Paul H. Ehrlich

Quantitative explanation for increased affinity shown by mixtures of monoclonal antibodies: importance of a circular complex.

Mixtures of some but not all monoclonal antibodies which bind to separate epitopes on human chorionic gonadotropin (hCG) show an increased affinity for the hormone. To find an explanation for the increase in affinity, we developed a mathematical model which predicts the quantities of intermediates formed when pairs of IgG1 mouse monoclonal antibodies having affinities of approximately 10(8) M-1 for hCG are mixed with the hormone. At low antibody concentrations (i.e. less than 1 nM or 0.15 micrograms/ml) analysis of possible antibody-hormone combinations, including linear and circular chains composed of less than 12 molecules of antibody and 12 molecules of hCG, suggests the increase in affinity is due to formation of a circular complex containing two molecules of antibody and two of hCG. Further, the model predicts that the circular complex will be the major species formed at antibody-antigen equivalence. This prediction is supported by experimental observations on the molecular weight of a new complex formed in the presence of hCG and the mixture of the monoclonal antibodies. In addition, based on experimental values of binding constants for individual antibodies to hCG, the model correctly quantifies the loss in complex observed in the presence of excess hCG antigen. At high antibody concentrations (i.e. greater than 10 nM or 1.5 micrograms/ml) the formation of linear chains of antibody hCG pairs becomes appreciable and contributes to the increase in apparent affinity of the mixture for hCG. These results suggest that the observed affinity of complex mixtures of antibody for antigens containing multiple epitopes calculated from Scatchard plots may not be related to the affinity or avidity of any of the antibody species for a given epitope.

Size isomers of testosterone-estradiol-binding globulin exist in the plasma of individual men and women

We isolated testosterone-estradiol-binding globulin TeBG rapidly and in high yield from pooled pregnancy plasma. It showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Both bands stained with three different monoclonal antibodies to TeBG, thus demonstrating their immunological similarity. Freshly drawn, individual sera, from men, women, and pregnant patients were submitted to microaffinity chromatography, a procedure which partially purifies TeBG in approximately 4 hr. The partially purified plasma was submitted to SDS PAGE, followed by immunoblotting. The blotted TeBG exhibited the same two bands seen in the isolated, purified protein. The size heterogeneity observed in TeBG purified to: proteolysis occurring during isolation; a peculiarity of pregnancy plasma; or heterogeneity attendant upon the use of pooled plasma for isolation.

Characterization of a human anti-hCG antiserum: A proposed standard for laboratories involved with the development of hCG vaccines

An antiserum (PC-81-1) was obtained from a man who developed antibodies against hCG during treatment for hypogonadism. The antiserum was unique in that its affinity for hCG was high (greater than 10(-10) M(-1] and its cross-reaction with hLH and the hCG-subunits was only 1-12.5% and 0.01%, respectively, of intact hCG. We propose that this human antiserum be used as a laboratory standard by investigators who are developing vaccines directed against hCG. The use of this standard in the proposed protocol will permit comparison of titers between laboratories. Lyophilized samples of antiserum PC-81-1 are available on request from the Population Council.

Immunochemistry of human chorionic gonadotropin.

In states of gestational trophoblastic disease, knowledge of the immunochemistry of human chorionic gonadotropin (hCG) is principally of value in relation to its use as a diagnostic tool and as a marker for the persistence of disease activity during chemotherapy (1, 2). Since these data are usually obtained through immunoassay of the hormone or its subunits, we have chosen to focus this review on that background information which relates to immunochemical recognition of the hormone. Numerous other summaries are available dealing with the biological role of the hormone and its action at target organ receptors (3-5).

Human Chorionic Gonadotropin Detection with Monoclonal Antibodies

Hormone production in cancer can result from the presence of transformed endocrine cells that continue to secrete or, as in the case of human chorionic gonadotropin, the hormone can also be considered an onco-developmental gene product and thus may be produced by many different types of tumors. The use of hormones as cancer markers has been previously reviewed (Sell and Wahren, 1982). This article will concentrate on hormone detection using monoclonal antibodies, particularly those aspects of monoclonal antibodies against human chorionic gonadotropin that differ from antisera.