Studies on the heterogeneity of anti-hapten antibodies by means of two-dimensional affinity electrophoresis

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Abstract
The molecular heterogeneity of rabbit anti-hapten antibodies has been investigated by two-dimensional affinity electrophoresis (2D-AEP). Anti-dansyl and anti-arsanilic diazo-antibodies were separated into several hundred IgG spots as in the case of anti-DNP antibodies. They were grouped into a number of monoclonal IgG families. At the beginning of immunization, IgG spots having low pI and low affinity were predominant but one or two weeks after immunization the IgG spots with high affinity and high pI increased. After the second or third immunization, the 2D-AEP patterns became stable and constant. Anti-arsanilic diazo and anti-DNP antibodies exhibited only weak cross-reactivity with other aromatic haptens. In contrast, anti-dansyl antibodies cross-reacted to a considerable degree with DNP-hapten. A few anti-dansyl IgG families which have cross-reactivity with DNP-hapten were separated. Their apparent dissociation constants to the haptens and their affinity ratios were calculated from their 2D-AEP patterns, according to the affinity theory.