Correlation of murine anti-dinitrophenyl antibody content as determined by ELISA, passive cutaneous anaphylaxis and passive hemolysis

Journal of Immunological Methods. 71 (1984) 229-239

229

Elsevier JIM03117

Correlation of Murine Anti-Dinitrophenyl Antibody Content as Determined by ELISA, Passive Cutaneous Anaphylaxis and Passive Hemolysis Soichiro Maekawa and Zoltan Ovary

1

Department of Pathology, New York University School of Medicine, New York, N Y 10016, U.S.A.

(Received 25 January 1984, accepted 12 March 1984)

Antibody contents of IgG1, IgG2a, IgG2b and IgE were measured by enzyme-linked immunosorbent assay (ELISA) in ascites and sera obtained from mice injected with hybridomas producing monoclonal anti-DNP antibodies. In addition, IgG1 and IgE antibodies from sera of immunized mice were also measured by ELISA. Concomitantly, antibody contents were also determined by passive cutaneous anaphylaxis (PCA) in mice for lgG1, IgG2a, lgG2b, by PCA in guinea pigs for IgG2a, by PCA in rats for IgE and by passive hemolysis (PL) for IgG2a and IgG2b. Good correlations were found in the investigated samples between ELISA and the biological determinations. Key words: EL1SA - passive cutaneous anaphylaxis - passive hemolysis

Introduction In the past some of the murine antibody isotypes were quantitated mainly by biological methods, such as passive cutaneous anaphylaxis (PCA) (Ovary, 1964) and c o m p l e m e n t fixation. The introduction of enzyme-linked i m m u n o s o r b e n t assay ( E L I S A ) (Engvall and Perlmann, 1971) greatly facilitated quantitative determinations of immunoglobulins and was rapidly applied to the determinations of murine antibodies. However; f o r the determinations of murine IgE, E L I S A could not be used as no murine myeloma protein of the IgE class existed. The construction of murine IgE producing h y b r i d o m a s (B6ttcher et al., 1978, 1980; Eshhar et al., 1980; Liu et al., 1980; R u d o l p h et al., 1981) made it possible to use E L I S A also for murine I g E antibodies (Hill and Liu, 1981). E L I S A measures quantitatively the a n t i b o d y protein, however it does not give any information on the biological activities of the antibody measured. For estimation of biological activities, P C A reactions and complement fixation remain important methods.

1 To whom correspondence should be addressed. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.

230 The correlation of the data obtained by ELISA and by biological methods is of interest for 2 reasons: (1) to see if in the future biological methods could still be used for antibody determinations; (2) to be able to estimate the antibody content in protein values determined by biological methods published in the past.

Materials and Methods

Solutions Sera, ascitic fluids and Evans blue dye (Eastman Kodak, Rochester, NY) were diluted in 0.15 M NaCI, when used for PCA. The buffers used for PL of dinitrophenyl (DNP)-coupled sheep erythrocytes (Stein et al., 1980) were previously described (Bloch et al., 1963). For ELISA, in addition to phosphate-buffered saline, pH 7.2, 0.15 M (PBS), the following solutions were freshly prepared: PBS with 0.05% Tween 20 (PBS-T; Sigma Chemical Co., St. Louis, MO); PBS with 10% fetal calf serum (Dutchland Lab., Denver, PA) and 0.02% NaN 3 (PBS-FCS; Fisher Scientific, Springfield, N J); PBS with 1% bovine serum albumin (PBS-BSA; Pentex BSA, Fraction V, Miles Lab., Elkhart, IN). Substrate solution: 10 mg o-phenylenediamine (Sigma) was dissolved in 100 ml double distilled water and 3/~1 of 30% H202 (Fisher Scientific, Fair Lawn, N J) were added. Animals BALB/c mice were obtained from Charles River Breeding Laboratories (Wilmington, MA), CB6F1 mice from Jackson Laboratories (Bar Harbor, ME). All mice were female and 8-12 weeks old at the beginning of immunizations and inoculations. Eight- to ten-week-old female CFW mice were obtained from Charles River Breeding Laboratories, Sprague-Dawley male rats 300 g weight and Hartley albino guinea pigs of both sexes 300 g weight were obtained from Camm Research Institute (Wayne, N J). These animals were used for PCA reactions. Antigens Dinitrophenylated ovalbumin (DNP-OVA), bovine serum albumin (DNP-BSA), keyhole limpet hemocyanin (DNP-KLH) and mouse serum albumin (DNP-MSA) were the same preparations as previously described (Kojima and Ovary, 1975). Immunizations BALB/c mice were immunized intraperitoneally (i.p.) with DNP-OVA or DNPKLH, as described (Kojima and Ovary, 1975), with slight modifications (i.e., the doses of antigens varied from 0.2 to 10 /,g and the dose of AI(OH) 3 used as adjuvant was 4 mg). Serum samples were taken from these mice at various intervals after immunization.

231

Hybridomas Hybridomas producing anti-DNP or anti-trinitrophenyl (TNP) monoclonal antibodies of the IgG1, IgG2a, IgG2b and IgE classes were previously described (Brttcher et al., 1980; Eshhar et al., 1980; Liu et al., 1980; Rudolph et al., 1981; Hirayama et al., 1982; Ovary, 1982). The antibody-producing parental strain was the BALB/c (D05-IGI:IgG1, D05-IC4:IgG2a, B53:IgE, ATCC 142 IGEL a2:IgE) or the CAF1 (NK:IgG1, NK:IgG2b, a generous gift of Dr. N. Klinman, Scripps Clinic and Research Foundation, La Jolla, CA), or the C57BL/6 (ATCC 141 IGEL b4:IgE). Both lines, ATCC 142 IGEL a2 (142a) and ATCC 141 IGEL b4 (141b), were obtained from the American Type Culture Collection (Rockville, MD). Serum samples containing monoclonal antibodies were taken from mice inoculated subcutaneously (s.c.) with 5 x 103 to 5 x 10 6 hybridoma cells in 0.1 ml or from mice inoculated i.p. with 2 x 10 6 tO 1 X 107 hybridoma cells in 0.2-1 ml. All hybridomas were injected into BALB/c mice except 141b, which was injected into CB6F1 mice. Ascitic fluids were taken from mice injected i.p. with hybridoma cells generally 6-8 days previously. Passioe cutaneous anaphylaxis (PCA) PCA reactions in mice, rats and guinea pigs were done as described (Ovary, 1964; Ovary et al., 1975). The sensitization period in mice was 1½ h, for rats 2 h, for guinea pigs 3 h. Mice were challenged with 0.5 mg DNP-MSA, guinea pigs with 0.4 mg and rats with 1 mg DNP-BSA. Passive hemolysis (PL) Passive hemolysis (complement fixation) was done as described (Bloch et al., 1963) with DNP-coated sheep erythrocytes prepared as described (Stein et al., 1980). ELISA (1) Standard protein preparations. Anti-DNP IgG1 (NK-1), IgG2a and IgG2b monoclonal antibodies obtained from ascitic fluids were first affinity purified on a DNP-BSA Sepharose 4B column as described (Ovary, 1982), then absorbed by the appropriate purified rabbit antibodies coupled to Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N J). The protein content was determined by micro-Kjeldahl and by optical density at 280 nm using 1.4 as conversion factor (Ovary, 1982). The monoclonal anti-DNP IgE used for standard protein was a generous gift of Dr. F.-T. Liu (Medical Biology Institute, La Jolla, CA) (Liu et al., 1980; Hill and Liu, 1981). (2) Rabbit antibodies to routine immunoglobulins. For production of rabbit anti-murine immunoglobulins, rabbits were immunized with myeloma proteins MOPC 21 (IgG1), UPC 10 (IgG2a), MOPC 195 (IgG2b) purchased from Litton Bionetics (Kensington, MD) and with our purified B53 preparation (IgE). All rabbit sera were rendered monospecific by absorption with the appropriate immunizing antigens coupled to Sepharose 4B as described (Hirayama et al., 1982). In addition, the anti-IgE serum was absorbed also with an anti-Dansyl monoclonal IgM preparation, a generous gift of Dr. F. Karush (University of Pennsylvania, Philadelphia, PA). No cross-reaction was observed with these antisera in ELISA.

232

(3) Peroxidase conjugated goat anti-rabbit IgG (GARIG-PO). This material was purchased from Cappel Laboratories (West Chester, PA). (4) Assay procedure. The assay procedure was described (Engvall and Perlmann, 1971; Hill and Liu, 1981). We used 96-well disposable, flexible, polyvinyl chloride microtitration V plates (Dynatech Laboratories, Alexandria, VA). The volume pipetted in each well was 0.05 ml, except for blocking the non-specific binding sites with PBS-FCS and for the substrate, when it was 0.1 ml. The antigen was DNP-BSA, and in preliminary experiments it was found that for determinations of IgG1, IgG2a 2 / , g / m l and of IgG2b, IgE 5 0 / , g / m l gave the optimal antigen coating. The samples and negative controls were diluted with PBS-BSA from 1/100 to 1/40,000. Eight to 10 dilutions of the standard protein were included in every assay. The optimal dilutions of our antisera in PBS-T were decided as follows: rabbit anti-IgGl: 1/1000; rabbit anti-IgG2a: 1/1000; rabbit anti-IgG2b: 1/250; rabbit anti-IgE: 1/750; GARIG-PO: 1/2500. For all washing procedures, PBS-T were used. The wells were incubated for 1 h at 37°C except for blocking when they were incubated overnight at 4°C. The incubation with the substrate was for 30 min at room temperature. The reaction was stopped with 0.1 ml 8 N H2SO 4. The absorbancy was determined at 488 nm by Artek ELISA Auto Reader (Artek System Corporation, Farmingdale, NY). (5) Quantitation of anti-DNP specific antibodies. Curves were constructed with every standard immunoglobulin isotype examined. The protein concentrations were plotted on the abscissa with logarithmic scale and the absorbances (after subtraction of the blank values) on the ordinate with arithmetic scale. The blank was the solution of PBS-BSA. An S shape curve was obtained. The minimum protein detectable (at the beginning of the ascending portion of the curve) and the maximum (at the end, when curve tends to become horizontal) are indicated by 2 small vertical bars (insert in the figures). To determine the antibody contents of the samples, in addition to the blank, 2 negative controls were included: one was a normal mouse serum, the other an ascitic fluid containing a monoclonal antibody from a different isotype than the sample to be titrated. Both controls were diluted to the same extent as the samples. From the different dilutions of the samples, those which had absorbances (after subtraction of the negative control values) falling on the ascending portion of the standard curve were used to calculate the antibody contents. Serum and ascites samples were examined simultaneously for antibody contents by ELISA, PCA and PL. The minimum antibody requirements for threshold reactions were calculated from the antibody protein concentrations of samples as determined by ELISA, and the last dilutions giving the biological reaction (threshold values). The correlation coefficients were calculated as described (Snedecor and Cochran, 1967). Results

Figs. 1-4 summarize the results. The protein values are plotted on the abscissa and the biological activities on the ordinate. In every figure the insert represents the

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ELISA standard curve. The sensitivity of ELISA (the range between which the protein concentration can be safely determined) will be given below for each immunoglobulin isotype.

IgG1 The sensitivity of ELISA for this immunoglobulin class was between 4 ng and 1 /xg standard protein/ml. Five ascites samples obtained from mice injected i.p. with anti-DNP IgGl-producing hybridoma NK-1, 1 ascites sample from hybridoma D05-IGI and 7 samples of sera from immunized mice were investigated. Specific IgG1 contents of the samples varied from 0.05 mg to 10.00 m g / m l by ELISA and from 80 to 8000 by PCA. Threshold values for PCA activity in mice were between 0.44 and 1.38 ~ g / m l . D05-IGI, contrary to other IgG1 antibodies, fixes complement and sensitizes the rat for PCA reactions (Hirayama et al.., 1982; Ovary, 1982). The threshold amount for PL was 0.98/~g/ml in our system (Ovary, 1982) and for sensitization of the rat was 97.60/~g/ml, 80 times more than the amount necessary to sensitize the mouse. For the sake of simplicity (and because it is only 1 sample) the titer of PL and rat PCA with D05-IGI are not represented in Fig. 1. The serum samples from immunized mice contained not only IgG1, but also IgG2a, IgG2b and IgE; all of these isotypes are capable of sensitizing the mouse.

234

The percentage of IgG2a + IgG2b to total IgG antibodies, calculated by the following equation: IgG2a + IgG2b × 100 = percentage IgG1 + IgG2a + IgG2b was 22% in one case, lower in others. In addition, the threshold values of PCA reactions in mice were 2-12 times higher with IgG2a or IgG2b than with IgG1 (results in Table I). The anti-DNP IgE content in these serum samples was less than 3% of the IgG1 content by ELISA and the PCA in rats was demonstrable only with very low dilutions, generally 1/40, at maximum 1/400 (not shown). Good correlation was obtained between PCA activities and the results with ELISA (Fig. 1). The correlation coefficient was 0.98 ( P < 0.01).

IgG2a The sensitivity of ELISA for this class was between 10 ng and 1.25 #g standard protein/ml. Eight samples of ascites from mice injected with anti-DNP monoclonal antibody producing hybridoma D05-IC4 were examined. Murine IgG2a sensitizes not only the mouse, but also the guinea pig for cutaneous anaphylaxis. It also fixes complement. The specific immunoglobulin contents of the samples were between 3.63 and 7.52 m g / m l by ELISA, between 4000 and 64,000 by PL, between 8000 and

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235

64,000 by PCA in guinea pigs and between 2000 and 8000 by PCA in mice. Threshold values were: for PCA in mice between 0.91 and 2.38/tg/ml, for PCA in guinea pigs between 0.12 and 0.60/tg/ml, for PL between 0.12 and 0.91 /~g/ml. The results are presented in Fig. 2. As can be seen, good correlations were found with antibody contents and each biological activity in the various samples. The correlation coefficients were 0.83 ( P < 0.05) for PL, 0.83 ( P < 0.05) for PCA in mice and 0.68 for PCA in guinea pigs. The P value for the last number was slightly greater than 0.05.

IgG2b The sensitivity of ELISA for this immunoglobulin class was between 10 ng and 5 ~ g / m l . Six samples of ascites from mice injected with anti-DNP monoclonal antibody producing hybridoma NK-2b were examined. This antibody class sensitizes the mouse for PCA reactions and fixes complement. The specific immunoglobulin contents of the samples were between 2.19 and 4.50 m g / m l by ELISA, between 2000 and 8000 by PL and between 400 and 3200 by PCA in mice. Threshold values were for PCA reaction between 1.40 and 5.50 # g / m l and for PL between 0.56 and 1.60 t~g/ml. The results are presented in Fig. 3. Good correlations between antibody contents and each biological activity of the different samples were obtained. The correlation

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236

coefficients were 0.83 ( P < 0.05) for PCA in mice and 0.75 for PL. The P value for the second number was slightly greater than 0.05.

IgE The sensitivity of ELISA for IgE was between 1 ng and 1 ~ g / m l . Samples from 6 ascites of mice injected i.p. with hybridoma B53, 142a or 141b. Twenty-one samples of sera from mice bearing hybridoma B53 or 142a and 2 samples of sera from immunized mice were examined. The specific IgE contents as determined by ELISA in the samples were between 3.00 and 850.00 ktg/ml and between 200 and 320,000 by PCA in rats. The threshold values for PCA reaction in rats were between 2.70 and 26.50 n g / m l . The results are presented in Fig. 4 and, as can be seen, good correlation was obtained with the protein amounts as determined by ELISA and rat PCA activities. The correlation coefficient was 0.81 ( P < 0.01). Table I summarizes the data concerning the sensitivity of ELISA with the different isotypes, the minimum and maximum antibody contents of the samples as determined by ELISA, the minimum and maximum antibody titers as determined by PCA and PL and the minimum antibody requirements for threshold reactions for PCA and for PL.

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SENSITIVITY OF ELISA, ANTIBODY CONTENT OF EXPERIMENTAL SAMPLES AND MINIMUM ANTIBODY REQUIREMENT FOR THRESHOLD REACTIONS OF PCA AND PL FROM MURINE IgG1, IgG2a, IgG2b AND IgE

TABLE I

238 Discussion

The sensitivity of ELISA for each immunoglobulin isotype with our reactants is between 1 and 10 n g / m l for the lower limit and 1 and 5 ~ g / m l for the upper limit. These limits are more than sufficient for quantitation of specific antibodies. Biological reactions such as PCA and PL (complement fixation) are very sensitive, but due to biological variations they are not very precise. When 2-fold dilutions are used, a difference less than 2 dilutions is not considered significant. In spite of these considerations it can be seen from the figures that in general there were good correlations between antibody contents as determined by ELISA and the titers as determined by the different biological reactions. There were 2 exceptions: PCA in guinea pigs with IgG2a and PL with IgG2b, which we will discuss below. Mice can be sensitized for PCA reactions even with short sensitization periods not only by IgG1, but also by IgE (unpublished observations) and also by IgG2a or IgG2b (Hirayama et al., 1982; Ovary, 1982). The amount of IgE, IgG2a a n d / o r IgG2b in our serum samples from immunized mice examined for IgG1 was negligible and was diluted out when IgG1 antibodies were examined by PCA in mice, which explains the good correlation between murine PCA and ELISA for IgG1. However, it is possible that in other samples the presence of other isotypes (if non-negligible amounts) could present a real obstacle for estimation of IgG1 content by PCA in mice. Only IgG2a can sensitize guinea pigs for PCA. It was expected that the correlation with the determinations by ELISA and PCA in guinea pigs would be better-than what we had found. PCA reactions have many variables and we think that this variability is the explanation that the correlation was not as good as expected. The estimations of IgG2a and IgG2b antibodies by complement fixation need special comment. As this biological activity is shared by several isotypes (IgM, IgG2a, IgG2b, IgG3, and some IgG1 (Ey et al., 1980; Hirayama et al., 1982; Ovary, 1982)), complement fixation is not a method of choice to estimate antibody content of any isotype in sera from immunized mice. Though complement fixation with monoclonal IgG2a and IgG2b had been expected to give very good correlations with antibody determinations by ELISA, it was found that with IgG2b the correlation was less good than expected. Here again, the variability of PL might be the explanation of the less perfect correlation observed. IgE estimation in rats was a widely used method for many years. Recently it was demonstrated that some monoclonal murine IgG1 antibodies might sensitize the rat for PCA reaction (Hirayama et al., 1982; Ovary, 1982). This fact was recently confirmed with some conventional antibodies when the sera were fractionated (Mota and Perini, 1983). However, with monoclonal IgG1 antibodies, about 10,000 times more was needed to sensitize the rat than the amount necessary from IgE (see Table I). We can conclude therefore that IgE estimation by PCA in rats may be safely used. The biological activities were correlated with class-specific antibody protein content determined by ELISA. Using these data, it is possible now to extrapolate

239 f r o m a certain p r o t e i n a m o u n t d e t e r m i n e d b y E L I S A to o b t a i n the c o r r e s p o n d i n g titer of the biological reaction. T h e s t u d y of the biological activity of the different m u r i n e isotypes is still i m p o r t a n t for the e l u c i d a t i o n of m a n y p r o b l e m s , a n d these c a n n o t b e investigated b y E L I S A . It must be e m p h a s i z e d , however, that m o r e than 1 i s o t y p e might c o n t r i b u t e to the s a m e biological reaction when sera from i m m u n i z e d mice are used. In these cases special m e t h o d s of a n t i b o d y p u r i f i c a t i o n s are necessary.

Acknowledgements T h e a u t h o r s wish to express their t h a n k s to Dr. N o r m a n K l i n m a n (Scripps Clinic a n d R e s e a r c h F o u n d a t i o n , L a Jolla, C A ) for the IgG1 ( N K - 1 ) - a n d I g G 2 b ( N K - 2 b ) p r o d u c i n g h y b r i d o m a s , to Dr. F u - T o n g Liu ( M e d i c a l Biology Institute, L a Jolla, C A ) for the a n t i - D N P IgE p r e p a r a t i o n , to Dr. F r e d K a r u s h ( U n i v e r s i t y of Pennsylvania, Philadelphia, PA) for the a n t i - D a n s y l m o n o c l o n a l I g M a n d to Mr. C s a b a de Szalay for his excellent technical help. This w o r k was s u p p o r t e d b y G r a n t s A I 03075 ( N I H ) , IM-346 ( A C S ) a n d N00014-83-k-0678 (Office of N a v a l Research). Dr. M a e k a w a is s u p p o r t e d b y a F e l l o w s h i p from the C a n c e r R e s e a r c h Institute, N e w York, NY.

References Bloch, K.J., F.M. Kourilsky, Z. Ovary and B. Benacerraf, 1963, J. Exp. Med. 117, 965. B~,ttcher, I., G. H~immerling and J.-F. Kapp, 1978, Nature (London) 275, 761. B~,ttcher, I., M. Ulrich, N. Hirayama and Z. Ovary, 1980, Int. Arch. Allergy Appl. Immunol. 61,248. Engvall, E. and P. Perlmann, 1971, Immunochemistry 8, 871. Eshhar, Z., M. Ofarim and T. Waks, 1980, J. lmmunoi. 124, 775. Ey, P.L., G.J. Russell-Jones and C.R. Jenkin, 1980, Mol. lmmunol. 17, 699. Hill, P.N. and F.-T. Liu, 1981, J. Immunol. Methods 45, 51. Hirayama, N., T. Hirano, G. K6hler, A. Kurata, K. Okumura and Z. Ovary, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 613. Kojima, S. and Z. Ovary, 1975, Cell. lmmunol. 15, 274. Liu, F.-T., J.W. Bohn, E.L. Ferry, H. Yamamoto, C.A. Molinaro, L.A. Sherman, N.R. Klinman and D.H. Katz, 1980, J. Immunol. 124, 2728. Mota, I. and A. Perini, 1983, Int. Arch. Allergy Appl. Immunol. 72, 199. Ovary, Z., 1964, Immunological Methods, CIOMS Symposium, ed. J.F. Ackroyd (Blackwell Scientific Publications, Oxford) p. 259. Ovary, Z., 1982, Int. Arch. Allergy Appl. Immunol. 69, 385. Ovary, Z., S.S. Caiazza and S. Kojima, 1975, Int. Arch. Allergy Appl. Immunol. 48, 16. Rudolph, A.K., P.D. Burrows and M.R. Wabl, 1981, Eur. J. Immunol. 11, 527. Snedecor, W.G. and G.W. Cochran, 1967, Statistical Method (The Iowa State University Press, Ames, IO) p. 172. Stein, K.E., C. Kanellopoulos-Langevin, D.I. Cohen and J.K. Inman, 1980, J. Immunol. Methods 37, 83.