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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.

482

APPLICATIONS

[56]

0 HN.,..IL~NH II

(CHa)4 - C - x

o Ill

0

0

X=-N-(CHa) 5 -C-OI

H X= - N-(CH2)~I H

o

o N- (CH2)3-C-OH

I¢ FIG. 1. Structure of different N-hydroxysuccinimide ester derivatives of biotin with

varying alkyl chains: |a, biotin-N-hydroxysuccinimide ester; |b, biotin-e-aminocaproyl-Nhydroxysuccinimide ester; Ie, biotin-e-aminocaproyl-,/-butyryl-N-hydroxysuccinimide ester.

on the usefulness of a kit. In addition, the potential health hazards associated with the use and disposal of radioactive compounds are incompatible with the development of simple assay procedures in many clinical laboratories and in developing countries. To avoid these drawbacks while retaining the specificity and sensitivity of an immunoassay, we explored the use of biotinylated probes (antibodies, antigens, or enzymes) in the development of immunoassays for polypeptide hormones and haptens. The biotinylated probes were prepared under mild conditions by conjugating N-hydroxysuccinimide ester derivatives of biotin (Fig. 1) to the various proteins (antibodies, antigens, or enzymes) via a peptide linkage. The biotinylated antibodies or enzymes were then evaluated in a solid-phase ELISA, using alkaline phosphatase as the enzyme and colorimetry at 405 nm as an end point. As the Nhydroxysuccinimide ester derivative of biotin possessing a long alkyl chain between the ureido ring of biotin and the carboxy terminus (compound le, see Fig. 1) led to the highest signal intensity, this derivative was chosen for all further biotinylations of proteins. 1 The biotinylated probes serve as primary labeled components in immunoassay procedures. Two types of formats have been adopted in our 1 F. Kohen, Y. Amir-Zaltsman, C. J. Strasburger, E. A. Bayer, and M. Wilchek, in "Complementary Immunoassays" (W. P. Collins, ed.), p. 57. Wiley, New York, 1987.

[56]

CHEMILUMINESCENT IMMUNOASSAYS

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studies: two-site or sandwich-type immunoassays ~-4and competitive-type immunoassays. 4 In both types of formats the high affinity for biotin and the four biotin-binding sites on avidin or streptavidin are exploited in order to amplify the sensitivity of the assay. The properties of the avidin-biotin complex have enabled the development of two different approaches in two-site immunoassays for polypeptide hormones. In both cases an immobilized antibody and a biotinylated antibody preparation are employed, each of which recognizes a different epitope of the antigen. In one approach, following immunological reaction of the immobilized and the biotinylated antibody with the antigen, conjugates comprising avidin or streptavidin covalently labeled with a given probe are employed.l,2 The latter probe can be an enzyme (e.g., alkaline phosphatase or horseradish peroxidase), a chemiluminescent agent, or a fluorescent marker. In the second approach, the use of conjugates is replaced by that of secondary probes. In this approach, unconjugated avidin or streptavidin is first applied followed by the addition of biotinylated enzyme (e.g., alkaline phosphatase, 1 penicillinase, 3 glucose-6-phosphate dehydrogenaseS.6). In both procedures, depending on the label used, the end point is determined by colorimetry, luminometry, disappearance of color, or time-resolved fluorescence. Assays based on these principles have been developed for human growth hormone (hGH) 1'2'4 and human chorionic gonadotropin (hCG). 5,6 In the competitive-type immunoassay for haptens, 4 a solid-phase antigen and the specific homologous biotin-labeled antibody are used. After the immunological reaction, labeled streptavidin is added, and the end point is determined depending on the type of marker attached to streptavidin. Figure 2 shows the approach used for haptens. Assays based on these principles have been developed for cortisol 4 and for estradiol. The use of the avidin-biotin interaction as a mediator in two-site sandwich-type and competitive immunoassays has several advantages: (1) biotinylation of proteins can be achieved with great facility under mild z C. J. Strasburger, Y. Amir-Zaltsman, and F. Kohen, 69th Endocrine Society Meeting, Indianapolis, Indiana, June, 1987, Abstr. No. 228. 3 y. Amir-Zaltsman, B. Gayer, E. A. Bayer, M. Wilchek, and F. Kohen, in "Non-Isotopic Immunoassay" (T. T. Ngo, ed.), p. 117. Plenum, New York, 1988. 4 C. J. Strasburger, Y. Amir-Zaltsman, and F. Kohen, in "Non-Radiometric Assays" (B. Albertson and F. Hazeltine, eds.), p. 79. Alan R. Liss, New York, 1988. 5 F. Kohen, E. A. Bayer, M. Wilchek, G. Barnard, J. B. Kim, W. P. Collins, I. Beheshti, A. Richardson, and F. McCapra, in "Analytical Applications of Bioluminescence and Chemiluminescence" (L. Kricka and T. P. Whitehead, eds.), p. 149. Academic Press, New York, 1984. 6 G. Barnard, E. A. Bayer, M. Wilchek, Y. Amir-Zaltsman, and F. Kohen, this series, Vol. 133, p. 284.

484

APPLICATIONS

[56]

+® B

Solid-phase Antigen

Biotinylated Antibody

Incubate I a.rrIB

+® B

Wash Add

B

B

B

~ ~ l Labeled Streptavidin

L

Chemilumineseent or Enzyme or Fluorescent marker

Determine end point FIG. 2. Schematic of a competitive immunoassay procedure for haptens using biotinyl antibody and labeled streptavidin as probes.

conditions, and the resulting conjugates show very little or no loss of activity1.4; (2) the end point in the assay is versatile (luminometry, colorimetry, time-resolved fluorescence); (3) mediation via the avidin-biotin interaction serves to amplify the sensitivity of the assay; and (4) reactive biotinyl derivatives, biotinylated enzymes, and enzyme-labeled avidin and streptavidin conjugates are all commercially available.

[56]

CHEMILUMINESCENT IMMUNOASSAYS

485

Owing to the commercial availability of reactive biotinyl derivatives (e.g., various N-hydroxysuccinimide ester derivatives of biotin), the biotinylation reaction can be performed with ease in any laboratory. In addition, the use of labeled streptavidin in the assay allows the design of a uniform immunoassay methodology that includes both haptens and polypeptide hormones. The choice of the label on streptavidin depends on the sensitivity desired and on the instrumentation at the disposal of the particular investigator. In the development of immunoassays for haptens and peptide hormones, we have mainly used chemiluminescent-labeled streptavidin in conjunction with measurement of the light yield in a luminometer as the end point. In order to compare this label with others, we also used enzyme- or fluorescent-labeled streptavidin conjugates. As representative examples, we report here a two-site immunochemiluminometric assay for human growth hormone (hGH) and a competitivetype immunoassay for cortisol based on the use of a chemiluminescentlabeled streptavidin as a probe. The use of streptavidin or acetylated avidin and biotinyl enzymes as probes in two-site assays has been described previously in this series 6 and in other publications. L3-5 Methods

Materials. Avidin-alkaline phosphatase conjugate, polyphenylalanine-polylysine (1:1), biotin-N-hydroxysuccinimide, biotin-e-aminocaproyl-N-hydroxysuccinimide, and biotin-e-aminocaproyl-y-butyryl-N-hydroxysuccinimide (Cat. No. 7304-1) were purchased from Bio-Makor (Rehovot, Israel); streptavidin from Cell-Tech (London); alkaline phosphatase (Type VII, from bovine intestine), Tween 20, p-nitrophenyl phosphate, and microperoxidase (MP-11) from Sigma Chemical Co. (St. Louis, MO); etched polystyrene balls from Northumbria Biologicals (England); Sepharose-protein A and disposable Sephadex G-25 PD-10 columns from Pharmacia (Uppsala, Sweden); 8% glutaraldehyde from Ted Bell Inc. (Tustin, CA); and 6-[N-(4-aminobutyl-N-ethyl)-2,3-dihydrophthalazine-l,4-dione] hemisuccinamide (ABEI-H) was a gift from LKB-Wallac (Turku, Finland). The following equipment was used to design and perform the assays described here under optimal conditions: UV monitor, e.g., LKB Uvicord 2138; luminometer, e.g., Berthold Clinilumat or Lumac M2500; reaction trays (20 or 60 wells) from Abbott; multiwash unit, e.g., Pentawash from Abbott; and horizontal shaker, e.g., Heidolph, 170-200 rpm. Preparation of Antibodies Specific polyclonal antibodies to steroids conjugated to bovine serum albumin are raised in rabbits and characterized in terms of titer, affinity,

486

APPLICATIONS

[561

and specificity by radioimmunoassay (RIA) procedures. The hybridoma technique of K6hler and Milstein 7 is used to generate monoclonal antibodies to steroids 8 and to peptide hormones. Polyclonal and monoclonal antibodies belonging to the IgG class are purified by affinity chromatography on Sepharoase-protein A.

Purification of Antibodies on Sepharose-Protein A Mouse IgG1 binds to protein A at pH 8.0, whereas mouse IgG of other subclasses as well as IgG from polyclonal rabbit antiserum are bound at pH 7.2. A Sepharose-protein A column (5 ml) is equilibrated with 0.1 M sodium phosphate buffer (pH 7.2 or 8.0), and 1 ml of ascitic fluid or 0.5 ml of antiserum, diluted with 0.5 ml of the respective buffer, is applied and allowed to react for a period of 30-60 min. The column is rinsed with the same buffer until baseline absorbance (A280)is regained in the effluent. For elution of the IgG fraction from the protein A column, the pH is lowered gradually by replacing the phosphate buffer with 0.1 M citrate buffers of pH 6, 4.5, and 3. The pooled IgG-containing peak is dialyzed against phosphate-buffered saline [(PBS) 10 m M phosphate, 150 m M NaC1 (pH 7.2)] and concentrated to 1-2 mg protein/ml over a P10 membrane in an Amicon concentrator. The preparation is stored at - 2 0 ° until use.

Biotinylation of Antibodies Biotin-e-aminocaproyl-y-butyryl-N-hydroxysuccinimide ester is dissolved in dry, amine-free dimethylformamide (DMF) at a concentration of 25 mg/ml (10/~1 of this solution is sufficient to label 1 mg of IgG, with a 75fold molar excess of biotin per mole of IgG in the reaction). The purified antibodies in PBS are made slightly basic (to pH 8.2-8.4) by adding 45 ~1 of 1 M Na2HPO4 (pH 9) for every milliliter of antibody solution. The above-described biotin-containing reagent (250/xg in 10 /zl of DMF) is then added to the antibody solution (1 mg protein/ml). The reaction mixture is stirred at room temperature for 1-6 hr or overnight at 4°. The reaction mixture is then applied to a Sephadex G-25 PD-10 column equilibrated with 25 m M Tris-HCl buffer (pH 7.1; Tris buffer), and the elution profile is monitored at 280 nm using an LKB Uvicord. The biotinylated antibody is eluted in the first peak (recovery -95%), and the second peak is discarded. The Sephadex G-25 column can be reused up to at least 20 times by regenerating it after use with 50 ml of 0.5 M sodium 7 G. K6hler and C. Milstein, Eur. J. Immunol. 6, 511 (1976). s F. Kohen and S. Lichter, in "Monoclonal Antibodies: Basic Principles, Experimental and Clinical Applications in Endocrinology" (G. Forti, M. B. Lipsett, and M. Serio, eds.), p. 87. Raven, New York, 1986.

[56]

CHEMILUMINESCENT IMMUNOASSAYS

487

chloride followed by 50 ml of Tris buffer. The column is stored after regeneration in PBS containing 0.05% sodium azide. For storage, 5/zl of 20% bovine serum albumin and 2/xl of 10% sodium azide are added per milliliter of biotinylated antibody. The biotinylated antibodies are then stored in aliquots of 1 ml at - 2 0 °. As refreezing of the biotinylated antibody is not recommended, the unused portion of the biotinylated antibody is stored at 4°.

Preparation of Active Ester of ABEI-H The active ester of ABEI-H is prepared by dissolving the hemisuccinamide derivative of ABEI (3.76 mg, 10 t~mol) in 140/xl of dry DMF. NHydroxysuccinimide (1.15 mg, 10/zM) in 20/xl of DMF is added, and after 5 min dicyclohexylcarbodiimide (3.09 mg, 15/zmol) in 40/zl of DMF is added to the solution containing ABEI-H. The vial is then covered with aluminum foil for light protection and kept overnight at 4 °. The activated ester is used in the next step without any further purification. When stored at 4 °, the active ester preparation can be used for at least 1 week.

Conjugation of Chemiluminescent Marker ABEI-H to Streptavidin Streptavidin (2 mg, 0.027 /xmol) is dissolved in 1 ml of 50 m M phosphate buffer (pH 8.3). To this solution the active ester derivative of ABEI-H in DMF (40/zl, 2/.~mol) is added. The reaction mixture is left overnight at 4 °. The precipitate formed is separated by centrifugation, and the supernatant is transferred to a Sephadex G-25 PD-10 column equilibrated with Tris buffer. On elution in the same buffer, three peaks absorb-' ing at 280 nm are obtained. The first peak represents the conjugate. The yield of chemiluminescent-labeled streptavidin is over 60%, and the incorporation ratio is 11-13 mol of the chemiluminescent marker per mole of streptavidin. The conjugate is stored at - 2 0 ° in Tris buffer containing 0.1% bovine serum albumin and 0.05% sodium azide. After thawing, the remaining conjugate is stored at 4 °. At an excess level of reagent, usually 10-20 ng of the streptavidin-ABEI-H conjugate is required per assay tube in our immunoassay procedures.

Preparation of Steroid-Protein Conjugates Steroid-protein conjugates are prepared in two steps. In the first step of the reaction, activated N-hydroxysuccinimide esters of carboxy derivatives of steroids (e.g., cortisol 21-hemisuccinate or cortisol-3-carboxymethyloxime) are prepared as described previously. 9 The activated ste9 F. Kohen, J. de Boever, and J. B. Kim, this series, Vol. 133, p. 387.

488

APPLICATIONS

[56]

roid derivative is then coupled to a protein carrier (ovalbumin or bovine serum albumin) using the above-described conditions for the preparation of chemiluminescent-labeled streptavidin. The conjugates are purified by gel filtration on Sephadex G-25 columns.

Immobilization of Proteins on Polystyrene Balls The following procedure is a modification of the methodology of Wood et al. 1°,11 Activation of Polystyrene Balls. The copolymer poly(phenylalaninelysine) is dissolved in water at 60° and stored at 1 mg/ml at 4 °. One thousand balls are transferred to a 500-ml Erlenmeyer flask and covered with 125 ml of water containing 3.5 mg of the copolymer (3.5/xg polymer/ ball). The coating process is allowed to proceed for 4 days at room temperature with occasional shaking (2 to 3 times/day). After coating, the balls are washed once with 0.15 M NaCI, washed 3 times with water, and dried under a stream of compressed air. The coated dry balls can be stored at room temperature for at least 6 months.

Covalent Coupling of Proteins to Activated Balls Glutaraldehyde activation. One hundred poly(phenylalanine-lysine)coated balls (equivalent to 14.3 g in weight) are covered in an Erlenmeyer flask with 20 ml of 0.4% glutaraldehyde in 50 m M phosphate buffer (pH 8.0). The reaction, during which aldehyde functions are formed on the eamino groups of the lysine residues, is allowed to proceed for 30 min at room temperature, with stirring of the flask every 5 min. After activation, the balls are washed twice with water, washed once with 0.15 M NaCI, and used immediately for the coupling of proteins. Protein coupling. The washed bails are poured into a solution of the desired protein (0.1-0.3 mg/100 balls) in 20 ml of 50 m M phosphate buffer (pH 8.0). The reaction is allowed to proceed for 2-3 hr at room temperature with occasional shaking (every 20 min) and overnight at 4 °, after which 10/~g bovine serum albumin per ball is added as the saturation reagent from a 200 mg/ml stock solution in 0.15 M NaC1 (saline). After a further 90 min at room temperature, the coupling solution is aspirated. Stabilization of glutaraldehyde linkages. The Schiff bases present in the balls prepared as above are reduced for 20 min at room temperature with a solution (20 ml) of freshly prepared 75 m M sodium borohydride in water. The formation of hydrogen bubbles indicates that the reaction is taking place, and the flask is shaken every 5 min. The sodium borohydride 10 W. G. Wood and A. Gadow, J. Clin. Chem. Clin. Biochem. 21, 789 (1983). 11 W. G. Wood, J. Braun, and U. Hantke, this series, Vol. 133, p. 354.

[56]

CHEMILUMINESCENT IMMUNOASSAYS

489

solution is aspirated, and the balls are washed 4 times with water. For storage the balls are covered with Tris buffer containing 2.5% bovine serum albumin and 0.05% sodium azide. At 4 ° in this solution the balls are stable for at least 2 years. Coating of Microtiter Plates. A solution of hGH (100/~1, 2.5/zg/ml) in 50 m M carbonate buffer (pH 9.6) is distributed into each well of a microtiter plate. The plate is incubated overnight at 4 ° and drained. A solution (100/~1) of 0.3% bovine serum albumin in saline is the added to each well to block additional binding sites, and the plate is left at room temperature for 2 hr. After draining and 1 wash step with 0.05% Tween 20 in saline, the plate is covered and can be stored dry at - 7 0 ° for at least 2 months.

Reagent Solutions Buffer A: 50 m M Tris-HCl (pH 7.4) containing, per liter, 5 g bovine serum albumin (BSA) and 0.5 g sodium azide Buffer B: buffer A, containing in addition, 0.5 g bovine y-globulin, 0.1 ml Tween 20, and 9 g NaC1 per liter Coating buffer: 50 m M sodium carbonate (pH 9.6) containing 0.1 g sodium azide per liter Wash solution: 9 g/liter NaC1 containing 0.05% (v/v) Tween 20 Heme catalyst: a stock solution of 1 mg/ml of microperoxidase (MP11, Sigma) is prepared in water; the stock solution can be stored for up to 6 months at 4 °, and a working solution is prepared freshly before each assay by diluting the stock solution 1 : 100 with distilled water Oxidant solution: a working solution is prepared freshly by mixing 0.1 ml of 30% hydrogen peroxide solution (Merck, Darmstadt, FRG) with 15 ml of distilled water

Evaluation of Biotinylated Proteins: Effect of Spacer Length Exemplified by Detection of Enzyme Activity The effect of the spacer length in various biotinyl derivatives (Fig. 1) on the signal in immunoassays involving avidin-biotin amplification was investigated by the following experiment. The biotin derivatives la (short chain) and Ic (long chain) are used to derivatize both alkaline phosphatase and a monoclonal antibody against human growth hormone (hGH) (clone 69, see below). The conjugates obtained are evaluated by incubating serial dilutions of the two biotinyl antibody derivatives in hGH-coated microtiter plates overnight 4 °, followed by sequential incubations for 30 min each at room temperature first with 100 ng of underivatized streptavidin and then with one of the two different biotinyl derivatives of alkaline phospha-

490

APPLICATIONS

[56]

1.6

"~

1.2

i--

Z

0,8

op,~ 0 V)

,x

0.~

I

1:1000

I

l

1:2000

I

I:~000

Biotinylated Ab Dilution

FIG. 3. Effect of spacer length of the biotinylated proteins on the signal intensity of the color development reaction, hGH-coated microtiter plates were incubated sequentially with different combinations of short- and long-chain derivatives of antibody 69 and of alkaline phosphatase. For experimental details, see text. Filled symbols represent the use of alkaline phosphatase derivatized with long-chain biotin (compound le, Fig. 1), whereas open symbols indicate the use of short-chain (la) biotinyl enzyme. Circles indicate long-chain (!¢) derivatized antibody, triangles short-chain (la) biotinylated antibody 69. tase at 1 : 1000 dilution. The incubation steps are carried out in a 0.1-ml volume of buffer B and separated by washing 3 times with 0.05% T w e e n 20 in saline. Substrate solution [1 mg/ml p-nitrophenyl phosphate in 10% diethanolamine (pH 9.6) containing 5 m M MgC12] is added, and the yellow color that develops is m e a s u r e d at 405 nm. The results (Fig. 3) indicate that significantly higher specific signals are obtained with the long alkyl chain derivatives of biotin. We therefore use the biotin derivative le in all subsequent experiments.

Two-Site lmmunochemiluminometric Assay for Human Growth Hormone Using Labeled Streptavidin as Probe 1,2,4 Reagents Monoclonal antibodies to h G H which bind to different epitopes on the H G H molecule: clone 518, affinity constant KA = 8 × 101° M - l ; clone 69, affinity constant KA = 2 × 109 M -1

[56]

CHEMILUMINESCENT IMMUNOASSAYS

491

hGH reference preparation: WHO International Laboratory for Biological Standards, Hampstead, London NW3 6RB, England Streptavidin-ABEI-H Serum specimens: from normal individuals or from patients undergoing growth hormone stimulation tests or submitted to the laboratory to assess growth hormone pituitary reserve; sera are stored at - 2 0 ° until assayed Purified monoclonal antibody (clone 518) coupled to polystyrene balls (as described above) serves as the capture antibody Monoclonal antibody to hGH (clone 69) biotinylated with compound I¢ (Fig. 1) Streptavidin labeled with ABEI-H (see text) is used as the chemiluminescent probe Assay buffer (buffer B) and reagents for initiating the light reaction are described above Procedure. A schematic diagram of the procedure is shown in Fig. 4, and details are presented in the legend. After the final incubation step of the reaction with ABEl-H-labeled streptavidin, the balls are washed 3 times. Each individual ball is then placed in a Lumacuvette, and sodium hydroxide (2 N, 250 ~1) is added to each tube. The tubes are incubated at 60° for 30 min. After cooling to room temperature and placing the tubes in the luminometer, 100/~1 of diluted microperoxidase and 100 ~1 of oxidant solution (0.2% H202) are injected sequentially into each individual tube. The chemiluminescence signal is integrated and recorded for 10 sec. Evaluation. A typical dose-response curve (Fig. 5) reflects the analog recording of the signal emanating from the photomultiplier tube. The sensitivity of the method (defined as the zero-standard signal plus 2 standard deviations) is 1.5 pg of hGH/tube or 0.03 mlU hGH/liter. This technique provides a working range of 0.04-100 mlU hGH/liter. The intraassay precision is 5.5%, and the interassay precision is 7.4%. The dynamics of hGH concentrations in basal plasma levels or under stimulation tests in normal patients are determined by conventional RIA and the two-site immunochemiluminometric assay. A good correlation between the two methods is consistently obtained (r = 0.97, n = 88). As an example, Fig. 6 shows the circadian profile of hGH levels in a female patient as determined by RIA and immunochemiluminometric assay. In addition, this method has been used to detect basal secretion and minor bursts in the circadian profile of hypopituitary patients whose plasma hGH levels are undetectable by conventional RIA methods.l,2,4 This method shows a gain in sensitivity by a factor of 10 in comparison with existing radio- or enzyme-immunoassay methods.

492

APPLICATIONS

[56]

50 #1 standard or sample +

50 /Jl horse serum or buffer

+ 100 /~l biotinylated anti-hGH (clone #69) diluted in assay buffer (I00 ng IgG/tube) +

1

anti-hGH (clone #518) coated ball (6.4 mm diameter)

t

incubate overnight at 4° wash 8 z (Penta-wash system)

+ 200 #1 ABEI-H labeled streptavidin diluted in assay buffer (20 ng/t'ube)

I

incubate for 40 minutes on horizontal rotator wash 3 x (Penta-wash system)

measure chemiluminescence in the alkaline microperoxidase 11202 system Fro. 4. Flow diagram of a two-site immunochemiluminometric assay for hGH. The biotinylated antibodies and chemiluminescent-labeled streptavidin are diluted in buffer B, The hGH standards (5(1~l/well) are prepared in buffer A, and 50 ~1 of horse serum is added to each well to compensate for matrix effects. The volume of the serum samples was 50 ~l/well, and each well received 50 ~l of buffer A.

Competitive-Type Immunoassays for Haptens Based on Biotinyl Antibody and Labeled Streptavidin as Probes Exemplified by Immunoassay of Salivary Cortisol Reagents P o l y c l o n a l r a b b i t a n t i s e r u m to c o r t i s o l - 3 - c a r b o x y m e t h y l o x i m e - b o vine serum albumin conjugate Ovalbumin-cortisol-3-carboxymethyloxime conjugate Streptavidin-ABEI-H The antibodies are purified on Sepharose-protein A and biotinylated u s i n g c o m p o u n d l e ( s e e F i g . I) as d e s c r i b e d in t h e t e x t . C o r t i s o l - 3 - c a r -

[56]

CHEMILUMINESCENT IMMUNOASSAYS

493

I000~/tul~e1 50C)oq/!

Z~Ol~/tube/

FIo. 5, Analog recording of a dose-response curve for hGH using biotinyl antibodies as primary probe, chemilurninescent-labeled streptavidin as secondary probe, and luminometry as an end point. The light generated on introduction of each sample is integrated for 10 sec and recorded in arbitrary light units.

9

~

....

o-----o measured by ICMA = =measured by RIA

I0.0~

fl

method

/l

5,0 2.5 0.0 . . . . . .

0

I0

-. . . . . . . .

:50 SampleNo. 20

40

50

FIG. 6. Circadian plasma hGH pattern in a patient (5 years accelerated growth) as determined by RIA and immunochemiluminometric assay. The RIA data were measured at the laboratory of A. Kowarski (Baltimore, MD). The low values plotted as zero for the RIA method represent measurements of less than 0,25 ng/ml, the detection limit of the RIA method.

494

APPLICATIONS

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10 #1 sample or standard + 200 #1 biotinylated polyclonal anti-cortisol

antibodies, diluted in assay buffer (30 ng IgG/tube) +

1

Cortisol-coated ball (6.4 mm diameter)

incubate for 40 minutes on horizontal rotator wash 3 x (Penta-wash system)

1 + 200 ~1 ABEI-H labeled streptavidin diluted in

assay buffer (20 ng/tube)

I

incubate for 40 minutes on horizontal rotator

wash 3 x (Penta-wash system)

measure chemiluminescence in the alkaline mieroperoxidase 1120 2 system

FIG. 7. Flow diagram for a competitive-type immunoassay for salivary cortisol using biotinyl antibody and chemiluminescent-labeled streptavidin as probes.

boxymethyloxime-ovalbumin conjugate is coupled to polystyrene balls and serves as the solid-phase antigen. Streptavidin labeled with ABEI-H is used as the chemiluminescent probe. For hapten assays buffer B (see above) is used throughout, and all the other reagents are the same as those used for the hGH assay. Immunoassay Procedure. A schematic diagram of the method is shown in Fig. 7. After the final incubation step, the balls are treated as described for the hGH assay. The light generated is integrated for I0 sec and recorded. Evaluation. A typical dose-response curve is shown in Fig. 8. The sensitivity of the method is less than 3 pg/tube and is comparable to that obtained by conventional RIA methods. For comparative purposes we have used two other end points in the cortisol assay (fluorometry or colorimetry, respectively, using streptavidin-fl-galactosidase or avidinalkaline phosphatase as the markers). Table I shows a comparison of the

[56]

CHEMILUMINESCENT IMMUNOASSAYS 41 I

L

I

n

~

495 u

1001 90 80 70 5O ~0

30 20 10

/i I 0

10

I

I

I

I

I

20

50

100

200

500

Cortiso[ (pg/tube) FIG. 8. Dose-response curve for cortisol using chemiluminescence as an end point. The value of B/Bo represents the ratio of the "bound" signal to the zero-dose bound signal.

three methods. We would like to stress that all these techniques can easily be set up in most laboratories and that the end point in a given assay will depend on the available instrumentation. Discussion

The use of biotinylated probes in immunoassays for haptens and peptide hormones is appealing for a variety of reasons: (1) the biotinylated enzymes and antibodies are stable reagents, and they can be prepared TABLE I COMPARISON OF THREE DIFFERENT END POINTS FOR MEASUREMENT OF CORTISOL BY SOLID-PHASE ANTIGEN LUMINESCENCE TECHNIQUE (SPALT) METHOD

Variable Detector response (light units or OD) Sensitivity (/zg/dl) Working range (/xg/dl) Reagent stability

Chemiluminescence~

End-point fluorometry b

Colorimetry c

23191-290

684-24.6

1.138-0.072

0.1 0.1-100

0.2 0.2-100 More than 2 years

0.2 0.2-100

System used: components of Fig. 2 in which L is a chemiluminescent marker. b System used: components of Fig. 2 in which L is/3-galactosidase. c System used: components of Fig. 2 in which L is alkaline phosphatase.

a

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easily since the reactive biotinyl derivatives are commercially available; (2) the end-point reaction in the assay can be varied according to the character of the avidin or streptavidin conjugate used in the method (colorimetry, fluorescence, or luminescence); and (3) the use of labeled avidin or streptavidin enables a uniform immunoassay methodology that comprises both small and large molecules. We have described here a two-site immunochemiluminometric assay for hGH and a competitive-type assay for cortisol. In both methods, chemiluminescent-labeled streptavidin serves as the marker. The metho d s can be applied for the measurement of other polypeptide hormones (e.g., luteinizing hormone, hCG, or thyroid-stimulating hormone) or haptens (e.g., estradiol). The two-site technique requires only a capture antibody and a biotin-labeled antibody directed against a different epitope of the antigen. On the other hand, the competitive-type assay for haptens requires a solid-phase antigen and a specific biotinylated homologous antibody. The present avidin-biotin-mediated immunochemiluminometric assay for hGH has a lower limit of sensitivity below 75 amol (attomoles) of hGH per sample. The dynamic range of the signal (zero-dose versus high-dose) obtained in the cortisol assay using a biotinylated polyclonal antibody shows a ratio of approximately 5 : I. However, when using the same assay technique in an estradiol assay employing a biotinylated monoclonal antibody, ~2 the dynamic range of the signal was increased to 20 : 1. This result suggests that the slope of a hapten assay can be improved by using biotinylated monoclonal antibodies instead of biotinylated polyclonal antibodies. Acknowledgments We are grateful to Drs. M. Wilchek, E. A. Bayer, G. Barnard, G. Messeri, J. Kostyo, Z. Zadik, Y. Zaltsman, and Mr. L. Toido for permission to quote collaborative work; to Mr. B. Resch and Dr. F. Berthold for the loan of a Clinilumat luminometer; to Dr. M. Pazzagli for the gift of rabbit anticortisol serum; to Dr. N. Moav for a generous gift of the monoclonal antibodies to hGH; and to Mrs. M. Kopelowitz for excellent secretarial assistance. C. J. S. is a Minerva Fellow at the Weizmann Institute of Science.

t2 C. J. Strasburger and F. Kohen, unpublished results.

Two-site and competitive chemiluminescent immunoassays.

[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 electro...
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