Incremental combining site filling of anti-polypeptide antibodies

Abstract

Antibodies involed in the ‘calcium dependent’ reaction of (Glu 60Ala 30Tyr 10) n anti-(Glu 60Ala 30Tyr 10) n have been fractioned by sequential precipitation with the glutamyl containing cross reacting polypeptides (Glu) n , (Glu 90Ala 10) n , (Glu 60Ala 40) n , in that respective order, and finally antigen. Isoelectric focusing and hydrogen exchange experiments have been performed on the parent preparation and the fractionated subpopulations. Although it had been reported earlier that the preparation contained four isoelectric species, as determined in gel experiments, the liquid column studies on the parent preparation and the subpopulations indicate that as many as 25 antibodies which are involved in calcium dependent interactions are elicited by (Glu 60Ala 30Tyr 10) n . The immunochemical basis for this heterogeneity of response is considered. From the hydrogen exchange studies done at high levels of antigen excess, where it has been shown previously that combining site sensitive hydrogen are blocked from exchange upon interaction with antigen, the extent of interaction of the cross reacting polypeptides with the parent preparation and the subpopulations have been determined. For the subpopulation isolated with poly Glu the hydrogen exchange studies show that 55 per cent of the combining site sensitive hydrogens are affected by interaction with poly Glu, 85% with (Glu 90Ala 10) n , with (Glu 60Ala 40) n and 100 per cent with the immunizing antigen. This incremental combining site filling of this subpopulation by the cross reacting polypeptides correlates with the light transparency of the respective specific precitates. Thus, that formed with poly Glu is transparent while those formed with the other polypeptides have increasing opacity, becoming typically white when formed with the immunizing antigen. The exchange studies done on the subpopulation isolated by precipitation with (Glu 90Ala 10) n indicate that the combining sites of these antibodies are mostly directed against determinants containing Glu and Tyr. For the subpopulation which requires the immunizing antigen for precipitation, the exchange studies show that the combining sites of these antibodies are most likely directed against unique sequences of Glu, Ala and Tyr. By comparing quantitative cross precipitin reaction data on the parent preparation with data obtained by hydrogen exchange, it is found that no correlation exists between the amount of antibody a cross reacting polypeptide precipitates and the percentage of ligand sensitive combining site hydrogens with which it interacts.