By Kumanogoh A., Kikutani H.
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The interaction of the multivalent macromolecular antigens may, for steric reasons, not result in all receptor sites being occupied. lSS Moreover, in precipitin inhibition studies, as the hapten displaces more and more antigen from antibody sites, the antigen itself will begin to contribute to the formation of the soluble complexes. When competitive binding radioimmunoassay or enzyme immunoassay are carried out with antisera, which almost always contain a heterogeneous spectrum of antibodies, the dilutions used are generally so high that one is working with only the fraction of antibodies of highest affinity.
Biophys. Acta 251, 427 (1971). 1r5 E. Engvall and P. Perlmann, J. Immunol. 109, 129 (1972). 17e D. R. Hoffman, J. Allergy Clin. lmmunol. 51, 303 (1973). 32 PRINCIPLES  AND METHODS (4000-8000 cpm) and allowed to come to equilibrium; the bound ligand is separated from the free ligand, and the proportion of free or of bound ligand is determined. For the system to be suitable, the receptor protein if added in sufficient quantity should precipitate 90-100% of the added ligand as determined by counting or enzyme assay.
A. Kabat, J. lmmunol. 108, 1232 (1972). l e o D. G. Streefkirk and C. P. J. Glaudemans, Biochemistry 16, 3760 (1977). 161 K. Takeo and S. Nakamura, Arch. Bioehem. Biophys. 153, 1 (1972). lnu V. Horesjf, M. Tich~, and J. Kocourek, Bioehirn. Biophys. Acta 499, 290 (1977). le~ S. Sugii, K. Takeo, and E. A. Kabat, J. lmmunol. 17,3, 1162 (1979). ~ V. Horesjf, M. Tich~, and J. Kocourek, Trends Bioehem. Sei. 4, 6 (1979).  PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS 31 probably be difficult to use affinity electrophoresis with heterogeneous mixtures of antibodies.