An antibody sandwich method for the radioimmunoassay of human α-fetoprotein

lmmunochemistry, 1974,Vol. II, pp. 771-775. PergamonPress. Printedin Great Britain

A N A N T I B O D Y S A N D W I C H M E T H O D FOR THE R A D I O I M M U N O A S S A Y OF H U M A N a - F E T O P R O T E I N MIHAI PECEC

and NICOLAE

VOICULETZ

Department of Nuclear Medicine, Institute of Oncology, Bucharest, Romania (First received 2 November 1973; in revised form 16 April 1974) Ahstract---A radioimmunoassay based on solid-phase techniques has been developed for human a-fetoprotein. The assay is carried out in three main steps: (1) equilibrium reaction between anti-a-fetoprotein serum and soluble a-fetoprotein; (2) adsorption of the free antibody fraction onto an a-fetoprotein + BSA immunoadsorbent; (3) titration of the adsorbed antibody by means of radiolabelled anti-IgG. An inhibition curve is constructed by referring to controls containing either no soluble antigen or normal rabbit serum instead of anti-a-fetoprotein. The method gives reproducible results over a broad concentration range; as little as 10 ng/ml of soluble antigen can be detected and the upper limit of the assay approaches the lowest amount of antigen which can be detected by immunodiffusion methods (8000 ng/ml). The method has some general advantages: (1) using an indirectly involved, labelled reagent avoids labelling damages of the particular immune reagents; (2) it allows to assay several antigen-antibody systems using the same labelled anti-IgG preparation; (3) the radiolysis phenomena are less critical than in other radioimmunoassay procedures; (4) it should be a sensitive assay, because of the amplification, the use of anti-IgG is performing; although this last point was not attained, firstly because the reagents that were used, were not especially prepared for use in radioimmunoassay procedures.

INTRODUCTION T h e a c c u r a t e d e t e r m i n a t i o n of a - f e t o p r o t e i n in h u m a n p a t i e n t s or n o r m a l s , as well as in l a b o r a t o r y a n i m a l s b e c a m e a v e r y actual p r o b l e m in t h e last years. S e v e r a l r a d i o i m m u n o a s s a y m e t h o d s w e r e p u b l i s h e d f o r h u m a n (Ruoslahti a n d Sepp/il~i, 1972; Silver et al., 1973) as well as f o r rat a - f e t o p r o t e i n s ( O a k e s et al., 1972; Sell, 1973; Sell a n d G o r d , 1973). All t h e s e m e t h o d s use t h e classical principle, originally i n t r o d u c e d b y Y a l o w a n d B e r s o n (1960). T h e y differ b e t w e e n e a c h o t h e r in the particular w a y to d e t e r m i n i n g the b o u n d / f r e e antigen ratio. W e r e p o r t in this p a p e r a m e t h o d w h i c h i n v o l v e s t h e d e t e r m i n a t i o n of t h e free a n t i b o d y fraction, at equilibrium, b y a d s o r b i n g it o n t o a n insolubilized antigen and assaying the adsorbed molecules by

m e a n s of r a d i o l a b e l l e d a n t i - I g G a n t i b o d i e s . A q u a n titative r e l a t i o n s h i p c o u l d b e e s t a b l i s h e d b e t w e e n t h e d e c r e a s e of the free a n t i b o d y f r a c t i o n a n d the c o n c e n t r a t i o n of soluble antigen originally p r e s e n t in the reaction. T h e p r e p a r a t i o n a n d the a s s a y of t h e r e a g e n t s are d e s c r i b e d a n d s o m e i m p o r t a n t f e a t u r e s of t h e a s s a y s y s t e m are discussed. MATERIALS AND METHODS a-FP* used in all the experiments was a human standard serum (Behringwerke AG, batch No. 7V), which contained 220 tzg a - F P per ml. It was used diluted in PBS-NCS, as needed. BSA was a commercial preparation (Behringwerke AG, batch no. 527 II), electrophoretically pure. PBS, Dulbecco's formula, without divalent cations (pH 7.4) and N C S were obtained from "Dr. I. Cantacuzino" Institute of Microbiology, Bucharest. Na 1~I, 'carrier free' solution in carbonate-bicarbonate buffer, 5 mCi/ml (ROTOP r, East-Germany) was diluted as needed in 0.1 M phosphate buffer (pH 8-0), immediately before use. Rabbit anti-human a-fetoprotein serum (Behringwerke AG, batches No. 2394A and 2260H) was used diluted in PBS-NCS. Goat anti-rabbit IgG serum was prepared by us through immunization of the animals with RIgG prepared by chromatography on DEAE-cellulose columns. It was checked for monospecificity by conventional immunoelectrophoresis. Immunoadsorbents: (a) Sepharose-RIgG immunoadsorbents were prepared according to Cuatrecasas et

*List of the abreviations used -FP BSA PBS NCS PBS-NCS NRS RIgG RIgG-Is a-FP-Is BSA-Is anti-a-FP anti-RIgG*

human a-fetoprotein bovine serumalbumin phosphate buffered saline normal calf serum PBS complemented by 10% NCS normal rabbit serum normal rabbit IgG rabbit IgG immunoadsorbent c~-fetoprotein immunoadsorbent adsorbent containing only BSA rabbit anti-human c~-fetoprotein serum radioiodinated, purified anti-rabbit IgG antibody 771

772

MIHAI P E C E C and N I C O L A E V O I C U L E T Z

a1.(1968), by mixing equal v o l u m e s of CNBr-activated Sepharose 2B and RIgG, 1% solution in PBS; the mixture was stirred for 24 hr at 4°C, thoroughly w a s h e d with PBS and 0.2 N HCI and stored at 4°C, until used. (b) BSA i m m u n o a d s o r b e n t s were prepared through glutardialdehyde crosslinking ( A v r a m e a s and T e r n y n c k , 1968). The protein mixtures contained BSA 50 mg/ml and either RIgG 2 m g / m l or a - F P 2 2 # g / m l , or any other protein than BSA. The polymerization was performed at pH 5.0 (0.1 M acetate buffer) by adding 20 gl of glutardialdehyde, 25% buffered solution (Merck Darmstadt), per ml of protein solution and reacting 24hr, at room temperature. T h e protein gels were subsequently homogenized in a Potter apparatus and washed three times with 100 v o l u m e s of P B S - N C S , by centrifugation. All the operation was repeated several times. Finally, the resulting s u s p e n s i o n s were diluted 1:8 or 1 : 10 (according with the initial volume of the gel) in P B S - N C S and stored at 4°C.

Purification and labelling of anti-RlgG antibodies The heat-decomplemented goat antiserum was adsorbed on a Sepharose-RIgG column, which was afterwards exhaustively w a s h e d with P B S and eluted by 0.2 N HCI. The eluting antibodies were immediately buffered at pH 8-0 using 0.3 M p h o s p h a t e buffer and 5 N N a O H . T h e y were subsequently concentrated by v a c u u m dialysis (Sartorius G.m.b.H. collodion bags) to 25 mg/ml, dialyzed against 500 volumes of 0.1 M p h o s p h a t e buffer p H 8.0, 48 hr at 4°C and finally, stored at - 20°C divided into small aliquots. The radioiodination was performed by the chloramineT method (Freeman, 1967): one atom of iodine per molecule of IgG and 0.01%0.8 mCi/mg of protein were introduced, After the addition of sodium metabisulphite, a small aliquot was used to determine the specific activity of the protein and the reaction yield; the remaining mixture was gelfiltered on a precalibrated column of BioGel A-0,5 m (BioRad Lab.), using PBS as the eluant. The radioactive material emerging from the column in the IgG region was collected and diluted to 25/xg/ml with P B S - N C S . It was stored at - 20°C for longer periods of time or at 4°C if it was to be used in the next 2-3 days. The free '~I content was determined immediately before use, by 10 per cent T C A precipitation.

incubated 2 hr, at 24°C, on a rotative mixer, then centrifuged and the s e d i m e n t s w a s h e d by 4-5ml of P B S - N C S . Two ml (50 txg, 10~ counts/min) of anti-RIgG* were added into each vial and all the vials were reincubated 2 hr, as before. Finally the vials were centrifuged, washed twice by 4.5 ml of P B S - N C S and counted.

Assay of soluble c~-FP On the basis of the previously described assays, we chose a combination that would yield in the absence of soluble a - F P , 45% saturation of the a - F P - I s and as m u c h as 20% saturation of the anti-RIgG*. One ml a m o u n t s of a n t i - a - F P , 1:250 dilution, were mixed with 1.0 ml of a - F P solutions, which concentration varied from 10 to 8000 ng/ml. Vials containing no c~-FP were run as 100 per cent adsorption controls and vials containing 1:250 N R S instead of anti-c~-FP were run as 0 per cent specific adsorption controls. The incubation was carried out for 24 hr, at 24°C, under continuous mixing; afterwards, 0.5 ml of c~-FP-Is were added into each vial and the samples processed as in the preceeding assay. RESULTS

Labelling and assay of anti-RIgG antibodies A l t h o u g h m a n y i o d i n a t i o n s w e r e d o n e , all t h e experiments reported here were carried out with a single batch of radioiodinated antibodies. This one d i s p l a y e d a n a c t i v i t y o f 20,000 c o u n t s / m i n / / x g o f p r o t e i n a n d c o n t a i n e d at t h e t i m e o f its p r e p a r a t i o n , 5 p e r c e n t f r e e ~:~I. T h i s o n e r a i s e d t o 8 p e r c e n t a f t e r 4 w e e k s , w h i l e s t o r e d at - 2 0 ° C . T h e r e s u l t s o f t h e a s s a y (Fig. 1) w e r e e x p r e s s e d as~

a d s o r b e d activitymgG-~ - a d s o r b e d activityBsA L. original activity x 100

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Assay of anti-RlgG* An a m o u n t of 12.5/xg (0.5 ml, 2.5 x 10~ counts/min) of radiolabelled antibody was mixed with increasing a m o u n t s of RIgG-Is in 16× 150ram fiat-bottomed, screwcapped glass vials and the final volume brought to 4.5 ml by P B S - N C S . Controls were run with BSA-Is instead of RlgG-ls. The vials were incubated 2 hr, at room temperature (24°C), on a rotative mixer, then centriguged 3 rain at 4000 g decanted and the sediments w a s h e d twice by 4.5 ml of P B S - N C S . Each vial was counted in a well type g a m m a scintillalion counter (FH 488+ F H 49, Frieseke and H o e p f n e r G.m.b.H.).

Sandwich assay of a-FP-ls A constant a m o u n t of i m m u n o a d s o r b e n t (0.5 ml, 1:8 suspension, v/v) was mixed with increasing a m o u n t s of a n t i - a - F P , 1:250 dilution, and the final volume brought to 4.5 ml by P B S - N C S . Controls were run either with l : 2 5 0 N R S or with no rabbit serum. The vials were

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Fig. 1. A s s a y of radioiodinated anti-rabbit IgG antibodies: 12.5/xg (0-5 ml, 2 . 5 / 1 0 5 counts/rain) were reacted with increasing a m o u n t s of RIgG-Is, final volume 4.5 ml, for 2 hr at 24°C, on a rotative mixer; controls were run with B S A - I s . After incubation the vials were centrifuged and washed twice by 4 . 5 m l v o l u m e s of P B S - N C S . (a) Percentage of specifically adsorbed anti-RIgG* (ordinate) in the presence of increasing a m o u n t s of i m m u n o a d s o r bent. (b) Percentage of the maximal specific adsorption plotted against the Iog,o value of the a m o u n t of i m m u n o a d s o r b e n t . ID/90 rain incubation 0 - - 2 hr incubation(l month later).

Antibody Sandwich Radioimmunoassay That percent of specifically adsorbed activity was plotted against the amount of immunoadsorbent used or its log10 value. In the first case (Fig. la), a typical 'saturation curve' with a well defined horizontal plateau, was obtained. When the results were plotted semilogarythmically, as percentages of that plateau value, the slope of the linear portion did not change significantly when testing at a 4week interval (Fig. lb). We could not compare the plateau values directly as we have changed, by time, the length of the incubation period. The value of this slope depends mainly on the equilibrium

773

later did not differ significantly from the one obtained in the preceeding assay, when the same amount of anti-RIgG* was incubated with an identical amount of BSA-Is. We attempted to increase the ratio value by more washes in the intermediate step (i.e. before the addition of anti-RIgG*) but, this actually lowered it because the control activity decreased in a lesser extent than that of the sample.

Assay of soluble a-Fp (Fig.3) The results were expressed by:

sample a d s o r p t i o n - 0 % specific adsorption control 100% adsorption c o n t r o l - 0% specific adsorption control x 100

constant of adsorption and we took its invariability as a proof that no important alterations in the antibody properties of the anti-RIgG* occured upon storage. The sample/control radioactivity ratios varied from 15 to 4.5, in inverse correlation with the amount of adsorbent used, if only one wash was performed and from 48 to 11 if two washes were done.

Assay of the a-FP-Is (Fig. 2) The results were expressed essentially as for the assay of anti-RIgG*. The shape of the curve is similar to that obtained in the preceeding assay but, the difference between duplicates is about three times greater and the sample/control activity ratio was constant over the whole range of anti-a-FP dilutions used, being as low as 4. Even the more diluted NRS control still adsorbed a two-fold greater amount of radioactivity than the same amount of a - F P - I s incubated with anti-RIgG* alone; the

The computed values actually represent the free antibody fraction in the soluble a - F P + anti-a-Fp system, at 24hr. When plotted against the log~0 value of the concentration of soluble a - F P solutions that were used, a linear relationship was obtained over the 10-8000 ng range. Regression curves were computed on the basis of the results obtained in four separate experiments, carried out at weekly intervals. They are: y=120.5-28.4841og~0 x and log~0 x = 4.188777- 0.0343 y. The correlation coefficient r = 0.988 and the mean experimental error (computed from the y to log~0 x regression curve), p ~ = 2.18(%); this value indicates that as little as 25 ng/ml can be successfully distinguished in the 10 to 200 ng range. The 100% control/0% specific adsorption control ratio was similar to that obtained in the assay of c~FP-Is, i.e. varied in the different experiments between 4 and 5. ~OO~

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Fig. 2. Sandwich assay of the a-FP immunoadsorbent: a-FP-Is 0.5 ml+ various amounts of 1:250 anti-a-FP, incubated 2hr at 24°C, on a rotative mixer; after centrifugation and washing 2.0ml anti-RIgG* (50/xg, 106counts/min) were added and the vials reincubated 2 hr, centrifuged and washed twice. (a) Percentage of specifically adsorbed anti-RlgG* (ordinate) in presence of increasing amounts of anti-a-FP (abscissa). (b) Percentage of the maximal specific adsorption of anti-RIgG* plotted against the Iog,ovalue of the amounts of anti-a-FP.

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Fig. 3. Assay of soluble c~-FP: To l ml of anti-o~-FP, diluted 1:250, standard dilutions of soluble a-FP were added in one ml of PBS-NCS and incubated 24 hr at 24°C. Afterwards, 0.5 ml of a-FP-Is were added and the vials incubated 2 hr under continuous mixing, centrifuged and washed once; 2.0 ml anti-RIgG* were added to the sediments and the vials reincubated 2 hr, centrifuged and washed twice. Percentage of specifically adsorbed antiRIgG* (ordinate) in presence of increasing amounts of soluble a-FP (abscissa), plotted semilogarythmically; dashed lines: ±2 S.D.

774

MIHAI PECEC and NICOLAE VOICULETZ DISCUSSIONS

The introduction of solid-phase coupled immunologic reagents allowed the development of radioimmunoassay techniques based on principles somewhat different than that of Yalow and Berson (Wide, 1971). Our method is similar to the immunoradiometric method introduced by Miles and Hales (1968) but, it differs from that one by the use of labelled anti-IgG antibodies instead of labelling the particular antibody. An almost identical approach was adopted by Wide et a/.(1971) who reported an allergen assay by using radiolabelled anti-IgE antibodies. The antibody sandwich method present some general advantages: (1) The fact that the radiolabelled reagent is not directly involved in the assay system avoids the damage of specific reagents by labelling procedures; the importance of radiolysis phenomena is also highly decreased because changes in the antibody properties of the anti-IgG preparation, if not too great, are expressed as systematic errors and automatically eliminated. This view is supported by the fact that we were able to use the same batch of radiolabelled anti-IgG over one month, in spite of some signs of radiolysis. (2) A potential advantage would be the possibility to deal with several antigen-antibody systems, while using the same radiolabelled anti-IgG preparation. (3) The sensitivity of the assay should be greater than of classical assay systems (Miles and Hales, 1968) and that of the immunoradiometric assay too. This is because the displacement of a single antibody molecule in the particular system, to be assayed, brings about the displacement of several anti-IgG molecules, according with the antigenic valence of the first. However, generally speaking, a multistep procedure increases the chance of possible experimental pitfalls. Therefore, the conditions for every step, in a particular case, have to be carefully established. The first step is the incubation of a constant amount of a n t i - a - F P with varying concentrations of soluble antigen. Because the separation of the free antibodies is made by an immunological procedure, there is particularly important to have relatively stable complexes formed. The choice of a 24hr incubation may be considered as a general recommendation (Wide, 1971) and it is supported in our particular case by the experience of Sell and Gord (1973). *This high adsorption capacity would have been probably overcome by using higher dilutions of anti-a-FP. But, this was not possible with the particular reagent we have used. This preparation proved to be monospecific by immunodiffusion and radioimmunoelectrophoretic criteria but, it was of rather low strength; in fact, it was not originally intended to be used in radioimmunoassay procedures.

The second and the third steps of the assay are solid-phase adsorptions, far more rapid reactions than those carried out in aqueous media. As for the later (Hunter, 1967) greater periods of incubation increase the stability of the antibody-adsorbent complexes but, in the presence of soluble immune complexes, this would also increase the chance of an antibody exchange between the two phases. We have then chosen the shortest time of incubation that allowed to obtain reproducible results with the particular set of reagents we have been using. The complexes were correspondingly easy to dissociate and more than one wash in the intermediate step was not beneficial. The adsorption of the free antibody molecules was performed in a relatively slight antigen ( a - F P Is) excess. This was done also to prevent an antibody exchange with soluble immune complexes. Adsorption in a greater antigen excess would increase the stability of the insoluble complexes (unpublished results), but we were thinking that the antigen was too rare (0.4%) in the insoluble matrix to allow for cooperative effects between adjacent molecules, so we limited ourselves to work at the lower-limit where the 1-st/2-d antibody ratio reaches a constant value (unpublished results). The main limiting factor, as regarding the sensitivity of the assay was the high unspecific adsorption. Because we have used unpurified a - F P standard and the a n t i - a - F P was made monospecific by adsorption with normal adult serum, one may inquire about the possibility of having to deal with antibodies to normal serum proteins. However, this is very unlikely because replacement of the usual diluent in the 100 per cent adsorption control by normal adult serum (l : 10 dilution in PBS) did not alter signifficantly the bound radioactivity; in addition, controls run without rabbit serum did not differ, no matter what particular adsorbent we were using. So the only black point is the high unspecific adsorption capacity of the protein gel.* It appears to us that further improvement in the sensitivity of the assay requires the change of the type of insoluble matrix. A positive finding is the reproducibility of the assay. Statistical calculations (Worthing and Geffner, 1955) showed a good correlation coefficient and a reasonably low value of the mean experimental error over a broad concentration range, which achieves the upper limit of the normal concentration range (Ruoslahti and Seppfil~i, 1972) and covers all the pathological range so far published, up to the lower limit of classical immunodiffusion methods (Silver et al., 1973). Further experimentation with purified reagents is needed in order to establish with better accuracy the optimal conditions for this type of assay.

Antibody Sandwich Radioimmunoassay REFERENCES

Avrameas S. and Ternynck T. (1968) Imrnunochemistry, 6, 53 Cuatrecasas P., Wilchek M. and Anfinsen C. B. (1968) Proc. natn. Acad. Sci. U.S.A. 61, 636 Freeman T. (1967) Handbook of Experimental Immunology ((Edited by Weir D. M.), Chap. 17. Blackwell, Oxford Hunter W. M. (1967) Handbook of Experimental Immunology (Edited by Weir D. M.), p. 629. Blackwell, Oxford. Miles L. E. M. and Hales C. N. (1968) Nature, Lond. 219, 186 Oakes D. D., Shuster J. and Gold P. (1972) CancerRes. 32, 2753. Ruoslahti E. and Sepp/il/i M. (1972) Nature, Lond. 235, 161

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Sell S. (1973) Cancer Res. 33, 100 Sell S. and Gord D. (1973) Immunochemistry, 10, 842 Silver H. K. B., Gold P., Feder S., Freedman S. O. and Shuster J. (1973) Proc. hath. Acad. Sci. U.S.A. 70, 526 Wide L. (1971) Radioimmunoassay Methods (Edited by Kirkham K. E. and Hunter W. M.), p. 405. Churchill & Livingstone, London. Wide L., Aronsson T., Faggerberg E. and Zetterstr6m O. (1971) Excerpta Medica International Congress Series No. 251, Allergology, Proc. VIII-th Eur. Congress of Allergology, p. 85. Worthing A. G. and Geffner J. (1955) Treatment of Experimental Data, Chap. 11-12. Wiley, New York Yalow R. S. and Berson S. A. (1960) J. clin. Invest. 39, 1157.