Journal of Immunological Methods, 150 (1992)23-32

23

© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$115.r,)1)

JIM06326

Amplification systems in immunoenzymatic techniques S. A v r a m e a s Unit~ d'Immunocytochimie, Institut Pasteur, 25 rue du Dr. Roux. 75724 Paris Cedex 15, France

(Accepted 12 February 1992) Key words: Antigen-antibody;Biotin-avidin;Fluorogenic luminescent substrate; Enzyme cascade; Non-specificsignal

Introduction Since the introduction, in 1966, of enzymes as markers for the labeling of antigens and antibodies (Avrameas and Uriel, 1966; Nakane and Pierce, 1966), immunoenzymatic techniques have been considerably developed and diversified. These methods are now routinely used for the localization of antigens (or antibodies) on tissues, for the detection of antigens (or antibodies) immobilized on various solid phases as well as for the titration of antibodies and for the precise measurement of antigens. Antigens a n d / o r antibodies are localized, detected a n d / o r titrated by means of various heterogeneous procedures. Antigens can be precisely measured using either heterogeneous or homogeneous assays (Avrameas and Guilbert, 1971a,b; Engvali and Perlmann, 1971; Van W e e m e n and Schuurs, 1971; Rubinstein et al., 1972). In homogeneous assays, there is no need to separate formed antigen-antibody immune complexes from the remaining free antigen and antibody (Rubinstein et al., 1972). In heterogeneous procedures, such separations are necessary and are associated with washing steps.

Correspondence to: S. Avrameas, Unit6 d'lmmunocytochimie, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.

These steps eliminate the excess reactants and substances that are present in biological media that might interfere with the assays. Heterogeneous procedures therefore, in principle, are much more sensitive than homogeneous assays. In general, heterogeneous immunoenzymatic procedures that are based on the use of enzymeantibody or enzyme-antigen conjugates, prepared according to several established protocols, and on the use of chromogenic substrates, are sufficiently sensitive for the routine detection, titration and quantitation of most constituents of biological interest. However, in order to detect constituents present in low amounts it is often necessary to devise procedures capable of strongly amplifying the enzymatic signal. Since all heterogeneous immunoenzymatic techniques involve, in their final step, the detection of an enzyme associated with a solid phase, essentially two approaches have been developed to obtain such an enzymatic signal amplification. The first includes procedures leading to a high accumulation of enzyme labels associated with the solid phase. The second consists of procedures that make use of enzyme substrates and substrate derivatives which give rise to reaction products detectable in minute amounts. The first is mainly employed for the localization or detection of cellular, or otherwise immobilized antigens, while the second is primarily applied to the titration or measurement of various humoral and cellular constituents.

Amplification systems based on accumulation of enzyme labels Procedures based on antigen-antibody interaction

In fact, the first of this series of amplification systems corresponds to the indirect or 'sandwich' procedure established by A. Coons for immunofluorescence (Coons et al., 1941; Coons, 1956). in order to detect a cellular antigen, a specific native unlabeled antibody, prepared in species A, is first allowed to react with the antigen and then, after washes, a fluorescein-labeled antibody, recognizing the immunoglobulins of species A is applied. Since the first antibody possesses many epitopes, it will bind more than one molecule of labeled antibody and thus results in an increase in the number of fluorescein molecules associated with the antigen. Similarly, enzyme-labeled anti-immunoglobulin antibodies are used in order to increase the enzymatic signal associated with the antigen. These procedures, because of their universality and simplicity are routinely employed in various fields, although their relationship with amplification systems is probably not, at first glance, evident. Modifications of this basic concept have resulted in the development of various immunoenzymatic procedures, initially designed for the localization of tissue antigens, leading to extensive amplification of the enzymatic signal. One of the simplest procedures consists of adding, after the enzyme-labeled antibody has been bound to the immobilized antigen, an unlabeled polyclonal antibody recognizing the epitopes of the enzyme used as the label, followed by the addition of free enzyme (Avrameas, 1969). In this way, the number of enzyme molecules associated with the revealing antibody are markedly increased. Similarly, adequately chosen unlabeled antibodies can be used to amplify the enzymatic signal. Initially, these procedures comprised five steps separated by washes (Avrameas, 1969; Masson, T. et ai., 1969; Sternberger and Cuculis, 1969): (1) incubation of the tissue with a specific antibody directed against the antigen to be detected and raised in species A; (2) incubation with an antibody recognizing the immunoglobulins of species A; (3) incubation with an anti-enzyme antibody also prepared in species A; (4) addition of the enzyme

marker; and (5) detection of the enzyme. These procedures are based on the principle that the anti-immunoglobulinantibody, when added in excess binds to the specific antibody associated with the cellular antigen only by one of its two active sites. The remaining free active site then acts as an acceptor for the anti-enzyme antibody that, in turn, reacts with subsequently added enzyme. These multi-step procedures were subsequently simplified by labeling with preformed soluble peroxidase-anti-peroxidase immune complex, known as the PAP procedure (Sternberger et al., 1970). These techniques have been refined by replacing polyclonal with monoclonal antibodies and further developed by using, in addition, other enzyme labels (Clark et al., 1982; Mason et ai., 1982; Ternynck et al., 1983; Cordell et al., 1984). Procedures based on such amplification systems have been reported to possess 2-50-fold increased sensitivity compared to conventional assays. These protocols are employed currently for the localization of cellular antigens, often for the detection, by immunoblotting-typeprocedures, of antigens immobilized on various solid phases, but only occasionally in quantitative enzyme immunoassays (Butler et al., 1978). In essence, the above-cited amplification procedures involve the preparation, through non-covalent antigen-antibody reactions, of hybrid antibodies able to distinguish between one site on an antigen and another on an enzyme. Hybrid antibody molecules possessing dual specificity can be prepared in fact by reduction and subsequent reoxidation of a solution containing antibodies of two different specificities and such an approach has been used to obtain hybrid anti-protein/ anti-enzyme antibodies useful in the immunocytochemical detection of antigens (Avrameas, 1969). More recently, in order to avoid the low yields obtained with these reduction-oxidation steps, hybrid antibodies were prepared by chemically coupling anti-enzyme and anti-protein antibodies (Guesdon et al., 1983). Hybrid antibodies prepared with monoclonal anti-enzyme antibodies were found to be highly effective and, by comparison to conventional enzyme-antibody conjugates, to be more sensitive (Guesdon et ai., 1983; Porstmann et al., 1984). At present, homogeneous populations of single hybrid monoclonal

antibody molecules possessing both anti-enzyme and anti-protein specificities are prepared by using a variant of the hybridization technology of K6hler and Milstein (Milstein and Cuello, 1983). Procedures based on at'idin (streptat,idin)-biotin interaction Avidin is a glycoprotein of 67 kDa found in egg white, and biotin, also known as vitamin H, is present in almost all cells, although in small amounts. Avidin has an extremely high affinity for biotin with an association constant of 10- t.s M (Wilcheck and Bayer, 1984). The avidin-biotin system has been used to develop immunoenzymatic procedures resulting in a large amplification of the enzymatic signal (Guesdon et al., 1979). Two methods have been devised. In the first a biotin-labeled antibody is allowed to react with the immobilized antigen and this step is followed, after washes, by the addition of avidin conjugated to an enzyme. In the second approach biotinlabeled antibody, native unlabeled avidin and biotin-labeled enzyme are used. Initially, this proeedure comprised four steps separated by washes: (1) incubation of the immobilized antigen with the biotinylated antibody; (2) incubation with unlabeled native avidin; (3) incubation with biotinylated enzyme; and (4) detection or measurement of the enzyme. This assay is based on the principle that avidin has four active sites. Because of steric hindrance all four combining sites are not involved in the interaction with the biotinylated antibody and the remaining free active sites act as aeceptors for the subsequently added biotinylated enzyme. This multi-step protocol has since been simplified by the preformation of soluble avidinbiotinylated enzyme complexes, a technique known as the ABC (avidin-biotin-complcx) procedure (Hsu et al., 1981). With these procedures, a relatively high non-specific signal is sometimes noted. This background is most often due to non-specific interactions induced by the positively charged avidin molecule (isoionic point 10). For this reason, at the present time, streptavidin, a neutral protein found in Streptomyces at:idinii and expressing the same characteristics as avidin, is used more and more in this type of amplification technique (Wilchek and Bayer, 1990).

Biotin can be coupled under mild conditions to macromolecules and, in most cases, a high number of biotin molecules can be introduced into antibodies and enzymes without affecting their biological activities. Since avidin has four active sites, its combination with such highly biotinylated molecules usually leads to considerable amplification. It must be remembered, however, that biotin~lation of even a few NH 2 groups in certain monoclonal antibodies sharply decreases their antigen-binding capacity. Techniques based on the avidin-biotin system have been reported to be 2-100-fold more sensitive than conventional procedures. They are currently used for the sensitive localization, detection and quantitation of various constituents of biological interest.

Amplification systems based on enzyme substrates

The most direct approach to the development of such methodologies is to use substrates giving rise to fluorescent products or light emission. Other approaches consist, mainly, of generating amplified signals by cyclic enzymatic reactions or by using labeled substrates. Procedures based on fluorogenic or l~minescent substrates Fluorescent compounds can be detected or measured even when present only in extremely small amounts. Using fluorimetry to increase the sensitivity of the enzyme immunoassays was proposed (Avrameas and Guilbert, 1971a) and incorporated early into immunoenzymatic technology (Ishikawa, 1973). The development of highly sensitive enzyme immunoassays started, however, with the use of 4-methylumbelliferyl-~-D-galactoside, the fluorogenie substrate of/3-galactosidase (Kato et al., 1975), and subsequently of 4-methylumbelliferyl phosphate the fluorogenic substrate of alkaline phosphatase (Shalev et ai., 1980). When hydrolyzed by the corresponding enzyme:, these non-fluorescent substances give rise to the intensively fluorescent compound, 4-methylumbelliferone (excitation wavelength 365 nm, emission 455 nm). Using these substrates different procedures have been developed for and ap-

plied to the measurement of various constituents. However, the increased sensitivity and lowered detection limits reported with these techniques varied considerably, ranging respectively, from 5to 100,000-fold and from 100 molecules to ng/ml. E. coli/3-galactosidase as the enzyme marker has the advantage, over calf intestinal alkaline phosphatase of allowing measurement at a slightly alkaline pH at which all mammalian /3-galactosidases are inactive (H/Ssli et al., 1978; Avrameas et al., 1979). In addition, the fluorogenic substrate of /3-galactosidase is much more stable than that of alkaline phosphatase, thus permitting incubation rimes of even several days (H0sli et al., 1978, Avrameas et al., 1979). Furthermore, kinetic rate measurement using automatic apparatuses is possible and can overcome the inconvenience of the spontaneous hydrolysis of the substrates (Kang et al., 1986). Light emission can be detected or quantified with high sensitivity. Early studies demonstrated the feasibility of measuring cell surface immunoglobulins or anti-thyroglobulin antibodies with enzyme immunoassays using horseradish peroxidase or glucose oxidase as the enzyme marker and either bioluminescence or chemiluminescence (Puget et al., 1977; Velan and Halmann, 1978). For a long time, enzyme immunoassays based on luminescence were not widely used, either because of the unusual reagents needed or because of the poor light emission. However, this changed upon the discovery of substances that enhance considerably the light emitted by the peroxidase oxidation of luminol in the presence of H 2 0 2 (Whitehead et al., 1983). These enhancer substances have enabled the development of sensitive immunoenzymatic techniques particularly suitable to immunoblotting-type procedures (Matthews et al., 1985). More recently, the preparation of chemiluminescent substrates for alkaline phosphatase and /3-galactosidase has led to the development of highly sensitive enzyme immunoassays (Bronstein et al., 1989; Schaap et al., 1989).

devised for alkaline phosphatase (Johannsson, 1985), The substrate nicotinamide adenir.e dinucleotide phosphate (NADP +) is converted by alkaline phosphatase into nicotinamide adenine dinucleotide (NAD +). This compound is reduced to N A D H by alcohol dehydrogenase in the presence of ethanol included in the reaction medium. In turn NADH, in the presence of diaphorase, is converted back into NAD ÷ with the simultaneous reduction of a tetrazolium salt also present in the reaction medium. This gives rise to an accumulation, of colored soluble formazan dye, which is proportional to the concentration of NAD ÷ generated by alkaline phosphatase. The newly formed N A D + enters into the reaction and is recycled many times. It has been reported that this procedure yields a 100-fold increase in sensitivity. A modification of this technique has been described by which the addition of semi-carbazide was found to increase assay sensitivity 250fold (Brooks et al., 1991). More recently, another enzyme-cascade procedure has been devised to measure alkaline phosphatascs. A masked inhibitor is converted into an active one by the catalytic action of alkaline phosphatase. This compound acts as a potent inhibitor of a second enzyme, liver carboxylesterase. Measurement of the residual esterase activity enables an estimation of the original alkaline phosphatase activity. The sensitivity of this procedure has been reported to be approximately 125-fold greater than that observed for the detection of alkaline phosphatase activity with p-nitrophenyl phosphate (Mize et al., 1989). A different approach, called catalyzed reported deposition, has been developed recently for the sensitive detection of peroxidase label present on solid phases (Bobrow et al., 1989). A histochemical substrate carrying biotin residues was prepared that, upon oxidation by peroxidase in the presence of H 2 0 2, gave rise to insoluble biotin derivatives that were detected with increased sensitivity by using streptavidin-enzyme conjugates.

Procedures based on a multi-enzyme cascade These techniques are based on the generation of amplified enzymatic signals by using multi-enzyme cascades. The first of these procedures was

Other procedures A radioactive enzyme substrate was employed to develop a sensitive technique called U S E R I A (Harris et al., 1979). Alkaline phosphatase was

used as the enzyme label and [3H]adenosine monophosphate as the substrate. Upon enzymatic reaction, this substrate gives [3H]adenosine and inorganic phosphate. The reaction products are separated by ion-exchange chromatography and the radioactivity is counted. When applied to the measurement of cholera toxin, this assay was reported to be 1000 times more sensitive thaw conventional enzyme immunoassays. It was estimated that as few as 600 molecules of toxin could be detected (Harris et al., 1979). Recently, an electrochemical enzyme immunoassay, using alkaline phosphatase as the enzyme label, has been devised (Jenkins et al., 1988). Alkaline phosphatase catalyzes the conversion of electro-inactive phenylphosphate into electro-active phenol that is then measured by liquid chromatography with electrochemical detection in a thin-layer flow cell. The detection limit for rabbit IgG with this procedure was reported to be 7.5 p g / m l (Jenkins et al., 1988). More recently, a protocol resulting in a 100fold more sensitive detection of peroxidase, applicable to immunoblotting-type assays has been described (Domingo and Marco, 1989). The histochemical substrate, 4-chloro-l-naphthol, after conversion by peroxidase in the presence of H zO2 into an insoluble product deposited on the solid phase, is visualized or photographed under ultraviolet light. The amplification noted is apparently due to the special ultraviolet absorption and the fluorescence-quenching properties of the insoluble reaction products of the substrate (Domingo and Marco, 1989).

Limits and potentials of amplification systems In addilion'to their specific antigen-antibody reactions that give rise to the specific signal, all immunological methods, including immunoenzymatic techniques, always involve non-specific interactions to greater or lesser degrees. The limiting factor in the development of sensitive immunoassays is the background noise induced by such non-specific interactions. It has to be kept in mind, however, that the specific antigen-antibody reaction, in fact, is the result of a series of nonspecific but programmed interactions occurring

between the active site of an antibody and a restricted area of an antigen. In other words, a specific antigen-antibody reaction necessarily involves non-specific interactions, and the reduction of an apparent background noise, beyond a given point, might also result in a decrease of the specific signal. This delicate balance, between specificity and background noise, which is inherent to all immunological ;.~.chniques and which becomes apparent when sufficiently sensitive procedures are used, may be the source of serious problems, particularly when sensitive amplifying systems are applied. The magnitude and nature of the non-specific signals vary with the immunological procedure under consideration. In immunoenzymatic teehT. niques, it appears that, by far, most of the background usually noted is due to the non-specific binding of the antibody-enzyme conjugate or the enzyme-amplifying construct to the solid phase. In principle, the sensitivity of all immunoenzymatic assays can be enhanced by increasing the concentration of the enzyme-antibody conjugate up to a given value. Before reaching this value, the specific signal increases proportionally more than the non-specific one but, after this point, an inverse relationship is observed. This value differs from one procedure to another and depends upon the purity of the reactants used to prepare the conjugate, the characteristics of the conjugate itself, and the solid phase used. Therefore, to ensure the specificity and sensitivity of immunocnzymatic assays, involving amplification systems, it is necessary to select with particular care the reagents to be used and to optimize all the reaction steps. In general, the non-specific binding of the enzyme-antibody conjugate (or the enzyme-amplifying construct) to the solid phase is considerably reduced by adding a protein to the reaction medium, for example bovine serum albumin, gelatin or appropriately selected whole serum, supplemented with a non-ionic detergent. By interacting with the hydrophobic sites present on the solid phases, proteins and detergents diminish the non-specific binding of the enzymeantibody conjugates. Several other approaches have also been explored to reduce the background in quantitative enzyme immunoassays (lshikawa et at., 1989). For

example, two-step coupling procedures, and especially those involving the selective use of thiol groups, produce conjugates of restricted heterogeneity with diminished non-specific binding capacities, particularly when Fab' fragments rather than whole lgG are used for coupling. Normal lgG, its fragments or a conjugate of these with bovine serum albumin, have been reported to be effective in reducing the non-specific binding, while the sensitivity of the assays was improved when incubations were carried out at 4-20°C (Ishikawa et al., 1989). In order to overcome the non-specific binding of the conjugate to the solid phase, a procedure called immune complex transfer immunoassay has been devised (Ishikawa et al., 1990) which uses an antigen, double-labeled with dinitrophenyl and biotin groups. The complex formed in the soluble phase between this antigen and an antibody is allowed to react with a solid phase coated with an anti-dinitrophenyl antibody. After washing, the adsorbed complex is eluted with dinitrophenyl-Llysine, transferred onto a streptavidin-coated solid phase and the immunoglobulins associated with it are measured by using an Fab'-enzyme conjugate. It is claimed that, compared to the other enzyme immunoassays, the detection limit of this procedure is 100,000-fold lower, while the non-specific binding is almost eliminated, and as few as 600 antigen molecules can be detected. It is almost impossible to objectively compare the various amplification systems thus far developed to detect or quantitate antigens and antibodies. For an objective evaluation, the same basic reagents, i.e., the same antibody and the same enzyme label have to he used in different laboratories and the results obtained have to be compared. Such comparative studies have not been performed and, therefore, it is not surprising to find such contrasting reports, describing increases in sensitivity ranging from 2- to 100,000-fold. The situation becomes even more complex if one considers that amplification procedures have often been developed in a given laboratory, and are routinely used only in that laboratory. Furthermore, equally often, amplification procedures have been reported once, using a prototype system, without being subjected to statistical analyses and calculation of intra-assay or

interassay coefficients of variation. Moreover, sensitivity and the detection limit are sometimes confused and considered to be equivalent concepts. One procedure, however, can be more sensitive than another but both might be able to detect the same limited concentration of an antigen or an antibody. In such a case, only the intensity and the rapidity of the enzymatic response are improved with the more sensitive procedure but the detection limit would remain unchanged.

Importance of antibody in developing amplification systems With the ultimate goal of having in hand a procedure enabling the quantitation, on a routine basis, of the immunoglobulins and antibodies synthesized by single immunocytes we have developed a certain number of amplification systems (Avrameas, 1969; HSli et al., 1978; Avrameas et al., 1979; Guesdon et al., 1979, 1983a,b; Labrousse et al., 1982; Ternynck et al., 1983). In our experience, those based on fluorogenic substrates have broad potentials because enzymes can be quantitated with a high degree of sensitivity while, because of fluorimetry, the assays can be dramatically miniaturized. At present, under the most optimal conditions we could define, we are able ~.o measure, in 2 txl serum samples, 8000 molecules or approximately 0.3 fg of human IgE with a coefficient of variation of 10%. The protocol devised for this purpose is based on the use of: (1) E. coli /3-galactosidase as the enzyme marker; (2) the fluorogenic substrate 4.methylumbelliferyl-/3-galactoside; (3) Terasaki plates with 10 p.I wells for the immobilization of antibodies; and (4) a spcctrofluorimeter hooked up to a microcomputer which automatically measures the fluorescence emitted in the wells of the Terasaki plates (Labrousse et al., 1982; Andr6 et al., 1983). During the course of these studies, we came to realize that, in fact, the characteristics of the antibody used constituted the main limiting factor in obtaining high sensitivity and high-performance enzyme immunoassays. The antibody reacts with the antigen through non-covalent bonds and, consequently, the anti-

gen-antibody complex formed corresponds to a reversible association. Therefore, the antibodyantigen reaction obeys the law of mass action and is given by the equation: Kt Ab+Ag ~ Ab-Ag K2 where Ab represents antibody; Ag antigen; and Ab-Ag the complex. Quantitatively, this equation is expressed as: K~

KI K2

IAb-Ag] lab]lag]

where l a b - Ag] is the concentration of tile complex; lab] is the concentration of the antibody; lag] is the concentration of the antigen; K I is the rate of the forward reaction; K 2 is the rate of the reverse reaction; K~ is the association constant at equilibrium. K I is usually higher than K 2 and this means that, once the complex is formed, the reaction is only slowly reversible. In the heterogeneous enzyme immunoassays, which include washes, it is this slow dissociation that allows the washes without significantly affecting the formed complex. This equation states that, at antigen and antibody concentrations below the association constant, no complex will be formed and antigen and antibody will be present only as free molecules. Thus, apparently, the limiting factor that determines the detection limit in all immunoassays is the binding affinity of the antibody for the antigen. Which marker substance is capable of being detected in minute amounts is only of secondary importance; even the most sensitive and sophisticated amplification system cannot detect complexes that have not been formed because of the too low concentrations of the reactants. The following two examples illustrate the limits imposed by the antibody used in sensitive enzyme immunoassays. The first example concerns an immunoenzymatic procedure for the titration of estradiol-17~ (Maurel et al., 1987). For this purpose, microtitration plates were coated with a specific polyclonal anti-estradiol antibody. The pla~.es were

incubated first with the sample containing the steroid, then an estradiol conjugate E. coli flgalactosidase was added and the tree hormone and the enzyme-bound estradiol were allowed to compete. After washes, the enzyme activity associated with the wells was revealed with either the chromogenic (o-nitrophenyl-/3-D-galactoside) or the fluorogenic substrate (4-methylumbelliferyl-/3D-galactoside) of /3-galactosidase. Using the fluorogenic substrate, the limit of detection was 1(3(I p g / m l and its range 100 pg to 100 ng/ml. The same values were obtained with the chromogenie substrate but 18 h of incubation were required, instead of the 2 h required for the fluorogenic substrate, l'hus, although the intensity and the rapidity of the assay were improved by using the fluorogenic rather than the chromogenic substrate, the detection limit was the same. Therefore, the concentration of estradiol in the samples seemed to be the limiting factor. When this limit was overcome by evaporating the samples in the wells before the addition of the estradiol-/3galactosidase conjugate, the sensiti'vity of the assay was markedly increased. The detection limit was now 1 p g / m l and the measurable range 1 pg to 10 ng/ml. Thus, in this case, a precisely defined amount of a given antibody with a known affinity constant limits the detection of the antigen in the sample rather than the sensitivity of the detection system as such. Fhe second example describes a sensitive enzyme immunoassay for the measurement, at the single cell level, of the immunoglobulins exposed on the surface of B lymphocytes. The assay makes use of Fab antibody fragments labeled with E. colt /3-galactosidase, and the fluorogenic substrate of this enzyme in combination with micromanipulations and ultramicroassays (HOsli et al., 1978; Avrameas et al., 1979). Lymphocyte suspensions were incubated with various concentrations of the Fab-/3-galaetosidase conjugate. The cells were then washed, allowed to adhere to a plastic film in the presence of stabilizing proteins and sugars, and finally lyophilizcd. Under a stereomicroscope, pieces of plastic bearing single cells were then excised and incubated in microwells, made in parafilm sheets and filled with the fluorogenic substrate of /3-galactosidase buffered at pH 8.5. After 24 h of incubation at 37°C, the

reaction mixture was adequately diluted a n d fluorescence was m e a s u r e d with a spectrofluorimeter. By this procedure, it was possible to d e t e r m i n e that 2000-20,000 molecules of K light c h a i n s were p r e s e n t on the surface of single lymphocytes. In principle, this m e t h o d associates all t h e conditions necessary to have an extremely sensitive assay. First, u n d e r t h e e x p e r i m e n t a l conditions established, it was possible to m e a s u r e easily 50 molecules o f /3-galactosidase. Second, t h e a n t i g e n to be m e a s u r e d , i.e., t h e s u r f a c e imm u n o g l o b u l i n was, by itself, p r e s e n t in an insoluble form. T h e r e f o r e , since t h e r e was no n e e d to use an immobilized antibody to c a p t u r e the antigen, o n e would expect that incubation with a high excess of Fab-/3-galactosidase conjugate would lead to an extremely low detection limit. It was found, however, that the Fab-/3-galactosidase conjugate c o n c e n t r a t i o n could not be increased over a given value, b e c a u s e , b e y o n d this level, the non-specific b i n d i n g increased too m u c h . U n d e r t h e most optimal conditions d e t e r m i n e d , approximately 1000 m o l e c u l e s o f the Fab-/3-galactosidase conjugate were b o u n d non-specifically to e a c h lymphocyte and this limited t h e accurate a n d reproducible d e t e c t i o n of s u r f a c e i m m u n o g l o b u lins to 2000 molecules. It is possible that even less t h a n 2000 m o l e c u l e s of K c h a i n s are p r e s e n t on the surface o f lymphocytes, b u t this value corres p o n d s to t h e detection limit i m p o s e d by t h e non-specific b i n d i n g of t h e conjugate. T o conclude, it c a n be a d v a n c e d that, in o r d e r to f u r t h e r develop amplification s y s t e m s e n a b l i n g t h e reliable a n d easy d e t e c t i o n o f h u n d r e d s and, p e r h a p s , of only a few molecules, studies exploring two d o m a i n s m u s t be u n d e r t a k e n . First, m o r e basic k n o w l e d g e is n e e d e d to clarify t h e differe n c e s b e t w e e n specific a n d non-specific binding. Better u n d e r s t a n d i n g in this area will certainly g e n e r a t e even m o r e sensitive p r o c e d u r e s . T h e second, a n d probably m o s t i m p o r t a n t step towards the d e v e l o p m e n t of ultrasensitive e n z y m e i m m u n o a s s a y s , is t h e p r e p a r a t i o n o f high quality antibodies, that is, t h o s e exhibiting high affinity a n d associated with low non-specific binding. It is m o r e t h a n probable that this goal will be r e a c h e d in t h e n e a r future by taking a d v a n t a g e of t h e a c h i e v e m e n t s m a d e in t h e field o f genetic engineering.

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Amplification systems in immunoenzymatic techniques.

Journal of Immunological Methods, 150 (1992)23-32 23 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$115.r,)1) JIM06326...
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