Abstract
Monoclonal antibodies (mAbs) of predetermined specificity were prepared by immunizing with a free (i.e., not conjugated to any carrier) synthetic peptide representing region 15–22 (site 1) of sperm whale myoglobin (SpMb). The cross-reactions of Mb variants with three mAbs were studied in order to determine whether such interactions are influenced by substitutions outsde the site. Finback whale Mb, which has no substitutions within region 15–22, showed lower cross-reactivity and relative binding affinity than the reference antigen, SpMb. Bottle-nose Atlantic dolphin myoglobin (BdMb) and badger myoglobin (BgMb), although they have identical substitutions within region 15–22 (Ala-15 to Gly and Val-21 to Leu), showed very different binding properties. The cross-reaction of BdMb was quite comparable to that of SpMb, while that of BgMb was much lower. Since the two proteins have identical structures in regions 15–22, the differences in their cross-reactivities are readily attributed to the effects of substitutions outside this region. Another pair of myoglobins, horse myoglobins (HsMb) and chicken myoglobin (ChMb), also have two identical substitutions (Ala-15 to Gly and Val-21 to Ile) within region 15–22, but possessed different cross-reactivity. The results indicate that the reaction of mAbs, whose specificity is precisely known and predetermined by the immunizing free peptide, can be markedly affected by substitutions outside the indicated binding region on the protein.
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Abaza, M. S. I., and Atassi, M. Z. (1992).J. Prot. Chem. 11, 433–444.
Andres, S. F., and Atassi, M. Z. (1970).Biochemistry 9, 2268–2275.
Arnheim, N. (1973). InThe Antigens (Sela, M., ed.), Academic Press, New York, pp. 377–416.
Arnon, R., and Maron, E. (1971).J. Mol. Biol. 61, 225–235.
Atassi, M. Z. (1967).Biochem. J. 102, 478–487.
Atassi, M. Z. (1970).Biochem. Biophys. Acta 221, 612–622.
Atassi, M. Z. (1975).Immunochemistry 12, 423–438.
Atassi, M. Z. (1978)Immunochemistry 15, 909–936.
Atassi, M. Z. (1980)Mol. Cell Biochem. 32, 21–44.
Atassi, M. Z., Habeeb, A. F. S. A., and Rydstedt, L. (1970).Biochim. Biophys. Acta 200, 184–187.
Atassi, M. Z., and Kazim, A. L. (1980).Biochem. J. 187, 163–172.
Atassi, M. Z., and Skalski, D. J. (1969).Immunochemistry 6, 25–34.
Atassi, M. Z., and Young, C. R. (1985).CRC Crit. Rev. Immunol. 5, 387–409.
Benjamin, D. C., Berzofsky, J. A., East, I. J., Gurd, F. R. N., Hannum, C., Leach, S. J., Margoliash, E., Michael, J. G., Miller, S. J., Todd, P. E., and Wilson, A. C. (1984).Annu. Rev. Immunol. 2, 67–101.
Berzofsky, J. A., and Berkower, I. (1989) InFundamental Immunology, 2nd ed. (Paul, W. E., ed.), Raven Press, New York, pp. 169–207.
Berzofsky, J. A., Buckenmeyer, G. K., Hicks, G., Gurd, F. R. N., Feldmann, R. J., and Minna, J. (1982).J. Biol. Chem. 257, 3189–3198.
Berzofsky, J. A., Buckenmeyer, G. K., Hicks, G., Killion, D. J., Berkower, I., Kohno, Y., Flanagan, M. A., Busch, M. R., Feldman, R. J. Minna, J., and Gurd, F. R. N. (1983). InProtein Conformation as Immunological Signal (Celada, F., Shumker, V., and Sercarz, E. E., eds.), Plenum Press, New York, pp. 165–180.
Berzofsky, J. A., Hicks, G., Fedorko, J., and Minna, J. (1980).J. Biol. Chem. 225, 11,188–11,191.
Dautrevaux, M., Boulanger, Y., Han, K.-K., and Biserte, G. (1969).Eur. J. Biochem. 11, 267–277.
Deconinck, M., Peiffers, S., Depretter, J. P., Paul, C., Schnek, A. G., and Leonis, J. (1975).Biochim. Biophys. Acta 386, 567–575.
DiMarchi, R. D., Wang, C.-C., Hemenway, J. B., and Gurd, F. R. N. (1978).Biochemistry 17, 1968–1970.
Dumur, V., Dautrevaux, M., and Han, K.-K. (1976).Biochim. Biophys. Acta 420, 376–386.
East, I. J., Todd, P. E., and Leach, S. J. (1980).Molecular Immunol. 17, 519–525.
East, I. J., Hurrel, J. G. R., Todd, P. E. E., and Leach, S. J. (1982).J. Biol. Chem. 257, 3199–3203.
Edmundson, A. B. (1965).Nature (London)205, 883–881.
Hannum, C. H., and Margoliash, E. (1985).J. Immunol. 135, 3303–3313.
Habeeb, A. F. S. A., and Atassi, M. Z. (1971).Biochim. Biophys. Acta 236, 131–141.
Jones, L. T., Castillo, O., and Lehmann, H. (1977).Biochim. Biophys. Acta 493, 460–464.
Kazim, A. L., and Atassi, M. Z. (1980).Biochem. J. 191, 673–680.
Koketsu, J., and Atassi, M. Z. (1974).Immunochemistry 11, 1–8.
Prager, E. M., Fainaru, M., Wilson, A. C., and Arnon, R. (1974).Immunochemistry 11, 153–156.
Romero-Herrera, A. E., and Lehmann, H. (1974).Biochim. Biophys. Acta 336, 318–323.
Schmitz, H. E., Atassi, H., and Atassi, M. Z. (1982).Mol. Immunol. 19, 1699–1702.
Schmitz, H. E., Atassi, H., and Atassi, M. Z. (1983a)Immunol. Commun. 12, 161–175.
Schmitz, H. E., Atassi, H., and Atassi, M. Z. (1983b)Mol. Immunol. 20, 719–726.
Takano, T. (1984).Methods and Applications in Crystallographic Computing (Hall, S. R., and Ashida, T., eds.), Clarendon Press, Oxford, pp. 262–272.
Tetaert, D., Han, K.-K., Plancot, M.-T., Dautrevaux, M., Ducastaing, S., Hombrados, I., and Neuzil, E. (1974).Biochim. Biophys. Acta 351, 317–324.
Todd, P. E. E., East, I. J., and Leach, S. J. (1982).Trends Biochem. Sci. 7, 212–216.
Twining, S. S., Lehmann, H., and Atassi, M. Z. (1980).Biochem. J. 191, 681–697.
Underwood, P. A. (1985).J. Immunol. Methods 85, 295–307.
van Regenmortel, M. H. V. (1988). InLaboratory Techniques in Biochemistry and Molecular Biology (Burdon, R. H., and van Knippenberg, P. H., eds.), Vol. 19, Elsevier Science Publishers, B.V. (Biomedical Division), pp. 1–39.
Watson, H. C. (1969). InProgress in Sterochemistry (Aylett, B. J., and Harris, M., eds.), Butterworth, London, pp. 299–333.
Young, C. R., and Atassi, M. Z. (1982).Immunol. Comm. 11, 9–16.
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Abaza, MS.I., Young, C.R. & Atassi, M.Z. Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 15–22 (antigenic site 1) of myoglobin. J Protein Chem 11, 445–454 (1992). https://doi.org/10.1007/BF01025021
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DOI: https://doi.org/10.1007/BF01025021