Journal of Immunological Methods, 7 (1975) 179--186 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
THE SPECIFICITY OF A SOLID PHASE RADIOIMMUNOASSAY FOR HUMAN IMMUNOGLOBULINS
R.P. EADY, J.C. CHAPPLE, D.W. HOUGH and G.T. STEVENSON Tenovus Research Laboratory, General Hospital, Southampton, S09 4XY, England
(Received 1 November 1974, accepted 8 November 1974)
The specificity of a solid phase radioimmunoassay for immunoglobulin antigens, human Fabp in the case illustrated, has been assessed. The anti-Fabp serum available reacted well on gel diffusion with all Fabp- and light chain-containing proteins. In contrast the radioimmunoassay, in which unlabelled test antigen competes with radiolabelled Fabp for anti-Fabp coupled to Sephadex beads, was sensitive only to proteins containing entire Fabp regions. A double antibody radioimmunoassay showed comparable or greater sensitivity for Fabp-containing proteins, but was much less specific. By using Fab3" as the labeled antigen, with only light chain determinants reacting with the anti-Fabp, the solid phase assay was rendered polyspecific in being sensitive to ali proteins containing light chain. Reasons for the specificity of the solid phase assay are discussed: steric factors can be expected in some cases to permit strong binding of antigen by multiple bonds, in other, to restrict the number of molecular determinants simultaneously available to antibody.
INTRODUCTION Ideally, the m e t h o d c h o s e n to separate a n t i b o d y - b o u n d antigen fron free antigen in r a d i o i m m u n o a s s a y s m u s t t o t a l l y separate the t w o f r a c t i o n i n s t a n t a n e o u s l y (Ekins and N e w m a n , 1970). F r o m the m a n y separatio; m e t h o d s available ( K i r k h a m and H u n t e r , 1971), the use o f a n t i b o d i e c o u p l e d to a solid m a t r i x has the advantage o f c o m p l e t e n e s s o f s e p a r a t i o r simplicity a n d speed. H o w e v e r the q u e s t i o n o f sensitivity o f the solid phas r a d i o i m m u n o a s s a y has o f t e n been raised. A r e n d s ( 1 9 7 1 ) r e p o r t e d considel able loss o f b o t h a n t i b o d y titre a n d assay sensitivity after c o u p l i n g a n t i b o d to cellulose. Loss o f sensitivity o f r a d i o i m m u n o a s s a y s using a solid phas s y s t e m has also been r e p o r t e d b y A r e n d s and R o o s ( 1 9 7 2 ) and H a r t ( 1 9 7 2 Wide ( 1 9 6 9 ) r e p o r t e d s o m e loss in a n t i b o d y titre after c o u p l i n g to S e p h a d e or cellulose, b u t a p p a r e n t l y little loss in assay sensitivity. R e c e n t l y , a d~ tailed c o m p a r i s o n o f the p r o p e r t i e s o f r a d i o i m m u n o a s s a y s using a n t i b o d c o u p l e d to Sepharose and the same a n t i b o d y in free s o l u t i o n was m a d e b B o l t o n and H u n t e r (1974). T h e y f o u n d , using low mol. w t ( < 2 4 0 0 ) antigen t h a t t h e r e was n o d i f f e r e n c e in sensitivity b e t w e e n the t w o systems. H o w e w s o m e loss in sensitivity o f the solid phase assay was observed w h e n larg~ mol. w t antigens were involved.
180 When using a solid phase radioimmunoassay to study the inhibition of binding of labelled human Fabp to anti-Fabp by structurally related antigens (Eady et al., 1973) we noted a very high specificity for the homologous antigen. We have expanded this observation so as to examine both the sensitivity and specificity of a radioimmunoassay using antibody coupled to Sephadex. Studies were carried out in two systems: the inhibition of binding of labelled homologous antigen (Fabp) to antibody, and the inhibition of binding of a labelled heterologous antigen (Fab3', sharing some determinants with Fabp) to the same antibody. A comparison was also made between solid phase and double antibody radioimmunoassay in the Fabp-anti-Fabp system. It was found that the solid phase method, using labelled homologous antigen, has a strong advantage where relative insensitivity to cross-reacting antigens is desirable. MATERIALS AND METHODS
Preparation of antibodies Randomly bred rabbits were injected subcutaneously in the four limbs with 1 mg of human Fabg incorporated into complete Freund's adjuvant (Difco). After 6 weeks, booster intravenous injections were given at monthly intervals using 0.2 mg human Fabp adsorbed on aluminium hydroxide. The IgG fraction was prepared from sera by precipitation with ammonium sulphate followed by DEAE-cellulose chromatography (Stevenson and Dorrington, 1970). The IgG fraction was shown to have antibody activity directed against both p and light chains by gel immunodiffusion (Ouchterlony, 1958).
Preparation of antigens Human IgM from pooled normal serum was prepared as described by Chaplin et al. (1965), with the insertion of preparative electrophoresis in a Pevikon block. Trace amounts of IgG were removed by passing the IgM through a column of Sepharose 4B coupled to IgG from rabbit anti-~/chain. Fabp was prepared from this IgM by digestion with papain (Eady et al., 1974). Human IgG was prepared from pooled normal serum as described by Stevenson and Dorrington (1970). Adsorption of any contaminating IgM was carried out using Sepharose 4B coupled to the IgG fraction from a rabbit anti-human IgM serum which had previously been absorbed with pooled human light chains. Fab~/ was prepared from this IgG fraction by peptic digestion (Frangione and Prelli, 1960). Human light chains were prepared from pooled normal IgG (Stevenson and Dorrington, 1970) and purified using Sepharose 4B coupled to IgG from rabbit anti-~, chain.
181
Radioiodination This was carried out by the chloramine T m e t h o d (Hunter, 1967), using carrier free t ~ s I (IMS/30 Radiochemical Centre, Amersham).
Solid phase radioimmunoassay Sephadex G-25 superfine (Pharmacia, Uppsala) was activated by the CNBr m e t h o d (Axen et al., 1967). The IgG fraction from the rabbit antiserum was coupled to the activated Sephadex by stirring for 24 hr at 4°C and pH 6.5. Loosely coupled protein was removed by washing with detergent-containing buffers and brief sonication, as described by Wide (1969). The assay was performed by adding 100 pg of 125 I-labelled antigen (50--100 pCi/pg) in 0.1 ml, to 1 ml of standard antigen or inhibiting antigen. I m m u n o s o r b e n t (1 ml) was added at the dilution necessary to bind 50--60% of the label in the absence of unlabelled antigen. The assay was carried out in the presence of carrier horse haemoglobin at 1.0 mg/ml. The assay tubes (Stevenson and Eady, 1973) were rotated about the horizontal axis for 48 hr at room temperature. Separation of the bound and free fractions and two washings of the solid phase were carried out by displacement filtration in the assay tubes. The buffer used for washing and for the assay was 0.05 M Na2 HPO4--KH2 PO4 pH 7.3, 0.45% NaC1, 0.05% NAN3, 0.5% Tween 20, (Sigma). The bound fractions were counted in a Wallac gamma scintillation spectrometer.
Double antibody radioimmunoassay This assay was performed by adding 100 pg of 12 s I-labelled antigen in 0.1 ml to 1 ml of standard antigen or other inhibiting antigen in the same assay tubes as used above. The IgG fraction from rabbit anti-human Fabp (0.1 ml) was added at the dilution necessary to bind 50--60% of the labelled antigen in the absence of unlabelled antigen. The tubes were allowed to stand at room temperature for 48 hr. To separate the bound and free fractions, use was made of an immunosorbent: Sepharose 4B coupled to the IgG from sheep anti-rabbit IgG which had previously been adsorbed with Sepharose 4B--human IgG (Den Hollander and Schuurs, 1971). The immunosorbent (1 ml, 2.5% v/v) was added to the assay tubes which were rotated about the horizontal axis for 18 hr at room temperature. The a m o u n t of immunosorbent used was shown to be capable of binding 80--95% of the 1251 label when the rabbit antibody was present in large excess over antigen. The separation of bound and free fractions was carried out by displacement filtration as above.
182 RESULTS B i n d i n g o f ~ 2 S i . h u m a n F a b p to r a b b i t a n t i - h u m a n F a b p
The solid phase radioimmunoassay for Fabp, illustrated in fig. la, was sensitive over the range 1.0--100 ng of Fabp. The inhibition curve given by h u m a n IgM, although displaced to the right, paralleled the inhibition curve for the homologous antigen. The inhibition curves given by human Fab% L chain and IgG were also displaced to the right, but in addition showed non-parallelism with respect to inhibition by Fabp. Bearing in mind the logarithmic scale on the abscissa, fig. l a is seen to depict an assay with a high degree of specificity for Fabp. Binding of'
2 S i _ h u m a n Fab'y t o r a b b i t a n t i - h u m a n F a b p
Various inhibition curves are illustrated in fig. lb. Those given by Fabp and Fab3' are close and parallel. The curve for IgG is also parallel and displaced to the right of the Fab7 curve to an extent suggesting that more than one Fab3' region in the IgG is reacting: because over much of the curves only about 1.5 times as much IgG as Fab3, is needed for a given degree of inhibition. The IgM curve is also parallel, but in contrast to the IgG curve is greatly displaced to the right, suggesting that only a fraction of the Fab# in the Bo I
-o
_B Bo
: a :: "
i b)
o9 I-O
:.
0-9
: ~ . ~
0.8
0.8
o.
i if
%
\
0-4
0"3"
I
"~
0-2 ~
"~
0.2 O'I
O'l I0
102
10 ~
° I
,5
10~
Antigen
,5,
,~
,5"
(ng/mll
Fig. 1. a) Inhibition of binding of 12 s I-labelled human-Fabp to Sephadex-coupled rabbit anti-human Fabp by the following unlabelled human proteins, b) Inhibition of binding of 12 s I-labelled human-Fab3' to Sephadex-coup]ed rabbit anti-human F a b t / b y the following unlabelled human proteins. (# ©) Fabp, (¢, e) IgM, (D []) Fab% (-m) IgG, (• A) light chain. Each point represents the mean of triplicate determinations.
183 B
(o)
_B Bo
Bo
I-0
1-0
0-9
~'~
0-9
0-8
0.8
0-7
0"7
0-6
0-6
(b}
2--..°
0-5 0-4
0-4
0"3
0"3
0-2
•
0"2
0-1
0-1 10
102
lOa
10'
10
I0~
I0I
10~
Antigen
(ng/ml) Fig. 2. Inhibition of binding of 12 s I-labelled human Fabp to rabbit anti-human Fab# by unlabelled human, (o ©) Fabp, (s o) IgM, (D D) Fab% (1 l) IgG, (/7 A) light chain. (a) Antibody coupled to Sephadex G-25; (b) antibody in free solution. Each point represents the mean of triplicate determinations. molecule can be reacting. Inhibition by free light chain was somewhat o u t of parallel to the inhibition by the ot he r antigens. Similar results to those depicted in fig. 1 have been f o u n d using a radioimmunoassay for h u m a n Fab%
Binding of 12 SI-human Fabp to rabbit anti-human Fabp: comparison o f solid phase and double antibody assays Fig. 2 compares the two assays. Although the dose--response curve for Fabp is slightly steeper using a double a n t i b o d y assay, the m i ni m um detectable a m o u n t of Fabp in b o t h assays is very similar. In b o t h assays the curves for inhibition of binding of 12 s I-Fabp to rabbit anti-Fabp by Fab% IgG and light chain were non-parallel to the curve for Fabp. However the degree of cross reactivity between Fabp and the o th e r antigens was greater using the double a n t i b o d y system: by a factor of a b o u t ten at low concentrations of the antigens. Fig. 2 shows th a t in bot h assays the inhibition curve of hum an IgM is parallel to that o f Fabp. However in the double ant i body assay, the displacem e n t o f IgM to the right is n o t observed. DISCUSSION One criterion of the specificity of a radioimmunoassay is the set of inhibition curves given by molecules with varying com pl em ent s of the antigenic determinants involved ( L a n d o n et al., 1968). Different classes of immunoglo-
184 bulins having c o m m o n light chain determinants provide a very convenient system for assessing the specificity of a radioimmunoassay. The h o m o l o g o u s solid phase assay using 1 z s I-Fabp was shown to be highly specific for the hom ol ogous antigen. The inhibition curves given by free light chain, Fab~ and IgG eventually flatten out after displacement of approx. 50% of the b o u n d 1 z s I-Fabp, which is consistent with the cross-reacting molecules being able to bind to antibodies to light chain determinants, but n o t to antibodies to Fdp determinants. However, the degree of displacem e n t of label by small amounts of the cross-reacting antigen is much less than would be expect ed were t hey able to c o m p e t e on equal terms with Fabp for antibodies to light chain determinants. The explanation for this p h e n o m e n o n is presumably the fact that Fabp can be bound to the solid phase simultaneously by two sets of determinants, giving an effective association co n s tan t much greater than can be realised by molecules with only one set of determinants. Results f r om the double antibody assay are consistent with this explanation. In this system the possibility of multiple bonding of an antigen molecule to a single particle does n o t arise and, as expected, at low concentrations of cross-reacting antigen equivalent displacement of labelled standard antigen f r om a n t i b o d y is observed. All Fabp determinants are present in IgM and this molecule is seen to displace all i 2 s I-Fabp from the solid phase antibody. The marked displacem e n t of the curve to the right, which was n o t observed using a double a n t i b o d y assay, indicates that steric factors are permitting only a fraction of the Fabp regions on the pentameric IgM molecules to react simultaneously with the solid phase antibody. In contrast with the specificity of the homologous solid phase assay, a heterologous solid phase assay constructed using 12 s I-Fab% which shares only light chain determinants with Fabp, yielded parallel inhibition curves for all light chain-containing immunoglobulins. The only except i on to this was free light chains which demonstrated slight non-parallelism for reasons n o t apparent. This heterologous assay is seen to represent a polyspecific assay of good sensitivity for all immunoglobulins containing light chain. Bolton and Hu n t er (1974) have shown that the solid phase radioimmunoassay for low mol. wt proteins can give the same sensitivity as assays where the an tib o d y is in free solution. We have confirmed this sensitivity and shown in addition t ha t a solid phase radioimmunoassay for immunoglobulins can be used in either a monospecific or a polyspecific form, with the f o r m e r exhibiting a notably higher specificity than is observed in the free a n t i b o d y system. One problem dem ons t r a t e d by our work is that steric factors can present a severe restriction in solid phase radioimmunoassays where very large mol. wt antigens such as IgM are involved. ACKNOWLEDGMENTS This work was supported by Tenovus, Cardiff, and the Wessex Regional
185
Hospital Board. We are grateful to Mrs Maureen Power for assistance in the preparation of the proteins.
REFERENCES Arends, J., 1971, Acta Endocrinol. 68, 425. Arends, J. and J. Roos, 1972, Scand. J. Clin. Lab. Invest. 29 Suppl. 126 Abst 14. Axen, R., J. Porath and S. Ernbach, 1967, Nature 214, 1302. Bolton, A.E. and W.M. Hunter, 1973, Biochim. Biophys. Acta 329, 318. Chaplin, H., S. Cohen and E.M. Press, 1965, Biochem. J. 95, 256. Den Hollander, F.C. and A.H.W.M. Schuurs, 1971, in: Radioimmunoassay methods, eds. K.E. Kirkham and W.M. Hunter (Churchill and Livingstone, Edinburgh) p. 419. Eady, R.P., D.W. Hough, P.J. Kilshaw and G.T. Stevenson, 1974, Immunology 26, 549. Frangione, B. and F. Prelli, 1960, J. Clin. Invest. 39, 1933. Hart, I.C., 1972, J. Endocrinol. 55, 51. Hunter, W.M., 1967, in: Handbook of experimental immunology, ed. D.M. Weir (Blackwell Scientific Publications, Oxford) p. 608. Kirkham, K.E. and W.M. Hunter, 1971, Radioimmunoassay methods (Churchill and Livingstone, Edinburgh) Session III, p. 243. Landon, J., J. Girard and F.C. Greenwood, 1968, in: Protein and polypeptide hormones, Part 1, ed. M. Margoulis. International Congress number 161 (Excerpta Medical Foundation, Amsterdam) p. 29. Ouchterlony, O., 1958, Prog. Allergy 5, 1. Porter, R.R., 1959, Biochem. J. 73, 119. Stevenson, G.T. and K.J. Dorrington, 1970, Biochem. J. 118, 703. Stevenson, G.T. and R.P. Eady, 1973, Analyt. Biochem. 54,300. Wide, L., 1971, in: Radioimmunoassay methods, eds. K.E. Kirkham and W.M. Hunter (Churchill and Livingstone, Edinburgh) p. 405.
THE SPECIFICITY OF A SOLID PHASE RADIOIMMUNOASSAY FOR HUMAN IMMUNOGLOBULINS
R.P. EADY, J.C. CHAPPLE, D.W. HOUGH and G.T. STEVENSON Tenovus Research Laboratory, General Hospital, Southampton, S09 4XY, England
(Received 1 November 1974, accepted 8 November 1974)
The specificity of a solid phase radioimmunoassay for immunoglobulin antigens, human Fabp in the case illustrated, has been assessed. The anti-Fabp serum available reacted well on gel diffusion with all Fabp- and light chain-containing proteins. In contrast the radioimmunoassay, in which unlabelled test antigen competes with radiolabelled Fabp for anti-Fabp coupled to Sephadex beads, was sensitive only to proteins containing entire Fabp regions. A double antibody radioimmunoassay showed comparable or greater sensitivity for Fabp-containing proteins, but was much less specific. By using Fab3" as the labeled antigen, with only light chain determinants reacting with the anti-Fabp, the solid phase assay was rendered polyspecific in being sensitive to ali proteins containing light chain. Reasons for the specificity of the solid phase assay are discussed: steric factors can be expected in some cases to permit strong binding of antigen by multiple bonds, in other, to restrict the number of molecular determinants simultaneously available to antibody.
INTRODUCTION Ideally, the m e t h o d c h o s e n to separate a n t i b o d y - b o u n d antigen fron free antigen in r a d i o i m m u n o a s s a y s m u s t t o t a l l y separate the t w o f r a c t i o n i n s t a n t a n e o u s l y (Ekins and N e w m a n , 1970). F r o m the m a n y separatio; m e t h o d s available ( K i r k h a m and H u n t e r , 1971), the use o f a n t i b o d i e c o u p l e d to a solid m a t r i x has the advantage o f c o m p l e t e n e s s o f s e p a r a t i o r simplicity a n d speed. H o w e v e r the q u e s t i o n o f sensitivity o f the solid phas r a d i o i m m u n o a s s a y has o f t e n been raised. A r e n d s ( 1 9 7 1 ) r e p o r t e d considel able loss o f b o t h a n t i b o d y titre a n d assay sensitivity after c o u p l i n g a n t i b o d to cellulose. Loss o f sensitivity o f r a d i o i m m u n o a s s a y s using a solid phas s y s t e m has also been r e p o r t e d b y A r e n d s and R o o s ( 1 9 7 2 ) and H a r t ( 1 9 7 2 Wide ( 1 9 6 9 ) r e p o r t e d s o m e loss in a n t i b o d y titre after c o u p l i n g to S e p h a d e or cellulose, b u t a p p a r e n t l y little loss in assay sensitivity. R e c e n t l y , a d~ tailed c o m p a r i s o n o f the p r o p e r t i e s o f r a d i o i m m u n o a s s a y s using a n t i b o d c o u p l e d to Sepharose and the same a n t i b o d y in free s o l u t i o n was m a d e b B o l t o n and H u n t e r (1974). T h e y f o u n d , using low mol. w t ( < 2 4 0 0 ) antigen t h a t t h e r e was n o d i f f e r e n c e in sensitivity b e t w e e n the t w o systems. H o w e w s o m e loss in sensitivity o f the solid phase assay was observed w h e n larg~ mol. w t antigens were involved.
180 When using a solid phase radioimmunoassay to study the inhibition of binding of labelled human Fabp to anti-Fabp by structurally related antigens (Eady et al., 1973) we noted a very high specificity for the homologous antigen. We have expanded this observation so as to examine both the sensitivity and specificity of a radioimmunoassay using antibody coupled to Sephadex. Studies were carried out in two systems: the inhibition of binding of labelled homologous antigen (Fabp) to antibody, and the inhibition of binding of a labelled heterologous antigen (Fab3', sharing some determinants with Fabp) to the same antibody. A comparison was also made between solid phase and double antibody radioimmunoassay in the Fabp-anti-Fabp system. It was found that the solid phase method, using labelled homologous antigen, has a strong advantage where relative insensitivity to cross-reacting antigens is desirable. MATERIALS AND METHODS
Preparation of antibodies Randomly bred rabbits were injected subcutaneously in the four limbs with 1 mg of human Fabg incorporated into complete Freund's adjuvant (Difco). After 6 weeks, booster intravenous injections were given at monthly intervals using 0.2 mg human Fabp adsorbed on aluminium hydroxide. The IgG fraction was prepared from sera by precipitation with ammonium sulphate followed by DEAE-cellulose chromatography (Stevenson and Dorrington, 1970). The IgG fraction was shown to have antibody activity directed against both p and light chains by gel immunodiffusion (Ouchterlony, 1958).
Preparation of antigens Human IgM from pooled normal serum was prepared as described by Chaplin et al. (1965), with the insertion of preparative electrophoresis in a Pevikon block. Trace amounts of IgG were removed by passing the IgM through a column of Sepharose 4B coupled to IgG from rabbit anti-~/chain. Fabp was prepared from this IgM by digestion with papain (Eady et al., 1974). Human IgG was prepared from pooled normal serum as described by Stevenson and Dorrington (1970). Adsorption of any contaminating IgM was carried out using Sepharose 4B coupled to the IgG fraction from a rabbit anti-human IgM serum which had previously been absorbed with pooled human light chains. Fab~/ was prepared from this IgG fraction by peptic digestion (Frangione and Prelli, 1960). Human light chains were prepared from pooled normal IgG (Stevenson and Dorrington, 1970) and purified using Sepharose 4B coupled to IgG from rabbit anti-~, chain.
181
Radioiodination This was carried out by the chloramine T m e t h o d (Hunter, 1967), using carrier free t ~ s I (IMS/30 Radiochemical Centre, Amersham).
Solid phase radioimmunoassay Sephadex G-25 superfine (Pharmacia, Uppsala) was activated by the CNBr m e t h o d (Axen et al., 1967). The IgG fraction from the rabbit antiserum was coupled to the activated Sephadex by stirring for 24 hr at 4°C and pH 6.5. Loosely coupled protein was removed by washing with detergent-containing buffers and brief sonication, as described by Wide (1969). The assay was performed by adding 100 pg of 125 I-labelled antigen (50--100 pCi/pg) in 0.1 ml, to 1 ml of standard antigen or inhibiting antigen. I m m u n o s o r b e n t (1 ml) was added at the dilution necessary to bind 50--60% of the label in the absence of unlabelled antigen. The assay was carried out in the presence of carrier horse haemoglobin at 1.0 mg/ml. The assay tubes (Stevenson and Eady, 1973) were rotated about the horizontal axis for 48 hr at room temperature. Separation of the bound and free fractions and two washings of the solid phase were carried out by displacement filtration in the assay tubes. The buffer used for washing and for the assay was 0.05 M Na2 HPO4--KH2 PO4 pH 7.3, 0.45% NaC1, 0.05% NAN3, 0.5% Tween 20, (Sigma). The bound fractions were counted in a Wallac gamma scintillation spectrometer.
Double antibody radioimmunoassay This assay was performed by adding 100 pg of 12 s I-labelled antigen in 0.1 ml to 1 ml of standard antigen or other inhibiting antigen in the same assay tubes as used above. The IgG fraction from rabbit anti-human Fabp (0.1 ml) was added at the dilution necessary to bind 50--60% of the labelled antigen in the absence of unlabelled antigen. The tubes were allowed to stand at room temperature for 48 hr. To separate the bound and free fractions, use was made of an immunosorbent: Sepharose 4B coupled to the IgG from sheep anti-rabbit IgG which had previously been adsorbed with Sepharose 4B--human IgG (Den Hollander and Schuurs, 1971). The immunosorbent (1 ml, 2.5% v/v) was added to the assay tubes which were rotated about the horizontal axis for 18 hr at room temperature. The a m o u n t of immunosorbent used was shown to be capable of binding 80--95% of the 1251 label when the rabbit antibody was present in large excess over antigen. The separation of bound and free fractions was carried out by displacement filtration as above.
182 RESULTS B i n d i n g o f ~ 2 S i . h u m a n F a b p to r a b b i t a n t i - h u m a n F a b p
The solid phase radioimmunoassay for Fabp, illustrated in fig. la, was sensitive over the range 1.0--100 ng of Fabp. The inhibition curve given by h u m a n IgM, although displaced to the right, paralleled the inhibition curve for the homologous antigen. The inhibition curves given by human Fab% L chain and IgG were also displaced to the right, but in addition showed non-parallelism with respect to inhibition by Fabp. Bearing in mind the logarithmic scale on the abscissa, fig. l a is seen to depict an assay with a high degree of specificity for Fabp. Binding of'
2 S i _ h u m a n Fab'y t o r a b b i t a n t i - h u m a n F a b p
Various inhibition curves are illustrated in fig. lb. Those given by Fabp and Fab3' are close and parallel. The curve for IgG is also parallel and displaced to the right of the Fab7 curve to an extent suggesting that more than one Fab3' region in the IgG is reacting: because over much of the curves only about 1.5 times as much IgG as Fab3, is needed for a given degree of inhibition. The IgM curve is also parallel, but in contrast to the IgG curve is greatly displaced to the right, suggesting that only a fraction of the Fab# in the Bo I
-o
_B Bo
: a :: "
i b)
o9 I-O
:.
0-9
: ~ . ~
0.8
0.8
o.
i if
%
\
0-4
0"3"
I
"~
0-2 ~
"~
0.2 O'I
O'l I0
102
10 ~
° I
,5
10~
Antigen
,5,
,~
,5"
(ng/mll
Fig. 1. a) Inhibition of binding of 12 s I-labelled human-Fabp to Sephadex-coupled rabbit anti-human Fabp by the following unlabelled human proteins, b) Inhibition of binding of 12 s I-labelled human-Fab3' to Sephadex-coup]ed rabbit anti-human F a b t / b y the following unlabelled human proteins. (# ©) Fabp, (¢, e) IgM, (D []) Fab% (-m) IgG, (• A) light chain. Each point represents the mean of triplicate determinations.
183 B
(o)
_B Bo
Bo
I-0
1-0
0-9
~'~
0-9
0-8
0.8
0-7
0"7
0-6
0-6
(b}
2--..°
0-5 0-4
0-4
0"3
0"3
0-2
•
0"2
0-1
0-1 10
102
lOa
10'
10
I0~
I0I
10~
Antigen
(ng/ml) Fig. 2. Inhibition of binding of 12 s I-labelled human Fabp to rabbit anti-human Fab# by unlabelled human, (o ©) Fabp, (s o) IgM, (D D) Fab% (1 l) IgG, (/7 A) light chain. (a) Antibody coupled to Sephadex G-25; (b) antibody in free solution. Each point represents the mean of triplicate determinations. molecule can be reacting. Inhibition by free light chain was somewhat o u t of parallel to the inhibition by the ot he r antigens. Similar results to those depicted in fig. 1 have been f o u n d using a radioimmunoassay for h u m a n Fab%
Binding of 12 SI-human Fabp to rabbit anti-human Fabp: comparison o f solid phase and double antibody assays Fig. 2 compares the two assays. Although the dose--response curve for Fabp is slightly steeper using a double a n t i b o d y assay, the m i ni m um detectable a m o u n t of Fabp in b o t h assays is very similar. In b o t h assays the curves for inhibition of binding of 12 s I-Fabp to rabbit anti-Fabp by Fab% IgG and light chain were non-parallel to the curve for Fabp. However the degree of cross reactivity between Fabp and the o th e r antigens was greater using the double a n t i b o d y system: by a factor of a b o u t ten at low concentrations of the antigens. Fig. 2 shows th a t in bot h assays the inhibition curve of hum an IgM is parallel to that o f Fabp. However in the double ant i body assay, the displacem e n t o f IgM to the right is n o t observed. DISCUSSION One criterion of the specificity of a radioimmunoassay is the set of inhibition curves given by molecules with varying com pl em ent s of the antigenic determinants involved ( L a n d o n et al., 1968). Different classes of immunoglo-
184 bulins having c o m m o n light chain determinants provide a very convenient system for assessing the specificity of a radioimmunoassay. The h o m o l o g o u s solid phase assay using 1 z s I-Fabp was shown to be highly specific for the hom ol ogous antigen. The inhibition curves given by free light chain, Fab~ and IgG eventually flatten out after displacement of approx. 50% of the b o u n d 1 z s I-Fabp, which is consistent with the cross-reacting molecules being able to bind to antibodies to light chain determinants, but n o t to antibodies to Fdp determinants. However, the degree of displacem e n t of label by small amounts of the cross-reacting antigen is much less than would be expect ed were t hey able to c o m p e t e on equal terms with Fabp for antibodies to light chain determinants. The explanation for this p h e n o m e n o n is presumably the fact that Fabp can be bound to the solid phase simultaneously by two sets of determinants, giving an effective association co n s tan t much greater than can be realised by molecules with only one set of determinants. Results f r om the double antibody assay are consistent with this explanation. In this system the possibility of multiple bonding of an antigen molecule to a single particle does n o t arise and, as expected, at low concentrations of cross-reacting antigen equivalent displacement of labelled standard antigen f r om a n t i b o d y is observed. All Fabp determinants are present in IgM and this molecule is seen to displace all i 2 s I-Fabp from the solid phase antibody. The marked displacem e n t of the curve to the right, which was n o t observed using a double a n t i b o d y assay, indicates that steric factors are permitting only a fraction of the Fabp regions on the pentameric IgM molecules to react simultaneously with the solid phase antibody. In contrast with the specificity of the homologous solid phase assay, a heterologous solid phase assay constructed using 12 s I-Fab% which shares only light chain determinants with Fabp, yielded parallel inhibition curves for all light chain-containing immunoglobulins. The only except i on to this was free light chains which demonstrated slight non-parallelism for reasons n o t apparent. This heterologous assay is seen to represent a polyspecific assay of good sensitivity for all immunoglobulins containing light chain. Bolton and Hu n t er (1974) have shown that the solid phase radioimmunoassay for low mol. wt proteins can give the same sensitivity as assays where the an tib o d y is in free solution. We have confirmed this sensitivity and shown in addition t ha t a solid phase radioimmunoassay for immunoglobulins can be used in either a monospecific or a polyspecific form, with the f o r m e r exhibiting a notably higher specificity than is observed in the free a n t i b o d y system. One problem dem ons t r a t e d by our work is that steric factors can present a severe restriction in solid phase radioimmunoassays where very large mol. wt antigens such as IgM are involved. ACKNOWLEDGMENTS This work was supported by Tenovus, Cardiff, and the Wessex Regional
185
Hospital Board. We are grateful to Mrs Maureen Power for assistance in the preparation of the proteins.
REFERENCES Arends, J., 1971, Acta Endocrinol. 68, 425. Arends, J. and J. Roos, 1972, Scand. J. Clin. Lab. Invest. 29 Suppl. 126 Abst 14. Axen, R., J. Porath and S. Ernbach, 1967, Nature 214, 1302. Bolton, A.E. and W.M. Hunter, 1973, Biochim. Biophys. Acta 329, 318. Chaplin, H., S. Cohen and E.M. Press, 1965, Biochem. J. 95, 256. Den Hollander, F.C. and A.H.W.M. Schuurs, 1971, in: Radioimmunoassay methods, eds. K.E. Kirkham and W.M. Hunter (Churchill and Livingstone, Edinburgh) p. 419. Eady, R.P., D.W. Hough, P.J. Kilshaw and G.T. Stevenson, 1974, Immunology 26, 549. Frangione, B. and F. Prelli, 1960, J. Clin. Invest. 39, 1933. Hart, I.C., 1972, J. Endocrinol. 55, 51. Hunter, W.M., 1967, in: Handbook of experimental immunology, ed. D.M. Weir (Blackwell Scientific Publications, Oxford) p. 608. Kirkham, K.E. and W.M. Hunter, 1971, Radioimmunoassay methods (Churchill and Livingstone, Edinburgh) Session III, p. 243. Landon, J., J. Girard and F.C. Greenwood, 1968, in: Protein and polypeptide hormones, Part 1, ed. M. Margoulis. International Congress number 161 (Excerpta Medical Foundation, Amsterdam) p. 29. Ouchterlony, O., 1958, Prog. Allergy 5, 1. Porter, R.R., 1959, Biochem. J. 73, 119. Stevenson, G.T. and K.J. Dorrington, 1970, Biochem. J. 118, 703. Stevenson, G.T. and R.P. Eady, 1973, Analyt. Biochem. 54,300. Wide, L., 1971, in: Radioimmunoassay methods, eds. K.E. Kirkham and W.M. Hunter (Churchill and Livingstone, Edinburgh) p. 405.
