Spontaneous re-aggregation of IgM subunits and restoration of antibody activity after reduction and alkylation of rabbit anti-Forssman antibody.

Highly purified rabbit IgM anti-Forssman antibody, after reduction in dilute solution with 2-mercaptoethanol and alkylation with iodoacetamide, showed no diminution and often some increase in haemagglutination of sheep erythrocytes. The capacity to cause complement dependent lysis was, however, destroyed. It is shown that despite reduction and alkylation reaggregation of the subunits occurs to a variable extent producing non-covalently bound aggregates with S values ranging from 7 to more than 20. The aggregates can bind specifically to and agglutinate erythrocytes with greater effectiveness as their size increases. This spontaneous re-aggregation is not prevented by the presence of gelatin, but fails to occur when the IgM antibody is reduced in the presence of a variety of other proteins. The effect of extraneous proteins is evident only when they are present at the time of reduction or are added within 10–15 minutes. It is suggested that following rupture of the interchain disulphide bonds, the IgM molecule can undergo a complex, time-dependent rearrangement into new stable forms which retain combining site activity but do not involve covalent bonds.