[55]

ENZYME IMMUNOASSAYS

469

can be radiolabeled, or the modified protein can be iodinated when desired. In this case (and for most amine-specific modifications), egg-white avidin is preferred over streptavidin because of its large number of lysines per subunit. Extensive modification of the lysines would also serve to reduce the high pI of the native protein, thereby reducing one potential source of nonspecific binding. Nonglycosylated avidin 8 is perhaps the protein of choice for such modifications since its employment precludes a second major contributor (the oligosaccharide component of the native avidin molecule) to nonspecific interactions. Another method of increasing the number of phenolic groups in avidin is to prepare conjugates with either polytyrosine or tyrosine-containing copolymers. 9 Chapters [56]-[64] in this volume illustrate the versatility of the avidin-biotin system in immunoassays. A more extensive literature summary providing details of the application of avidin-biotin technology in immunoassays and diagnostics is presented in [3] in this volume. 8 y. Hiller, E. A. Bayer, and M. Wilchek, this volume [6]. 9 M. Wilchek, J. Solid-Phase Biochem. 5, 193 (1980).

[55] A v i d i n - B i o t i n S y s t e m in E n z y m e I m m u n o a s s a y s B y THI~RI~SE TERNYNCK a n d STRATIS AVRAMEAS

Avidin is a 66,000-Da glycoprotein present in egg white. One of its properties is the ability to bind with a high affinity (dissociation constant 10-15 M) to the small molecule biotin (or vitamin H).1,2 Biotin is easily linked to proteins, which modifies little if any of their biological activities, and, when bound, retains its high affinity for avidin. The avidin-biotin system has been used to develop various procedures) In immunoassays, in order to detect or quantify a constituent, biotinylated antibodies are allowed to react with the constituent, and then avidin (linked to a marker substance) is added. A procedure based on this principle was initially developed for the immunocytochemical localization of erythrocyte surface antigens; in this case, the biotin-labeled antibody was revealed by ferritin-labeled avidin. 3-5 N. M. Green, Biochem J. 89, 585 (1963). 2 N. M. Green, Adv. Protein Chem. 29, 85 (1975). 3 M. Wilchek and E. A. Bayer, lmmunol. Today 5, 39 (1984). 4 H. Heitzmann and F. M. Richards, Proc. Natl. Aead. Sci. U.S.A. 71, 3537 (1974). 5 E. A. Bayer, M. Wilchek, and S. Skutelsky, FEBS Lett. 68, 240 (1976).

METHODS IN ENZYMOLOGY, VOL. 184

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

470

APPLICATIONS

Enzyme t Biotin

Immobilized Antigen

Immobilized Antigen

Biotinylated Antibody

Biotinylated Antibody

[55]

A

Enzyme-Labeled Avidin

Avidin

Biotin-Labeled Enzyme

FIG. 1. Detection of antigens by the labeled avidin-biotin (LAB) system (A) or by the bridged avidin-biotin (BRAB) system (B).

The same principle was subsequently employed in immunofluorescence 6 and immunoenzymatic techniques. 7 In these latter procedures, two protocols for linking enzymes to proteins were described. In the first, the enzyme is covalently linked to avidin. The labeled antibody (or antigen) is allowed to react with the antigen (or the antibody), and, after washing, enzyme-labeled avidin is added. After further incubation and washing, the enzyme-associated antigen is stained histochemically or measured spectrophotometrically (Fig. 1A). In the following, this procedure is referred to as the labeled avidin-biotin (LAB) technique. In the second procedure, biotin-labeled antibody (or antigen), biotinlabeled enzyme, and native unlabeled avidin are used. To quantify or to detect an antigen, the biotin-labeled antibody is allowed to react with the antigen. After washing to eliminate excess labeled antibody, avidin is added. After incubation and washing, the biotin-labeled enzyme is added. This step, after an additional incubation and washing, is followed by histochemical staining or measurement of the enzyme associated with the antigen by conventional procedures. This procedure, referred to as the bridged avidin-biotin (BRAB) technique (Fig. 1B), is based on the princi6 M. H. Heggeness and J. F. Ash, J. Cell Biol. 73, 783 (1977). 7 J.-L. Guesdon, T. Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979).

[55]

ENZYME IMMUNOASSAYS

471

pie that avidin possesses four active sites, not all of which react with the biotin residues associated with the antigen-biotinylated antibody complex. Thus, the remaining free active sites can operate as acceptors for another biotin-labeled protein secondarily added to the system. Since the first papers on the avidin-biotin system appeared, the use of streptavidin as an alternative to the highly positively charged avidin has been introduced. Streptavidin is a biotin-binding protein extracted from bacteria of the species Streptomyces which has an affinity comparable to that of avidin 2 and has been shown to be as useful as avidin. 8 Streptavidin is less basic (pI 6.5) than avidin and has no carbohydrate residues, 2 thus limiting nonspecific reactions with acidic groups or lectins, respectively.

Experimental Procedures

Materials Biotinyl-N-hydroxysuccinimide ester (BNHS) Avidin from egg white Streptavidin from Streptomyces avidinii Peroxidase, fl-galactosidase, alkaline phosphatase, glucose oxidase Double-distilled glycerol Dimethylformamide fl-Galactosidase anti-mouse Ig conjugate, prepared by the one-step glutaraldehyde method 9,1°

Buffers and Solutions Phosphate-buffered saline (PBS): 0.15 M NaCI, in 10 m M potassium phosphate buffer (pH 7.4) Saturated ammonium sulfate solution: dissolve by boiling 750 g of solid ammonium sulfate in 1 liter of distilled water and allow the solution to cool 0.1 M BNHS solution: dissolve I mg of biotinyl-N-hydroxysuccinimide ester in 30/zl of dimethylformamide

Biotinylation of Polyclonal Purified Antibodies Antibodies are biotinylated using BNHS as detailed in Table I.

s F. M. Finn, N. lwata, G. Titus, and K. Hofmann, Hoppe-Seyler's Z. Physiol. Chem. 362, $679 (1981). 9 S. Avrameas, Immunochemistry 6, 43 (1969). t0 S. Avrameas, T. Ternynck, and J.-L. Guesdon, Scand. J. lmmunol. 8, suppl. 7, 7 (1981).

472

APPLICATIONS

[55]

TABLE I GENERAL PROCEDURE FOR BIOTINYLATION OF ANTIBODIES Step

Procedure

1

Prepare a solution of 2 mg of antibodies in 1 ml of sodium bicarbonate solution and dialyze overnight at 4 ° against the same solution Add l0/~l of 0.1 M B N H S (in dimethylformamide) Incubate 1 hr at room temperature Dialyze against PBS overnight at 4 ° Add an equal volume of glycerol (for storage) Store at 4 or - 2 0 °

2 3 4 5 6

Biotinylation of Monoclonal Antibodies in Hybridoma Culture Supernatant Precipitate 10-20 ml of hydridoma culture supernatant with an equal volume of saturated ammonium sulfate solution. Centrifuge (3000 g, 20 min, 4°) then wash the pellet once with a 40% solution of saturated ammonium sulfate in distilled water. Centrifuge (3000 g, 20 min, 4°) and suspend the pellet in 1 ml of distilled water and dialyze overnight at 4° against PBS and then against 0. I M bicarbonate solution. Proceed as in step 2 (Table I). Comments. We have shown (Ref. 7 and Table II) that (1) the number of biotin molecules fixed per molecule of antibody depends on the molar ratio of BNHS per amino group during the coupling reaction; (2) the capacity of the biotin-labeled antibody preparation to bind to avidin depends on the number of biotin moieties introduced into the molecule; and (3) compared to native antibodies, the antigen-binding capacity of biotinlabeled antibodies is not significantly modified by the number of biotin molecules incorporated (see Table II, the enzyme immunoassay results with 11.4 and 114/.d of biotin). Figure 2 shows the reactivity of monoclonal antihapten antibody, prepared against the trinitrophenyl (TNP) group, in relation to the degree of biotinylation. It is evident from Fig. 2 that a maximum level of reactivity is already obtained with a concentration of 1 mM BNHS (curve 3 in Fig. 2) and that the use of higher concentrations does not further modify the reactivity of the biotinylated antibody. In addition, these preparations give the same signal as the unlabeled preparation when revealed by an anti-mouse immunoglobulin conjugate (data not shown). For these reasons, there is no risk in adding an excess of biotin and thus in working without knowing the exact IgG content of

[55]

ENZYME IMMUNOASSAYS

473

TABLE II ANTIGEN-BINDING CAPACITY OF SHEEP ANTI-RABBIT IMMUNOGLOBULIN ANTIBODIES BIOTINYLATED WITH VARIOUS AMOUNTS OF BNHS a

BNHS b (tzl)

BNHS/amino group (molar ratio)

Biotin labeling c (%)

Activity d (OD)

0 1.14 11.4 45.6 114

0 0.1 1 4 10

0 26 42 95 100

0 0.44 1.04 n.d. e 1.12

Data from Ref. 7. Biotin-N-hydroxysuccinimide ester at 34 mg/ml (0.1 M solution) in dimethylformamide. The quantities are given for 2 mg of purified polyclonal anti-mouse Ig antibodies in 1 ml of 0.1 M bicarbonate solution. Percentage of blocked amino groups determined according to Habeeb's method [A. F. S. A. Habeeb, Anal. Biochem. 14, 328 (1966)]. Optical densities obtained by incubating solutions of biotinylated antibodies at 1 /xg/ml with Ig-coated plates followed by avidin labeled with fl-galactosidase, read at 414 nm. Not determined.

the preparation. However, as far as monoclonal antibodies are concerned, it is well known that their physicochemical properties vary from one antibody to another and that one cannot predict in advance whether the binding capacity of one monoclonal antibody will be modified or totally abolished by chemical modifications, such as those produced by the labeling. In this case, preliminary assays must be performed to assess the remaining antibody activity after the biotin labeling of a monoclonal antibody by testing its antigen-binding capacity using a second labeled antibody (usually enzyme-labeled polyclonal rabbit or sheep anti-mouse immunoglobulin antibodies).

Biotinylation of Antigens Dialyze 1 ml of protein at 2 mg/ml in 0.1 M bicarbonate solution overnight at 4 °. To each 0.25-ml fraction of protein, add 20, 10, 5, or 2/zl of 10 mM BNHS (1 mg of BNHS in 300/~1 of dimethylformamide). Proceed as in step 3 (Table I). Test by enzyme-immunoassay using antibodycoated plates (see Fig. 3).

474

APPLICATIONS

[55]

2

1;8o 1;4o 1;2o 1/lo BIO'nN-ANTIBODY DILUTIONS FIG. 2. Antigen-bindingcapacity of various preparations of biotinylated anti-TNP monoclonal antibodies. The culture supernatant of a monoclonal anti-TNP antibody was precipitated as described in the text. Biotinylation was performed by adding 5 (curve 1), 10 (2), 20 (3), or 40/zl (4) of 10 mM BNHS to 200-/zlsamples (20-100/zg/ml antibody). Samples were tested by enzyme immunoassay on TNP-ovalbumin-coated plates and revealed with peroxidase-labeled avidin (Table IV).

Comments. As we have shown for bovine serum albumin (BSA), 7 the antibody-binding capacity of an antigen may decrease with increasing biotin substitutions. In addition, since biotin substitution depends on the B N H S / a m i n o group molar ratio and since the number of amino groups in a given protein is not always cited in the literature (this could be difficult to estimate in nonspecialized laboratories), it is therefore suggested that each antigen be tested to determine the optimal amount of B N H S needed for the coupling reaction following the general scheme given above. Figure 3 gives an example corresponding to the biotinylation of actin. It can be seen that the greater the biotin substitution, the higher the signal, and in this particular case the extent of biotinylation of actin does not modify its antibody-binding capacity. Biotinylation of Enzymes Dissolve 2 mg of peroxidase or glucose oxidase in 0.5 ml of 0.1 M c a r b o n a t e - b i c a r b o n a t e buffer (pH 9.5) or dialyze 2 mg of/3-galactosidase

[55]

ENZYME IMMUNOASSAYS

475

2 rOl O~

>. I--

1.5

1

11

:33

100

360

BIOTIN-ACT'IN CONCENTRATIONS (~/ml) FIG. 3. Antibody-bindingcapacityof various biotinylatedactin preparations. Biotinylation was performedby adding 5 (curve 1), 10 (2), or 20 ~1 (3) of 10 mM BNHS to 250-1xl samplesof actin at 2 mg/ml.Preparationswere testedby enzymeimmunoassayson antiactin monoclonal antibody-coatedplates with peroxidase-labeledavidin (Table IV).

(usually kept in saturated ammonium sulfate) against this buffer. Add 20/zl of 0.1 M BNHS and proceed as in step 4 (Table I). Comments. We have shown (Ref. 7 and data summarized in Table III) that the enzymes commonly used in immunoenzymatic technique (i.e., peroxidase, glucose oxidase, alkaline phosphatase, and fl-galactosidase) can be labeled with biotin. Our results indicate, however, that some enzymes lose some of their catalytic activity after biotinylation. Indeed, the activity of alkaline phosphatase (from Escherichia coli) is highly affected even at low substitution: 35% of the activity is lost after addition of 3.4 biotin molecules per molecule, whereas that of alkaline phosphatase from calf intestine remains unchanged. The same loss of activity is observed when /3-galactosidase is 80% substituted. At this level of substitution, neither glucose oxidase nor peroxidase activity appears to be modified (Table III). Thus, for enzyme assays, glucose oxidase, peroxidase, /3galactosidase, or alkaline phosphatase from calf intestine are recommended.

Coupling of Avidin or Streptavidin to Enzymes Avidin or streptavidin is coupled to enzymes by the one-step glutaraldehyde procedure. 9 Mix 4 mg of either peroxidase or/3-galactosidase

476

APPLICATIONS

[55]

TABLE III CATALYTICACTIVITYOF ENZYMESBIOTINYLATEDWITHVARIOUSAMOUNTSOF BNHS~ BNHS/amino group b (molar ratio) Enzyme and parameters Glucose oxidase Labeling (%)c Activity (%)d Peroxidase Labeling (%) Activity (%) Alkaline phosphatase (E. coli) Labeling (%) Activity (%) /3-Galactosidase (E. coli) Labeling (%) Activity (%)

0.05

0.1

4

1.4 n.d.

2.8 95

0.5

1

2

5

10

35 n.d. e

50 100

71 100

84 100

100

68 100

72 100

90 35

100 35

100 35

74 78

84 60

3.4 65 55 95

100

80 100

Data from Ref. 7. b Biotin-N-hydroxysuccinimideester at 34 mg/ml (0.1 M solution) in dimethylformamide. The quantities are given for 2 mg of purified polyclonalanti-mouse Ig antibodies in l ml of 0.1 M bicarbonate solution. c Percentage of blocked amino groups determined according to Habeeb's method [A. F. S. A. Habeeb, Anal. Biochem. 14, 328 (1966)]. d Enzyme activity versus native enzyme. e Not determined.

with 2 mg of avidin (or streptavidin) in a final volume of 1 ml of 0.1 M phosphate buffer (pH 6.8). Add 50/zl of a 1% glutaraldehyde solution in the same buffer and incubate for 3 hr at room temperature (below 24°). Add 50 /.d o f 2 M lysine in phosphate buffer for 30 min and dialyze overnight against PBS at 4 °. Centrifuge for 10 min in an E p p e n d o r f centrifuge ( - 1 0 , 0 0 0 g). Add an equal volume of glycerol and store at 4 or - 2 0 °. Comments. The coupling of avidin to enzymes using glutaraldehyde, which interacts with protein amino groups, decreases its positively charged groups and thus its nonspecific binding (see below). H o w e v e r , as in all protein coupling procedures, the conditions described here should be followed carefully because extensive cross-linking can occur, which often decreases the biological activity of the macromolecules.

Chemical Modification o f Avidin Mix 1 ml of avidin (2 mg/ml) in 0.1 M phosphate buffer (pH 7.4) with 1 ml of 4% (w/v) paraformaldehyde in the same buffer. Incubate for 2 hr at

[55]

ENZYME IMMUNOASSAYS

477

room temperature and dialyze against PBS overnight at 4°. Add 2 ml of glycerol. Comments. Avidin is a basic protein (pI 10) and thus could bind to other macromolecules by electrostatic interactions, leading to nonspecific reactions. Treatment with formaldehyde (which blocks amino groups) results in a preparation of avidin possessing low nonspecific binding characteristics. Furthermore, the formaldehyde-treated avidin preparation is stable in solution, whereas native avidin is not and must be kept frozen in small aliquots. Glutaraldehyde treatment of avidin also decreases its nonspecific binding but at the same time can decrease its specifc biotin binding. We have shown that incubation with biotin-labeled compounds under alkaline (pH 9) or high ionic strength (0.5 M NaCI) conditions decreases nonspecific reactions. 7 However, such nonphysiological conditions could effect the specific binding in some antigen-antibody systems. To overcome the disadvantage of the nonspecific binding of avidin, the use of streptavidin was introduced. Streptavidin has the same biotinbinding properties as avidin 8 and can be labeled with biotin or enzymes in exactly the same manner as avidin.

Quantitative Enzyme Immunoassay Media and Substrates

Washing medium: PBS-Tween 20 (0.1%) Saturating medium: PBS-gelatin (0.5%) or PBS-BSA (1%) Diluting medium: PBS-Tween-gelatin (0.5%) Alkaline phosphatase substrate: 1 mg/ml solution of p-nitrophenylphosphate (Sigma Chemical Co., St. Louis, MO) in 0.1 M Tris-HCl buffer (pH 8.2 for E. coli phosphatase or pH 9.8 for calf intestinal phosphatase), containing 1.5 M NaCI; stop the reaction with 1 N NaOH and read at 414nm. fl-Galactosidase substrate: 0.8 mg/ml of o-nitrophenyl-fl-D-galactopyranoside (Sigma) in 0.1 M phosphate buffer (pH 7.4) containing 0.1 M mercaptoethanol; stop the reaction with 2 M sodium carbonate and read at 414nm. Peroxidase substrate: 0.5 mg/ml of o-phenylenediamine (Sigma) in sodium citrate-citric acid buffer (pH 5.5) containing 0.03% HzO2; stop the reaction with 2 N sulfuric acid and read at 492 nm. Procedure. Enzyme immunoassay (EIA) using the avidin-biotin system is performed using the protocol described in Table IV. Comments. The coating of plates is usually performed with proteins diluted in 0.1 M carbonate-bicarbonate buffer (pH 9.5) at a concentration

478

APPLICATIONS

[55]

TABLE IV GENERAL PROCEDURE FOR ENZYME IMMUNOASSAY Step

9.1 9.2

10 11 12

Procedure Coat the plates with 100/zl of antigen or antibody preparation (usually at 5/xg/ ml) in 0.1 M carbonate-bicarbonate buffer (pH 9.5), 2 hr at 4 ° Keep the plates at 4 ° until used Discard the coating solution and incubate with the saturating medium, 30-60 min at 37° Wash 3 times Incubate with the first antibody (or antigen) diluted in diluting medium, 60-120 min at 37° Wash 3 times Incubate with the biotinylated second antibody, 60 min at 37° Wash 3 times Proceed with one of the following two avidin-biotin systems LAB (labeled avidin-biotin) procedure: Incubate with enzyme-labeled avidin (1/zg/ml), 30 min at 37° BRAB (bridged avidin-biotin) procedure: Incubate with the paraformaldehyde-treated avidin (2-5/zg/ml), 30 min at 37°, wash 3 times, incubate with biotinylated enzyme (l /xg/ml), 30 min at 37° Wash Incubate with the appropriate substrate solution until the desired color intensity is obtained. Stop the enzymatic activity and read the optical density at 492 nm

of 5/zg/ml. However, the optimal coating concentration, especially for antigens, may vary considerably. In such cases, this concentration has to be determined for each system. Furthermore, the carbonate-bicarbonate buffer (pH 9.5) can be replaced by PBS if some proteins are found to be unstable at alkaline pH. The BRAB procedure using native avidin does not present any particular advantage. It does not significantly increase the sensitivity, while it includes an additional step. The specific type of enzyme used for labeling avidin is not critical and is rather a matter of personal choice. As in all these immunoassays, the first antibody determines the sensitivity of the assay rather than the detection system used. EIAs performed with the avidin-biotin system are at least as sensitive as those using conventional enzyme-antibody conjugates. The use of biotinylated antigen has been shown to be useful in competitive assays and has good sensitivity.ll 11 R. Rappuoli, P. Leoncini, P. Tarli, and P. Neri, Anal. Biochern. 118, 168 (1981).

[55]

ENZYME IMMUNOASSAYS

479

Immunocytochemical Assays Media and Substrate

Washing medium: PBS Diluting medium: PBS-gelatin (0.5%) or PBS-BSA (1%) Substrate for enzyme detection Alkaline phosphatase (calf intestine) substrate: mix 1 volume of 0.4 mg/ml of naphthol AS-MX phosphate (sodium salt, Sigma) in 0.2 M Tris-HCl buffer (pH 8.2), with 1 volume of 6 mg/ml of Fast Red TR salt (Sigma) in distilled water and incubate for 20 min; the reaction product is red Peroxidase substrate: prepare a solution of diaminobenzidine (Sigma) at 0.5 mg/ml in 0.1 M Tris-HCl buffer (pH 7.6) containing 0.03% H202 and incubate for 7-10 min; the reaction product is brown Procedure. Following the general procedure described in Table V the avidin-biotin system can be applied to the immunocytochemical detection of antigens. The substrate solutions are prepared, filtered, and used

TABLE V GENERAL PROCEDUREFOR IMMUNOCYTOCHEMICALASSAYS Step

7.1 7.2

8

9 10 11

Procedure Prepare tissue sections or cells using standard methods Fix the tissue (with paraformaldehyde, acetone, alcohol, etc.) or deparaffinize and hydrate tissue sections of wax-embedded material exactly as described in other procedures; treat the sections to reduce the endogenous enzyme activity Add appropriate dilutions of the first antibody and incubate, 30-60 min Wash Incubate with biotinylated second antibody (10 ~g/ml for purified antibody or previously determined dilutions for hybridoma supernatants), 30-60 min Wash Proceed with one of the following two avidin-biotin systems: LAB (labeled avidin-biotin) procedure: Incubate with the avidin-enzyme preparation (10-20/zg/ml), 30 rain BRAB (bridged avidin-biotin) procedure: Incubate with paraformaldehydetreated avidin (10/~g/ml), 15-30 min, wash, incubate with biotinylated enzyme (20/.~g/ml), 15-30 min Wash Stain with appropriate substrate solution Counterstain with suitable histological stain Mount with a suitable medium

480

APPLICATIONS

[55]

immediately. 9 After the incubation, slides are washed with distilled water and mounted with a microscope mounting medium. Comments. As for EIA, streptavidin often gives better results than avidin, although formaldehyde treatment of avidin consistently decreases the nonspecific background. The LAB procedure is more rapid than that using BRAB, because an additional step is not required. However, the BRAB procedure often allows more sensitive immunocytochemical detection of antigen. To shorten the BRAB procedure, the use of preformed avidin-biotinylated enzyme complexes (ABC) has been proposed.12 This procedure, in our hands, results in less sensitive detection of antigens than the sequential BRAB procedure. This is probably due to the size of the complex, which would penetrate with increased difficulty into the tissues. Furthermore, the preparation of the ABC is critical, and it is preferable to use a commercially available preparation. As was pertinently commented, ~3,~4some points need to be stressed for the use of the biotin-avidin system in immunocytochemistry. Biotin is an ubiquitous compound; it is a vitamin required by all living cells, and thus it is present in all tissues and body fluids either in a circulating form or bound to proteins. Consequently, high levels of "nonspecific" binding of avidin or streptavidin can be obtained. Furthermore, avidin is a glycoprotein and thus could bind to lectins sometimes present in tissues.

Immunoblotting Media and Substrate

Saturating medium: PBS-BSA (1%) Washing medium: PBS-Tween 20 (0.1%) Staining solutions for transferred proteins: 0.2% Ponceau S (Serva) in 3% trichloroacetic acid Substrates for enzyme detection: the same as for immunocytochemical assays; the incubation time indicated can, however, be increased until the desired intensity is obtained Procedure. The avidin-biotin system has been successfully used for detecting antigens, after their electrophoretic separation and transfer onto nitrocellulose sheets either by electrophoresis ~5 or by diffusion under pressure, 16 using the protocol outlined in Table VI. 12 S. 13 E. t4 R. 15 H. 16 G.

M. Hsu, L. Raine, and H. Fanger, J. Histochem. Cytochem. 29, 577 (1981). A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980). E. Morris and C. B. Saelinger, Irnrnunol. Today 5, 127 (1984). Towbin, T. Staehelin, and F. Gordon, Proc. Natl. Acad. Sci. U.S.A. 72, 313 (1974). Peltre, J. Lapeyre, and B. David, Immunol. Lett. 5, 127 (1982).

[56]

C H E M I L U M I N E S C E IMMUNOASSAYS NT

481

TABLE VI GENERAL PROCEDURE FOR IMMUNOBLOTTING Step

Procedure

1

Separate proteins by electrophoresis either according to their molecular weights [sodium dodecyl sulfate (SDS)-polyacrylamide gel] or according to their pl (isoelectrofocusing) Transfer the proteins from the gel onto nitrocellulose filters either electrophoretically~5(polyacrylamide gel) or by diffusion under pressure 16(agarose gel) Assess the protein transfer by staining with Ponceau S Saturate the nitrocellulose sites by incubation in the saturating solution, 30 min at 37° Wash Incubate the sheets with the biotinylated antibody (or antigen) at the optimal concentration previously determined (1-20 ttg/ml), 60 rain at 37° Wash Incubate with enzyme-labeled avidin (or streptavidin) (1-10/xg/ml), 15-30 min at 37° Wash Incubate with the appropriate substrate until the desired color intensity is obtained Wash with distilled water Keep dry

2 3 4 5 6 7 8 9 10 11 12

Comments. This procedure has been used with success with biotinylated monoclonal antibodies to characterize their reactivity on electrophoretically separated proteins and with biotinylated antigens to determine the pl of antibodies after separation by isoelectrofocusing.

[56] T w o - S i t e and Competitive

Chemiluminescent Immunoassays By

CHRISTIAN

J. STRASBURGER

and

FORTUNE

KOHEN

Introduction C o n v e n t i o n a l i m m u n o a s s a y p r o c e d u r e s for h a p t e n s a n d for p e p t i d e hormones require the preparation, purification, and characterization of the antigen or antibody labeled with a radioisotope or enzyme, thus rend e r i n g e a c h p a r t i c u l a r a s s a y t e d i o u s to d e v e l o p a n d l i m i t e d in s c o p e . F u r t h e r m o r e , t h e s h o r t half-life a n d r a d i o l y s i s - i n d u c e d d a m a g e o f t h e 125Il a b e l e d a n t i b o d y o r a n t i g e n limit a s s a y s e n s i t i v i t y a n d i m p o s e a t i m e limit

METHODS IN ENZYMOLOGY, VOL. 184

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.