plates designed for covalent binding, no expensive and unstable cross-linking reagents are required. Furthermore, any immobilized antibodies have their antigen-binding sites facing away from the solid phase, so allowing maximal antigen binding. Bromoacetyl-functionalized polystyrene beads exhibit up to a 10-fold greater capacity for protein binding than unmodified polystyrene [16]. Moreover, no detectable dissociation occurs, unlike that observed with simple adsorption. The bromoacetyl coating is produced by chlorosulfonating the aromatic ring of the polystyrene surface followed by sulfonamide formation using excess di- or tri-amine and, lastly, reaction of the amine groups with bromoacetyl bromide. Proteins or other biologically active materials can be attached to the metal surface by first adding a linking coordinating polymer, such as polyimine, polythiol or poly(iminoethene)N-dithiocarboxylate. After chemisorption of these polymers to the metal surface, the amino or thiol groups that are accessible can be used to link proteins or other ligands either directly or through bifunctional reagents (see above). Using this method, the active ester derivative of biotin and rabbit IgG were immobilized with the aid of bifunctional cross-linkers such as SMCC or glutaraldehyde [P3.].
for about 75% of the total [1.]. The use of chromogenic substrates in enzyme immunoassays has been reviewed by Porstmann and Porstmann [17]. Recent developments include a stabilized chromogenic substrate solution for use with HRP, containing both 3,3',5,5'-tetramethylbenzidine and peroxide (Transgenic Sciences Inc.). A chemiluminogenic assay for HRP labeling has been greatly enhanced by the inclusion of some phenolic compounds to the substrate solution, including piodophenol, 1,6-dibromo-2-naphthol, firefly luciferin and 6-hydroxybenzothiazole [18]. In addition, novel chemiluminogenic enzyme substrates based on 1,2-dioxetanes for use with alkaline phosphatase and 13-galactosidase have been described [19]. These substrates are 3-(2'-spiroadamantane)-4-methoxy-4-(3'phosphoryloxy)phenyl-l,2-dioxetane (AMPPD), also known as 3-(4-methoxyspiro 1,2-dioxetane-3,2'tricyclo[3.31.1] decan-4-yl)phenyl phosphate, and 3-(2'-spiroadamantane)-4-methoxy-4- (3'- 13-D-galactopyranoyloxy) phenyl-l,2-dioxetane (AMPGD). The dephosphorylation of AMPPD by alkaline phosphatase generates a chemiluminescent glow which lasts for longer than 10minutes, and 0.01 attomol levels of alkaline phosphatase can be measured [19]. The synthetic and purification procedures for these chemilumigenic substrates have been described [p4o., P5.o]. A further chemiluminescent substrate is luminol, oxidised by xanthine oxidase. The chemiluminescence generated by this reaction can be greatly enhanced by the presence of iron-ethylenediamine tetra-acetic acid and sodium perborate in alkaline buffer [20]. Inclusion of folic acid or azahypoxanthine in the substrate solution eliminates inhibition of xanthine oxidase by allopurinol, a widely used hypo-uricemic agent [20]. In contrast, a highly sensitive colorimetric substrate system has been developed, for use with alkaline phosphatase [21]. NADP is used as a substrate, and alcohol dehydrogenase and diaphorase as the two auxiliary second enzymes that provide the amplification. The dephosphorylation of NADP by alkaline phosphatase produces NAD which serves as a substrate for alcohol dehydrogenase. A consequence of alcohol dehydrogenase catalysis is the reduction of NAD to NADH which, in the presence of diaphorase, reduces the colorless iodonitrotetrazolium violet to highly colored formazan. Concomitantly, NADH is oxidized to NAD, and can thus be recycled. The net result of this cycling of NAD and NADH between alcohol dehydrogenase and diaphorase in the presence of their respective substrates is an accumulation of formazan. Using this amplification system, as little as 0.005 attomol (approximately 3000 enzyme molecules) can be assayed, In this issue, Brodelius (pp 23-29) also discusses amplification by enzyme cycling.
Substrate systems New techniques and detection systems The last ten years have seen the increasing use of enzymes as non-isotopic labels in immunoassays. Horseradish peroxidase (HRP) and alkaline phosphatase are the two most widely used enzyme labels, accounting
Surface-enhanced resonance Raman scattering (SERRS) has been used to measure binding between biomolecules with mutual affinity, including antigen-antibody inter-
Immunoassay
actions [33]. Great increases in the intensity of Raman light scattering are observed when molecules are brought into close proximity with certain metal surfaces. These surface-enhanced Raman scattering effects can be made more intense if the frequency of the excitation light is the same as a major absorption band of the molecule being illuminated. This can result in greater than seven orders of magnitude enhancement in the observed intensity of Raman light scattering. This phenomenon is partially the result of electromagnetic field enhancements, and the signal diminishes rapidly at distances greater than 50-100A from the metal surface. Consequently, molecules a few hundred angstroms away from the metal surface make a negligible contribution to the observed signal. SERRS-active molecules, such as 2,4-dinitrifluorobenzene, 2-[4'-hydroxyphenylazo] benzoic acid, 4-dimethylaminoazobenzene-4'-isothiocyanate and erythrosin, can be conjugated to biomolecules as a reporter group. For instance, pdimethylaminobenzene (DAB) was covalently attached to an antibody to human thyroid-stimulating hormone (TSH), without loss of antibody activity [33]. The resulting conjugate has been used in a sandwich assay for TSH that employs silver surfaces coated with anti-TSH antibody to capture TSH antigen. The captured antigen, in turn, traps DAB-labeled antiTSH antibody. A linear relationship was observed between the intensity of the resultant SERES signals and the TSH antigen concentration over a range of 4-60 ~tlU ml-1. As well as their simplicity, SERRS immunoassays have several other advantages over enzyme-labeled immunoassays. First, the reporter dye molecules are both inexpensive and stable. Second, no signal development time is required (read-out begins immediately upon illumination), and yet the signal cannot over-develop (compared with optical absorbance) or self-quench (compared with fluorescence), but can be enhanced by increasing the number of reporter groups. Third, because reporters bound at the metal surface scatter light with molar etficiencies several orders of magnitude greater than those remaining in solution, it is not necessary to remove the bound reporters before reading the signal. Recently, Pharmacia Biosensor AB have introduced a real-time biospecific interactions analytical device based on surface plasmon resonance and a microfluidic system. When a light is focused on the interface of thin metal/glass layers at an incident angle, the free electrons of the thin metal layer at the metal sample interface are resonantly excited into oscillations. The quanta of the oscillations of surface electrons at the boundary as produced by light excitation are called plasmon. Light energy transfered to the surface electrons causes a decrease in the intensity of the reflected light. The incident angle of the light that excites the surface electrons is sensitive to the refractive index of the sample medium in contact with the thin metal layer. Changes in the refractive index of the meduim at the surface due to immunochemical binding reactions or other biospecific interactions will therefore result in changes in the angle of the incident light that can provide a maximal energy transfer from the light to the surface electrons (i.e. a minimal intensity of the reflected
Ngo
light). This system combines the advantages of a flow injection analysis with an optical monitoring unit based on surface plasmon resonance techniques. Interactions are detected as they occur on the surface of the sensor. A single injection analysis takes about 5--10min. New sensors
A silicon sensor-based filtration immunoassay, using a biotin-labeled capturing membrane able to detect antigens at sub-fmol level, has been described [22oo,23]. In a typical sandwich immunoassay, the target antigen sample is mixed with two sets of antibodies, one set labeled with biotin, and the other set with fluorescein, as well as streptavidin and urease-conjugated anti-fluorescein antibody. After the formation of 'pentameric' complexes (streptavidin: biotinyl-antibody: target antigen: fluorescein labeled-antibody: urease coniugated antifluorescein), the solution is filtered through a biotinlabeled membrane. Any unbound reagents are washed free, and the membrane is then pressed against a silicon sensor in a reader containing a solution of urea. The hydrolysis of urea by urease to give ammonia occurs in a microvolume of 0.5 pl, producing a significant pH change which in turn causes a change in surface potential on the sensor. The sensor is prepared from a silicon wafer coated with a proprietary insulating monolithic top layer and the silicon, insulator and electrolyte act as a capacitor. The circuit is closed by connecting the sensor, via an ammeter, and a device for varying the voltage across the capacitor, to an electrode in the electrolyte solution. Current is measured across the capacitor at various potentials. Measurements are made by addressing the back of the sensor with light from one of an army of light-emitting diodes. The photoresponse, together with the reverse potential of the device, causes the interface between the silicon and the insulating layer to charge up. This transient response results in a burst of current flowing in the external circuit. By modulating the light, an alternating current is generated which flows vertically through the device at that point and is measured by the ammeter; no direct current passes through the sensor. The magnitude of the alternating current depends upon the bias potential across the sensor. Accordingly, any chemical reaction that changes to the surface potential of the sensor will change this current [24]. Further discussion on new immtmosensors can be found in the review by Guilbault (this issue, pp 3-8). New devices
A novel enzyme immunoassay device (known under the trade mark of accuPINCH Test) has been developed by Hycor for rapid screening of drugs in human urine. The test has a limit of detection of several.ng per ml, and provides a preliminary indication of the presence of a particular drug. The enzyme immunoassay is competitive, using limiting reagent levels. First, an aliquot of urine specimen is added to the 'separation disk', situated at the bottom of a test well. This disc is coated with immobilized antibodies that bind the drug. Several drops of wash solu-
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Analytical biotechnolosy tion are then added and, after absorption, this is followed by exactly one drop of enzyme-labeled drug. The principle of the test is that the drug in the specimen competes with the enzyme-labeled drug for antibody sites on the separation disc. If the antibody sites are blocked on the separation disc, soluble enzyme-labeled drug is instead transferred to the 'detection disc' which is pressed against the disc two minutes later. The detection disc conrains a chromogen, and thus any color change indicates the presence of the drug. In the absence of drug, however, no color change occurs because the enzyme-labeled drug will bind soley to the separation disk. This device was developed using technology disclosed in a US patent [p6o]. Enzyme-labeling is also used as part of a novel immunochromatographic assay device [p7o]. The device consists of a strip of falter paper in a chromatographic development jar that contains a buffered substrate solution. Several clearly separated zones are present on the paper strip. The lowest zone, closest to the development solution, is usually a sample-receiving zone, and the next is the enzyme-labeled antibody zone. As the enzymelabeled antibody is not covalently bound to the paper, it can move as the result of capillary action of the development solution. The uppermost zone is an indicator zone, composed of immobilized antibodies covalently bound to the paper. The assay is initiated by sample application at the base of the strip, followed by placing the strip in the buffered substrate solution at the bottom of the chromatographic development jar. As the buffered substrate solution migrates up the strip, sample from the sample-receiving zone is mixed with the enzyme-labeled antibody, forming complexes. The complexes are then carried to the indicator zone, where the antigen of the complex is bound by the immobilized antibody which effectively stops further upward migration of the complexed antigen. Any free enzyme-labeled antibodies, continue to migrate upward with the solution above the indicator zone. A substrate must be selected that migrates slower than either the antigen or the enzyme-labeled antibody. In this way, the substrate ~/1 only be able to contact enzyme held back because of antigen-antibody reaction in the indicator zone. Consequently, the indicator zone will only become colored if antigen is present in the sample.
levels [26]. Genes were constructed to allow expression of two separate polypeptide fragments; enzyme-donor (ED) and enzyme-acceptor (EA). These fragments were designed so that, when mixed together, they spontaneously recombine to form active ~3-galactosidase. Ligands, in this case vitamin B12, can be attached chemically to the ED peptide to control the extent of recombination with the EA by their interaction with the anti-ligand antibody. Analyte ligands in the sample compete with the ligand-ED conjugate for a limiting amount of antibody binding sites. The third homogeneous enzyme immunoassay detects carcinoembryonic antigen (CEA) [27]. Hybrid bispecific monoclonal antibodies reacting with CEA and with the E. coli ffgalactosidase were produced by hybridoma fusion or chemical linkage of half-antibodies. The original anti-J3-galactosidase antibody was shown to protect the enzyme from thermal denaturation. By combining this antibody with two different non-competing anti-CEA antibodies, two antibody populations that were useful for homogeneous enzyme immunoassays could be selected. The activity of a number of enzymes entrapped in surfactant-reversed micelles in organic solvents can be modulated by the degree of hydration, i.e. the water/surfactant molar ratio [28,29]. The activity of some enzymes is greatly increased. Maximal enzyme activity is observed when the radius of the inner cavity of reversed micelles is equaled to that of the globular protein entrapped. Thus, it appears that ermyme activity is controlled by the ratio of miceUe to enzyme size. In an immunoassay situation, the effective dimension of the enzyme-labeled analyte in a reversed micelle can be altered by adding specific anti-analyte antibodies. These bind the enzyme-labeled analyte and form complexes with dimensions larger than the original conjugate; consequently, the activity of the enzyme is expected to decrease. The decrease in the enzyme activity should be restored by addition of the ligand. This prediction was realized by using a HRP-labeled thyroxine conjugate in an anti-thyroxine antibody system. A standard curve for thyroxine in the range of 2-20 nM was established [29].
Reduction of non-specific binding New principles Three notable homogeneous enzyme immunoassays have been developed in the past year. The first, which detects plasma protein C, uses labeled antibody [25]. The assay is based on a unique property of HRP; in the presence of excess hydrogen peroxide (the enzyme substrate), the oxidation of the chromogen is catalyzed much faster by an aggregated enzyme than by a monomer. In the presence of antigens, HRP-labeled antibodies form high molecular weight aggregates. A highly sensitive and rapid homogeneous enzyme immunoassay has been described that uses recombinant fragments of ~-galactosidase to determine vitamin B12
Conventional enzyme immunoassays for antibodies in serum are carried out by incubating the serum with immobilized antigens (Group five immunoassays; see above) to trap specificIgs. The trapped Igs are then reacted with enzyme-labeledanti-Ig antibody to estimate the amount of antibodyspecificallybound. The sensitivity of this assay is limited by non-specific binding of other Igs in the serum to the solid phase. In order to overcome this limitation, a novel, highly sensitive immunecomplex transfer enzyme immunoassay has been developed [30,31,32"]. The assay is performed as follows; an antigen is labeled with a hapten, such as a dinitrophenyl group (DNP), and the resulting DNP-labeled antigen is incubated with the test serum and an anti-DNP antibody
Immunoassay Ngo immobilized on the solid phase. After washing the solid phase to remove much of the nonspecifically bound protein, the DNP-labeled antigens are displaced from the solid base using a high concentration of DNP-lysine. The eluted solution is incubated with solid phase anti-antigen antibodies, which bind specifically. After a further brief wash, the solid phase is exposed to an enzyme-labeled anti-Ig antibody. The amount of enzyme label bound to the solid phase indicates the quantity of specific antibody in the test serum. The scheme shown in Fig. 1 depicts the reactions involved in the measurement of human anti-thyroglobulin IgG. Using such a procedure, the detection limit of the antibody in serum is 5.5 ngm1-1
tion signal of enzyme immunoassays [34]. In one example, biotin-conjugated phenolic compounds were used as reporter molecules and their deposition was catalyzed by HRP-labeled reagents. This, in turn, promoted the capture of a large number of streptavidin-labeled enzyme conjugates, resulting in amplification of the enzymatic signal.
Sample amplification
The detection of biospecific interactions including antibody-antigen reactions, with eLlipsometry, is greatly amplified by using nanometer-size silica particles [35]. Another means of amplifying the detection signal in ellipsometric measurements would be to use enzymelabeled reagent to cause the deposition of insoluble products on the detector surface from soluble enzyme substrates, as previously described [ps°o]. In a liposomebased immunoassay, it has been demonstrated that avidin molecules are able to bind membrane-bound biotin on groups of liposomes derived from biotinylated phospholipid molecules. The resultant liposome-attached avidin is also able to bind additional biotins or biotinylated antibodies from the solution [36]. These derivatized liposomes were highly reactive toward multivalent antigens; a linear increase in light scattering was recorded between 1 and 10 pmol of antigen. The data show that liposomes carrying biotinylated phospholipid could be a useful generic reagent for immunoassays. A further advantage of liposomes is that each one can entrap up to 100 000 marker enzyme molecules. This amplification enhances signal detection. Antigens on a solid phase can also be amplified by using polyacrylic polyhydrazide polymers [37]. The procedure involves periodate oxidation of the carbohydrate groups of antibodies that are already immunochemically bound to antigens attached to the solid phase. In this way, the complexes of antigen and bound oxidized IgG are exposed to polyhydrazide polymers. After rinsing, the solid phase is placed in contact with a periodate oxidized giyco-enzyme such as HRP, and
The detection signal of an immunoassay can be enhanced by enzymatically amplifying the number of reporter molecules, or by physically amplifying the size and therefore the quantity and signal of reporter molecules. For example, in immunoassays that use piezoelectric oscillator detection, the change in resonant frequency of the detector can be correlated with the amount of mass change on the surface of the quartz crystal [P8°.]. If the quartz crystal microbalance and the coated layer obey rigid-layer behavior, the mass change can be determined from the frequency change using the Sauerbry relationship: df = - ( 2 fo dm)/(A Jpu), where df is the measured frequency shift, fo the parent frequency of the quartz crystal, dm the mass change, A the piezoelectrically active area, p the density of quartz and u the shear modulus. The mass change can be amplified enzymatically. Thus, in a quartz crystal microbalance (QCM) immunoassay, the detection of the binding of an analyte to an antibody-coated surface on a QCM is amplified by an enzyme-labeled conjugate. This conjugate is capable of catalyzing the conversion of soluble substrates to insoluble products that accumulate on the surface of the QCM and therefore lead to a greater mass change. Consequently there is a greater change in the resonant frequency [P8°-]. A novel signal amplification method based on catalyzed reporter deposition has been used to enhance the detec-
The detection signal of immunoassays can also be enhanced by using mass-amplified reagents. For example, in a piezoelectric crystal-based specific binding assay, colloidal sol was used as a label in order to increase the mass of binding reagents and to amplify the response to analyte [p9o].
H Anti-TG + SP-Anti-DNP TG-DNP
H.Anti-TG:TG-DNP:SP-Anti-DNP
DNP-Lysine
SP-Anti-TG ,,~,,~,........
H.Anti-TG:TG-DNP
HRP-R-Anti H.IgG (H.Anti-TG:TG-DNP):SP-Anti-TG
Fig. 1. Use of the technique of im[(H.Anti-TG:TG-DN P):SP-Anti-TG]:HRP-R-Anti Substrate
lgG
Color
munocomplex enzyme imrnunoassay to measure levels of human antithyroglobulin IgG. DNP, dinitrophenyl; H, human; HRP, horseradish peroxidase; Ig, irnmunoblobulin; R, rabbit; SP, solid phase; TG, thyroglobulin.
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Analytical biotechnology the amount of enzyme that can bind is measured with the usual peroxidase substrates.
sion Protein of Protein A and Neomycin Phosphotransferase II in Two-chamber-well Mierotiter Plates. Anal B t ~ 1990, 187:89-93. 12.
KEMENYDM, CHALLACOMBESJ: Microtitre Plates and Other Solid-phase Supports. In ELISA and Other Solid Phase lmmunoassays edited by Kemeny DM, Challacombe SJ. Chichester: John Wiley & Sons, 1988, 31-55.
13.
RASMUSSENSE: Solid Phases and Chemistries. In Comp/ementary Immunoassay~ edited by Collins WP. Chichester: John Wiley & Sons, 1988, 43--55.
14.
GRAVESHCB: The Effect of Surface Charge on Non-specilic Binding of Rabbit Immunoglobulin-G in Solid-phase Immunoassays. J Immunol Methods 1988, 111:157-166.
15.
MCGINIAYPB, BARDSLEYWG: The Kinetics of Adsorption of Human Immunoglobulin-G to Polyvinyl Chloride Enzymelinked Immunoassorbent-Assay Vessel Wells. B/ochem J 1989, 261:715-720.
16.
PETERMANJH, TARCHAPJ, CHO VP, BUTLERJE: The Im~unochemistry of Sandwich-ELISAs, IV: The Antigen Capture Capacity of Antibody Covalently Attached to Bromoacetyl Surface-functionalized Polystyrene. J Immunol Methods 1988, 111:271-275.
17.
PORSTMANNB, PORSTMANNT: Chromogenic Substrates for Enzyme lmmunoassay. In NontsotoIn'c Immunoassay edited by Ngo TF. New York: Plenum Press, 1988, 57-84.
18.
KRICKALJ, STOTT RAW, THORPE GHG: Enhanced Chemiluminescence Enzyme Immunoassays. In Complementary Immunoassays edited by Collins WP. Chichester: John Wiley & Sons, 1988, 169-179.
19.
BRONSTEINI, EDWARDS B, VOYTAJC: 1,2-Dioxetanes: Novel Chemilumineseent Enzyme Substrates. Applications to Immunoassays. J Biolumin Chemilumin 1989, 4:99-111.
20.
BARETA, FERT V, AUMAI~ J: Application of Long-term Enhanced Xanthine Oxidase-induced Luminescence in Solidphase Immunoassays. Anal B/ocbem 1990, 187:20-26. JOHANNSSONA, BATES DI2 ELISA and Other solid Phase Immunoassays, edited by Kemeny DM, ChaUacombe SJ. Chichester: John Wiley & Sons, 1988, 85-106.
Conclusion
Increasing numbers of immunoassays are being dev-eloped using non-isotopic labels, such as enzymes, fluorescent molecules, and chemi- and bio-luminescent molecules. Highly sensitive non-labeled immunoassay techniques are emerging rapidly. Efforts are continuing to be directed towards speeding up, simplifying, and automating assay protocols.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •• of outstanding interest 1. GODLINGJP: A Decade of Deveinpmem in Immunoassay • Methodology. Clin Gbem 1990, 36:1408-1427. A succint and comprehensive summary of immunoassay methods developed in the last 10 years, showing the future trends.
2.
NGO TF: Enzyme-mediated Immunoassay: an Overview. In Enzym~ mediated lmmunoassay edited by Ngo TT, Leuhoff HM. New York: Plenum Press, 1985, pp 3-32.
3.
GOULDBJ, MARKSV: Recent Developments in.Enzyme Immunoassays in Nonisotopic lmmunoasay, edited by Ngo "IT. New York: Plenum Press, 1988, pp 3-26.
4.
NGO TT: Enzyme Systems and Enzyme Conjugates for Solid-phase ELIS& In Immunochemistry of Solic#phase Immunoassay edited by Butler JE. Beca Raton: CRC Press, 1991.
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VOGELC-W: (ed) Immunoconjugates, Antibody Conjugates in Radioimaging and Therapy of Cancer. Oxford: Oxford University Press, 1987.
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ZALLrrSKYMR, GARG PK, FRIEDMAN HS, BIGNER DD: Labeling Monodonal Antibodies and F(ab)'2 Fragments with AParticle-emitting Nuclide Astatine-211: Preservation of Immunoreactivity and In Vivo Localizing Capacity. Proc Natl Acad Sci USA 1989, 86:7149-7153.
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ISHIKAWAE, HASHIDA S, KOHNO T, TANAKA K: Methods for Enzyme-labeling of Antigens, Antibodies and their Fragments. In Nonisotopic Immunoassay edited by Ngo TT. New York: Plenum Press, 1988, 27-55.
PETERHANSA, MECKLENBURGM, MEUSSDOERFFERF, MOSBACHK: A Simple Competitive Enzyme-linked Immunosorbent Assay UsIng Antigen-~-Galactosidase Fusions. Anal Biochem 1987, 163:470-475. LINDBLADH C, PERSSON M, BULOW L, STAHL S, MOSBACH K: The Design of a Simple Competitive ELISA Using Human Proinsulin-alkaline Phosphatase Conjugates Prepared by Gene Fusion. Biochem Biophys Res Commun 1987, 149:607~14.
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KOBATAKEE, NISmMOmY, IKAmYAMAY, AIZAW^M, KATOS: Application of a Fusion Protein, Metapyrocatechase/Protein A to an Enzyme Immunoassay. Anal Biochem 1990, 186:14-18.
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HUNGERHD, FLACHMEmRC, SCI-LM~TG, BEHRENDTG, COUTELLE C: Ultrasensitive Enzymatic Radioimmunoassay Using a Fu-
OLSONJD, PANF~ PR, ARMENTAR, FEMMEL i ~ , MEmCK H, GUMPRZJ, GOLTZM, ZUK RF: A silicon Sensor-based Filtration Immunoassay using Biotin-mediated Capture. J Immun Meth 1990, 134:71-79. Methods for preparing the biotin membrane, the assay principle and the light-addressable potenUometric sensor are described and discussed in detail. 23.
BmGGSJ, KUNGVT, GOMEZB, KASPERKC, NAGAINISPA, 1VIASINO RS, RICE IS, ZUK RF, CHAZAROSSIANVE: Sub-femtomole Quantitation of Proteins with Threshold, for the Biopharmaceutical industry. Biotechniques 1990, 9:598-606.
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HAFEMANDG, PARCEJW, MCCONNELL HM: Light-addressable Potentiometric Sensor for Biochemical Systems. Sc/ence 1988, 240:1182-1185.
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SUZUKIK, HAYASHIT, KOYAMAM, YOSHIMURAT, SHIMAMOTOM, KIMURAN, KITAM: Rapid Homogeneous Enzyme Immunoassay of Plasma Protein C. Clin C~'m Acta 1989, 184:227-234.
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KHANNAPI, DWORSCHACKRT, MANNINGWB, HARMSJD: A New Homogeneous Enzyme Immunoassay Using Recombinant Enzyme Fragments. Clin Chim Acta 1989, 185:231-240.
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GOROG G, GANDOIa~ A, PARADISl G, ROLLEm E, KLASEN E, DESSI V, STROM R, CELADAF: Use of Bispecific Hybrid Antibodies for the Development of a Homogeneous Enzyme Immunoassay. J Immunol Methods 1989, 123:131-140.
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MARTINEKK, LEVASHOVAV, KLYACHKO N, KHMELNITSKI YI~ BEREZlN IV: Micellar Enzymology. Eur J Biochem 1986, 155:453--468.
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KABANOVAV, KHRUTSKAYAi i , EREMIN SA, KLYACHKONL, I.EVASHOVAV: A New Way in Homogeneous Immunoassay: Reversed MiceUar Systems as a Medium for Analysis. Anal Biocbem 1989, 181:145-148.
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KOHNOT, ISHIKAW^E: A Novel Enzyme Immunoassay of Anti-insulin IgG in Guinea Pig Serum. Biochem Biopbys Res Commun 1987, 147:644-649.
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ISHIKAWAE, HASHIDAS, TANAKAK, KOHNO T: Methodological Advances In Enzymology. Development and Applications of Ultrasensitive Enzyme Immunoassays for Antigens and Antibodies. Clin Chim Acta 1989, 185:223-230.
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KOHNOT, MrrSUKAW^T, MATSUKURAS, TSUNETOSHIY, ISHIKAWA E: Measurement of Anti-thyroglobulin IgG in Serum by Novel and Sensitive Immune Complex Transfer Enzyme Immunoassay. Clin Biochem 1989, 22:277-284. Theory and practical details of the development of sensitive enzyme immunoassays for quantifying specific antibodies, as exemplified by the measurement of anti-thyroglobulin IgG in serum. 33.
ROHR "rE, CO'I'rON T, FAN N, TARCHA PJ: Immunoassay Employing Surface-enhanced Raman Spectroscopy. Anal BR~hem 1989, 182:388-398.
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BOBROWMN, HARRISTD, SHAUGHNESSYK, LrlT GJ: Catalyzed Reporter Deposition, a Novel Method of Signal Amplification. Application to Immunoassays. J Immunol Methods 1989, 125:279-285.
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MANDENIUSCF, MOSBACHK: Detection of Biospecific Interactions using Amplified Elipsometry. Anal Biochem 1988, 170:68-72.
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PLANTAL, BR1ZGIS M, I.OCASIO-BROWN I., DURST RA: Generic Lip•some Reagent for Immunoassays. Anal Biochem 1989, 176:420-426.
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HEIMGARTNERU, KOZULIC B, MOSBACH K: Polyacrylic Polyhydrazides as Novel Reagents for Detection of Antibodies in Immunoblotting Assays. J Immunol Methods 1990, 132:239-245.
Annotated patents • 0,
of interest of outstanding interest
P1 •
DEANRT, BOUTIN RH, WEBER RW: Bifunctional Coupling Agents and Radionuclide-labeled Compositions Prepared Therefrom. PCT 1989, W • 89/12625.
Preparation and application of bifunctional coupling agents that have a protein reactive group and protected •hi•Is which, upon deprotection can chelate radionuclldes. P2 KENTENJH, CASADEIJ, POWELL MJ: Luminescent Chimeric j Proteins. PCT 1989, W • 89/09393. Photoproteins have been fused genetically with binding proteins and used in highly sensitive immunoassays. P3 CHADWICKAT, HUDSON MJ: Bonding to Metal Surfaces. • Eur Pat Appl 1989, Pub No. 0 3339 821. Interposed coordinating polymers were first bonded to the metal surface, and then biologically active molecules were linked directly or through crosslinking reagents to other parts of the polymer, P4 •0
EDWARDS B, BRONSTEIN I, LAIRD A, VOYTA JC: Novel Chemiluminescent Fused Polycyclic Ring-containing 1,2Dioxetanes and Assays In which they are used. PCT 1990, W • 9O/00164. Procedures for preparing novel chemiluminogenic enzyme substrates and their applications in biological binding reactions. P5 EDWARDSB, VOYTAJC: Purification of Stable Water-soluble •0 Dioxetane. PCT 1990, W • 90/02742. Chromatographic procedures for purifying chemiluminogenic enzyme subs•rates. P6 GUIREPE, CHUDZIK SJ: Field Assay for Ligands. 1989. US • Pat No. 4,826,759. Reagents were kept apart by depositing them individually in separated zones on a strip. The reagents could be brought in contact by appropriate folding of the strip. P7 BAKERTS, PERRY MJ, FLEMING IM: Binding Assay Device. • 1989. UK Pat GB 2191578. An immunochromatrographic assay method performed on a chromat•gram support with several defined zones, one being the sample receiving zone. Other zones include binding and indicator zones. P8 ~le
WARDMD, EBERSOLE RC: Enzymatically Amplified Piezoelectric Specific Binding Assay. 1989. PCT W • 89/09937. mass change on the surface of a quartz crystal balance was amplified by deposition of insoluble products, generated bT~enzymatic conversion of soluble subs•rates by ermyme-labeled reagents. The change in resonant frequency is consequently amplified. P9 WARDMD, EBERSOLE RC: Piezoelectric Specific Binding As• say with Mass Amplified Reagents. 1989. PCT W • 89/09938. A piezoelectric crystal-based specific binding assay in which a colloidal sol is used to amplify the mass of binding reagents and, thererfore, proportionally increases the change in the resonant frequency.
TY Ngo, BioProbe International Incorporation, 14272 Franklin Avenue, Tustin, California 91680, USA.
109
Introduction Immunoassay is a quantitative analytical technique that is characterized by a very high degree of sensitivity and specificity. It is based on the interaction between an antibody and its complementary antigen or hapten. Immunoassays can be classified into six groups [1o]. Group one includes the classic 'competitive' immunoassays of antigens or haptens, using limiting amounts of both antibody and labeled analyte. Group two is similar to Group one in that all reagents are present in limiting amounts. The difference is that the labeled reagent is the antibody rather than the analyte, and the immobilized reagent is a derivative of the analyte instead of the antibody. Nephelometric, turibidimetric and gravimetric precipitation assays comprise Group three. These assays involve a quantification of the sizes of molecular complexes and aggregates formed through the antibody-antigen interactions. Group four includes assays in which all reagents are used in excess. One example is a two-site sandwich immunoassay in which both the immobilized antibody and labeled antibody are used in excess. Similarly, in the one-site immunoenzymometric assay for hapten, a calculated excess of labeled antibody is first incubated with the analyte. Any labeled antibody that is not occupied by analyte is removed by the addition of an excess of immobilized analyte to which it binds. The amount of labeled antibody with bound analyte is then quantified. Group five includes assays for specific antibodies using immobiliT.ed antigens and labeled secondary antibodies. Finally, group six consists of labeled reagent immunoassays that do not require separation of bound from unbound reagents before the intensity of the signal is measured. These assays are generally known as separation-free or homogeneous assays, and involve the signal from the labeled reagent being modulated by the immunochemical binding reaction [2,3].
All immunoassays that require a separation of either antibody or antigen-bound labeled reagents from the unbound labeled reagents will have, at some points during the assay, a heterogeneous phase to facilitate the separation process. Accordingly, such assays are termed heterogeneous assays. With the possible exception of assays in Group three, all immunoassays require, in addition to antibody, labeled reagents. Additionally, heterogeneous assays require a solid phase. This review will focus on recent developments in novel detection systems and new assay principles as well as methods for preparing labeled reagents and solid phases.
Label conjugation A direct covalent attachment of monomeric labels to the amino groups of either antibody or antigen can be achieved for labels carrying any of the following groups: aldehyde, carboxyiate, isocyanate, imidate, epoxide, aziridine, activated halogen, sulfonyi chloride or 2-alkoxy N-methylpyridiniu [4]. For example, labels which carry maleimide groups can be reacted with the thiol groups of a protein. Similarly, the histidine or tyrosine groups of a protein can be linked to labels carrying/>aminophenyl groups. The advent of hybridoma technology has enabled the production of large quantities of monoclonal antibodies with defined specificities. This has created an opportunity for using monoclonal antibodies in vivo for either radio-immunotherapeutic or tumor imaging applications [5]. For instance, monodonal antibodies reactive with human glioma were successfully labeled with the 0t-emitting nuclide astatine 211At [6]. Although At is a halogen, proteins that are labeled with 211At using the procedure commonly used for protein iodination do not give a stable product. A new procedure of labeling employs N-succinimidyt, 31211At] astatobenzoate to acyiate the protein to give a stable product of high specific activ-
Abbreviations AMPPD--3•(2••spir•adamantane)•4-meth•xy•4-(3•-ph•sph•ry••xy)pheny•-1•2-di•xetane; CEA-carcinoembryonic antigen; DAB---p-dimethylaminobenzene; DNP~dinitrophenyl; EA~nzyme-acceptor; ED enzyme-donor; ELISA--enzyme-linked immunosorbent assay; HRP--horseradish peroxidase;Ig--immunoglobulin; Q ~ u a r t z crystal microbalance; SERRS---surface-enhanced resonance Raman scattering; SMCC---succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate; TSH--thyroid-stimulating hormone. 102
~) Current Biology Ltd ISSN 0958-1669
ImmunoassayNgo 103 ity (4 mCi/mg protein), within the time-frame consistent with the half-life of the radionuclide. The label, N-succinimidyl 31211At]astatobenzoate, was synthesized by reacting N-succ'mimidyl 3-(trimethylstannyl)benzoate with 211At in the presence of t-butyl hydroperoxide [6]. The radio-labeling of antibody with metal ion can be carried out with the aid of a bifunctional reagent which reacts with the antibody at one end whilst allowing a metal ion to chelate to the other end. A series of heterobifunctional reagents have been prepared. The new cross-linking reagents are capable of reacting at one end of the reagent with thiol groups of a protein, whilst the thiol groups at the other end of the reagent are protected. Upon deprotection, metal ions such as technetium-99m can chelate to these thiol groups [plo]. The labeling of antibody or antigen with polymeric and poly-functional molecules, such as enzymes or phycobiliproteins, is mostly carried out by using suitable cross-linking reagents. The use of homobifunctional reagents such as glutaraldehyde or bisimidate tends to result in a heterogeneous mixture containing molecular species of different sizes and specific activities. Ishikawa [7] has developed a procedure that uses heterobifunctional cross-linking reagents to prepare enzyme-labeled reagents of high specific activity and with a narrow molecular size distribution. The use of heterobifunctional cross-linking reagents allows a reaction sequence to be more easily controlled. The most useful of these reagents are succinimidyt-4(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and its more water-soluble sulfo-derivative (sulfo-SMCC), N-succinimidyl (4-iodoacetyi) aminobenzoate, succinimidyl-m-maleimidobenzoate and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). In addition to chemical methods for preparing labeled immunochemical reagents, certain enzyme-labeled antigens can be prepared by using gene fusion methods. For example, Peterhans et al. [8] have carried out the in-frame fusion of the gene encoding the amino-terminal 461 bp of human interferon-0t2 with the gene for Escherichia coli J3-galactosidase. The expression of this fused protein in E. coli produced a conjugate that was enzymatically active as well as immunochemically reactive towards a monoclonal antibody directed against the amino-terminal region of human interferon
produced a fusion protein of Protein A and neomycin phosphotransferase II in E. coll. This conjugate serves as an enzyme-labeled second antibody reagent by virtue of the Protein A moiety, and it has been used to establish an ultrasensitive enzymatic radioimmunoassay with a detection level of 10 femtogram (1 pg ml-1). Procedures for preparing luminescent chimeric proteins have been described [P2"]. These consist of a photoprorein (aequorin) and a second protein such as a light or heavy chain immunoglobulin (Ig), an antigenic peptide, avidin, or Protein A. For example, Lindbladh, Mosbach and B/ilow have recently prepared a genetically fused Protein A and luciferase conjugate and demonstrated its use in a bioluminescent immunoassay (personal communication). The advantages of using gene fusion techniques for producing enzyme-labeled antigens are the ability to precisely control the stoichiometric ratio of enzyme and antigen, the ease with which the conjugate can be produced in large amounts, and the relatively low cost of such a production process. For further discussion on genetic fusion techniques, see the reviews by Brodelius (this issue, pp 23-29) and Scouten (this issue, pp 37-43).
Solid phases The solid phase on which the antibodies or antigens are immobilized is one of the most important elements in heterogeneous immunoassays because it contributes towards the reproducibility, accuracy, precision and sensitivity of an assay [12]. The most popular solid phases used for enzyme immunoassays are microwell plates or strips, which are usually made of polyvinylchloride or polystyrene. Antibodies or protein antigens are mostly immobilized by passive adsorption. The mechanism of protein adsorption to solid phases is not very well understood. It is postulated that the first stage involves redistribution of the functional groups in order to overcome potential repulsive forces between the protein molecules and the solid phase. The adsorption of a protein to the solid phase requires the exposure of the hydrophobic functional groups of the protein. The second event in the adsorption process is thought to involve van der Waals' interactions brought about by local dipoles that occur in the contact zone. These interactions result in the dehydration and subsequent binding of the protein to the solid phase. The release of water or other solvent molecules from the surface of a protein leads to a considerable gain in translational entropy which could be the driving force in the passive adsorption of protein to a solid phase [13]. Gamma-irradiation of polystyrene surface can increase protein adsorptivity. Polar and hydrophobic polystyrene surfaces, however, have been found to achieve the best protein adsorption [13]. The effect of the surface charge of a solid phase on the non-specific binding of rabbit IgG in solid phase immunoassays has been investigated [14]. The binding capacity was found to increase with increasing positive charges on the solid phase surface. Kinetic
104 Analyticalbiotechnology studies on the adsorption of human IgG to potyvinylchloride microwells demonstrate that binding to the solid phase is controlled by rate-limiting diffusion onto the surface, followed by a rapid and irreversible adsorption [ 15]. Although the use of passive adsorption as a technique to immobilize antibody onto a solid phase has been significantly developed since its first introduction by Catt and Tregear in 1967, the concern over the stability of the non-covalently bound antibody has continued. This concern has stimulated research aimed at improving the bond stability between the immobilized protein and the solid phase. For example, Nunc Inc. have introduced microwell dishes which are coated with a secondary amine, methyl amine. Any ligand carrying a carboxylate group can thus be linked covalently with the aid of a condensing reagent such as a water soluble carbodiimide. Bio-Products Ltd recently introduced microwells with a hydrophilic surface of reactive aldehyde groups that enable the binding of amino group-containing ligands. Covalent immobilization of a protein through its amino acid residues always runs the risk of inactivating the biological function of the protein. Whenever possible it is, therefore, desirable to attach the protein via its non-amino acid portion, such as carbohydrate groups in the case of a glycoprotein. BioProbe International Inc. have introduced microwell plates (Avidplate-HZ) with hydrophilic hydrazide surface groups capable of binding to the Fc region of oxidized Igs. Unlike other microwell plates designed for covalent binding, no expensive and unstable cross-linking reagents are required. Furthermore, any immobilized antibodies have their antigen-binding sites facing away from the solid phase, so allowing maximal antigen binding. Bromoacetyl-functionalized polystyrene beads exhibit up to a 10-fold greater capacity for protein binding than unmodified polystyrene [16]. Moreover, no detectable dissociation occurs, unlike that observed with simple adsorption. The bromoacetyl coating is produced by chlorosulfonating the aromatic ring of the polystyrene surface followed by sulfonamide formation using excess di- or tri-amine and, lastly, reaction of the amine groups with bromoacetyl bromide. Proteins or other biologically active materials can be attached to the metal surface by first adding a linking coordinating polymer, such as polyimine, polythiol or poly(iminoethene)N-dithiocarboxylate. After chemisorption of these polymers to the metal surface, the amino or thiol groups that are accessible can be used to link proteins or other ligands either directly or through bifunctional reagents (see above). Using this method, the active ester derivative of biotin and rabbit IgG were immobilized with the aid of bifunctional cross-linkers such as SMCC or glutaraldehyde [P3.].
for about 75% of the total [1.]. The use of chromogenic substrates in enzyme immunoassays has been reviewed by Porstmann and Porstmann [17]. Recent developments include a stabilized chromogenic substrate solution for use with HRP, containing both 3,3',5,5'-tetramethylbenzidine and peroxide (Transgenic Sciences Inc.). A chemiluminogenic assay for HRP labeling has been greatly enhanced by the inclusion of some phenolic compounds to the substrate solution, including piodophenol, 1,6-dibromo-2-naphthol, firefly luciferin and 6-hydroxybenzothiazole [18]. In addition, novel chemiluminogenic enzyme substrates based on 1,2-dioxetanes for use with alkaline phosphatase and 13-galactosidase have been described [19]. These substrates are 3-(2'-spiroadamantane)-4-methoxy-4-(3'phosphoryloxy)phenyl-l,2-dioxetane (AMPPD), also known as 3-(4-methoxyspiro 1,2-dioxetane-3,2'tricyclo[3.31.1] decan-4-yl)phenyl phosphate, and 3-(2'-spiroadamantane)-4-methoxy-4- (3'- 13-D-galactopyranoyloxy) phenyl-l,2-dioxetane (AMPGD). The dephosphorylation of AMPPD by alkaline phosphatase generates a chemiluminescent glow which lasts for longer than 10minutes, and 0.01 attomol levels of alkaline phosphatase can be measured [19]. The synthetic and purification procedures for these chemilumigenic substrates have been described [p4o., P5.o]. A further chemiluminescent substrate is luminol, oxidised by xanthine oxidase. The chemiluminescence generated by this reaction can be greatly enhanced by the presence of iron-ethylenediamine tetra-acetic acid and sodium perborate in alkaline buffer [20]. Inclusion of folic acid or azahypoxanthine in the substrate solution eliminates inhibition of xanthine oxidase by allopurinol, a widely used hypo-uricemic agent [20]. In contrast, a highly sensitive colorimetric substrate system has been developed, for use with alkaline phosphatase [21]. NADP is used as a substrate, and alcohol dehydrogenase and diaphorase as the two auxiliary second enzymes that provide the amplification. The dephosphorylation of NADP by alkaline phosphatase produces NAD which serves as a substrate for alcohol dehydrogenase. A consequence of alcohol dehydrogenase catalysis is the reduction of NAD to NADH which, in the presence of diaphorase, reduces the colorless iodonitrotetrazolium violet to highly colored formazan. Concomitantly, NADH is oxidized to NAD, and can thus be recycled. The net result of this cycling of NAD and NADH between alcohol dehydrogenase and diaphorase in the presence of their respective substrates is an accumulation of formazan. Using this amplification system, as little as 0.005 attomol (approximately 3000 enzyme molecules) can be assayed, In this issue, Brodelius (pp 23-29) also discusses amplification by enzyme cycling.
Substrate systems New techniques and detection systems The last ten years have seen the increasing use of enzymes as non-isotopic labels in immunoassays. Horseradish peroxidase (HRP) and alkaline phosphatase are the two most widely used enzyme labels, accounting
Surface-enhanced resonance Raman scattering (SERRS) has been used to measure binding between biomolecules with mutual affinity, including antigen-antibody inter-
Immunoassay
actions [33]. Great increases in the intensity of Raman light scattering are observed when molecules are brought into close proximity with certain metal surfaces. These surface-enhanced Raman scattering effects can be made more intense if the frequency of the excitation light is the same as a major absorption band of the molecule being illuminated. This can result in greater than seven orders of magnitude enhancement in the observed intensity of Raman light scattering. This phenomenon is partially the result of electromagnetic field enhancements, and the signal diminishes rapidly at distances greater than 50-100A from the metal surface. Consequently, molecules a few hundred angstroms away from the metal surface make a negligible contribution to the observed signal. SERRS-active molecules, such as 2,4-dinitrifluorobenzene, 2-[4'-hydroxyphenylazo] benzoic acid, 4-dimethylaminoazobenzene-4'-isothiocyanate and erythrosin, can be conjugated to biomolecules as a reporter group. For instance, pdimethylaminobenzene (DAB) was covalently attached to an antibody to human thyroid-stimulating hormone (TSH), without loss of antibody activity [33]. The resulting conjugate has been used in a sandwich assay for TSH that employs silver surfaces coated with anti-TSH antibody to capture TSH antigen. The captured antigen, in turn, traps DAB-labeled antiTSH antibody. A linear relationship was observed between the intensity of the resultant SERES signals and the TSH antigen concentration over a range of 4-60 ~tlU ml-1. As well as their simplicity, SERRS immunoassays have several other advantages over enzyme-labeled immunoassays. First, the reporter dye molecules are both inexpensive and stable. Second, no signal development time is required (read-out begins immediately upon illumination), and yet the signal cannot over-develop (compared with optical absorbance) or self-quench (compared with fluorescence), but can be enhanced by increasing the number of reporter groups. Third, because reporters bound at the metal surface scatter light with molar etficiencies several orders of magnitude greater than those remaining in solution, it is not necessary to remove the bound reporters before reading the signal. Recently, Pharmacia Biosensor AB have introduced a real-time biospecific interactions analytical device based on surface plasmon resonance and a microfluidic system. When a light is focused on the interface of thin metal/glass layers at an incident angle, the free electrons of the thin metal layer at the metal sample interface are resonantly excited into oscillations. The quanta of the oscillations of surface electrons at the boundary as produced by light excitation are called plasmon. Light energy transfered to the surface electrons causes a decrease in the intensity of the reflected light. The incident angle of the light that excites the surface electrons is sensitive to the refractive index of the sample medium in contact with the thin metal layer. Changes in the refractive index of the meduim at the surface due to immunochemical binding reactions or other biospecific interactions will therefore result in changes in the angle of the incident light that can provide a maximal energy transfer from the light to the surface electrons (i.e. a minimal intensity of the reflected
Ngo
light). This system combines the advantages of a flow injection analysis with an optical monitoring unit based on surface plasmon resonance techniques. Interactions are detected as they occur on the surface of the sensor. A single injection analysis takes about 5--10min. New sensors
A silicon sensor-based filtration immunoassay, using a biotin-labeled capturing membrane able to detect antigens at sub-fmol level, has been described [22oo,23]. In a typical sandwich immunoassay, the target antigen sample is mixed with two sets of antibodies, one set labeled with biotin, and the other set with fluorescein, as well as streptavidin and urease-conjugated anti-fluorescein antibody. After the formation of 'pentameric' complexes (streptavidin: biotinyl-antibody: target antigen: fluorescein labeled-antibody: urease coniugated antifluorescein), the solution is filtered through a biotinlabeled membrane. Any unbound reagents are washed free, and the membrane is then pressed against a silicon sensor in a reader containing a solution of urea. The hydrolysis of urea by urease to give ammonia occurs in a microvolume of 0.5 pl, producing a significant pH change which in turn causes a change in surface potential on the sensor. The sensor is prepared from a silicon wafer coated with a proprietary insulating monolithic top layer and the silicon, insulator and electrolyte act as a capacitor. The circuit is closed by connecting the sensor, via an ammeter, and a device for varying the voltage across the capacitor, to an electrode in the electrolyte solution. Current is measured across the capacitor at various potentials. Measurements are made by addressing the back of the sensor with light from one of an army of light-emitting diodes. The photoresponse, together with the reverse potential of the device, causes the interface between the silicon and the insulating layer to charge up. This transient response results in a burst of current flowing in the external circuit. By modulating the light, an alternating current is generated which flows vertically through the device at that point and is measured by the ammeter; no direct current passes through the sensor. The magnitude of the alternating current depends upon the bias potential across the sensor. Accordingly, any chemical reaction that changes to the surface potential of the sensor will change this current [24]. Further discussion on new immtmosensors can be found in the review by Guilbault (this issue, pp 3-8). New devices
A novel enzyme immunoassay device (known under the trade mark of accuPINCH Test) has been developed by Hycor for rapid screening of drugs in human urine. The test has a limit of detection of several.ng per ml, and provides a preliminary indication of the presence of a particular drug. The enzyme immunoassay is competitive, using limiting reagent levels. First, an aliquot of urine specimen is added to the 'separation disk', situated at the bottom of a test well. This disc is coated with immobilized antibodies that bind the drug. Several drops of wash solu-
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Analytical biotechnolosy tion are then added and, after absorption, this is followed by exactly one drop of enzyme-labeled drug. The principle of the test is that the drug in the specimen competes with the enzyme-labeled drug for antibody sites on the separation disc. If the antibody sites are blocked on the separation disc, soluble enzyme-labeled drug is instead transferred to the 'detection disc' which is pressed against the disc two minutes later. The detection disc conrains a chromogen, and thus any color change indicates the presence of the drug. In the absence of drug, however, no color change occurs because the enzyme-labeled drug will bind soley to the separation disk. This device was developed using technology disclosed in a US patent [p6o]. Enzyme-labeling is also used as part of a novel immunochromatographic assay device [p7o]. The device consists of a strip of falter paper in a chromatographic development jar that contains a buffered substrate solution. Several clearly separated zones are present on the paper strip. The lowest zone, closest to the development solution, is usually a sample-receiving zone, and the next is the enzyme-labeled antibody zone. As the enzymelabeled antibody is not covalently bound to the paper, it can move as the result of capillary action of the development solution. The uppermost zone is an indicator zone, composed of immobilized antibodies covalently bound to the paper. The assay is initiated by sample application at the base of the strip, followed by placing the strip in the buffered substrate solution at the bottom of the chromatographic development jar. As the buffered substrate solution migrates up the strip, sample from the sample-receiving zone is mixed with the enzyme-labeled antibody, forming complexes. The complexes are then carried to the indicator zone, where the antigen of the complex is bound by the immobilized antibody which effectively stops further upward migration of the complexed antigen. Any free enzyme-labeled antibodies, continue to migrate upward with the solution above the indicator zone. A substrate must be selected that migrates slower than either the antigen or the enzyme-labeled antibody. In this way, the substrate ~/1 only be able to contact enzyme held back because of antigen-antibody reaction in the indicator zone. Consequently, the indicator zone will only become colored if antigen is present in the sample.
levels [26]. Genes were constructed to allow expression of two separate polypeptide fragments; enzyme-donor (ED) and enzyme-acceptor (EA). These fragments were designed so that, when mixed together, they spontaneously recombine to form active ~3-galactosidase. Ligands, in this case vitamin B12, can be attached chemically to the ED peptide to control the extent of recombination with the EA by their interaction with the anti-ligand antibody. Analyte ligands in the sample compete with the ligand-ED conjugate for a limiting amount of antibody binding sites. The third homogeneous enzyme immunoassay detects carcinoembryonic antigen (CEA) [27]. Hybrid bispecific monoclonal antibodies reacting with CEA and with the E. coli ffgalactosidase were produced by hybridoma fusion or chemical linkage of half-antibodies. The original anti-J3-galactosidase antibody was shown to protect the enzyme from thermal denaturation. By combining this antibody with two different non-competing anti-CEA antibodies, two antibody populations that were useful for homogeneous enzyme immunoassays could be selected. The activity of a number of enzymes entrapped in surfactant-reversed micelles in organic solvents can be modulated by the degree of hydration, i.e. the water/surfactant molar ratio [28,29]. The activity of some enzymes is greatly increased. Maximal enzyme activity is observed when the radius of the inner cavity of reversed micelles is equaled to that of the globular protein entrapped. Thus, it appears that ermyme activity is controlled by the ratio of miceUe to enzyme size. In an immunoassay situation, the effective dimension of the enzyme-labeled analyte in a reversed micelle can be altered by adding specific anti-analyte antibodies. These bind the enzyme-labeled analyte and form complexes with dimensions larger than the original conjugate; consequently, the activity of the enzyme is expected to decrease. The decrease in the enzyme activity should be restored by addition of the ligand. This prediction was realized by using a HRP-labeled thyroxine conjugate in an anti-thyroxine antibody system. A standard curve for thyroxine in the range of 2-20 nM was established [29].
Reduction of non-specific binding New principles Three notable homogeneous enzyme immunoassays have been developed in the past year. The first, which detects plasma protein C, uses labeled antibody [25]. The assay is based on a unique property of HRP; in the presence of excess hydrogen peroxide (the enzyme substrate), the oxidation of the chromogen is catalyzed much faster by an aggregated enzyme than by a monomer. In the presence of antigens, HRP-labeled antibodies form high molecular weight aggregates. A highly sensitive and rapid homogeneous enzyme immunoassay has been described that uses recombinant fragments of ~-galactosidase to determine vitamin B12
Conventional enzyme immunoassays for antibodies in serum are carried out by incubating the serum with immobilized antigens (Group five immunoassays; see above) to trap specificIgs. The trapped Igs are then reacted with enzyme-labeledanti-Ig antibody to estimate the amount of antibodyspecificallybound. The sensitivity of this assay is limited by non-specific binding of other Igs in the serum to the solid phase. In order to overcome this limitation, a novel, highly sensitive immunecomplex transfer enzyme immunoassay has been developed [30,31,32"]. The assay is performed as follows; an antigen is labeled with a hapten, such as a dinitrophenyl group (DNP), and the resulting DNP-labeled antigen is incubated with the test serum and an anti-DNP antibody
Immunoassay Ngo immobilized on the solid phase. After washing the solid phase to remove much of the nonspecifically bound protein, the DNP-labeled antigens are displaced from the solid base using a high concentration of DNP-lysine. The eluted solution is incubated with solid phase anti-antigen antibodies, which bind specifically. After a further brief wash, the solid phase is exposed to an enzyme-labeled anti-Ig antibody. The amount of enzyme label bound to the solid phase indicates the quantity of specific antibody in the test serum. The scheme shown in Fig. 1 depicts the reactions involved in the measurement of human anti-thyroglobulin IgG. Using such a procedure, the detection limit of the antibody in serum is 5.5 ngm1-1
tion signal of enzyme immunoassays [34]. In one example, biotin-conjugated phenolic compounds were used as reporter molecules and their deposition was catalyzed by HRP-labeled reagents. This, in turn, promoted the capture of a large number of streptavidin-labeled enzyme conjugates, resulting in amplification of the enzymatic signal.
Sample amplification
The detection of biospecific interactions including antibody-antigen reactions, with eLlipsometry, is greatly amplified by using nanometer-size silica particles [35]. Another means of amplifying the detection signal in ellipsometric measurements would be to use enzymelabeled reagent to cause the deposition of insoluble products on the detector surface from soluble enzyme substrates, as previously described [ps°o]. In a liposomebased immunoassay, it has been demonstrated that avidin molecules are able to bind membrane-bound biotin on groups of liposomes derived from biotinylated phospholipid molecules. The resultant liposome-attached avidin is also able to bind additional biotins or biotinylated antibodies from the solution [36]. These derivatized liposomes were highly reactive toward multivalent antigens; a linear increase in light scattering was recorded between 1 and 10 pmol of antigen. The data show that liposomes carrying biotinylated phospholipid could be a useful generic reagent for immunoassays. A further advantage of liposomes is that each one can entrap up to 100 000 marker enzyme molecules. This amplification enhances signal detection. Antigens on a solid phase can also be amplified by using polyacrylic polyhydrazide polymers [37]. The procedure involves periodate oxidation of the carbohydrate groups of antibodies that are already immunochemically bound to antigens attached to the solid phase. In this way, the complexes of antigen and bound oxidized IgG are exposed to polyhydrazide polymers. After rinsing, the solid phase is placed in contact with a periodate oxidized giyco-enzyme such as HRP, and
The detection signal of an immunoassay can be enhanced by enzymatically amplifying the number of reporter molecules, or by physically amplifying the size and therefore the quantity and signal of reporter molecules. For example, in immunoassays that use piezoelectric oscillator detection, the change in resonant frequency of the detector can be correlated with the amount of mass change on the surface of the quartz crystal [P8°.]. If the quartz crystal microbalance and the coated layer obey rigid-layer behavior, the mass change can be determined from the frequency change using the Sauerbry relationship: df = - ( 2 fo dm)/(A Jpu), where df is the measured frequency shift, fo the parent frequency of the quartz crystal, dm the mass change, A the piezoelectrically active area, p the density of quartz and u the shear modulus. The mass change can be amplified enzymatically. Thus, in a quartz crystal microbalance (QCM) immunoassay, the detection of the binding of an analyte to an antibody-coated surface on a QCM is amplified by an enzyme-labeled conjugate. This conjugate is capable of catalyzing the conversion of soluble substrates to insoluble products that accumulate on the surface of the QCM and therefore lead to a greater mass change. Consequently there is a greater change in the resonant frequency [P8°-]. A novel signal amplification method based on catalyzed reporter deposition has been used to enhance the detec-
The detection signal of immunoassays can also be enhanced by using mass-amplified reagents. For example, in a piezoelectric crystal-based specific binding assay, colloidal sol was used as a label in order to increase the mass of binding reagents and to amplify the response to analyte [p9o].
H Anti-TG + SP-Anti-DNP TG-DNP
H.Anti-TG:TG-DNP:SP-Anti-DNP
DNP-Lysine
SP-Anti-TG ,,~,,~,........
H.Anti-TG:TG-DNP
HRP-R-Anti H.IgG (H.Anti-TG:TG-DNP):SP-Anti-TG
Fig. 1. Use of the technique of im[(H.Anti-TG:TG-DN P):SP-Anti-TG]:HRP-R-Anti Substrate
lgG
Color
munocomplex enzyme imrnunoassay to measure levels of human antithyroglobulin IgG. DNP, dinitrophenyl; H, human; HRP, horseradish peroxidase; Ig, irnmunoblobulin; R, rabbit; SP, solid phase; TG, thyroglobulin.
107
108
Analytical biotechnology the amount of enzyme that can bind is measured with the usual peroxidase substrates.
sion Protein of Protein A and Neomycin Phosphotransferase II in Two-chamber-well Mierotiter Plates. Anal B t ~ 1990, 187:89-93. 12.
KEMENYDM, CHALLACOMBESJ: Microtitre Plates and Other Solid-phase Supports. In ELISA and Other Solid Phase lmmunoassays edited by Kemeny DM, Challacombe SJ. Chichester: John Wiley & Sons, 1988, 31-55.
13.
RASMUSSENSE: Solid Phases and Chemistries. In Comp/ementary Immunoassay~ edited by Collins WP. Chichester: John Wiley & Sons, 1988, 43--55.
14.
GRAVESHCB: The Effect of Surface Charge on Non-specilic Binding of Rabbit Immunoglobulin-G in Solid-phase Immunoassays. J Immunol Methods 1988, 111:157-166.
15.
MCGINIAYPB, BARDSLEYWG: The Kinetics of Adsorption of Human Immunoglobulin-G to Polyvinyl Chloride Enzymelinked Immunoassorbent-Assay Vessel Wells. B/ochem J 1989, 261:715-720.
16.
PETERMANJH, TARCHAPJ, CHO VP, BUTLERJE: The Im~unochemistry of Sandwich-ELISAs, IV: The Antigen Capture Capacity of Antibody Covalently Attached to Bromoacetyl Surface-functionalized Polystyrene. J Immunol Methods 1988, 111:271-275.
17.
PORSTMANNB, PORSTMANNT: Chromogenic Substrates for Enzyme lmmunoassay. In NontsotoIn'c Immunoassay edited by Ngo TF. New York: Plenum Press, 1988, 57-84.
18.
KRICKALJ, STOTT RAW, THORPE GHG: Enhanced Chemiluminescence Enzyme Immunoassays. In Complementary Immunoassays edited by Collins WP. Chichester: John Wiley & Sons, 1988, 169-179.
19.
BRONSTEINI, EDWARDS B, VOYTAJC: 1,2-Dioxetanes: Novel Chemilumineseent Enzyme Substrates. Applications to Immunoassays. J Biolumin Chemilumin 1989, 4:99-111.
20.
BARETA, FERT V, AUMAI~ J: Application of Long-term Enhanced Xanthine Oxidase-induced Luminescence in Solidphase Immunoassays. Anal B/ocbem 1990, 187:20-26. JOHANNSSONA, BATES DI2 ELISA and Other solid Phase Immunoassays, edited by Kemeny DM, ChaUacombe SJ. Chichester: John Wiley & Sons, 1988, 85-106.
Conclusion
Increasing numbers of immunoassays are being dev-eloped using non-isotopic labels, such as enzymes, fluorescent molecules, and chemi- and bio-luminescent molecules. Highly sensitive non-labeled immunoassay techniques are emerging rapidly. Efforts are continuing to be directed towards speeding up, simplifying, and automating assay protocols.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •• of outstanding interest 1. GODLINGJP: A Decade of Deveinpmem in Immunoassay • Methodology. Clin Gbem 1990, 36:1408-1427. A succint and comprehensive summary of immunoassay methods developed in the last 10 years, showing the future trends.
2.
NGO TF: Enzyme-mediated Immunoassay: an Overview. In Enzym~ mediated lmmunoassay edited by Ngo TT, Leuhoff HM. New York: Plenum Press, 1985, pp 3-32.
3.
GOULDBJ, MARKSV: Recent Developments in.Enzyme Immunoassays in Nonisotopic lmmunoasay, edited by Ngo "IT. New York: Plenum Press, 1988, pp 3-26.
4.
NGO TT: Enzyme Systems and Enzyme Conjugates for Solid-phase ELIS& In Immunochemistry of Solic#phase Immunoassay edited by Butler JE. Beca Raton: CRC Press, 1991.
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VOGELC-W: (ed) Immunoconjugates, Antibody Conjugates in Radioimaging and Therapy of Cancer. Oxford: Oxford University Press, 1987.
22. ..
6.
ZALLrrSKYMR, GARG PK, FRIEDMAN HS, BIGNER DD: Labeling Monodonal Antibodies and F(ab)'2 Fragments with AParticle-emitting Nuclide Astatine-211: Preservation of Immunoreactivity and In Vivo Localizing Capacity. Proc Natl Acad Sci USA 1989, 86:7149-7153.
7.
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ISHIKAWAE, HASHIDA S, KOHNO T, TANAKA K: Methods for Enzyme-labeling of Antigens, Antibodies and their Fragments. In Nonisotopic Immunoassay edited by Ngo TT. New York: Plenum Press, 1988, 27-55.
PETERHANSA, MECKLENBURGM, MEUSSDOERFFERF, MOSBACHK: A Simple Competitive Enzyme-linked Immunosorbent Assay UsIng Antigen-~-Galactosidase Fusions. Anal Biochem 1987, 163:470-475. LINDBLADH C, PERSSON M, BULOW L, STAHL S, MOSBACH K: The Design of a Simple Competitive ELISA Using Human Proinsulin-alkaline Phosphatase Conjugates Prepared by Gene Fusion. Biochem Biophys Res Commun 1987, 149:607~14.
10.
KOBATAKEE, NISmMOmY, IKAmYAMAY, AIZAW^M, KATOS: Application of a Fusion Protein, Metapyrocatechase/Protein A to an Enzyme Immunoassay. Anal Biochem 1990, 186:14-18.
11.
HUNGERHD, FLACHMEmRC, SCI-LM~TG, BEHRENDTG, COUTELLE C: Ultrasensitive Enzymatic Radioimmunoassay Using a Fu-
OLSONJD, PANF~ PR, ARMENTAR, FEMMEL i ~ , MEmCK H, GUMPRZJ, GOLTZM, ZUK RF: A silicon Sensor-based Filtration Immunoassay using Biotin-mediated Capture. J Immun Meth 1990, 134:71-79. Methods for preparing the biotin membrane, the assay principle and the light-addressable potenUometric sensor are described and discussed in detail. 23.
BmGGSJ, KUNGVT, GOMEZB, KASPERKC, NAGAINISPA, 1VIASINO RS, RICE IS, ZUK RF, CHAZAROSSIANVE: Sub-femtomole Quantitation of Proteins with Threshold, for the Biopharmaceutical industry. Biotechniques 1990, 9:598-606.
24.
HAFEMANDG, PARCEJW, MCCONNELL HM: Light-addressable Potentiometric Sensor for Biochemical Systems. Sc/ence 1988, 240:1182-1185.
25.
SUZUKIK, HAYASHIT, KOYAMAM, YOSHIMURAT, SHIMAMOTOM, KIMURAN, KITAM: Rapid Homogeneous Enzyme Immunoassay of Plasma Protein C. Clin C~'m Acta 1989, 184:227-234.
26.
KHANNAPI, DWORSCHACKRT, MANNINGWB, HARMSJD: A New Homogeneous Enzyme Immunoassay Using Recombinant Enzyme Fragments. Clin Chim Acta 1989, 185:231-240.
27.
GOROG G, GANDOIa~ A, PARADISl G, ROLLEm E, KLASEN E, DESSI V, STROM R, CELADAF: Use of Bispecific Hybrid Antibodies for the Development of a Homogeneous Enzyme Immunoassay. J Immunol Methods 1989, 123:131-140.
28.
MARTINEKK, LEVASHOVAV, KLYACHKO N, KHMELNITSKI YI~ BEREZlN IV: Micellar Enzymology. Eur J Biochem 1986, 155:453--468.
Immunoassay Ngo 29.
KABANOVAV, KHRUTSKAYAi i , EREMIN SA, KLYACHKONL, I.EVASHOVAV: A New Way in Homogeneous Immunoassay: Reversed MiceUar Systems as a Medium for Analysis. Anal Biocbem 1989, 181:145-148.
30.
KOHNOT, ISHIKAW^E: A Novel Enzyme Immunoassay of Anti-insulin IgG in Guinea Pig Serum. Biochem Biopbys Res Commun 1987, 147:644-649.
31.
ISHIKAWAE, HASHIDAS, TANAKAK, KOHNO T: Methodological Advances In Enzymology. Development and Applications of Ultrasensitive Enzyme Immunoassays for Antigens and Antibodies. Clin Chim Acta 1989, 185:223-230.
32. 00
KOHNOT, MrrSUKAW^T, MATSUKURAS, TSUNETOSHIY, ISHIKAWA E: Measurement of Anti-thyroglobulin IgG in Serum by Novel and Sensitive Immune Complex Transfer Enzyme Immunoassay. Clin Biochem 1989, 22:277-284. Theory and practical details of the development of sensitive enzyme immunoassays for quantifying specific antibodies, as exemplified by the measurement of anti-thyroglobulin IgG in serum. 33.
ROHR "rE, CO'I'rON T, FAN N, TARCHA PJ: Immunoassay Employing Surface-enhanced Raman Spectroscopy. Anal BR~hem 1989, 182:388-398.
34.
BOBROWMN, HARRISTD, SHAUGHNESSYK, LrlT GJ: Catalyzed Reporter Deposition, a Novel Method of Signal Amplification. Application to Immunoassays. J Immunol Methods 1989, 125:279-285.
35.
MANDENIUSCF, MOSBACHK: Detection of Biospecific Interactions using Amplified Elipsometry. Anal Biochem 1988, 170:68-72.
36.
PLANTAL, BR1ZGIS M, I.OCASIO-BROWN I., DURST RA: Generic Lip•some Reagent for Immunoassays. Anal Biochem 1989, 176:420-426.
37.
HEIMGARTNERU, KOZULIC B, MOSBACH K: Polyacrylic Polyhydrazides as Novel Reagents for Detection of Antibodies in Immunoblotting Assays. J Immunol Methods 1990, 132:239-245.
Annotated patents • 0,
of interest of outstanding interest
P1 •
DEANRT, BOUTIN RH, WEBER RW: Bifunctional Coupling Agents and Radionuclide-labeled Compositions Prepared Therefrom. PCT 1989, W • 89/12625.
Preparation and application of bifunctional coupling agents that have a protein reactive group and protected •hi•Is which, upon deprotection can chelate radionuclldes. P2 KENTENJH, CASADEIJ, POWELL MJ: Luminescent Chimeric j Proteins. PCT 1989, W • 89/09393. Photoproteins have been fused genetically with binding proteins and used in highly sensitive immunoassays. P3 CHADWICKAT, HUDSON MJ: Bonding to Metal Surfaces. • Eur Pat Appl 1989, Pub No. 0 3339 821. Interposed coordinating polymers were first bonded to the metal surface, and then biologically active molecules were linked directly or through crosslinking reagents to other parts of the polymer, P4 •0
EDWARDS B, BRONSTEIN I, LAIRD A, VOYTA JC: Novel Chemiluminescent Fused Polycyclic Ring-containing 1,2Dioxetanes and Assays In which they are used. PCT 1990, W • 9O/00164. Procedures for preparing novel chemiluminogenic enzyme substrates and their applications in biological binding reactions. P5 EDWARDSB, VOYTAJC: Purification of Stable Water-soluble •0 Dioxetane. PCT 1990, W • 90/02742. Chromatographic procedures for purifying chemiluminogenic enzyme subs•rates. P6 GUIREPE, CHUDZIK SJ: Field Assay for Ligands. 1989. US • Pat No. 4,826,759. Reagents were kept apart by depositing them individually in separated zones on a strip. The reagents could be brought in contact by appropriate folding of the strip. P7 BAKERTS, PERRY MJ, FLEMING IM: Binding Assay Device. • 1989. UK Pat GB 2191578. An immunochromatrographic assay method performed on a chromat•gram support with several defined zones, one being the sample receiving zone. Other zones include binding and indicator zones. P8 ~le
WARDMD, EBERSOLE RC: Enzymatically Amplified Piezoelectric Specific Binding Assay. 1989. PCT W • 89/09937. mass change on the surface of a quartz crystal balance was amplified by deposition of insoluble products, generated bT~enzymatic conversion of soluble subs•rates by ermyme-labeled reagents. The change in resonant frequency is consequently amplified. P9 WARDMD, EBERSOLE RC: Piezoelectric Specific Binding As• say with Mass Amplified Reagents. 1989. PCT W • 89/09938. A piezoelectric crystal-based specific binding assay in which a colloidal sol is used to amplify the mass of binding reagents and, thererfore, proportionally increases the change in the resonant frequency.
TY Ngo, BioProbe International Incorporation, 14272 Franklin Avenue, Tustin, California 91680, USA.
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