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Anti-Hapten Antibodies of Restricted Heterogeneity: Studies on Binding Properties and Component Chains¹

From the Department of Biochemistry Research, Roswell Park Memorial Institute, ² Buffalo, New York 14203

Abstract

Antibodies against p-azobenzoate (Xp) from two rabbits that exhibited restricted heterogeneity were studied by isoelectric focusing of whole antibody and of light and heavy chains.³ One antibody (2709) showed two light chain bands and five heavy chain bands. Binding studies on 2709 showed two homogeneously binding components with a 100-fold difference in binding constant for p-iodobenzoate. The other antibody (2883) had about 15 components detectable by isoelectric focusing with three major light and five heavy chain bands. A sample of antibody 2883 was fractionated by sucrose gradient isoelectric focusing and seven of the fractions were further characterized. Binding studies with four haptens indicated that fractions A, B and C contained antibodies with similar binding properties and fractions F and G contained other antibodies with similar binding properties. Isoelectric focusing of mildly reduced and alkylated samples of the fractions showed that the antibodies in fractions A, B and C gave a light chain band not found in the other fractions and that the antibodies in F and G gave a large amount of the third light chain band. Although some of the fractions gave a single significant band on gel isoelectric focusing, each of them gave at least three heavy chain bands. This indicated that post-synthetic charge modifications are not necessarily symmetrical. Isoelectric focusing of mildly reduced and alkylated antibodies has much higher resolution than disc electrophoresis of either light or heavy chains.

Footnotes

¹ This work was supported in part by Grant No. AI-3962 from the National Institutes of Allergy and Infectious Diseases and in part by a grant from the General Research Support Fund of Roswell Park Memorial Institute.

³ Abbreviations used are: Xp, p-azobenzoate; BGG, bovine globulin; ovalb, ovalbumin; RSA, rabbit serum albumin; NRS, normal rabbit serum; K_rel, relative binding constant; K₀, hapten binding constant; a, heterogeneity index; and pI, the pH of the isoelectric point.

² A unit of the New York State Department of Health.