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A highly sensitive enzyme-linked immunosorbent assay for idiotype-bearing antibodies
Journal oflrnmunologicalMethods, 69 (1984) 51-59
51
Elsevier JIM03033
A Highly Sensitive Enzyme-Linked Immunosorbent Assay for Idiotype-Bearing Antibodies Gabriella Di Felice, Carlo Pini, G i n o Doria 1 and Luciano Adorini 1 Laboratory of Immunology, Istituto Superiore di Sanitgt, and i ENEA- Euratom lmmunogenetics Group, Laboratory of Pathology, Centro Rieerche Energetiche Casaeeia, Rome, Italy
(Received 17 August 1983, accepted 16 December 1983)
A sensitive and specific immunoenzyme assay (EL1SA) for quantitation of total and cross-reactive idiotype-bearing (CRI) anti-ABA antibodies is described. Total anti-ABA antibodies are directly assessed in ABA-BGG coated polyvinyl wells with enzyme-labelled rabbit anti-mouse immunoglobulins. By interpolation on a standard curve absorbance values give the concentration of anti-ABA antibodies with a sensitivity of 30 ng/ml, CRI + antibodies are quantitated by inhibition of enzyme-labelled monoclonal CRI + antibody binding to solid-phase coated rabbit anti-CRI immunoglobulins. The concentration of CRI + antibodies, evaluated by interpolation on a standard inhibition curve, can be measured at the level of 10 ng/ml. This highly sensitive, rapid, specific and reproducible assay is easily used, with minor modifications, to detect specific antibodies in any idiotype system. Key words: antibody idiotypes - EL1SA - cross-reactive idiotypes - azobenzene arsonate
Introduction Analysis of idiotypes, the antigenic structures associated with the variable regions of specific antibodies, has been useful, it has been instrumental in probing the genetics of antibody diversity, the expression of the antibody repertoire during ontogeny, and the idiotypic network and its immunoregulatory activities (Greene et al., 1982). A well characterized idiotypic system is that associated to antibodies specific for the hapten azobenzene arsonate (ABA) induced in strain A and A L / N mice (Kuettner et al., 1972). All mice of these strains, when immunized with ABA-protein conjugates produce anti-ABA antibodies, 20-70% of which bear a serologically defined major idiotypic marker, the cross-reactive idiotype (CRI). A second idiotypic marker (minor CRI), present on 5-10% of anti-ABA antibodies induced in strain A and A L / N mice, has also been defined (Gill-Pazaris et al., 1981). Several methods have been described to detect idiotype bearing antibodies, either by direct binding or binding inhibition assays (Ju et al., 1977; Owen and Nisonoff, 1978; Gill-Pazaris et al., 1979; Marshak-Rothstein et al., 1980; Hirai et al., 1981; 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
52 Margolies et al., 1981; Walker and Morahan, 1981), all employing iodinated reagents. A recent study makes use of immunoenzymatic assays to detect anti-ABA and CRI + antibodies but does not evaluate the many variables involved (Hornbeck and Lewis, 1983). We report the development of a quantitative enzyme immunoassay which, besides avoiding potential radiation hazard, allows rapid semiautomated total anti-ABA and CRI + antibody determination with high sensitivity and reproducibility. This method, with minor modification, may be used to detect specific antibodies in any idiotypic system.
Materials and Methods
Induction of anti-ABA antibodies Three-month-old A / J mice were immunized intraperitoneally with 100 /~g of ABAz000-keyhole limpet haemocyanin (KLH) in CFA. ABA-protein conjugates were prepared as previously described (Adorini et al., 1981). Mice were bled weekly from the retroorbital plexus and all sera were stored at - 2 0 ° C and individually tested.
Quantitation of total anti-ABA antibodies by ELISA (a) Plate coating. PVC plates (Falcon 3912) were coated overnight at 4°C with 0.2 ml/well of a solution containing different concentrations (2.5-100 #g/ml) of ABA7s-BGG or ABA64-OVA dissolved in 0.05 M carbonate buffer, pH 9.6, 0.02% NaN 3. (b) Sample incubation. After washing the plates 3 times in PBS containing 0.05% Tween 20 (Merck, Darmstadt) and 0.02% sodium azide (PBS/Tween/NaN3), sera diluted in the same buffer were added (0.2 ml/well) in duplicate and plates incubated for 2 h at room temperature (20°C). Normal A / J serum and PBS/Tween/NaN 3 were used as negative controls. Monoclonal anti-ABA antibodies at known concentration were used as reference standard. All samples and controls were also added to a plate previously incubated overnight with PBS/Tween/NaN 3 instead of antigen solution. After incubation, plates were washed as above. (c) Conjugate incubation. Rabbit anti-mouse immunoglobulins (Behring) were conjugated to alkaline phosphatase (Sigma, St. Louis, MO) according to Engvall and Perlmann (1972). The conjugate was added (0.2 ml/weU) diluted 1:300 in PBS/Tween/NaN 3 as determined by checkboard titration, and plates were incubated overnight at 4°C. (d) Substrate incubation. After the conjugate incubation, plates were washed as above and incubated with 0.2 lad/well of 1 mg/ml paranitrophenyl phosphate (Sigma), in 0.1 M carbonate buffer, pH 9.8, 10 -3 M MgCI 2. The reaction was stopped after 30 min by adding 50 #l/well of 2 M NaOH. Absorbance at 405 nm was read by Dynatech automatic 96-well reader.
53
(e) Quantitative anti-ABA antibodies evaluation. Absorbance values of P B S / T w e e n / N a N 3 coated plates were subtracted from absorbance values of antigen coated plates and the antibody concentration in serum samples was estimated by interpolation on a standard curve obtained with different concentrations (0.035-1.25 t~g/ml) of monoclonal anti-ABA antibody 36-65 (gift of Prof. M. Gefter, M.I.T., Cambridge, MA) (Marshak-Rothstein et al., 1980). The final concentration of anti-ABA antibodies was weighted by the dilution factor selected for each sample. Quantitation of CR1 ÷ anti-ABA antibodies by ELISA inhibition (a) Plate coating. PVC plates (Falcon 3912) were coated overnight at 4°C with 0.2 ml/well of 2.5 ~tg/ml of rabbit anti-CRI antibodies in 0.05 M carbonate buffer, pH 9.6, 0.02% NaN 3, (b) Sample incubation. After 3 washings with P B S / T w e e n / N a N 3 , serum samples appropriately diluted were added (0.2 ml/well). Two different incubation times were tested: 2 h at room temperature or overnight at 4°C. Normal A / J serum and P B S / T w e e n / N a N 3 were used as negative controls. Monoclonal anti-ABA CRI ÷ antibody 36-65 at known concentration was used as reference standard. After incubation, plates were washed as above. (c) Conjugate incubation. 0.2 ml/well alkaline phosphatase labelled monoclonal anti-ABA CRI + antibodies 36-65 diluted 1:200 in P B S / T w e e n / N a N 3 were added and incubated either for 2 h at room temperature or overnight at 4°C. (d) Quantitative evaluation of CRI ÷ antibodies. Mean optical densities obtained for each concentration of the standard (CRI ÷ monoclonal antibody 36-65) were divided by the mean optical densities obtained when no inhibitor was added (wells filled with P B S / T w e e n / N a N 3 in step (b). For both reference standard and serum samples, the percentage inhibition increased as a sigmoid function of the amount of inhibitor added. Quantitation of CRI ÷ antibodies was obtained by interpolation on the probit transformation of the sigmoid curve.
Preparation of rabbit anti-CRI antibodies A rabbit was immunized in the footpads with 2 mg of monoclonal anti-ABA CRI ÷ antibody 36-65 emulsified in CFA. After 1 month, the rabbit received a second injection of monoclonal antibody 36-65 in IFA and after 3 months a third one intramuscularly. Forty days later the rabbit was bled, and the serum absorbed on immobilized normal A / J immunoglobulins to remove anti-isotypic and anti-allotypic antibodies. The A / J immunoglobulin column was prepared by coupling to Sepharose 4B the 50% ammonium sulphate precipitate from normal A / J ascitic fluid induced according to Tung et al. (1976). After each application, the A / J column was eluted with 3.5 M MgC12 and re-equilibrated in PBS. The absorbed rabbit antiserum was then applied to a Protein A-Sepharose column (Pharmacia, Uppsala) to separate immunoglobulins from other serum components. After this treatment the protein A immunoadsorbent eluate was concentrated, dialyzed against PBS and tested for anti-idiotypic specificity.
54
Results
Determination of anti-ABA antibodies To estimate the total anti-ABA antibody concentration in A / J mice by ELISA, the ability of 2 ABA-protein conjugates in coating PVC plates was evaluated. As shown in Fig. 1, binding of alkaline phosphatase-labelled anti-ABA antibody 36-65 was higher on plates coated with ABA-BGG than on plates coated with the same concentration (2.5-100 /Lg/ml) of ABA-OVA. No binding could be demonstrated on plates coated with BGG (data not shown). Coating the plates with a superoptimal antigen concentration resulted in lower binding capacity, as already demonstrated for other ELISA systems (Engvall and Perlmann, 1972). ABA-BGG was selected as solid-phase antigen in subsequent experiments. To quantitate the anti-ABA antibody concentration, the ELISA was calibrated with monoclonal antibody 36-65. As shown in Fig. 2, the dose-response curve was linear in the 1.25-0.035 /~g/ml antibody concentration range which was therefore considered the optimal range for this assay. Determination of CRI + antibodies (a) Specificity of rabbit anti-CRI antibodies. To evaluate the specificity of the
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Fig. 1. Binding of alkaline phosphatase-labelled monoclonal anti-ABA antibody 36-65 to polyvinyl wells coated with different concentrations of ABA-BGG (zx) or ABA-OVA (O).
55
rabbit anti-CRI antibodies they were tested by ELISA inhibition assay before and after adsorption on A / J immunoglobulins (Fig. 3). Inhibition experiments were carried out with either monoclonal anti-CRI (gift of Prof. M. Gefter) (MarshakRothstein, 1981) or rabbit anti-CRI antibodies, both alkaline phosphatase-labelled. It is evident that absorbed anti-CRI antibodies selectively inhibit the binding of the enzyme-labelled monoclonal anti-CRI, whereas no interaction between CRI ÷ antibody coated on the solid phase and the enzyme-labelled rabbit anti-mouse immunoglobulins was observed. These rabbit anti-CRI antibodies were therefore CRI-specific and after purification by Protein A-Sepharose affinity chromatography they were absorbed on the solid-phase to quantitate CRI ÷ antibodies.
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Fig. 2. Titration of affinity purified monoclonal anti-ABA antibody 36-65 on A B A - B G G coated polyvinyl wells. The reaction was developed with enzyme-labelled rabbit anti-mouse immunoglobulins.
56
(b) Quantitation of CR1 + anti-ABA antibodies. Fig. 4 (left panel) demonstrates that the interaction between rabbit anti-CRI and enzyme-labelled monoclonal CRI ÷ antibody 36-65 is inhibited by increasing concentrations of unlabelled monoclonal CRI ÷ antibodies. When the sigmoid curve obtained from inhibition data is analyzed by probit transformation a linear relation is obtained between amount of inhibitor and probit values (Fig. 4, right panel). Two different incubation conditions for the inhibitor and the alkaline phosphatase-labelled CRI were compared: in the first, both inhibitor and conjugate were incubated at room temperature for 2 h; in the second, both inhibitor and conjugate were incubated at 4°C overnight (data not shown). Satisfactory results (Fig. 4) were obtained with the first incubation condition. The data indicate that, under the conditions used, as little as 10 n g / m l of CRI ÷ antibodies may be detected. The primary anti-ABA and CRI ÷ antibody response was evaluated by quantitative ELISA in A / J mice injected with ABA-KLH. Results in Fig. 5 show that both anti-ABA and CRI ÷ antibodies are detectable 7 days after immunization. The anti-ABA antibody response reaches a plateau level 3 weeks after priming, whereas the proportion of CRI ÷ antibodies seems to increase up to the last bleeding, 38 days
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Fig. 3. Inhibition by unabsorbed rabbit anti-CRI antibody of binding of enzyme-labelled monoclonal anti-CRl (O e ) and of enzyme-labelled rabbit anti-mouse immunoglobulins (A . . . . . . A) to CRIcoated plates; and inhibition by normal A / J immunoglobulin-absorbed rabbit anti-CRl antibody of binding of enzyme-labelled monoclonal anti-CRl ( O C)) and of enzyme-labelled rabbit anti-mouse immunoglobulins (zx. . . . . . zx) to CRl-coated plates. Solid line represents binding of enzyme-labelled
monoclonal anti-CRI to CRI and broken line represents binding of enzyme-labelled rabbit anti-mouse immunoglobulins to CRI.
57
after immunization. The average maximal anti-ABA antibody concentration attained is 7 mg/ml; 40% of this response is represented by CRI ÷ antibodies. Discussion ABA-specific and CRI-bearing antibodies have been quantitated by several authors (Ju et al., 1977; Owen and Nisonoff, 1978; Gill-Pazaris et al., 1979; Marshak-Rothstein et al., 1980; Hirai et al., 1981; Margolies et al., 1981; Walker and Morahan, 1981) with radioimmunological methods. To detect anti-ABA antibodies, an indirect binding assay is usually performed in tubes or in 96-well plates coated with ABA protein, and binding is revealed by 125I-labelled rabbit anti-mouse immunoglobulins antibody. This procedure is used to quantitate total anti-ABA antibodies by testing simultaneously different concentrations of anti-ABA affinity purified antibody and using the standard curve obtained to evaluate by interpolation the antibody concentration. This assay is able to detect about 100 ng/ml of anti-ABA antibody (Ju et al., 1977). To evaluate CRI ÷ antibodies, the interaction between affinity purified rabbit anti-CRI and ~25I-labelled anti-ABA CRI ÷ antibody is inhibited by the CRI present
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Fig. 4. Quantitation of CRI ÷ anti-ABA antibodies. Plates were coated with 0.5 /~g/well of rabbit anti-CRI and binding of enzyme-labelled CRI was inhibited by different concentrations of CRI + monoclonal antibody 36-65. Standard inhibition curve (left panel) and probit transformation (fight panel) are shown. Density values of non-inhibited controls were in the range of 0.8-1.2 optical densities.
58
in the serum sample. A standard inhibition curve is obtained by affinity purified CRI ÷ antibodies, and the CRI concentration in the sample is evaluated by interpolation. Results are usually expressed in terms of the inhibitor dilution giving 50% inhibition. The sensitivity of this assay has been estimated, by the use of a highly inhibitory monoclonal antibody in the standard radioimmunoassay for CRI, to be about 140 n g / m l (Barrett and Nisonoff, 1982). In the sensitive and specific assay for quantitation of total and CRI ÷ anti-ABA antibodies described here 125I-labelled antibodies are substituted with alkaline phosphatase-labelled reagents.To determine the concentration of anti-ABA antibodies, antigen (ABA-BGG) was adsorbed onto the solid phase and mouse sera and enzyme-labelled rabbit anti-mouse antibodies sequentially added. The absorbance values give, by interpolation on a standard curve, the concentration of total anti-ABA antibodies, which can be detected at the 30 n g / m l level. Quantitative evaluation of CRI ÷ antibodies was based on an inhibition assay in which a highly specific rabbit anti-CRI antibody was adsorbed on the plates and revealed by addition of enzyme-labelled CRI ÷ antibody. This reaction was inhibited by CRI 10
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Fig. 5. Time-response curve of anti-ABA and CRI + antibodies induced in A / J mice immunized i.p. with 100 #g ABA-KLH-CFA. Mice (5 mice at each time interval) were bled weekly and serum samples tested for total anti-ABA ( O O) and CRI + (A. . . . . . zx) antibody concentration. Normal sera from A//J mice were simultaneously tested for total anti-ABA ( ) and CRI +( . . . . . . ) antibodies. Results are expressed as arithmetic means with standard errors.
59
present in the serum sample. The concentration of CRI + antibodies in the sample was quantitated by interpolation on a standard inhibition curve obtained with monoclonal CRI + antibody as inhibitor. This immunoenzymatic assay detects CRI bearing antibodies at a concentration as low as 10 ng/ml. We have used this enzyme-linked assay to detect both total anti-ABA and CRI ÷ antibodies in sera from A / J mice injected with a single dose of ABA-KLH. Seven days after priming, CRI bearing antibodies could be measured although no anti-ABA antibodies were detected. This could be explained by the initial relatively low affinity of anti-ABA antibodies not allowing stable binding of antibodies to the solid-phase antigen. In contrast, the high affinity of the purified rabbit anti-CRI antibody adsorbed on the plate permits stable binding of CRI + antibodies. From day 14 after priming an increase in both total anti-ABA and CRI ÷ bearing antibodies was observed; the antibody production observed in our experiments was in the upper range determined by conventional radioimmunoassay. From the data presented it is evident that the use of an immunoenzymatic procedure has definite advantages as compared with classical radioimmunoassay, including higher sensitivity, the possibility of complete automation and elimination of radiolabelled reagents with short half-life and potential danger to the operator.
Acknowledgements The authors thank Prof. M. Gefter for the gift of monoclonal antibodies. This work was supported by Istituto Pasteur-Fondazione Cenci Bolognetti.
References Adorini, L., C. Pini, G. D'Agostaro, G. Di Felice, C. Mancini, L.V. Pozzi, S. Vietri and G. Doria, 1981, J. Immunol. 127, 1018. Barrett, M.C. and A. Nisonoff, 1982, Eur. J. Immunol. 12, 977. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109, 129. Gill-Pazaris, L.A., A.R. Brown and A. Nisonoff, 1979, Ann. Immunol. 130 C, 199. Gill-Pazaris, L.A., E. Lamoyi, A.R. Brown and A. Nisonoff, 1981, J. Immunol. 126, 75. Greene, M.I., M.J. Nelles, M.S. Sy and A. Nisonoff, 1982, Adv. Immunol. 32, 253. Hirai, Y., E. Lamoyi, Y. Dohi and A. Nisonoff, 1981, J. Immunol. 126, 71. Hornbeck, P.V. and G.K. Lewis, 1983, J. Exp. Med. 157, 1116. Ju, S.T., A. Gray and A. Nisonoff, 1977, J. Exp. Med. 145, 540. Kuettner, M.G., A. Wang and A. Nisonoff, 1972, J. Exp. Med. 135, 579. Margolies, M.N., A. Marshak-Rothstein and M.L. Gefter, 1981, Mol. Immunol. 18, 1065. Marshak-Rothstein, A., M. Siekevitz, M.N. Margolies, M. Mudgett-Hunter and M.L. Gefter, 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 1120. Marshak-Rothstein, A., M.N. Margolies, J.D. Benedetto and M.L. Gefter, 1981, Eur. J. Immunol. 11,565. Owen, F.L. and A. Nisonoff, 1978, J. Exp. Med. 148, 182. Tung, A., S.T. Ju, S. Sato and A. Nisonoff, 1976, J. Immunol. 116, 676. Walker, I.D. and G. Morahan, 1981, Stand. J. Immunol. 13, 433.
51
Elsevier JIM03033
A Highly Sensitive Enzyme-Linked Immunosorbent Assay for Idiotype-Bearing Antibodies Gabriella Di Felice, Carlo Pini, G i n o Doria 1 and Luciano Adorini 1 Laboratory of Immunology, Istituto Superiore di Sanitgt, and i ENEA- Euratom lmmunogenetics Group, Laboratory of Pathology, Centro Rieerche Energetiche Casaeeia, Rome, Italy
(Received 17 August 1983, accepted 16 December 1983)
A sensitive and specific immunoenzyme assay (EL1SA) for quantitation of total and cross-reactive idiotype-bearing (CRI) anti-ABA antibodies is described. Total anti-ABA antibodies are directly assessed in ABA-BGG coated polyvinyl wells with enzyme-labelled rabbit anti-mouse immunoglobulins. By interpolation on a standard curve absorbance values give the concentration of anti-ABA antibodies with a sensitivity of 30 ng/ml, CRI + antibodies are quantitated by inhibition of enzyme-labelled monoclonal CRI + antibody binding to solid-phase coated rabbit anti-CRI immunoglobulins. The concentration of CRI + antibodies, evaluated by interpolation on a standard inhibition curve, can be measured at the level of 10 ng/ml. This highly sensitive, rapid, specific and reproducible assay is easily used, with minor modifications, to detect specific antibodies in any idiotype system. Key words: antibody idiotypes - EL1SA - cross-reactive idiotypes - azobenzene arsonate
Introduction Analysis of idiotypes, the antigenic structures associated with the variable regions of specific antibodies, has been useful, it has been instrumental in probing the genetics of antibody diversity, the expression of the antibody repertoire during ontogeny, and the idiotypic network and its immunoregulatory activities (Greene et al., 1982). A well characterized idiotypic system is that associated to antibodies specific for the hapten azobenzene arsonate (ABA) induced in strain A and A L / N mice (Kuettner et al., 1972). All mice of these strains, when immunized with ABA-protein conjugates produce anti-ABA antibodies, 20-70% of which bear a serologically defined major idiotypic marker, the cross-reactive idiotype (CRI). A second idiotypic marker (minor CRI), present on 5-10% of anti-ABA antibodies induced in strain A and A L / N mice, has also been defined (Gill-Pazaris et al., 1981). Several methods have been described to detect idiotype bearing antibodies, either by direct binding or binding inhibition assays (Ju et al., 1977; Owen and Nisonoff, 1978; Gill-Pazaris et al., 1979; Marshak-Rothstein et al., 1980; Hirai et al., 1981; 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
52 Margolies et al., 1981; Walker and Morahan, 1981), all employing iodinated reagents. A recent study makes use of immunoenzymatic assays to detect anti-ABA and CRI + antibodies but does not evaluate the many variables involved (Hornbeck and Lewis, 1983). We report the development of a quantitative enzyme immunoassay which, besides avoiding potential radiation hazard, allows rapid semiautomated total anti-ABA and CRI + antibody determination with high sensitivity and reproducibility. This method, with minor modification, may be used to detect specific antibodies in any idiotypic system.
Materials and Methods
Induction of anti-ABA antibodies Three-month-old A / J mice were immunized intraperitoneally with 100 /~g of ABAz000-keyhole limpet haemocyanin (KLH) in CFA. ABA-protein conjugates were prepared as previously described (Adorini et al., 1981). Mice were bled weekly from the retroorbital plexus and all sera were stored at - 2 0 ° C and individually tested.
Quantitation of total anti-ABA antibodies by ELISA (a) Plate coating. PVC plates (Falcon 3912) were coated overnight at 4°C with 0.2 ml/well of a solution containing different concentrations (2.5-100 #g/ml) of ABA7s-BGG or ABA64-OVA dissolved in 0.05 M carbonate buffer, pH 9.6, 0.02% NaN 3. (b) Sample incubation. After washing the plates 3 times in PBS containing 0.05% Tween 20 (Merck, Darmstadt) and 0.02% sodium azide (PBS/Tween/NaN3), sera diluted in the same buffer were added (0.2 ml/well) in duplicate and plates incubated for 2 h at room temperature (20°C). Normal A / J serum and PBS/Tween/NaN 3 were used as negative controls. Monoclonal anti-ABA antibodies at known concentration were used as reference standard. All samples and controls were also added to a plate previously incubated overnight with PBS/Tween/NaN 3 instead of antigen solution. After incubation, plates were washed as above. (c) Conjugate incubation. Rabbit anti-mouse immunoglobulins (Behring) were conjugated to alkaline phosphatase (Sigma, St. Louis, MO) according to Engvall and Perlmann (1972). The conjugate was added (0.2 ml/weU) diluted 1:300 in PBS/Tween/NaN 3 as determined by checkboard titration, and plates were incubated overnight at 4°C. (d) Substrate incubation. After the conjugate incubation, plates were washed as above and incubated with 0.2 lad/well of 1 mg/ml paranitrophenyl phosphate (Sigma), in 0.1 M carbonate buffer, pH 9.8, 10 -3 M MgCI 2. The reaction was stopped after 30 min by adding 50 #l/well of 2 M NaOH. Absorbance at 405 nm was read by Dynatech automatic 96-well reader.
53
(e) Quantitative anti-ABA antibodies evaluation. Absorbance values of P B S / T w e e n / N a N 3 coated plates were subtracted from absorbance values of antigen coated plates and the antibody concentration in serum samples was estimated by interpolation on a standard curve obtained with different concentrations (0.035-1.25 t~g/ml) of monoclonal anti-ABA antibody 36-65 (gift of Prof. M. Gefter, M.I.T., Cambridge, MA) (Marshak-Rothstein et al., 1980). The final concentration of anti-ABA antibodies was weighted by the dilution factor selected for each sample. Quantitation of CR1 ÷ anti-ABA antibodies by ELISA inhibition (a) Plate coating. PVC plates (Falcon 3912) were coated overnight at 4°C with 0.2 ml/well of 2.5 ~tg/ml of rabbit anti-CRI antibodies in 0.05 M carbonate buffer, pH 9.6, 0.02% NaN 3, (b) Sample incubation. After 3 washings with P B S / T w e e n / N a N 3 , serum samples appropriately diluted were added (0.2 ml/well). Two different incubation times were tested: 2 h at room temperature or overnight at 4°C. Normal A / J serum and P B S / T w e e n / N a N 3 were used as negative controls. Monoclonal anti-ABA CRI ÷ antibody 36-65 at known concentration was used as reference standard. After incubation, plates were washed as above. (c) Conjugate incubation. 0.2 ml/well alkaline phosphatase labelled monoclonal anti-ABA CRI + antibodies 36-65 diluted 1:200 in P B S / T w e e n / N a N 3 were added and incubated either for 2 h at room temperature or overnight at 4°C. (d) Quantitative evaluation of CRI ÷ antibodies. Mean optical densities obtained for each concentration of the standard (CRI ÷ monoclonal antibody 36-65) were divided by the mean optical densities obtained when no inhibitor was added (wells filled with P B S / T w e e n / N a N 3 in step (b). For both reference standard and serum samples, the percentage inhibition increased as a sigmoid function of the amount of inhibitor added. Quantitation of CRI ÷ antibodies was obtained by interpolation on the probit transformation of the sigmoid curve.
Preparation of rabbit anti-CRI antibodies A rabbit was immunized in the footpads with 2 mg of monoclonal anti-ABA CRI ÷ antibody 36-65 emulsified in CFA. After 1 month, the rabbit received a second injection of monoclonal antibody 36-65 in IFA and after 3 months a third one intramuscularly. Forty days later the rabbit was bled, and the serum absorbed on immobilized normal A / J immunoglobulins to remove anti-isotypic and anti-allotypic antibodies. The A / J immunoglobulin column was prepared by coupling to Sepharose 4B the 50% ammonium sulphate precipitate from normal A / J ascitic fluid induced according to Tung et al. (1976). After each application, the A / J column was eluted with 3.5 M MgC12 and re-equilibrated in PBS. The absorbed rabbit antiserum was then applied to a Protein A-Sepharose column (Pharmacia, Uppsala) to separate immunoglobulins from other serum components. After this treatment the protein A immunoadsorbent eluate was concentrated, dialyzed against PBS and tested for anti-idiotypic specificity.
54
Results
Determination of anti-ABA antibodies To estimate the total anti-ABA antibody concentration in A / J mice by ELISA, the ability of 2 ABA-protein conjugates in coating PVC plates was evaluated. As shown in Fig. 1, binding of alkaline phosphatase-labelled anti-ABA antibody 36-65 was higher on plates coated with ABA-BGG than on plates coated with the same concentration (2.5-100 /Lg/ml) of ABA-OVA. No binding could be demonstrated on plates coated with BGG (data not shown). Coating the plates with a superoptimal antigen concentration resulted in lower binding capacity, as already demonstrated for other ELISA systems (Engvall and Perlmann, 1972). ABA-BGG was selected as solid-phase antigen in subsequent experiments. To quantitate the anti-ABA antibody concentration, the ELISA was calibrated with monoclonal antibody 36-65. As shown in Fig. 2, the dose-response curve was linear in the 1.25-0.035 /~g/ml antibody concentration range which was therefore considered the optimal range for this assay. Determination of CRI + antibodies (a) Specificity of rabbit anti-CRI antibodies. To evaluate the specificity of the
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Fig. 1. Binding of alkaline phosphatase-labelled monoclonal anti-ABA antibody 36-65 to polyvinyl wells coated with different concentrations of ABA-BGG (zx) or ABA-OVA (O).
55
rabbit anti-CRI antibodies they were tested by ELISA inhibition assay before and after adsorption on A / J immunoglobulins (Fig. 3). Inhibition experiments were carried out with either monoclonal anti-CRI (gift of Prof. M. Gefter) (MarshakRothstein, 1981) or rabbit anti-CRI antibodies, both alkaline phosphatase-labelled. It is evident that absorbed anti-CRI antibodies selectively inhibit the binding of the enzyme-labelled monoclonal anti-CRI, whereas no interaction between CRI ÷ antibody coated on the solid phase and the enzyme-labelled rabbit anti-mouse immunoglobulins was observed. These rabbit anti-CRI antibodies were therefore CRI-specific and after purification by Protein A-Sepharose affinity chromatography they were absorbed on the solid-phase to quantitate CRI ÷ antibodies.
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Fig. 2. Titration of affinity purified monoclonal anti-ABA antibody 36-65 on A B A - B G G coated polyvinyl wells. The reaction was developed with enzyme-labelled rabbit anti-mouse immunoglobulins.
56
(b) Quantitation of CR1 + anti-ABA antibodies. Fig. 4 (left panel) demonstrates that the interaction between rabbit anti-CRI and enzyme-labelled monoclonal CRI ÷ antibody 36-65 is inhibited by increasing concentrations of unlabelled monoclonal CRI ÷ antibodies. When the sigmoid curve obtained from inhibition data is analyzed by probit transformation a linear relation is obtained between amount of inhibitor and probit values (Fig. 4, right panel). Two different incubation conditions for the inhibitor and the alkaline phosphatase-labelled CRI were compared: in the first, both inhibitor and conjugate were incubated at room temperature for 2 h; in the second, both inhibitor and conjugate were incubated at 4°C overnight (data not shown). Satisfactory results (Fig. 4) were obtained with the first incubation condition. The data indicate that, under the conditions used, as little as 10 n g / m l of CRI ÷ antibodies may be detected. The primary anti-ABA and CRI ÷ antibody response was evaluated by quantitative ELISA in A / J mice injected with ABA-KLH. Results in Fig. 5 show that both anti-ABA and CRI ÷ antibodies are detectable 7 days after immunization. The anti-ABA antibody response reaches a plateau level 3 weeks after priming, whereas the proportion of CRI ÷ antibodies seems to increase up to the last bleeding, 38 days
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1,5 o 1,0
0,5 • A--
1
--
-
.&----
0 --
-.A--
2
LOG2 RABBIT ANTI-CRI
3
0 --
--
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4 DILUTION
Fig. 3. Inhibition by unabsorbed rabbit anti-CRI antibody of binding of enzyme-labelled monoclonal anti-CRl (O e ) and of enzyme-labelled rabbit anti-mouse immunoglobulins (A . . . . . . A) to CRIcoated plates; and inhibition by normal A / J immunoglobulin-absorbed rabbit anti-CRl antibody of binding of enzyme-labelled monoclonal anti-CRl ( O C)) and of enzyme-labelled rabbit anti-mouse immunoglobulins (zx. . . . . . zx) to CRl-coated plates. Solid line represents binding of enzyme-labelled
monoclonal anti-CRI to CRI and broken line represents binding of enzyme-labelled rabbit anti-mouse immunoglobulins to CRI.
57
after immunization. The average maximal anti-ABA antibody concentration attained is 7 mg/ml; 40% of this response is represented by CRI ÷ antibodies. Discussion ABA-specific and CRI-bearing antibodies have been quantitated by several authors (Ju et al., 1977; Owen and Nisonoff, 1978; Gill-Pazaris et al., 1979; Marshak-Rothstein et al., 1980; Hirai et al., 1981; Margolies et al., 1981; Walker and Morahan, 1981) with radioimmunological methods. To detect anti-ABA antibodies, an indirect binding assay is usually performed in tubes or in 96-well plates coated with ABA protein, and binding is revealed by 125I-labelled rabbit anti-mouse immunoglobulins antibody. This procedure is used to quantitate total anti-ABA antibodies by testing simultaneously different concentrations of anti-ABA affinity purified antibody and using the standard curve obtained to evaluate by interpolation the antibody concentration. This assay is able to detect about 100 ng/ml of anti-ABA antibody (Ju et al., 1977). To evaluate CRI ÷ antibodies, the interaction between affinity purified rabbit anti-CRI and ~25I-labelled anti-ABA CRI ÷ antibody is inhibited by the CRI present
100
J
O
13
/ []
'~
5
o-
50
,y..,[] I
i0
I
100 MONOCLONAI
I
t
I
I
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i0
i00
i000
ANTI-AB# 36-65 AN~EIBODY CONCENTRATION
(NG/ML)
Fig. 4. Quantitation of CRI ÷ anti-ABA antibodies. Plates were coated with 0.5 /~g/well of rabbit anti-CRI and binding of enzyme-labelled CRI was inhibited by different concentrations of CRI + monoclonal antibody 36-65. Standard inhibition curve (left panel) and probit transformation (fight panel) are shown. Density values of non-inhibited controls were in the range of 0.8-1.2 optical densities.
58
in the serum sample. A standard inhibition curve is obtained by affinity purified CRI ÷ antibodies, and the CRI concentration in the sample is evaluated by interpolation. Results are usually expressed in terms of the inhibitor dilution giving 50% inhibition. The sensitivity of this assay has been estimated, by the use of a highly inhibitory monoclonal antibody in the standard radioimmunoassay for CRI, to be about 140 n g / m l (Barrett and Nisonoff, 1982). In the sensitive and specific assay for quantitation of total and CRI ÷ anti-ABA antibodies described here 125I-labelled antibodies are substituted with alkaline phosphatase-labelled reagents.To determine the concentration of anti-ABA antibodies, antigen (ABA-BGG) was adsorbed onto the solid phase and mouse sera and enzyme-labelled rabbit anti-mouse antibodies sequentially added. The absorbance values give, by interpolation on a standard curve, the concentration of total anti-ABA antibodies, which can be detected at the 30 n g / m l level. Quantitative evaluation of CRI ÷ antibodies was based on an inhibition assay in which a highly specific rabbit anti-CRI antibody was adsorbed on the plates and revealed by addition of enzyme-labelled CRI ÷ antibody. This reaction was inhibited by CRI 10
/
0.i
- i// A/
0.01
r
I
I
I
I
I
7
14
21
28
35
42
DAYS AFTER IMr~UNIZATION
Fig. 5. Time-response curve of anti-ABA and CRI + antibodies induced in A / J mice immunized i.p. with 100 #g ABA-KLH-CFA. Mice (5 mice at each time interval) were bled weekly and serum samples tested for total anti-ABA ( O O) and CRI + (A. . . . . . zx) antibody concentration. Normal sera from A//J mice were simultaneously tested for total anti-ABA ( ) and CRI +( . . . . . . ) antibodies. Results are expressed as arithmetic means with standard errors.
59
present in the serum sample. The concentration of CRI + antibodies in the sample was quantitated by interpolation on a standard inhibition curve obtained with monoclonal CRI + antibody as inhibitor. This immunoenzymatic assay detects CRI bearing antibodies at a concentration as low as 10 ng/ml. We have used this enzyme-linked assay to detect both total anti-ABA and CRI ÷ antibodies in sera from A / J mice injected with a single dose of ABA-KLH. Seven days after priming, CRI bearing antibodies could be measured although no anti-ABA antibodies were detected. This could be explained by the initial relatively low affinity of anti-ABA antibodies not allowing stable binding of antibodies to the solid-phase antigen. In contrast, the high affinity of the purified rabbit anti-CRI antibody adsorbed on the plate permits stable binding of CRI + antibodies. From day 14 after priming an increase in both total anti-ABA and CRI ÷ bearing antibodies was observed; the antibody production observed in our experiments was in the upper range determined by conventional radioimmunoassay. From the data presented it is evident that the use of an immunoenzymatic procedure has definite advantages as compared with classical radioimmunoassay, including higher sensitivity, the possibility of complete automation and elimination of radiolabelled reagents with short half-life and potential danger to the operator.
Acknowledgements The authors thank Prof. M. Gefter for the gift of monoclonal antibodies. This work was supported by Istituto Pasteur-Fondazione Cenci Bolognetti.
References Adorini, L., C. Pini, G. D'Agostaro, G. Di Felice, C. Mancini, L.V. Pozzi, S. Vietri and G. Doria, 1981, J. Immunol. 127, 1018. Barrett, M.C. and A. Nisonoff, 1982, Eur. J. Immunol. 12, 977. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109, 129. Gill-Pazaris, L.A., A.R. Brown and A. Nisonoff, 1979, Ann. Immunol. 130 C, 199. Gill-Pazaris, L.A., E. Lamoyi, A.R. Brown and A. Nisonoff, 1981, J. Immunol. 126, 75. Greene, M.I., M.J. Nelles, M.S. Sy and A. Nisonoff, 1982, Adv. Immunol. 32, 253. Hirai, Y., E. Lamoyi, Y. Dohi and A. Nisonoff, 1981, J. Immunol. 126, 71. Hornbeck, P.V. and G.K. Lewis, 1983, J. Exp. Med. 157, 1116. Ju, S.T., A. Gray and A. Nisonoff, 1977, J. Exp. Med. 145, 540. Kuettner, M.G., A. Wang and A. Nisonoff, 1972, J. Exp. Med. 135, 579. Margolies, M.N., A. Marshak-Rothstein and M.L. Gefter, 1981, Mol. Immunol. 18, 1065. Marshak-Rothstein, A., M. Siekevitz, M.N. Margolies, M. Mudgett-Hunter and M.L. Gefter, 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 1120. Marshak-Rothstein, A., M.N. Margolies, J.D. Benedetto and M.L. Gefter, 1981, Eur. J. Immunol. 11,565. Owen, F.L. and A. Nisonoff, 1978, J. Exp. Med. 148, 182. Tung, A., S.T. Ju, S. Sato and A. Nisonoff, 1976, J. Immunol. 116, 676. Walker, I.D. and G. Morahan, 1981, Stand. J. Immunol. 13, 433.