The use of phycobiliproteins as fluorescent labels in immunoassay

Journal of Immunological Methods, 92 (1986) 1-13

1

Elsevier JIM 04055 Review article

The use of phycobiliproteins as fluorescent labels in immunoassay Mel N. K r o n i c k Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, U.S.A.

(Received16 April 1986, accepted 30 April 1986) Key words: Fluorescenceimmunoassay; Flow cytometry; Phycobiliprotein;Phycoerythrin;Allophycocyanin

Introduction The efficiency of photosynthesis in many species of algae is enhanced significantly by the presence of a class of light-harvesting proteins, the phycobiliproteins, that contain multiple bilin prosthetic groups (Glazer, 1981, 1984). The phycobiliproteins are present as aggregates in particles called phycobilisomes that lie near the chlorophyll reaction centers. In their native configuration, light energy absorbed by the phycobiliproteins is efficiently transferred to the chlorophyll. When the phycobiliproteins are purified and isolated, however, the proteins become highly fluorescent because the molecules no longer have any nearby acceptors to which to transfer the absorbed energy. The spectroscopic properties of the phycobiliproteins, as discussed below, exhibit several unique qualitative and quantitative features. Oi, Stryer, and Glazer realized that these features made phycobiliproteins ideal candidates for use as fluorescent labels in immunoassay and demonstrated in an elegant series of experiments that the Abbreviations: AIDS, acquired immunodeficiency syndrome; ARC, AIDS-relatedcomplex; FETI, fluorescenceexcitation transfer immunoassay; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; LGL, large granular lymphocyte; NK, natural killer; PCFIA, particle concentration fluorescence immunoassay; PCB, phycocyanobilin;PE, phycoerythrin; PEB, phycoerythrobilin; PUB, phycourobilin; SMCC, succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate; SMPB, succinimidyl-4-(p-maleimidophenyl)butyrate; SPDP, succinimidyl-3-(2-pyridyldithio)propionate;7AAD, 7amino-actinomycinD.

use of phycobiliproteins as covalently linked fluorescent tags was possible and practical and could result in many advantages when compared to conventional labels such as fluorescein (Oi et al., 1982).

Useful properties of the phycobiliproteins The absorption and fluorescence spectra of four prototypical phycobiliproteins'are shown in Fig. 1. The three main classes of phycobiliproteins are shown: phycoerythrin, phycocyanin, and allophycocyanin. The different classes differ significantly in their protein structure and pigment content as noted in Table I. R-phycoerythrin, isolated •from the higher red macroalga, Gastroclonium coulteri, has more phycourobilin pigment relative to phycoerythrobilin pigment than does the Bphycoerythrin, from the unicellular red alga, Porphyridium cruentum. The R and B prefixes refer to historical nomenclature related to the algal source. Extinction coefficients can be as great as 2.4 x 10 6 M - a c m -x, some 30 times that of the frequently used label fluorescein while the quantum efficiencies can be as high as 0.8, as good as or better than conventional synthetic labels (Hemmila, 1985). This enormous extinction coefficient and high quantum efficiency can increase the sensitivity of fluorescent assays. The molecular weights of the phycobiliproteins can be large, but as Oi et al. clearly demonstrated, this does not appear to interfere with the practical usefulness of phycobiliproteins (Oi et al., 1982).

0022-1759/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)

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Fig. 1. Absorption and fluorescence spectra of four prototypical phycobiliproteins, a: R-phycoerythrin; b: B-phycoerythrin; c C-phycocyanin; and d: allophycocyanin. TABLE I PHYSICAL PROPERTIES OF TYPICAL PHYCOBILIPROTEINS Source

Structure Pigment content a

Molecular weight Absorption hmax(nm) ~max(M -1 cm -1) Fluorescence hm~x(nm) Quantum yield References

B-phycoerythrin

R-phycoerythrin

C-phycocyanin

Allophycocyanin

P orphyridium cruentum

Gastroclonium coulteri

A nabaena oariabilis

A nabaena variabilis

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110 000

546 2.4 × 106

566 2.0 × 106

614 5.8 × 105

650 7.0 X 105

575 0.59 Glazer and Hixson (1977) Grabowski and Gantt (1978) Glazer (1985)

574 0.85 Oi et al. (1982) Klotz and Glazer (1985)

643 0.51 Glazer et al. (1973) Bryant et al. (1976)

660 0.68 Cohen-Bazire et al. (1977), Bryant et al. (1976)

" PEB = phycoerythrobilin, PUB = phycourobilin, PCB = phycocyanobilin (see Glazer, 1985, for structures and linkages).

The specific wavelength positions and shapes of the spectra of the phycobiliproteins result in many of their advantages in immunoassay. Soini and Hemmila (1979) clearly illustrated the fact that fluorescent background most often limits the sensitivity of fluorescent measurements and that the background due to endogenous fluorescent molecules in most biological environments is most severe with excitation and emission at the blue end of the visible spectrum. The orange to red fluorescence of the phycobiliproteins, especially in conjunction with their large extinctidn coefficients and quantum efficiencies, thus directly overcomes many of the limitations of conventional fluorescent tags. Also, the broad absorption band of the phycoerythrin results in a significant separation of excitation and emission wavelengths, the Stokes shift, so that phycoerythrin fluorescence can be easily discriminated from that of other dyes (like fluorescein) or from endogenous fluorescent molecules in samples. This, in turn, allows the use of phycoerythrin and fluorescein together in systems where two independent molecular species can be probed with only one excitation wavelength. Oi et al. (1982) first reported this use for the simultaneous detection of two independent cell surface markers and Houghton (1985) described a similar technique for the immunoassay of two independent species dissolved in solution. The spectral relationship of the individual phycobiliproteins relative to each other (which exists because of their natural function as energy transfer systems) was taken advantage of by Glazer and Stryer (1983) to produce a totally new fluorescent species, the 'tandem' conjugate. By covalently linking allophycocyanin to phycoerythrin, they produced a conjugate that efficiently absorbs light at blue-green wavelengths via the phycoerythrin moiety, transfers energy via an intermolecular resonant process to allophycocyanin, and then emits light in the red at the allophycocyanin emission peak. Such a species has an enormous (almost 200 nm) Stokes shift which further removes signal from background and makes possible three-color simultaneous analyses with one excitation source as discussed below (Chen, 1985). The fortuitous positions of the phycobiliproteins' spectra in the visible wavelength region enhance the utility of phycobiliproteins in systems

utilizing laser excitation sources. The optical properties of lasers can be effectively utilized to deliver large amounts of light into very small volumes (Sepaniak, 1985). This is the main reason, for example, that lasers are the standard light source in flow cytometry, where cells in a flowing st'ream of liquid pass through a light source in single file (Parks and Herzenberg, 1984; Parks et al., 1986). The argon ion laser, the usual laser used in flow cytometry systems, emits at 488 nm where phycoerythrin strongly absorbs. The helium-neon laser, emitting at 633 nm, can offer additional technical advantages from a cost and reliability point of view, and works well for excitation of the phycobiliprotein allophycocyanin (Shapiro et al., 1983). A new version of the helium-neon laser, emitting at 543 nm, may also become popular as a phycoerythrin excitation source. The unique physical structures of the phycobiliproteins result in still more advantages. As noted in Table I, each phycobiliprotein contains as many as 34 individual bilin pigments. Detailed studies of protein structure, pigment structure, and modes of pigment attachment have been performed on many of the phycobiliproteins (Frank et al., 1978; Sidler et al., 1981; Fuglistallefet al., 1983; Lundell et al., 1984; Schoenleber et al., 1984a,b; Glazer and Klotz, 1985; Nagy et al., 1985; Schirmer et al., 1985; Sidler et al., 1985). The protein structure holds the pigment molecules in a very specific configuration so that very little fluorescence quenching occurs. Conventional fluorescent dyes packed into such a small volume would severely quench each other's fluorescence. Also, the protein structure of each phycobiliprotein enfolds the pigment molecules in such a fashion that the structure isolates the pigments from the protein's external environment. The fluorescence of phycobiliproteins is thus independent of pH over a broad range (from approximately pH 5 to pH 9) and immune from collisional quenching from molecules or ions likely to be found in typical use environments.

Sources of phycobiliproteins Phycobiliproteins appear in very high concentration in many algal species but must be ex-

4

TABLE

II

EXAMPLES

OF PURIFICATION

METHODS

Protein

Source organism

References

B-phycoerythrin

Porphyridium cruentum

R-phycoerythrin

Gastroclonium coulteri

C-phycocyanin C-phycocyanin Allophycocyanin Allophycocyanin

Anabaena variabilis Spirulina platensis Anabaena variabilis Spirulinaplatensis

Glazer and Hixson (1977) Gant and Lipschultz (1974) Oi et al. (1982) KIotz and Glazer (1985) Hardy (1986) Bryant et al. (1976) Hardy (1986) Bryant et al. (1976) Hardy (1986)

tensively purified to be useful. Many different protocols can be found in the literature for the different types of phycobiliproteins (see Table II). Unicellular red algae (e.g., Porphyridium cruenturn) or cyanobacteria (e.g., Anabaena variabilis) can be grown relatively easily in laboratory scale cultures. The red macroalgae such as Gastroclonium coulteri are harvested from their natural marine environments where permitted by law or cultured in large tanks or farms. The general features of the purification procedures are all similar. Single cell sources are broken open using a French press, homogenized, or, for cyanobacteria, enzymes like lysozyme are used to dissolve the cell envelope. Purification from macroalgae begins by homogenization in a high speed blender. Cell debris is removed by centrifugation and then ammonium sulfate is added in sufficient amounts to precipitate the phycobiliproteins. One or more ion exchange steps, either on resins or hydroxylapatite, are then used to produce proteins near homogeneity, and then gel filtration or crystallization is used as a final clean-up step. High purity phycobiliproteins are now available from several commercial sources so that it should not be necessary for those desiring starting material for conjugation to expend the time and effort necessary to purify their own phycobiliproteins. Utilization of phycobiliproteins

as labels

The rapid acceptance of the use of phycobiliproteins as labels has, in part, been due to the

rather straightforward techniques used to couple phycobiliproteins to the specific binding molecule of interest. Oi et al. (1982) clearly showed that the use of such techniques preserved the unique spectral properties of the phycobiliproteins and the unique binding properties of the antibody, avidin, drug, etc., to which the phycobiliprotein was linked. In most cases, techniques utilized for many years in protein chemistry can be directly applied because the phycobiliproteins are stable, hydrophilic proteins (Means and Feeney, 1971; Glazer, 1976). Usually, heterobifunctional reagents, which enable independent activation of the two molecules to be coupled, are used to link covalently the phycobiliprotein to the specific binding molecule (Carlsson et al., 1978; Yoshitake et al., 1979; Haugland, 1985). Fig. 2 lists several of the most commonly used heterobifunctional reagents and schematically illustrates the activation reaction mode of the particular heterobifunctional reagent.

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Fig. 3 shows a flowchart of the two most popular reaction schemes used to link a molecule (typically another protein) to a phycobiliprotein. Both approaches shown involve derivatizing one or more of the many lysyl c-amino groups on the phycobiliprotein with reagents that yield free sulfhydryl groups. These sulfhydryls then are linked to specific reactive groups previously placed on the molecule to be labeled. In one approach, the reactive groups in question are formed by derivatizing with a reagent like SPDP that leaves an - S - S - b o n d with a good leaving group. The subsequent linkage of the sulfhydryls on the phycobiliproteins to the SPDP derivative thus results in a disulfide bond. The alternative approach uses reagents like SMCC or SMPB to leave maleimide groups on the molecules to be labeled. The maleimides will react with the sulfhydryls on the

phycobiliproteins to yield thioether linkages. Other variations on these methods are possible including reduction of endogenous disulfides of antibodies (Parks et al., 1984). All the heterobifunctional reagents shown in Fig. 2 are commonly available from various chemical supply vendors. Specific conjugation protocols have been given by several authors (Oi et al., 1982; Kronick and Grossman, 1983; Hardy, 1986). In general, any chemistry used to conjugate a protein or hapten to an enzyme or to a carrier protein can be used so long as the conjugation conditions do not denature the phycobiliproteins. Exact concentrations and molar ratios will depend upon the particular pair of molecules to be conjugated and the heterobifunctional reagent chosen. Increases in yield of conjugate may be accompanied by increases in nonspecific binding and some optimization is almost always required. It is recommended to start the optimization with reaction parameters that primarily yield relatively well characterized conjugates of one phycobiliprotein molecule to one specific-binding molecule, even if overall efficiency is low. As efficiency is increased, a statistical mix of conjugates of three or more molecules will be obtained in addition to the one-to-one conjugates. Although most work has been done by coupling to antibodies, phycobiliproteins can also be coupled to small molecules by derivatizing the small molecule into an active ester, a sulfonyl halide, or some other species compatible with aqueous chemistry at near-neutral pH. Oi et al. (1982) coupled biotin to phycoerythrin for use as a second-step labeling reagent. Winfrey and Wagman (1984) and Khanna (1985) described the labeling of phycoerythrin with a digoxin derivative for use in a clinical assay for digoxin. Because conjugations are almost never run with 100% efficiency it is usually desirable to purify the conjugates of interest away from the unconjugated starting materials. Unconjugated specific binding molecules can block binding sites and hence reduce sensitivity. Unconjugated phycobiliproteins can result in additional fluorescence background and also reduce sensitivity. Gel filtration has proven the most common and general technique and works extremely well where phycobiliproteins are coupled to large molecules such as antibodies

(Oi et al., 1982; Kronick and Grossman, 1983). Agarose-based media such as Bio-Gel A-1.5 m (Bio-Rad Laboratories, Richmond, CA) work extremely well. HPLC gel filtration techniques have also been used (Oi et al., 1982) but this author and others (Kronick, unpublished results) have observed rapid degradation of column performance, probably due to non-specific interactions with the phycobiliproteins. In a typical purification such as described by Kronick and G.rossman (1983) for purification of phycoerythrin-IgG conjugates, two distinct colored bands will appear. The faster band contains the high molecular weight conjugates and the slower band, the unconjugated phycobiliproteins. Ion exchange chromatography using hydroxylapatite has also proven very useful for conjugate purification, These methods, as opposed to gel filtration, must be specifically developed for each conjugate pair because retention properties depend so strongly on chemical composition. Hardy (1986) has shown some examples for antibody conjugates and Glazer and Stryer (1983) used hydroxylapatite to purify 'tandem' conjugates consisting of phycoerythrin linked to allophycocyanin. In both instances the conjugate has an affinity for hydroxylapatite between that of each of the two starting materials. The generality of the gel filtration techniques must be balanced against the inevitable dilution which results and also the increased time required. Affinity chromatography in conjunction with gel filtration chromatography may be the method of choice when attempting purification of conjugates of phycobiliproteins with low molecular weight drugs or hormones. Gel filtration will remove the small unconjugated molecules and affinity chromatography will concentrate the conjugates and remove excess phycobiliproteins. Researchers and commercial producers of phycobiliprotein conjugates have used storage conditions similar to those common ones in practice for other fluorescent or enzyme conjugates. The conjugates are typically stored in liquid form in neutral buffer in the dark at 2-8°C. Sodium azide at concentration from 0.02%-0.10% is added as an anti-microbial agent. Gelatin, bovine serum albumin or an equivalent stabilizing agent is usually added in concentrations of 0.1-1.0 mg/ml, especially if con-

jugates are dilute. Commercial producers usually certify three months stability if these conditions are utilized. Stability has been observed for storage times of up to a year (Kronick, unpublished results).

Applications to cell labeling and flow cytometry The most immediate and dramatic application of phycobiliproteins has been to the multicolor fluorescent labeling of cell surface molecules. By using fluorescein conjugated to an antibody specific for one cell surface molecule and phycoerythrin conjugated to an antibody specific for a second cell surface molecule, the simultaneous occurrence of two different markers can be observed with a single excitation source. A simple fluorescent microscope with fluorescein excitation optics is all that is required to observe two-color staining. Applications for the observation of various subsets of human peripheral lymphocytes become possible immediately. For example, fluorescein-labeled anti-Leu 2 antibodies will specifically bind to suppressor T cells and cause them to glow yellowish-green under the microscope. Phycoerythrin-labeled anti-Leu 3 antibodies will specifically bind to helper T cells in the same population and cause the helper T cells to glow orange. Thus, examination of T cell subset ratios, an important parameter in diagnosing immune deficiency diseases such as AIDS (acquired immunodeficiency syndrome), is easily possible using a simple fluorescent microscope (Becton Dickinson, 1985). Pizzolo and Chilosi (1984) used this same technique to investigate cellular heterogeneity in lymph node sections. Even more powerful applications are made possible when flow cytometry is combined with the multi-color fluorescent labeling. As was described above, flow cytometry characterizes a population of cells on a cell-by-cell basis. The use of two independent, fluorescently tagged antibodies directed against two independent cell surface markers allows identification of discrete populations defined by the presence of the two markers. A clear example is shown in Fig. 4 for two-color staining of human peripheral blood lymphocytes from both a healthy subject and from a patient

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Fig. 4. Two dimensional contour plot showing data from a two-color flow cytometric analysis of human peripheral blood lymphocytes from a healthy subject (a) and from an ill patients exhibiting AIDS-related complex (b). The horizontal axis plots levels of green fluorescence from fluorescein-labeled anti-Leu 7 antibody. The vertical axis plots levels of yellow-orange fluorescence from a phycoerythrin-labeled antiLeu 2 antibody. Only two-color flow cytometric analysis could clearly identify the marked increase in the population in (b) which stains with both antibodies simultaneously. Courtesy of Becton Dickinson Immunocytometry Systems, Mountain View, CA.

exhibiting AIDS-related complex (ARC). The figure shows contour plots, the commonly used method to display two-color flow cytometric data. The position on the plot is determined by the simultaneous intensity of stains of the anti-Leu 7 antibody conjugated to fluorescein isothiocyanate (FITC) and anti-Leu 2 antibody conjugated to

phycoerythrin (PE). The height of the contour shows the relative number of cells of a particular green (FITC) and yellow-orange (PE) fluorescence. The anti-Leu 2 antibody is specific for T cytotoxic suppressor cells and the anti-Leu 7 antibody is specific for certain subsets of LGL (large granular lymphocyte) and NK (natural killer) cells and also certain T cell subsets. In the upper right hand comer of plot (b) is a population of cells that stains strongly positive for both anti-Leu 2 and anti-Leu 7. The two-color flow cytometry data thus identifies a significant increase in the doubly positive cell population for the ill patient. Such a population increase could not be uniquely (or easily) seen without the two-color measurement technique. The two-color analysis technique described above has now become a standard weapon in the arsenal of the immunologist. Zoumbros et al. (1985), for example, have used two-color analysis to correlate hematopoietic suppression in aplastic anemia with the population of activated T suppressor cells. Lewis et al. (1985) were able to use two color analysis to identify a disproportionate expansion of a minor T cell subset in patients with AIDS. Ryan (1985) has reported that he can identify very small numbers of aberrant cells in a population of lymphocytes. This enabled him to monitor successfully the remission of pediatric patients with certain leukemias. The technique of two-color flow cytometry using phycoerythrin conjugates has, in fact, become so common place that the original work of Oi et al. (1982) that first demonstrated the use of phycobiliproteins for two-color flow cytometry is often no longer even referenced. Before phycobiliproteins, two-color flow cytometry had been done with two laser excitation sources instead of one. Thus, when two color flow cytometry became possible using fluorescein and phycoerythrin tags with just one laser, researchers soon realized that three-color flow cytometry was possible by using two lasers. Three-color flow cytometry allows further definition of additional cell populations and increased analysis speeds because more parameters can be investigated at once. Two groups pioneered this technique. Hardy et al. (1983) used an argon ion laser at 488 nm to excite two different antibodies labeled with fluorescein

and phycoerythrin, respectively, and a dye laser at 615 nm to excite an allophycocyanin-labeled antibody. Their three-color analyses were used to establish relationships of B cell populations in normal and immunodeficient mice. Lanier and Loken (1984) used the argon ion laser at 488 nm to excite phycoerythrin- and fluorescein-labeled antibodies and a dye laser at 600 nm to excite biotinylated antibodies that had been labeled indirectly with Texas Red-avidin. Lanier and Loken used this three-color technique to analyze the coexpression of cell surface antigens on human peripheral lymphocytes. Hardy et al. (1984) then combined these two three-color methods to do simultaneous four-color flow cytometry. A dye laser operating at 605 nm was used to excite both Texas Red and allophycocyanin labels and the argon ion laser at 488 nm was used as described above. The four-color technique made possible studies of complicated murine B cell differentiation linkages. All the above three- and four-color techniques involved use of two lasers. The development of the 'tandem' conjugate concept by Glazer and Stryer (1983) made possible three-color flow cytometry with just one excitation source. As described above, 'tandem' conjugates consist of two different phycobiliproteins covalently coupled to provide for very efficient inter-molecular energy transfer. When phycoerythrin is linked to allophycocyanin, a synthetic tag is created that can absorb light at 488 nm efficiently and yet emit efficiently with a peak at 660 nm. This allows the 488 nm line of an argon ion laser to excite simultaneously fluorescein (emitting with a 525 nm peak), phycoerythrin (emitting with a 575 nm peak), and the ' tandem' conjugate (emitting with a 660 nm peak). When each of these tags is used to label a different antibody, three-color flow cytometry becomes possible with a single excitation wavelength. Chen (1985) described such an application to the threecolor classification of human peripheral blood lymphocytes and obtained results very similar to those Lanier and Loken (1984) obtained using two lasers. A related but different three-color technique has been developed by Rabinovitch and co-workers to give unique and very detailed information about the internal biology of a cell (Rabinovitch, 1985;

Rabinovitch et al., 1986a). They utilized the DNA-binding fluorescent dye, 7-amino-actinomycin D (7AAD). The spectral properties of 7AAD allow single, argon ion laser excitation at 488 nm for the measurement of DNA content and cell cycle simultaneously with two cell surface markers labeled with fluorescein- and phycoerythrin-conjugated antibodies. 7AAD fluoresces with a peak at 660 nm. The researchers showed with this three-color method that mouse Ly-1 ÷ B cells, the subset that has been associated with autoantibody production, are greatly enriched for cells in the S phase of the cell cycle. They also demonstrated that 7AAD staining of human blood peripheral lymphocytes depends upon cell activation and chromatin conformation. Still another multi-color technique incorporating phycobiliproteins has been developed that provides additional and profound insights into biology of cellular activation events (Rabinovitch et al., 1986b). Two lasers were used. One ion laser was used in the ultraviolet to excite indo-1, a new dye whose fluorescence shifts from violet to blue depending upon intracellular Ca 2÷. A second ion laser was used to excite fluorescein- and phycoerythrin-labeled antibodies specific for different cell surface markers. This novel approach allowed Rabinovitch and his collaborators to demonstrate the heterogeneous nature of the intracellular Ca2+ response to mitogenic stimuli within populations of peripheral blood lymphocytes. Quantitation of the surface immunofluorescent labels showed that some of this heterogeneity is related to cellular immuno-phenotype. This work should further the understanding of the role of intracellular calcium ion as a trigger of cellular responses to activating stimuli.

Application to immunoassay of soluble antigens or antibodies The enormous extinction coefficients and high quantum yields of the phycobiliproteins provide the means to enhance the sensitivity of a wide variety of fluorescence-based immunoassay techniques. The inherent advantages are well-illustrated in the recent work of Mathies and Stryer (1986) which attempted single molecule detection

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Fig. 5. Fluorescence spectral plot of a flowing stream of a 5 x 1 0 -13 M solution of B-phycoerythrin. Also shown is the R a m a n spectrum of water under same experimental conditions. Experimental parameters corresponded to an average of 3.2 molecules of phycoerythrin in an illuminated volume of 10 pl. The phycoerythrin emission peak is clearly visible from the background and the R a m a n water band at 625 nm. From Mathies and Stryer (1986), by permission.

using phycoerythrin. Fig. 5 shows the fluorescence spectrum they obtained from a flowing 5 X 10 -13 M solution of B phycoerythrin. The average number of phycoerythrin molecules in the illuminated region was three and the signal can be seen to still be significantly above the inherent Raman scattering background. The extent to which this spectroscopic advantage can be utilized in practical immunoassays depends heavily on the particular immunoassay technique and the inherent fluorescent background problems associated with each technique. The sensitivity limits on fluorescence always come back to the background problems described by Soini and Hemmila (1979). Comparison to other techniques and labels must always be done in the context of the actual assay conditions. For example, comparison of phycobiliproteins with polymeric tags such as those used by Hassan et al. (1979) or Hirschfeld (1976) must take into account quenching and non-specific binding. Similarly, comparison with time resolved fluorescent tags (Hemmila, 1985) must take into account the chemiluminescent and phosphorescent backgrounds in a particular assay format. The fluorescence excitation transfer immunoassay (FETI) or fluorescence quenching immtmoassay described by Ullman et al. (1976) and Ullman

and Khanna (1981) take full advantage of the unique properties of phycobiliproteins in a manner reminiscent of the energy-transfer role these proteins play in nature. In FETI~the concentration of a particular hapten or antigen is measured by monitoring the extent of fluorescence quenching observed when the molecule of interest competes with a fluorescently tagged version of the same molecule for binding sites on quencher-labeled specific antibody. The assay is inherently a homogeneous one and no separation steps are required. Thus, a great emphasis is placed on discrimination of signal from sample-induced background. Phycoerythrin, which serves in nature as an energy donor, can perform the same role in FETI. Its longer excitation and emission wavelengths relative to fluorescein help to minimize the effect of the serum-generated fluorescence which falls off significantly at the red end of the visible spectrum (Soini and Hemmila, 1979). Kronick and Grossman (1983) first used phycoerythrin in energy transfer immunoassays for human IgG in a model system. Subsequently, practical commercial clinical assays for digoxin were developed and have been described by Winfrey and Wagman (1984) and Khanna (1985). The commercial assays show a sensitivity of better than 10 -1° M which represents almost an order of magnitude improvement over performance possible with conventional dyes in the FETI format. Significant enhancement of sensitivity in conventional immunoassays can be achieved by straight-forward substitution of phycoerythrinlabeled antibodies for antibodies labeled with conventional dyes like fluorescein. Kronick and Grossman (1983), for example, achieved about a 6-fold increase in sensitivity in a sandwich assay which utilized latex beads as the solid phase. Phycoerythrin-labeled rabbit anti-human IgG was used instead of fluorescein-labeled antibody in an assay protocol like that of Curry et al. (1975). A sensitivity level of 10 -11 M was obtained using a commercial fluorimeter. The advantage relative to fluorescein is moderated by the fact that one can label an antibody with several fluoresceins. Nonspecific binding and inadequate rejection of scattering in the commercial fluorimeter also compromise ultimate sensitivity. Kronick and Grossman also had some evidence that the large

10 phycoerythrin tag caused steric interference with antibody binding sites and further reduced sensitivity. The technique of particle concentration fluorescence immunoassay (PCFIA) provides another approach to increased sensitivity (Jolley et al., 1984). In PCFIA, competition or sandwich assays are run using antibody-labeled latex beads. To enhance sensitivity and ease of use, the beads, after incubation with sample and labeled reagents, are concentrated on filters in the bottoms of small wells. The concentrated beads are rinsed and t h e n read in their concentrated state to improve signal to background ratios by as much as 1000 times. Francis et al. (1985) recently described the assays for human IgG in which phycoerythrin-labeled antibodies were substituted for fluorescein-labeled antibodies in a simple sandwich assay format. This simple substitution resulted in a 4-fold improvement in sensitivity. The increase is surely significant although it also probably suffers from some limitations due to one or more of the same factors faced by Kronick and Grossman: light scattering picked up as fluorescence, non-specific binding and steric interference. It should be noted that Francis et al. were able to achieve their improved results with a lower labeled antibody concentration by using phycoerythrin instead of fluorescein. With appropriate optical filters, it should be possible to use PCFIA assays using the same multi-color tricks employed in flow cytometry. This would permit simultaneous assay of two or more analytes in a mixture. Also, it has been demonstrated by Bethell and Buck (1986) and Francis et al. (1985) that the PCFIA technology can be used to measure cell staining. In such applications, the cells become the solid phase instead of the latex beads and phycoerythrin-labeled antibodies can be successfully used to increase sensitivity significantly. An additional example of sensitivity enhancement using phycoerythrin-labeled antibodies was provided by the work of Houghton (1985). A factor of five enhancement in sensitivity was realized substituting phycoerythrin for a fluorescein label in a novel separation scheme based upon polymer chemistry. The polymer chemistry scheme (Nowinski and Hoffman, 1985) allowed immunometric sandwiches to be formed in solution and

then separated from unbound label by inducing polymerization of the monomer-labeled antibody participating in each complete sandwich. The polymer aggregates so formed incorporated labeled antibody to the extent complete sandwiches are formed. The aggregates were assayed for fluorescence using a flow cytometer. By use of two independent labeled antibodies, one labeled with fluorescein and the other with phycoerythrin, simultaneous analysis of human IgG and IgM was accomplished in a fashion totally analogous to two-color flow cytometry.

Conclusion As demonstrated by the results presented in this review, phycobiliprotein labels have already made significant contributions to the field of immunoassay both in terms of increased sensitivity and in terms of simplifying multi-color analyses of cells and molecules. Patents have already issued on applications of phycobiliproteins (Stryer et al., 1985; Stryer and Glazer, 1985) and several companies are selling phycobiliprotein conjugates as research reagents or in diagnostic kits. It is true, however, that the expected sensitivity enhancement based solely on comparison of spectral properties has not been achieved. As researchers gain experience in using phycobiliprotein reagents, one can expect this discrepancy to become less, if not disappear entirely. The use of optimal conjugation methodologies and appropriate stabilizers, surfactants, and assay protocols should minimize effects of steric interference and non-specific binding. Similarly, instrument parameters such as optimal filters will improve with time as users gain experience with particular applications. Assuming improvements can be made in areas like non-specific binding, the fundamental limitations of sample or matrix-induced endogenous fluorescent background still remain. Although the longer wavelength excitation and emission of phycoerythrin (relative to fluorescein) help somewhat, the use of allophycocyanin labels with their far red absorption and emission may become even more important. Loken et al. (1985) have taken advantage of the potential of allophycocyanin labels in a recent study using helium-neonexcita-

11

tion at 633 nm for flow cytometry of alveolar macrophage cells. These cells exhibit the same problems described in depth by Soini and Hemmila (1979): excitation at the blue end of the spectrum tends to excite endogenous fluorescence. Loken et al. used streptavidin-allophycocyanin conjugates and were able to obtain results equivalent to those obtained using an argon ion laser and phycoerythrin conjugates, thus demonstrating the advantages of working at the red end of the spectrum. It is important to note that the less powerful (albeit more reliable and less expensive) helium-neon laser gave such good results in the spectral region where photomultiplier tubes have less efficiency and when allophycocyanin was substituted for phycoerythrin, a protein with a much higher extinction coefficient. Two limitations of using phycobiliproteins need to be addressed in order for phycobiliproteins to make even further inroads into practical applications: the difficulty of conjugation of the protein and the inherent stability of the proteins. As discussed in detail above, phycobiliproteins are proteins and thus require the use of heterobifunctional reagents, with all their idiosyncracies, to form linkages. Because the coupling is never perfectly controlled, purification procedures are usually required to isolate well characterized conjugates. Fundamental improvements that could address the difficulties of conjugation would go a long way to improve all protein-protein conjugations including both phycobiliprotein and enzyme labeling. Protein stability is important because the complex structures of the phycobiliproteins must be preserved in order to preserve their impressive fluorescent properties. These structures, in the purified natural molecules, are held together through non-covalent bonds. At high dilutions, the potential exists for the protein to fall apart into its subunits. The ~, subunit of the phycoerythrin appears to stabilize phycoerythrin sufficiently to permit dilutions to less than 10 -12 M with no problems (Glazer, 1985; Oi et al., 1982). Allophycocyanin, however, has been observed to change its fluorescence excitation and emission spectra upon dilution beyond 10 -8 M (Kronick and Robison, unpublished results). Ong and Glazer, while investigating subunit interactions, discovered a novel cross-linking protocol that gives

allophycocyanin stability out to the limits of detection at 10 -13 M (Ong and Glazer, 1985; Kronick and Robison, unpublished results). One another stability issue, photodestruction, also needs to be addressed. Mathies and Stryer (1986) showed that on a per molecule basis, phycoerythrin is about twice as stable to photodestruction as fluorescein. Clearly this is acceptable since fluorescein is the most frequently used fluorescent label. Improvements, however, would be most welcome and the unique, protected environment of the phycobiliprotein chromophores would appear to offer a potential foundation upon which some novel approaches to increasing photostability could be built.

Acknowledgements The author gratefully acknowledges continued support of Applied Biosystems in the pursuit of understanding and applying phycobiliproteins as labels. Paul Grossman and Debra Robison have helped significantly in our efforts. There have been many useful discussions with Lubert Stryer, David Parks, and Randy Hardy at Stanford University and especially with Alex Glazer of the University of California at Berkeley.

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