Detection of Immune Complexes Using ¹²⁵I Goat Anti(human IgG) Monovalent (Fab') Antibody Fragments

Summary

Separation and identification of immune complexes in serum were based on sucrose gradient velocity sedimentation of samples labeled with radioactive monovaent antibody (Fab' fragments) to human immunoglobulin. In vitro complexes formed from tetanus toxoid and human antitoxoid (TATC) appeared in the gradient as a “radiopeak” sedimenting at lOS rather than the normal 75 position for human IgG (HulgG). These complexes were stable under the conditions of the gradient. They could be dissociated at low pH and separated by filtration into two fractions: one which sedimented at the 75 position of HuigG when centrifuged with ¹²⁵I Fab, and another which reformed TATC after incubation with fresh tetanusimmune globulin and centrifugation with 125I Fab. As little as 13 µg IgG could be detected easily by this technique; complexed IgG was quantified on the assumption that its interaction with 125I Fab was similar to that of free IgG. A similar 10–115 radiopeak appeared in samples of sera from 5 cancer patients and 1 patient with Weber-Christian disease. Similar peaks were not seen in samples of sera from 10 normal healthy controls. A study of serial serum samples from 1 cancer patient showed a 10-11S radiopeak in preoperation serum that was significantly reduced 1 week after removal of the tumor. In serum taken 1 month after surgery, a small peak reappeared; recurrent disease was found subsequently. This method may have potential for detecting and isolating tumor antigens and the corresponding antibody for diagnostic tests and basic immunologic studies.