Radioimmunoassays for the measurement of the A and B chains of insulin were developed using antisera against S-sulfonated, oxidized and reduced derivatives of the chains. Antisera against S-sulfonated A chain were found to react with S-sulfonated A chain, but not with the oxidized or reduced A chain whereas antisera against the reduced or oxidized A chains reacted with all three (S-sulfonated, oxidized and reduced) A chain derivatives. Antisera against all three B chain derivatives reacted equally well with B chain preparations whether S-sulfonated, oxidized or reduced. Because of greater stability radioiodinated oxidized chain -preparations were employed in the assay rather than radioiodinated S-sulfonated chains.
Although the A and B chain immunoassays were specific for one or more antigenic sites on the A and B chain respectively, they were not specific for the chains alone since insulin, proinsulin, and convertible and nonconvertible fractions of proinsulin cross-reacted in both assays, especially in the B chain assay.
Plasma interfered in both assays in a nonspecific and nonuniform fashion. With this consideration, no evidence has been found that the chains of insulin were present in the: (1) plasma of fasted man, baboon and pig, (2) plasma of man and pig after oral glucose, (3) plasma of baboon and pig after intravenous insulin, or (4) perfusate of isolated rat liver after insulin administration.