[125I] protein A: A tracer for general use in immunoassay

Journal oflmmunological Methods, 24 (1978) 269--285 © Elsevier/North-Holland Biomedical Press

['2sI]PROTEIN

A: A TRACER

FOR GENERAL

269

USE IN IMMUNOASSAY

JOHN J. LANGONE Laboratory o f lmmunobiology, National Cancer Institute, National Institutes o f Health, Bethesda, MD 20014, U.S.A. (Received 20 April 1978, accepted 6 June 1978)

An immunoassay method was developed in which 125I-labeled Protein A ([125 I]PA) of high specific activity (100 Ci/mmole) and functional activity (t>85%) served as a general tracer. Antigen (or hapten) was immobilized by covalent binding to a solid bead support. Aliquots of the appropriate beads were incubated with antibody (either purified IgG fraction or whole antiserum), washed with buffer, then incubated with [12SI]PA. The amount of [12sI]PA bound to the antibody-coated beads was a measure of antibody binding. The ability of antigen (or hapten) in the fluid phase to inhibit the binding of antibody under optimal conditions, measured as inhibition of [I~'5I]PA binding, served as the basis for quantification in the assay. The method was applied to 3 antigens (human chorionic gonadotropin (HCG), human immunoglobulin M (IgM) and goat immunoglobulin G (IgG)) and to methotrexate as an example of a hapten. Optimal assay conditions were developed and, in each case, picomole levels or less of the homologous ligand could be detected. Antibody specificity was determined by measuring the ability of compounds related to the antigen or hapten to act as inhibitors. Levels of HCG in urine from pregnant women and levels of IgM in normal human sera were determined by this method. The assay required only approximately 3 h to perform, gave accurate reproducible results, and was at least as sensitive as other available immunoassay methods (e.g. radioimmunoassay). INTRODUCTION

I m m u n o a s s a y s w h i c h u t i l i z e r a d i o l a b e l e d l i g a n d s (e.g. r a d i o i m m u n o a s s a y , RIA), or radiolabeled antibody (immunoradiometric assay) are among the m o s t s e n s i t i v e a n d s p e c i f i c a n a l y t i c a l m e t h o d s a v a i l a b l e ( f o r r e v i e w s , see Berson and Yalow, 1971; Odell and Daughaday, 1971; Butler and Beiser, 1973; Sonksen, 1974; Rose and Friedman, 1976). These assays, especially R I A , a r e c o m m o n l y u s e d in t h e c l i n i c a n d t h e r e s e a r c h l a b o r a t o r y t o d e t e c t b i o l o g i c a l l y i m p o r t a n t m o l e c u l e s in p h y s i o l o g i c a l f l u i d s a t t h e n a n o m o l e level o r l o w e r ( O d e l l a n d D a u g h a d a y , 1 9 7 1 ; R o s e a n d F r i e d m a n , 1 9 7 6 ) . O n e o f t h e c r i t i c a l c o m p o n e n t s o f t h e s e a s s a y s is t h e r a d i o l a b e l e d t r a c e r . C o n sidering RIA, a specific radiolabeled ligand must be prepared for each assay. G e n e r a l l y , '2sI a n d 3H a r e t h e n u c l i d e s o f c h o i c e s i n c e h i g h e r s p e c i f i c a c t i v i t i e s a r e p o s s i b l e c o m p a r e d t o '4C. S i n c e t h e s p e c i f i c a c t i v i t y o f t h e r a d i o l a b e l e d l i g a n d is o n e d e t e r m i n a n t o f a s s a y s e n s i t i v i t y , a c t i v i t y in t h e C i / m m o l e r a n g e is d e s i r a b l e . H o w e v e r , s o m e c o m p o u n d s a r e u n s t a b l e w h e n

270 radiolabeled and lose immunoreactivity while others are labile to the oxidizing agents used in the chloramine T or lactoperoxidase iodination methods (Greenwood, 1971; Hunter, 1974). In many cases, hapten derivatives containing tyramine or structurally related groups that permit the introduction of ~2Sl must be prepared and purified. Results presented in this report demonstrated that ~2SI-labeled Protein A ([~2sI]PA; specific activity >100 Ci/mmole) could be prepared routinely and that the functional activity, i.e., the ability of the labeled product to bind IgG, was >~85% for at least 4 weeks. Based on the competition between IgG in the fluid phase and rabbit IgG bound to a solid support for a limited a m o u n t of [~sI]PA, the specificity of PA for IgG from 12 species was determined on a quantitative basis. The ability of [12sI]PA to react specifically with the Fc region of IgG from certain species without affecting the ability of antibody to bind antigen served as the basis for a general immunoassay procedure in which [~SI]PA was the only radiolabeled tracer necessary. The assay was simple to perform, was at least as sensitive as other immunoassay methods (e.g. RIA), and required less than 3 h to perform. Optimal conditions were established and assays developed for human chorionic gonadotropin (HCG), human IgM, and goat IgG. Development of an assay for the cancer chemotherapeutic agent methotrexate demonstrated the applicability of the method to analysis of haptens as well as antigenic macromolecules. The appropriate assay was used to measure HCG equivalents in urine from pregnant women and the concentration of IgM in normal human sera. MATERIALS AND METHODS

Reagents Protein A was purchased from Pharmacia Fine Chemicals, Inc., Piscataway, NJ. Rabbit or goat IgG bound to agarose {approximately 3 pg IgG/mg beads), agarose beads functionalized with free carboxyl groups (Immunobeads), and Affi-gel 701 (polyacrylamide beads functionalized with free amino groups) were purchased from Bio-Rad Laboratories, Rockville Centre, NY. Chromatographically pure IgG fractions listed in Table 2, human IgM (HuIgM), and the IgG fractions of rabbit antisera to human chorionic gonadotropin, human IgM {heavy chain specific) and goat IgG {heavy and light chain) were purchased from Cappel Laboratories, Downington, PA. Whole rabbit anti-methotrexate serum was kindly supplied by Dr. Lawrence Levine, Department of Biochemistry, Brandeis University, Waltham, MA. Human chorionic gonadotropin (HCG) was purchased from Calbiochem, La Jolla, CA, and methotrexate (MTX) was supplied by the Drug Evaluation Branch of the National Cancer Institute, NIH, Bethesda, MD. 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and compounds tested as inhibitors in the immunoassays were the highest quality reagents available and purchased from commercial sources.

271

Buffers Isotonic veronal buffered saline, pH 7.2, containing 0.1% gelatin, 0.001 M Mg 2+ and 0.00015 M Ca 2÷ (VBS-gel) was prepared as described in Rapp and Borsos (1970). Isotonic phosphate-buffered saline (PBS), pH 7.2, was obtained from the NIH Media Unit.

lodination o f Pro tein A ([12si]PA) The general procedure used to prepare ~2SI-labeled PA was described in Langone et al. (1977).

Immobilized HCG, HulgM and methotrexate HCG (0.50 mg) and HuIgM (1.0 mg) were coupled to Immunobeads by the following procedure. The protein in 0.5 ml of 0.003 M phosphate, pH 6.35, was added to 100 mg of Immunobeads suspended in 9.0 ml of the same buffer. EDAC (6 mg added as 0.3 ml of a 20 mg/ml solution in buffer) was added and the mixture allowed to rock overnight at 4°C. The tubes were centrifuged, the supernatant fluids decanted and the beads washed with three 10 ml fractions of cold PBS. Ten milliliters of cold 5 M guanidine hydrochloride, pH 7.2, was added and the beads centrifuged immediately. After two additional similar washings, the beads were washed 8 times with 10 ml portions of PBS and allowed to stand in this buffer for 2 h at 4°C. The beads were washed twice with 10 ml portions of VBS-gel and stored in 25 ml of VBS-gel containing 0.04% sodium azide. Methotrexate (300 pg) was coupled to Affi-gel 701 (500 mg) by the same general procedure.

Protein binding assay for fluid phase IgG The general procedure to measure fluid phase IgG has been reported (Langone et al., 1977).

Immunoassay procedure VBS-gel was used to make all dilutions. In the general immunoassay procedure, 0.1 ml of diluted antibody, 0.1 ml of test sample (or buffer to determine maximum binding) and 0.05 ml of the appropriate bead suspension were incubated at 30°C for 45--60 rain (except in kinetics experiments; see Results). The beads were washed with two 3 ml portions of VBS-gel, then incubated for an additional 60 min at 30°C with 0.1 ml of [~2Sl]PA (approximately 38,000 counts/min (cpm) unless otherwise specified). The beads were washed twice with 3 ml portions of VBS-gel and bound [~2sI]PA determined by counting in a Packard model 5360 auto gamma spectrometer. The counter efficiency was 69%. Control tubes included [~2sI]PA plus beads, antibody alone plus [12sI]PA and inhibitor plus beads treated with [12sI]PA. RESULTS

12Sl-labeled Protein A ([12sI]PA) The Bolton--Hunter reagent (Bolton and Hunter, 1973) (Amersham-

272 TABLE 1 SPECIFIC ACTIVITY OF [12SlJPROTEIN A PREPARATIONS a No. of iodinations

Specific activity (Ci/mmole)

10 9

Mean ± S.D.

Range

114 ± 21 108 ± 10

8.t--165 8,1--118

a 50 ~g of Protein A was iodinated with 0.2 mCi Bolton--llunter reagent as described in Langone et al. (1977).

S e a r l e , > 1 4 0 0 C i / m m o l e ) w a s u s e d t o i o d i n a t e P r o t e i n A ( L a n g o n e e t al., 1 9 7 7 } . T h e r e s u l t s o f 10 i o d i n a t i o n s p e r f o r m e d d u r i n g 1 y e a r a r e s u m m a r i z e d in T a b l e 1. T h e d a t a s h o w e d t h a t a s p e c i f i c a c t i v i t y o f a t l e a s t 1 0 0 C i / m m o l e c o u l d b e o b t a i n e d r o u t i n e l y . W h e n t h e s i n g l e i o d i n a t i o n w h i c h gave an abnormally high specific activity (165 Ci/mmole) was disregarded, the standard error for the remaining 9 samples was only -10~, of the mean (108 Ci/mmole).

Relative specificity of Protein A for IgG from different species I n an e a r l i e r r e p o r t ( L a n g o n e e t al., 1 9 7 7 ) , w e d e s c r i b e d a q u a n t i t a t i v e a s s a y for fluid phase IgG based on the inhibition of binding of [12sI]PA to rabbit I

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Fig. 1. Inhibition of [125IJPA binding to 20 PI~ of rabbit IgG Immunobeads by differing amounts of rabbit (e o), swine (,; :~), mouse (A-A), and rat (:.-,.) IgG. Out of 37,000 cpm [125I]PA added, approximately 10,800 epm were bound under the optimal assay conditions described in Langone et al. (1977).

273 TABLE 2 I N H I B I T I O N O F BINDING O F [12sI ] P R O T E I N A TO I M M O B I L I Z E D R A B B I T IgG BY IgG F R O M D I F F E R E N T S P E C I E S .

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lIorse Sheep Goat Rat Chicken

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N a n o g r a m s IgG required to inhibit by 50% 60 60 60 135 290 3000 4500

5000 40,000 :-"100,000 a > 100,000 b > 100,000 b

at this level. b < 15% i n h i b i t i o n at this l e v e l

a 45% inhibition

IgG b o u n d covalently to agarose beads. It was d e m o n s t r a t e d that human, guinea pig and rabbit IgG were equally efficient inhibitors. To furt her characterize the specificity of PA on a quantitative basis, the IgG fractions from several o t h e r species were tested in the same assay. The curves in Fig. 1 show representative results for IgG from rat, mouse, pig and rabbit. Approximately 60 ng o f rabbit IgG and 140 ng of swine IgG were required to give 50% inhibition of Protein A binding and as little as 12 or 20 ng gave 20% inhibition, respectively. In contrast, 5 pg of mouse IgG gave 50% inhibition while even 100 ~g o f rat IgG failed to inhibit significantly. The data in Table 2 summarize the results obtained for the 12 species tested and showed that the specificity of Protein A for IgG varied by a factor of over 103. The data were expressed as the q u a n t i t y o f IgG required to inhibit the binding of ['2SI]PA to rabbit IgG beads by 50% under conditions which we have shown to be optimal (Langone et al., 1977).

Immunoassay for goat IgG It was considered possible that the IgG preparations that were ineffective inhibitors might bind [12sI]PA in the absence o f rabbit IgG. Based on the results shown in Fig. 2A, this was n o t the case. G oat IgG (a p o o r inhibitor, see Table 2) bound to agarose beads was tested for its ability to bind ['2sI]PA. These results were compared to the binding observed when rabbit IgG beads were used. Under the test conditions, m a x i m u m binding (approximately 15,000 cpm) to the rabbit IgG beads was obtained when an a m o u n t o f beads equivalent o f 0.4 ~g IgG was used. In contrast, the goat IgG beads failed to bind a significant a m o u n t of ['2sI]PA even when an equivalent o f 2.4 ~g goat IgG was tested. Thus, the data in Table 2 gave a quantitative

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Fig. 2. A: b i n d i n g o f [ i 2 S I J P A ( 1 8 , 0 0 0 c p m a d d e d ) to differing a m o u n t s of r a b b i t (:~ ---(:) o r goat ( e - e ) IgG [ m m u n o b e a d s . A l i q u o t s of b e a d s were i n c u b a t e d for 6 0 rain at 3 0 ° C w i t h [ 1 2 5 I ] P A in a t o t a l v o l u m e o f 0.3 ml VBS-gel. T h e b e a d s were w a s h e d twice with 3 ml p o r t i o n s o f cold VBS-gel b e f o r e t h e n u m b e r o f c p m b o u n d was d e t e r m i n e d . A relative bead c o n c e n t r a t i o n o f 1 c o r r e s p o n d s to 0 . 0 0 9 ktg of r a b b i t IgG or 0.04 p g goat IgG. B: i n h i b i t i o n of b i n d i n g o f r a b b i t a n t i - g o a t IgG (0.1 ml of a 1 : 1500 d i l u t i o n ) to goat IgG I m m u n o b e a d s by differing a m o u n t s of goat (:~ <:), s h e e p (dP-e), h u m a n (A . . . . &), swine ( I m), h o r s e (:". • --,".), or r a b b i t ( - - r) [gG. A n a m o u n t (50 pl) o f goat IgG I m m u n o b e a d s e q u i v a l e n t to a p p r o x i m a t e l y 0.04 ~g goat IgG was used. O u t of 3 7 ; 5 0 0 c p m [12.; 1]PA a d d e d , 1 1 , 4 0 0 c p m were b o u n d u n d e r t h e assay c o n d i t i o n s .

275 measure o f Protein A specificity for IgG from the 12 species tested. Based on these observations a 'sandwich' t y p e immunoassay for goat IgG was developed using the IgG fraction of rabbit antiserum to goat IgG and [12sI]PA as the tracer. Optimal bead and rabbit a n t i b o d y concentrations were determined as detailed below for the HCG assay. The ability of differing amounts of goat IgG and IgG from o t h e r species to inhibit the binding o f rabbit anti-goat IgG to the beads was determined indirectly by measuring the inhibition o f [~2sI]PA binding (see Materials and Methods). T he inhibition curves are shown in Fig. 2B. Only 13 ng of goat IgG gave 50% inhibition o f [~25I]PA binding and 4.5 ng gave 15% inhibition. Sheep IgG also was an effective c o m p e t i t o r since only 40 ng was required for 50% inhibition. In contrast, 10,000 ng of human, horse, rabbit or pig IgG gave no more than 20% inhibition. T he general applicability of this m e t h o d was d e m o n s t r a t e d by developing assays for HCG, HuIgM and m e t h o t r e x a t e . In each case, [12sI]PA was used as the radiolabeled tracer.

Immunoassay for HCG HCG bound to agarose beads was prepared as described in Materials and Methods. The ability o f differing concentrations of the IgG fraction of rabbit anti-HCG to bind to di f f er e nt amounts o f the beads is shown in Fig. 3. With a n t i b o d y diluted 1 : 250 a m a x i m u m binding of [12sI]PA (approxi m at el y 25,500 cpm o u t of 37,000 cpm added) was achieved when 0.05 mg o f beads was used. As expected, as more dilute a n t i body was used, the m axi m um n u m b e r of cpm bound (as an indicator o f bound HCG specific IgG) also decreased. Even when the a n t i b o d y was diluted 1 : 6750 over 3500 cpm o f added radioactivity was bound to the beads. In the absence of a n t i b o d y , there was no significant binding o f [12sI]PA at any of the bead concentrations tested. In the n e x t expe r i m e nt , the binding of differing amounts of ['2sI]PA to 4 different co n cent r at i ons of HCG beads that had been pretreated with a 1 : 250 dilution of anti-HCG was determined. T he results are shown in Fig. 4. The highest a m o u n t of beads tested (0.04 mg) bound up to 50,000 cpm at the highest c o n c e n t r a t i o n ( 1 00, 000 cpm) of [~25I]PA. However, excess [12sI]PA was n o t present under these conditions since the binding curve did n o t plateau. When 0.013 mg of beads was used, addition of a p p r o x i m a t e l y 35,000 cpm gave m a x i m u m binding (16,000 cpm), i.e. [~2sI]PA was in excess since addition o f more radiolabel did n o t increase the binding significantly. As expected, with less beads less [~2sI]PA was required to give m a x i m u m binding. Even when only 0.0014 mg of beads was used, a m a x i m u m o f 2000 cpm was b o u n d c om pared to a background binding of 300 cpm to the beads in the absence of antibody. Based on these results, 3 8 , 0 0 0 - - 4 0 , 0 0 0 cpm [12sI]PA generally was added to 0.10--0.013 mg beads (50 ul) to give binding of a p p r o x i m a t e l y 15,000 cpm against a background o f ab o u t 300 cpm. Under these conditions, or if fewer beads were used, ['25I]PA was in excess.

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Fig. 3. Binding of [1251]PA (0.1 ml, 38,000cpm added) to differing amounts of HCG beads that were treated with rabbit anti-HCG diluted either 1 : 250 (e o), 1 : 750 (~', .:~), 1 : 250 (/'./.), or 1 : 6750 (& A). Binding of [125I]PA to beads in the absence of antibody (- . . . . . :~) also was determined. The beads were incubated with antibody for 60 min at 30°C, washed, then incubated with [12SI]PA for 60 min at 30°C before the number of cpm bound was determined. A relative concentration of 1 = 2.5 /lg HCG beads.

Kinetics of antibody and [~2sI]PA binding In t h e e x p e r i m e n t s described so far, beads were i n c u b a t e d with a n t i b o d y for 60 min at 30°C. T h e y were t h e n washed with b u f f e r and i n c u b a t e d with [~2sI]PA for 60 min at 30°C b e f o r e the n u m b e r o f c p m b o u n d was determined. The results o f kinetics e x p e r i m e n t s s h o w n in Fig. 5 indicated t h a t these c o n d i t i o n s were sufficient to give m a x i m u m binding o f a n t i b o d y to the beads (Fig. 5A) and o f [ ' 2 s I ] P A to the a n t i b o d y - t r e a t e d beads (Fig. 5B). In Fig. 5A aliquots c o n t a i n i n g 0.04, 0 . 0 1 3 , or 0 . 0 0 4 3 mg HCG beads (50 ~l) were i n c u b a t e d with 0.1 ml o f anti-HCG (1 : 250) at 30°C. A t differing times, samples were washed twice with 3 ml VBS-gel, then i n c u b a t e d with 0.1 ml [ ' 2 s I ] P A ( 3 8 , 0 0 0 c p m ) for 60 min at 30°C. A f t e r the beads were washed with buffer, the a m o u n t o f [~2sI]PA b o u n d was d e t e r m i n e d . T h e curves s h o w e d t h a t for each c o n c e n t r a t i o n o f beads tested, a 15 min incubation period was sufficient t o give m a x i m u m a n t i b o d y binding. Similarly, to o b t a i n t h e curves in Fig. 5B, the same 3 c o n c e n t r a t i o n s o f HCG beads were

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i n c u b a t e d with anti-HCG for 60 min at 30~C. T h e beads were washed, then i n c u b a t e d at 30°C for differing times with [ I : s I ] P A ( 3 8 , 0 0 0 c p m ) . F o r each c o n c e n t r a t i o n o f beads, m a x i m u m binding o f [~2sI]PA to the a n t i b o d y c o a t e d beads was achieved b y 1 5 - - 2 0 min.

Inhibition o f anti-HCG binding by HCG and related compounds T h e curves in Fig. 6 d e m o n s t r a t e d the ability o f differing a m o u n t s o f HCG to inhibit the binding o f anti-HCG to the HCG beads, using [12sI]PA as the tracer. In Fig. 6A, the e f f e c t o f varying the c o n c e n t r a t i o n s o f a n t i b o d y was d e t e r m i n e d using a c o n s t a n t a m o u n t o f HCG beads. Mixtures c o n t a i n i n g 0 . 0 1 3 mg o f beads and serial dilutions o f HCG were i n c u b a t e d with a n t i b o d y diluted either 1 : 250, 1 : 750, or 1 : 2 2 5 0 . The beads were washed, incub a t e d with [ 1 : s I ] P A and the r a d i o a c t i v i t y b o u n d was d e t e r m i n e d after a 60 min i n c u b a t i o n at 30°C. A t the highest a n t i b o d y c o n c e n t r a t i o n tested (1:250), approximately 14,200cpm [ I : s I ] P A was b o u n d and 7 0 n g (0.07 IU) o f HCG gave 50% i n h i b i t i o n o f a n t i b o d y binding. T h e sensitivity o f the assay increased as the a n t i b o d y c o n c e n t r a t i o n was decreased. When

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Fig. 5. A: k i n e t i c s o f b i n d i n g o f r a b b i t a n t i - H C G (1 : 250) to e i t h e r 4.3 (A :'), 13 (~. --e), or 4 0 p g (c~ o) HCG b e a d s as m e a s u r e d by [12SI]PA ( 3 8 , 0 0 0 c p m a d d e d ) b i n d i n g . O t h e r details are given in t h e text. B: k i n e t i c s o f b i n d i n g o f [ 1 2 s l ] P A ( 3 8 , 0 0 0 c p m a d d e d ) t o e i t h e r 4.3 (/\ /',), 13 ( ~ o), or 40 ttg (• ~) HCG b e a d s t h a t had b e e n p r e t r e a t e d w i t h r a b b i t a n t i - H C G (1 : 2 5 0 ) b y i n c u b a t i o n at 30°C for 60 rain. O t h e r details are given in t h e t e x t .

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-3 N A N O G R A M S HCG A D D E D ( : I.U. X 10 )

Fig. 6. Inhibition of the binding of rabbit anti-HCG to HCG beads by differing amounts of HCG as measured by inhibition of [12sI]PA binding. A: effect of varying antibody concentration on assay sensitivity. Inhibition curves obtained using 13 ~g of HCG beads and anti-HCG diluted 1 : 250 (v. =;14,200 cpm bound), 1 : 750 (© o;8000cpm bound) or 1 : 2250 (A ~; 4250 cpm bound). B: effect of varying bead concentration on assay sensitivity. Inhibition curves obtained usinganti-HCG diluted 1 : 2 5 0 and either 40 (," - : , ; 23,500 cpm bound), 13 ( ; e; 14,200 cpm bound), or 4.3/lg (:'~ /~; 6000 cpm bound) HCG beads. In each case, 38,000 cpm [12SI]PA was added.

a n t i b o d y d i l u t e d 1 : 2 2 5 0 was u s e d , o n l y 2 0 n g ( 0 . 0 2 I U ) gave 5 0 % i n h i b i t i o n a n d 5 n g ( 0 . 0 0 5 I U ) gave 20% i n h i b i t i o n . U n d e r t h e s e c o n d i t i o n s , m a x i m u m b i n d i n g was 4 2 5 0 c p m . T h e c u r v e s s h o w n i n Fig. 6 B , d e m o n strated the effect of varying the concentration of HCG beads while holding t h e a n t i b o d y c o n c e n t r a t i o n c o n s t a n t (1 : 2 5 0 ) o n t h e s e n s i t i v i t y o f t h e assay.

280 TABLE 3 INHIBITION OF ANTI-HCG BINDING TO HCG BEADS BY ttORMONES RELATED TO HCG .

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.

Compound

ng required for 50% inhibition

HCG Human luteinizing hormone (LH)

70 25

a-Subunit of LH

50

Human follicle-stimulating hormone (FSH) .

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180

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.

At the highest a m o u n t of beads tested (0.04 mg), a p p r o x i m a t e l y 2 3 , 5 0 0 cpm [12SI]PA was b o u n d a n d 50% i n h i b i t i o n was o b t a i n e d w i t h a p p r o x i m a t e l y 2 0 0 n g { 0 . 2 0 I U ) o f H C G . T h e s e n s i t i v i t y was i n c r e a s e d b y d e c r e a s i n g t h e b e a d c o n c e n t r a t i o n . W h e n 0 . 0 0 4 3 m g was u s e d , o n l y 4 5 n g ( 0 . 0 4 5 I U ) o f H C G gave 50% i n h i b i t i o n ; m a x i m u m b i n d i n g was 6 0 0 0 c p m ) . F u r t h e r r e d u c t i o n o f t h e b e a d c o n c e n t r a t i o n t o 0 . 0 0 1 4 m g gave n o s i g n i f i c a n t c h a n g e in the sensitivity (data not shown). The relative ability of human luteinizing hormone (LH), the a-subunit of LH, and follicle-stimulating h o r m o n e (FSH) to i n h i b i t the b i n d i n g of antiH C G (1 : 2 5 0 ) t o H C G b e a d s ( 0 . 0 1 3 rag) was d e t e r m i n e d . T h e r e s u l t s in

TABLE 4 LEVELS OF HCG EQUIVALENTS IN URINE Subject a .

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D.S. C.B. E.M. C.L. D.L. Da.L. M.B. J.L.

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.

Concentration of HCG equivalents (pg/ml = IU/ml) b .

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6.8 ± 0.8 15.8 ± 0.5 13.5 -- 0.0 <0.05 c <0.05 c <0.05 c <0.05 (: < 0.05 c

a D.S. and C.B. were in the second trimester of pregnancy, E.M. in the third trimester. C.L., D.L. and Da.L. were females who were not pregnant; M.B. and J.L. were male subjects, b Urine samples from pregnant women were centrifuged at 3000 x g for 5 min, diluted 1 : 30 and 1 : 90 and 0.1 ml al!quots analyzed in duplicate as described in the text. Undiluted samples (0.1 ml) from other subjects were analyzed. e Not detected in undiluted urine (i.e. < 10% inhibition in the immunoassay).

281

Table 3 show that these c o m p o u n d s are effective inhibitors. This good crossreactivity normally is found since the a-subunit is a c o m m o n structural feature (Jacobs and Lawton, 1974). The HCG assay was used to measure immunoreactive material in the urine o f 3 pregnant women. T he results in Table 4 show that HCG reactivity is detected easily in 0.1 ml of samples diluted 1 : 30--1 : 90 (35--55% inhibition). In contrast, no significant inhibition was observed when 0.1 ml of undiluted urine from males or from nonpregnant women was tested. The data were given as HCG equivalents since LH showed strong cross-reactivity (Table 3) and also is produced during pregnancy (Jacobs and Lawton, 1974).

Immunoassay /'or human IgM The procedure described for the d e v e l o p m e n t of the HCG assay was used to establish optimal conditions for an assay for human IgM. The ability of differing amounts of human IgM and human IgG to inhibit the binding of the lgG fraction of rabbit anti-human IgM is shown in Fig. 7. A p p r o x i m a t e l y 60 ng o f IgM gave 50% inhibition and 15 ng gave 15% inhibition. In contrast, 16,000 ng o f IgG was required to give 50% inhibition and approxi m at el y 3500 ng failed to inhibit significantly. The possibility that the inhibition was due to IgM as a trace c o n t a m i n a n t in the IgG preparation has n o t been excluded. This assay was used to det er m i ne levels of IgM in the sera of 5 normal individuals. In addition, the corresponding levels of IgG were measured

r

-I

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.

]

100 z F, z

8O

.<

60

_g40 b-20 I

10

[

!

I

100

1,000

10,000

100

000

NANOGRAMS ADDED

Fig. 7. Inhibition of rabbit a n t i h u m a n IgM (1 : 200) binding to IgM beads (13 ~lg) by differing a m o u n t s of either h u m a n IgM (~. e) or h u m a n IgG (.=) o), based on inhibition of [125I]PA binding. In the absence of inhibitor, binding was 9 6 0 0 e p m out of 38,000 epm added.

282 TABLE 5 LEVELS OF IgM AND IgG IN NORMAL HUMAN SERA a Serum No.

1 2 3 •1 5

Concentration (mg/ml ± S.E.) IgM b

IgG

0.57 1.15 1.00 1.74 1.11

12.0 ~ 0.3 11.5 t 0.2 8.3 ± 0.7 9.5 ± 0.0 11.3" 0.4

, 0.04 : 0.20 ± 0.06 -' 0.06 ' 0.06

c

a Sera were diluted 1 : 4500 for IgM, or 1 : 9000 for IgG and assayed as described in the text. Similar results were obtained for IgM with a 1 : 1500 dilution and for IgG with a 1 : 27,000 dilution. A standard curve for IgM or IgG was included in the assay. b Reported value (Roitt, 1971): 0.50--2.0 mg/ml. c Reported value (Roitt, 1971): 8--16 mg/ml.

using t h e assay b a s e d on t h e i n h i b i t i o n o f [12sI]PA b i n d i n g to r a b b i t lgG I m m u n o b e a d s (see a b o v e a n d L a n g o n e et al., 1 9 7 7 ) . T h e d a t a are r e p o r t e d in T a b l e 5. T h e IgM c o n c e n t r a t i o n s r a n g e d f r o m 0.57 t o 1.74 m g / m l a n d a g r e e d well w i t h t h e r e p o r t e d values ( R o i t t , 1 9 7 1 ) . T h e IgG levels r a n g e d b e t w e e n 8 . 3 - - 1 2 . 0 m g / m l a n d also w e r e c o n s i s t e n t with r e p o r t e d n o r m a l levels ( R o i t t , 1 9 7 1 ) .

Immunoassay for methotrexate In t h e e x p e r i m e n t s d e s c r i b e d so far, i m m u n o a s s a y s were d e v e l o p e d for a n t i g e n i c m a c r o m o l e c u l e s using t h e IgG f r a c t i o n s o f a n t i s e r a . F u r t h e r m o r e , to p r e p a r e t h e a n t i g e n - b e a d c o m p l e x e s , each a n t i g e n was b o u n d c o v a l e n t l y t h r o u g h free a m i n o g r o u p s t o c a r b o x y l g r o u p s on t h e b e a d . To d e m o n s t r a t e m o r e general a p p l i c a t i o n o f this m e t h o d , an assay was d e v e l o p e d f o r t h e h a p t e n m e t h o t r e x a t e ( M T X ) . M T X was c o u p l e d to k e y h o l e l i m p e t h e m o c y a n i n a n d i n j e c t e d i n t o r a b b i t s to p r o d u c e a n t i - M T X a n t i b o d i e s ( L e v i n e a n d P o w e r s , 1 9 7 4 ) . T h e w h o l e s e r u m was u s e d in t h e assay a l o n g w i t h MTX b o u n d t h r o u g h its c a r b o x y l g r o u p s to t h e free a m i n o g r o u p s o f Affi-gel 701. As in t h e e x p e r i m e n t s h o w n in Fig. 3, t h e b i n d i n g o f [12sI]PA to d i f f e r i n g a m o u n t s o f MTX b e a d s t h a t w e r e t r e a t e d with w h o l e a n t i s e r u m at d i l u t i o n s ranging b e t w e e n 1 : 2 5 0 and 1 : 6 7 5 0 or w i t h b u f f e r in the a b s e n c e o f a n t i s e r u m was d e t e r m i n e d ( d a t a n o t s h o w n ) . U n d e r t h e c o n d i t i o n s t e s t e d , up to 2 3 , 5 0 0 c p m were b o u n d ( a n t i s e r u m 1 : 2 5 0 a n d 4.0 mg b e a d s ) . Based on t h e s e results, an o p t i m a l set o f c o n d i t i o n s was e s t a b l i s h e d ( 3 3 0 ~g b e a d s t r e a t e d w i t h antiMTX d i l u t e d 1 : 1 2 0 0 b o u n d 1 2 , 5 0 0 c p m ) , a n d t h e a b i l i t y o f folic acid a n d s t r u c t u r a l l y r e l a t e d c o m p o u n d s t o i n h i b i t a n t i b o d y b i n d i n g was d e t e r m i n e d . T h e i n h i b i t i o n curves in Fig. 8 s h o w e d t h a t a p p r o x i m a t e l y 8 0 0 pg o f M T X gave 50% i n h i b i t i o n o f b i n d i n g a n d as l i t t l e as 100 pg gave 15% i n h i b i t i o n . In

283 100

|0o -

~

eO

i

~ LEUCOVORIN

•

N-6-Me FAH4

40

St 2O

0.01

0.10

1.0

10

100

1.000

10.000

NANOGRAMSADDED

Fig. 8. Inhibition of binding of rabbit anti-methotrexate (1 : 1200) to methotrexate beads (330pg) by differing amounts of methotrexate (o ©), aminopterin (~ o), leucovorin (~, ~), folic acid (m --), 7,8-dihydrofolic acid (FAH2; o ~), 5,6,7,8-tetrahydrofolic acid (FAH4; v ~), and N-5-methyltetrahydrofolic acid (N-5-MeFAH4; • •). In the absence of inhibitor, binding was 12,500 cpm out of 38,600 cpm added.

c o n t r a s t , greater t h a n 1 0 , 0 0 0 ng o f folic acid, 7 , 8 - d i h y d r o f o l i c acid (FAH2), 5 , 6 , 7 , 8 - t e t r a h y d r o f o l i c acid (FAH~) and N - 5 - m e t h y l t e t r a h y d r o f o l i c acid were required for 50% inhibition. Even 1 0 , 0 0 0 ng o f folinic acid (leucovorin), the drug used in 'rescue' t r e a t m e n t o f c a n c e r patients a f t e r administration o f high doses o f MTX, failed t o inhibit significantly. A f t e r MTX, a m i n o p t e r i n was the m o s t effective c o m p e t i t o r : 100 ng gave 50% inhibition. This result is e x p e c t e d , since a m i n o p t e r i n resembles MTX m o r e closely t h a n the o t h e r folic acid analogs tested. T h e inhibition curve for MTX was t h e same if d e t e r m i n e d in t h e presence o f h u m a n serum o r urine {data n o t p r e s e n t e d ) . G e n e r a l l y , w h e n c o n c e n t r a t e d serum (0.1 ml o f < 1 : 100 dilution) was a n a l y z e d b y this m e t h o d significant binding o f [~2sI]PA to the t u b e o c c u r r e d p r e s u m a b l y d u e to IgG binding non-specifically to the glass. When this o c c u r r e d the beads simply were t r a n s f e r r e d to new t u b e s b e f o r e t h e y were c o u n t e d . DISCUSSION Protein A binds specifically t o the Fc region o f IgG isolated f r o m several species ( F o r s g r e n and SjSquist, 1 9 6 6 ; Kronvall and F r o m m e l , 1 9 7 0 ) w i t h o u t affecting t h e ability o f the a n t i b o d y to bind antigen. Based on this p r o p e r t y , an i m m u n o a s s a y m e t h o d was d e v e l o p e d in which [~:sI]PA served as a general tracer. [~2sI]PA was p r e p a r e d r o u t i n e l y with a r e p r o d u c i b l e specific activity o f over 100 C i / m m o l e . T h e f u n c t i o n a l activity was over 85% and r e m a i n e d c o n s t a n t for at least 4 weeks. A l t h o u g h we did n o t a t t e m p t to o p t i m i z e i o d i n a t i o n c o n d i t i o n s , we have s h o w n ( L a n g o n e et al., 1977} t h a t excess B o l t o n - - H u n t e r reagent was present. In c o n t r a s t t o this p r o c e d u r e , in which

284 the e-amino groups of lysine residues were labeled under extremely mild conditions (Bolton and tIunter, 1973), ot her workers have iodinated PA by the chloramine T m e t h o d (Dorval et al., 1975) or by use of lactoperoxidase (Biberfeld et al., 1975). However, overiodination of tyrosine residues by these two procedures led to significant loss of functional activity (Dorval et al., 1975). The immunoassay m e t h o d was applied to proteins (HCG, HuIgM and goat IgG) which were bound to agarose beads by amide linkages joining free amino groups o f the protein to carboxyl groups on the bead. This same support (Immunobeads) should also serve for haptens which have free amino groups but no carboxyl functions. The ability to bind m e t h o t r e x a t e via its carboxyl group(s) to the free amino groups of Affi-gel 701 and to use these beads in an assay for MTX de m ons t r at ed that the general m e t h o d could be e x t e n d ed to carboxyl-substituted haptens. This assay for MTX was at least as sensitive as the available RIAs for MTX (Levine and Powers, 1974; Raso and Schreiber, 1975). The a m o u n t of beads {100--500 mg) prepared with 5 nmoles HCG, 1.1 nmoles lgM, or 200 nmoles MTX was sufficient for 8000, 2000 and 1500 tests, respectively, under optimal conditions. In the presence of 0.04% sodium azide, the beads were stored at 4°C in buffer for at least 2 mo n th s w i t hout loss of activity. Once the appropriate antigen (hapten) beads and ant i body were available, the assay was simple to perform and gave highly reproducible results. Although the IgG fractions of antisera were used in the assays for the proteins, whole antiserum worked perfectly well as d emo n s tr at ed in the assay for m e thot rexat e. Thus, general use of whole serum should offer no disadvantages. Antibodies prepared in rabbits were used since rabbit IgG reacted well with ['2sI]PA (Fig. 1 and "Fable 2) and rabbit is a convenient laboratory animal for purposes of antibody production. However, since IgG from ot her species (e.g. guinea pig) reacts well with PA {'Fable 2), these sera should also be useful. This immunoassay m e t hod was at least as sensitive as other immunoassay methods {e.g. RIA). In each assay described, pmole levels of the homologous ligand were d etec t e d and the ability of c o m p o u n d s related to the antigen (or hapten) to inhibit a n t i b o d y binding also was determined. The assay for human IgM was used to analyze 5 normal sera and the IgM levels found to agree with reported values. Similarly, the HCG assay was used to analyze urine from pregnant women. Immunoreactive material, considered as HCG equivalents since luteinizing h o r m o n e also was detected by the antibody, ranged between 6.8 and 15.8 pg/ml urine. In contrast, no activity was found in undiluted samples of urine from females who were not pregnant, nor in the urine from males. In addition to elevated levels found during pregnancy, high levels of HCG often are associated with several types of cancer (Bagshawe, 1974) {e.g. gestational and t e r a t o m a t o u s choriocarcinoma). Quantitative measures o f this and ot her t u m o r associated products may be useful in the diagnosis of cancer and in the monitoring of drug t r e a t m e n t (Bagshawe, 1974).

285 T h e general use o f [12sI]PA as a t r a c e r will be e s p e c i a l l y u s e f u l in t h e d e v e l o p m e n t o f i m m u n o a s s a y s f o r c o m p o u n d s f o r which r a d i o l a b e l e d d e r i v a t i v e s are n o t r e a d i l y a v a i l a b l e . As an e x a m p l e , we have d e v e l o p e d a s e n s i t i v e a n d s p e c i f i c i m m u n o a s s a y for l e u c o v o r i n ( f o l i n i c a c i d ) , a folic acid d e r i v a t i v e u s e d to ' r e s c u e ' c a n c e r p a t i e n t s t r e a t e d w i t h high d o s e s o f m e t h o t r e x a t e {data to be r e p o r t e d ) . In a d d i t i o n t o t h e d e t e c t i o n o f t u m o r assoc i a t e d p r o d u c t s (e.g. H C G ) , o n e p o t e n t i a l a p p l i c a t i o n o f this m e t h o d is d e t e c t i o n o f (IgG) a u t o a n t i b o d y or a n t i b o d y t o d r u g s in p h y s i o l o g i c a l fluids, using a p p r o p r i a t e b e a d s to a b s o r b t h e a n t i b o d y w h i c h w o u l d t h e n b i n d [J2sI] PA. T h e s e a p p l i c a t i o n s c u r r e n t l y are u n d e r i n v e s t i g a t i o n . REFERENCES Bagshawe, K.D., 1974, in: Radioimmunoassays and Saturation Analysis, ed. P.II. Sonksen (Brit. Med. Bull. 30, No. 1) p. 68. Berson, S.A. and R. Yalow, 1971, in: Immunobiology, eds. R.A. Good and A.W. Fisher (Sinauer Associates, Inc., Stamford, CT). Biberfeld, P., V. Ghet, ie and J. SjSquist, 1975, J. Immunol. Methods 6,249. Bolton, A.E. and W.M. Hunter, 1973, Biochem, J. 133, 529. Butler, Jr., V.P. and S.M. Beiser, 1973, Adv. Immunol. 17, 255. Dorval, G., K.I. Welsh and H. Wigzell, 1975, J. Immunol. Methods 7,237, Forsgren, A. and J. Sj/squist, 1966, J. Immunol. 97,822. Greenwood, F.C., 1971, in: Principles of Competitive Protein Binding Assays, eds. W.E. Odell and W.H. Daughaday (Lippincott, Philadelphia, PA) p. 288. Hunter, W.M., 1974, in: Radioimmunoassay and Saturation Analysis, ed. P.H. Sonksen (Brit. Med. Bull. 30, No.l) p. 18. Jacobs, H.S. and N.F. Lawton, 1974, in: Radioimmunoassay and Saturation Analysis, ed. P.II. Sonksen (Brit. Med. Bull. 30) p. 55. Kronvall, G. and D. Frommel, 1970, Immunochemistry 7, 124. Langone, J.J., M.D.P. Boyle and T. Borsos, 1977, J. Immunol. Methods 18, 281. Levine, L. and E. Powers, 1974, Res. Commun. Chem. Pathol. Pharmacol. 9,543. Odell, W.E. and W.H. Daughaday, eds., 1971, Principles of Competitive Protein Binding As,says (Lippincott, Philadelphia, PA). Rapp, H.J. and T. Borsos, 1970, Molecular Basis of Complement Action (AppletonCentury-Crofts, New York). Raso, V. and R. Schreiber, 1975, Cancer Res. 35, 1407. Roitt, I., 1971, Essential Immunology (Blackwell Scientific Publications, London). Rose, N.R. and H. Friedman, eds., 1976, Manual of Clinical Immunology, Section (7 (American Society for Microbiology, Washington, DC) p. 169. Sonksen, P.H., ed., 1974, Radioimmunoassay and Saturation Analysis (Brit. Med. Bull. 30, No. 1 ).