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Contaminating antibodies in anti-IgE antisera
Journal of Immunological Methods, 13 (1976) 91--95 91 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
CONTAMINATING ANTIBODIES IN ANTI-IgE ANTISERA *
GEORGE M. BERNIER ** and O. ROSS McINTYRE Departments o f Medicine, Case Western Reserve University at University Hospitals of Cleveland, Cleveland, Ohio, and Dartmouth Medical School, Hanover, New Hampshire, USA
(Received 20 April 1976, accepted 1 May 1976) Antisera directed against IgE globulin from a variety of commercial and non-commercial sources were found to contain antibodies that reacted in Ouchterlony double diffusion with some X-type light chains. These antisera cross reacted with a small percentage of ~type myeloma globulins as well. Absorption studies suggested that the cross reacting antibodies were directed against antigenic determinants present on the light chain of the myeloma globulin used for immunization. These findings may partially explain some of the difficulties encountered in immunologic measurement of IgE globulin in body fluids. INTRODUCTION T h e m e a s u r e m e n t o f i m m u n o g l o b u l i n E in b o d y fluids b y various i m m u nologic t e c h n i q u e s is largely d e p e n d e n t u p o n t h e s p e c i f i c i t y o f t h e anti-IgE a n t i s e r u m . Because n o r m a l IgE is d i f f i c u l t t o isolate in p u r e f o r m a n d b e c a u s e IgE m y e l o m a p r o t e i n s are rare, t h e m a j o r i t y o f anti-IgE a n t i s e r a curr e n t l y available have b e e n p r e p a r e d b y i m m u n i z a t i o n w i t h relatively f e w s o u r c e s o f IgE. T h e IgE m y e l o m a globulin o f o n e p a t i e n t in p a r t i c u l a r , P.S. ( O g a w a et al., 1 9 6 9 ) , has b e e n used b y m a n y investigators a n d c o m m e r c i a l s o u r c e s in this c o u n t r y t o i m m u n i z e a n i m a l s f o r t h e p r o d u c t i o n o f anti-IgE antisera. O n e d a n g e r i n h e r e n t in utilizing a single m y e l o m a globulin as t h e s o u r c e o f m u l t i p l e a n t i s e r a is t h e p o s s i b i l i t y t h a t relatively u n c o m m o n antigenic d e t e r m i n a n t s o t h e r t h a n t h e desired o n e (IgE in this case) m a y be p r e s e n t a n d give rise to n o n - s p e c i f i c a n t i b o d i e s d i f f i c u l t to d e t e c t b y conv e n t i o n a l m e a n s . C u r r e n t l y , t h e m e a s u r e m e n t o f IgE a p p e a r s d i f f i c u l t to s t a n d a r d i z e b e t w e e n l a b o r a t o r i e s f o r various reasons ( P o l m a r , 1 9 7 5 ) . T h e e v i d e n c e p r e s e n t e d in this r e p o r t indicates o n e r e a s o n m a y be t h a t m a n y c o m m e r c i a l and n o n - c o m m e r c i a l anti-IgE a n t i s e r a c o n t a i n a n t i b o d i e s directed against light c h a i n d e t e r m i n a n t s p r e s e n t in t h e i m m u n i z i n g m y e l o m a globulin. T h e s e a n t i b o d i e s a p p e a r t o r e c o g n i z e a s u b p o p u l a t i o n o f ~ chains, p o s s i b l y r e l a t e d to a s u b - g r o u p o f h u m a n i m m u n o g l o b u l i n m o l e c u l e s , t h e Oz s u b - g r o u p (Ein, 1 9 6 8 ) . * Supported by USPHS grants AM 14700, CA 04326 and CA 17864. * * Scholar, Leukemia Society of America.
92 MATERIALS AND METHODS A n t i s e ra
Anti-IgE antisera were produced by one of us (G.B.) by immunizing New Zealand white rabbits with agar gel-isolated (Bernier, 1973) IgE m y e l o m a globulin P.S. The antisera were absorbed with normal human serum, or, as noted below, with the Bence--Jones protein of patient P.S. Several commercially prepared anti-IgE antisera were purchased. The companies and lot numbers were: Hyland, Costa Mesa, California, goat, lot 8271M007A1; Meloy, Falls Church, Virginia, goat, lot A10932331; Miles Labs, Kankakee, Illinois goat, lot P1001, 61-081; Kallstead Labs, Chaska, Minnesota, equine, lot 126G031. In addition, two anti-IgE antisera prepared against P.S. IgE m y e l o m a globulin by other investigators were examined. Anti-IgE (N.D.), a rabbit antisera prepared against a different IgE m y e l o m a globulin was also tested. Rabbit antisera specific for other immunoglobulins and for free X chain have been previously described (Fermin et al., 1974). A n tigens
Myeloma glc~bulins (including three of the IgE class) and Bence--Jones proteins of known chain composition were tested in Ouchterlony double diffusion. Bence--Jones proteins of X t y p e including several of defined Oz type (Ein, 1968; Putnam, 1969) were studied. Plasma from patient P.S. was obtained by plasmaphoresis and stored at --20 ° C. Urine was collected fresh and stored frozen at --20 ° C. Papain digestion of m y e l o m a globulins was performed at 37°C for 2 h, using a 100 : 1 ratio of myeloma globulin to enzyme (Worthington Biochemical, Freehold, New Jersey), in EDTA and cysteine. Immunodiffusion studies were carried out at room temperature in 1% agarose gels, in 0.15 M glycine buffer, pH 8.0 containing 0.003 M sodium azide and 0.0001 M disodium ethylene-diaminetetraacetic acid (Fermin et al., 1974). RESULTS All of the antisera tested reacted with the three IgE m y e l o m a globulins in agar double diffusion. In addition each reacted with one or more X m y e l o m a proteins of IgG or IgA t y p e or free X chains. In some cases, the precipitation line in agar was a strong one, and in others faint (fig. 1). Of 55 non-IgE proteins tested, 9 reacted with 6 of the 7 antisera employed. The Meloy goat anti-human IgE, lot no. A 1 0 9 3 2 3 3 1 did not react with m y e l o m a globulins and reacted with only two of the free light chains tested. Several of the X m y e l o m a proteins that reacted with anti-IgE (P.S.) were tested with anti-IgE
93
Fig. 1. Double diffusion in agar showing reactivity between 2 k Bence--Jones proteins (1 mg/ml) at 8 and 10 o'clock, 2 IgG k myeloma globulins (4 mg/ml) at 2 and 4 o'clock, and 2 different commercial anti-IgE antisera. P.S. m y el o m a globulin at the top and normal serum diluted 1 : 10 at bottom. Antiserum A reacts with at least 3 non-IgE antigens. Antiserum B gives strong precipitation arcs with the 4 myeloma proteins and with normal serum.
94
Fig. 2. A) Trough: Anti-IgE antiserum prepared against IgE myeloma globulin P.S., and adsorbed with normal human serum, reacting in immunoelectrophoresis with the serum of a myeloma patient A.J. (top}. Both IgG " ~ myeloma globulin (7 mobility) and free chain (~ mobility) react. Visible precipitation does not occur with the antigen below, normal human serum. B) Trough: Same antiserum absorbed with P.S. Bence--Jones protein. Top: myeloma serum A.J.; Bottom: P.S. serum. Following absorption with P.S. Bence-Jones protein, the antiserum no longer reacts with the IgG • ~ globulin nor with the free chain, but does react with the IgE myeloma globulin P.S.
(N.D.). In no i n s t a n c e did t h e y react. T h a t t h e a n t i s e r a w e r e r e a c t i n g w i t h the ~ light c h a i n was s u p p o r t e d b y t h e s e o b s e r v a t i o n s : 1) T h e a n t i s e r a t h a t r e a c t e d w i t h IgG • ~ m o l e c u l e s reacted w i t h t h e p a p a i n - p r o d u c e d F a b f r a g m e n t o f o n e o f t h e s e m y e l o m a globulins. 2) T h e a n t i s e r a r e a c t e d w i t h t h e u r i n a r y free light chain o f p a t i e n t P.S. 3) W h e n t h e r e a c t i v e m y e l o m a globulins w e r e o b t a i n e d f r o m p a t i e n t s w h o e x c r e t e d B e n c e - - J o n e s p r o t e i n s , t h e u r i n a r y free light chains also r e a c t e d w i t h t h e anti-IgE antisera. 4) When t h e antisera were a b s o r b e d w i t h t h e urin a r y free light chain o f p a t i e n t P.S. or a n y o t h e r r e a c t i v e free ~ light chains, r e a c t i v i t y w i t h n o n - I g E m y e l o m a globulins was a b o l i s h e d (fig. 2). Since a p p r o x i m a t e l y 20% o f ~ m y e l o m a p r o t e i n s r e a c t e d w i t h t h e anti-
95 sera, the possibility existed that the reactive antigen was the Oz determinant, known to be present on approximately 20% of k m y e l o m a proteins. All six Bence--Jones proteins known by amino acid sequence or by antigenic testing to be Oz-- failed to react with the antisera; two of two Oz+ k light chains interfered with the precipitation lines formed by the antisera and the reactive k chains, but neither formed strong precipitation lines themselves. The relationship, therefore, between the reactive antigen and the Oz determinant is uncertain but these studies suggest that the antigens were related. It is also possible that the antigen being detected represents variable region subtype (Milstein, 1967). DISCUSSION Because of the extremely low concentrations of IgE in body fluids, estimations of this immunoglobulin are generally made by radioimmunoassay rather than by diffusion in agar, and reactions of partial identity are not readily appreciated. The findings presented in this report may provide a partial explanation for the difficulties in standardizing IgE measurements from laboratory to laboratory. Since the methods for absorbing contaminating antibodies from anti-IgE antisera differ widely, it is possible that m a n y antiIgE antisera contain antibodies to the determinants cited here. One published m e t h o d (Ishizaka and Ishizaka, 1974) used for producing specific antiIgE antisera involved immunizing with the P.S. m y e l o m a globulin and absorption with the Fab fragment of the same globulin. Such a procedure would certainly remove the antibodies discussed in this report. If the P.S. antigen is used for specifically isolated anti-IgE in preparing antibodies for radioimmunoassays, it is unlikely that exclusion of contaminating antibodies can be achieved w i t h o u t a specific absorption step employing P.S. Fab or Bence Jones protein. ACKNOWLEDGMENTS We are grateful to Dr. Richard Wistar for supplying Bence--Jones proteins of known Oz type, and Claire Johnson for technical assistance.
REFERENCES Bernier, G.M., 1973, Immunol. Methods 3, 99. Ein, D., 1968, Proc. Natl. Acad. Sci. U.S. 60,982. Fermin, E.A., C.A. Johnson, R.E. Eckel and G.M. Bernier, 1974, J. Lab. Clin. Med. 83, 681. Ishizaka, T. and K. Ishizaka. 1974, J. Immunol. 112, 1078. Milstein, C., 1967, Nature 216, 330. Ogawa, M., S. Kochwa, C. Smith, K. Ishizaka and O.R. McIntyre, 1969, New Eng. J. Med. 281, 1217. Polmar, S.H., 1975, in: Laboratory diagnosis of immunologic disorders, eds. G.N. Vyas, D.P. Stites and G. Brecher (Grune and Stratton, Inc.) p. 213. Putnam, F.W., 1969, Science 163,633.
CONTAMINATING ANTIBODIES IN ANTI-IgE ANTISERA *
GEORGE M. BERNIER ** and O. ROSS McINTYRE Departments o f Medicine, Case Western Reserve University at University Hospitals of Cleveland, Cleveland, Ohio, and Dartmouth Medical School, Hanover, New Hampshire, USA
(Received 20 April 1976, accepted 1 May 1976) Antisera directed against IgE globulin from a variety of commercial and non-commercial sources were found to contain antibodies that reacted in Ouchterlony double diffusion with some X-type light chains. These antisera cross reacted with a small percentage of ~type myeloma globulins as well. Absorption studies suggested that the cross reacting antibodies were directed against antigenic determinants present on the light chain of the myeloma globulin used for immunization. These findings may partially explain some of the difficulties encountered in immunologic measurement of IgE globulin in body fluids. INTRODUCTION T h e m e a s u r e m e n t o f i m m u n o g l o b u l i n E in b o d y fluids b y various i m m u nologic t e c h n i q u e s is largely d e p e n d e n t u p o n t h e s p e c i f i c i t y o f t h e anti-IgE a n t i s e r u m . Because n o r m a l IgE is d i f f i c u l t t o isolate in p u r e f o r m a n d b e c a u s e IgE m y e l o m a p r o t e i n s are rare, t h e m a j o r i t y o f anti-IgE a n t i s e r a curr e n t l y available have b e e n p r e p a r e d b y i m m u n i z a t i o n w i t h relatively f e w s o u r c e s o f IgE. T h e IgE m y e l o m a globulin o f o n e p a t i e n t in p a r t i c u l a r , P.S. ( O g a w a et al., 1 9 6 9 ) , has b e e n used b y m a n y investigators a n d c o m m e r c i a l s o u r c e s in this c o u n t r y t o i m m u n i z e a n i m a l s f o r t h e p r o d u c t i o n o f anti-IgE antisera. O n e d a n g e r i n h e r e n t in utilizing a single m y e l o m a globulin as t h e s o u r c e o f m u l t i p l e a n t i s e r a is t h e p o s s i b i l i t y t h a t relatively u n c o m m o n antigenic d e t e r m i n a n t s o t h e r t h a n t h e desired o n e (IgE in this case) m a y be p r e s e n t a n d give rise to n o n - s p e c i f i c a n t i b o d i e s d i f f i c u l t to d e t e c t b y conv e n t i o n a l m e a n s . C u r r e n t l y , t h e m e a s u r e m e n t o f IgE a p p e a r s d i f f i c u l t to s t a n d a r d i z e b e t w e e n l a b o r a t o r i e s f o r various reasons ( P o l m a r , 1 9 7 5 ) . T h e e v i d e n c e p r e s e n t e d in this r e p o r t indicates o n e r e a s o n m a y be t h a t m a n y c o m m e r c i a l and n o n - c o m m e r c i a l anti-IgE a n t i s e r a c o n t a i n a n t i b o d i e s directed against light c h a i n d e t e r m i n a n t s p r e s e n t in t h e i m m u n i z i n g m y e l o m a globulin. T h e s e a n t i b o d i e s a p p e a r t o r e c o g n i z e a s u b p o p u l a t i o n o f ~ chains, p o s s i b l y r e l a t e d to a s u b - g r o u p o f h u m a n i m m u n o g l o b u l i n m o l e c u l e s , t h e Oz s u b - g r o u p (Ein, 1 9 6 8 ) . * Supported by USPHS grants AM 14700, CA 04326 and CA 17864. * * Scholar, Leukemia Society of America.
92 MATERIALS AND METHODS A n t i s e ra
Anti-IgE antisera were produced by one of us (G.B.) by immunizing New Zealand white rabbits with agar gel-isolated (Bernier, 1973) IgE m y e l o m a globulin P.S. The antisera were absorbed with normal human serum, or, as noted below, with the Bence--Jones protein of patient P.S. Several commercially prepared anti-IgE antisera were purchased. The companies and lot numbers were: Hyland, Costa Mesa, California, goat, lot 8271M007A1; Meloy, Falls Church, Virginia, goat, lot A10932331; Miles Labs, Kankakee, Illinois goat, lot P1001, 61-081; Kallstead Labs, Chaska, Minnesota, equine, lot 126G031. In addition, two anti-IgE antisera prepared against P.S. IgE m y e l o m a globulin by other investigators were examined. Anti-IgE (N.D.), a rabbit antisera prepared against a different IgE m y e l o m a globulin was also tested. Rabbit antisera specific for other immunoglobulins and for free X chain have been previously described (Fermin et al., 1974). A n tigens
Myeloma glc~bulins (including three of the IgE class) and Bence--Jones proteins of known chain composition were tested in Ouchterlony double diffusion. Bence--Jones proteins of X t y p e including several of defined Oz type (Ein, 1968; Putnam, 1969) were studied. Plasma from patient P.S. was obtained by plasmaphoresis and stored at --20 ° C. Urine was collected fresh and stored frozen at --20 ° C. Papain digestion of m y e l o m a globulins was performed at 37°C for 2 h, using a 100 : 1 ratio of myeloma globulin to enzyme (Worthington Biochemical, Freehold, New Jersey), in EDTA and cysteine. Immunodiffusion studies were carried out at room temperature in 1% agarose gels, in 0.15 M glycine buffer, pH 8.0 containing 0.003 M sodium azide and 0.0001 M disodium ethylene-diaminetetraacetic acid (Fermin et al., 1974). RESULTS All of the antisera tested reacted with the three IgE m y e l o m a globulins in agar double diffusion. In addition each reacted with one or more X m y e l o m a proteins of IgG or IgA t y p e or free X chains. In some cases, the precipitation line in agar was a strong one, and in others faint (fig. 1). Of 55 non-IgE proteins tested, 9 reacted with 6 of the 7 antisera employed. The Meloy goat anti-human IgE, lot no. A 1 0 9 3 2 3 3 1 did not react with m y e l o m a globulins and reacted with only two of the free light chains tested. Several of the X m y e l o m a proteins that reacted with anti-IgE (P.S.) were tested with anti-IgE
93
Fig. 1. Double diffusion in agar showing reactivity between 2 k Bence--Jones proteins (1 mg/ml) at 8 and 10 o'clock, 2 IgG k myeloma globulins (4 mg/ml) at 2 and 4 o'clock, and 2 different commercial anti-IgE antisera. P.S. m y el o m a globulin at the top and normal serum diluted 1 : 10 at bottom. Antiserum A reacts with at least 3 non-IgE antigens. Antiserum B gives strong precipitation arcs with the 4 myeloma proteins and with normal serum.
94
Fig. 2. A) Trough: Anti-IgE antiserum prepared against IgE myeloma globulin P.S., and adsorbed with normal human serum, reacting in immunoelectrophoresis with the serum of a myeloma patient A.J. (top}. Both IgG " ~ myeloma globulin (7 mobility) and free chain (~ mobility) react. Visible precipitation does not occur with the antigen below, normal human serum. B) Trough: Same antiserum absorbed with P.S. Bence--Jones protein. Top: myeloma serum A.J.; Bottom: P.S. serum. Following absorption with P.S. Bence-Jones protein, the antiserum no longer reacts with the IgG • ~ globulin nor with the free chain, but does react with the IgE myeloma globulin P.S.
(N.D.). In no i n s t a n c e did t h e y react. T h a t t h e a n t i s e r a w e r e r e a c t i n g w i t h the ~ light c h a i n was s u p p o r t e d b y t h e s e o b s e r v a t i o n s : 1) T h e a n t i s e r a t h a t r e a c t e d w i t h IgG • ~ m o l e c u l e s reacted w i t h t h e p a p a i n - p r o d u c e d F a b f r a g m e n t o f o n e o f t h e s e m y e l o m a globulins. 2) T h e a n t i s e r a r e a c t e d w i t h t h e u r i n a r y free light chain o f p a t i e n t P.S. 3) W h e n t h e r e a c t i v e m y e l o m a globulins w e r e o b t a i n e d f r o m p a t i e n t s w h o e x c r e t e d B e n c e - - J o n e s p r o t e i n s , t h e u r i n a r y free light chains also r e a c t e d w i t h t h e anti-IgE antisera. 4) When t h e antisera were a b s o r b e d w i t h t h e urin a r y free light chain o f p a t i e n t P.S. or a n y o t h e r r e a c t i v e free ~ light chains, r e a c t i v i t y w i t h n o n - I g E m y e l o m a globulins was a b o l i s h e d (fig. 2). Since a p p r o x i m a t e l y 20% o f ~ m y e l o m a p r o t e i n s r e a c t e d w i t h t h e anti-
95 sera, the possibility existed that the reactive antigen was the Oz determinant, known to be present on approximately 20% of k m y e l o m a proteins. All six Bence--Jones proteins known by amino acid sequence or by antigenic testing to be Oz-- failed to react with the antisera; two of two Oz+ k light chains interfered with the precipitation lines formed by the antisera and the reactive k chains, but neither formed strong precipitation lines themselves. The relationship, therefore, between the reactive antigen and the Oz determinant is uncertain but these studies suggest that the antigens were related. It is also possible that the antigen being detected represents variable region subtype (Milstein, 1967). DISCUSSION Because of the extremely low concentrations of IgE in body fluids, estimations of this immunoglobulin are generally made by radioimmunoassay rather than by diffusion in agar, and reactions of partial identity are not readily appreciated. The findings presented in this report may provide a partial explanation for the difficulties in standardizing IgE measurements from laboratory to laboratory. Since the methods for absorbing contaminating antibodies from anti-IgE antisera differ widely, it is possible that m a n y antiIgE antisera contain antibodies to the determinants cited here. One published m e t h o d (Ishizaka and Ishizaka, 1974) used for producing specific antiIgE antisera involved immunizing with the P.S. m y e l o m a globulin and absorption with the Fab fragment of the same globulin. Such a procedure would certainly remove the antibodies discussed in this report. If the P.S. antigen is used for specifically isolated anti-IgE in preparing antibodies for radioimmunoassays, it is unlikely that exclusion of contaminating antibodies can be achieved w i t h o u t a specific absorption step employing P.S. Fab or Bence Jones protein. ACKNOWLEDGMENTS We are grateful to Dr. Richard Wistar for supplying Bence--Jones proteins of known Oz type, and Claire Johnson for technical assistance.
REFERENCES Bernier, G.M., 1973, Immunol. Methods 3, 99. Ein, D., 1968, Proc. Natl. Acad. Sci. U.S. 60,982. Fermin, E.A., C.A. Johnson, R.E. Eckel and G.M. Bernier, 1974, J. Lab. Clin. Med. 83, 681. Ishizaka, T. and K. Ishizaka. 1974, J. Immunol. 112, 1078. Milstein, C., 1967, Nature 216, 330. Ogawa, M., S. Kochwa, C. Smith, K. Ishizaka and O.R. McIntyre, 1969, New Eng. J. Med. 281, 1217. Polmar, S.H., 1975, in: Laboratory diagnosis of immunologic disorders, eds. G.N. Vyas, D.P. Stites and G. Brecher (Grune and Stratton, Inc.) p. 213. Putnam, F.W., 1969, Science 163,633.