Characterization of a major human antibody clonotype (1A) by monoclonal antibodies to combining siteassociated idiotopes

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Abstract
To identify major human antibody clonotypes with specificity to N‐acetyl‐D‐glucosamine (GlcNAc), affinity‐purified antibody preparations of different human individuals were analyzed by isoelectric focusing (IEF). A major clonotype (1A) was identified representing 7% of all anti‐GlcNAc antibodies of one donor (no. 371). Anti‐GlcNAc antibodies of donor 371 were used as immunogen to prepare monoclonal anti‐idiotopic antibodies (mAb). Two anti‐idiotopic mAb were specific for the major clonotype 1A, as shown by blotting of the anti‐GlcNAc antibodies after IEF in thin‐layer agarose to nitrocellulose filters and staining with either iodinated antigen or iodinated anti‐idiotopic mAb. The two anti‐idiotopic mAb 13F15 and 10F59 were further analyzed with respect to the antigenic determinant which they recognize. Both were directed to combining site‐related determinants as shown by inhibition studies. Although 13F15 has a 500‐fold higher binding capacity (relative affinity) equal amounts do not inhibit more than 50% of 10F59 binding, suggesting that the two mAb detect two closely related, but not identical, idiotopes in the antigen combining site of clone 1A. Although clonotype 1A is a unique antibody exclusively found in IEF of donor 371, the idiotope 1A.1, defined by mAb 13F15, is a recurrent determinant detectable on different anti‐GlcNAc spectrotypes in various human sera.

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