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

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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.

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