Development of the ultra sensitive sandwich assay of human inhibin and it's clinical applocations

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Inhibin shows molecular heterogeneity and there exist not only biologically active dimers but also immunologically active and biologically inactive monomers in human body fluid. It is also widely recognized that several proteins including follistatin and alpha 2 macroglobulin binding to inhibin and structurally related activin interfere with the assay of inhibin.In order to establish a highly sensitive and specific sandwich assay of biologically active inhibin, we investigated the character of human inhibin, produced a variety of antibodies against inhibin and studied several methods of labeling the antibodies.First we established a large scale purification system for inhibin from human follicular fluid. It was then possible to obtain the required amount of inhibin for a standard in the assay kit. Recombinant human inhibin B was expressed, purified and characterized and we confirmed that this recombinant inhibin is useful as an inhibin B assay standard (Hasegawa et al.J Reprod Develop … More 42 : 23-25,1996. Inhibin and Activin pp139-154,1996. Hasegawa et al. Sexual Differentiation and Maturation pp139-154,1996).Next, we investigated labeling methods using many kinds of polyclonal and monoclonal antibodies. Using an anti alpha subunit polyclonal antibody coated on a bead and anti beta_A subunit monoclonal antibody labeled by ^<125>I,we established an inhibin A radioimmunometric assay (Inhibin A IRMA) (Hasegawa et al.Inhibin, Activin, and Follistatin, pp104-117,1997). Changes in inhibin A concentrations in circulating blood during normal human pregnancy and abnormal pregnancy such as pregnancy induced hypertension and intrauterine growth retardation were clarified using this inhibin A IRMA (Mizunuma et al. Inhibin, Activin, and Follistatin pp151-161,1997). Because this inhibin A IRMA is not sensitive enough to measure the inhibin concentrations in circulating blood in non-pregnant women, further investigation has been done in order to improve its sensitivity. The polyclonal antibody was purified by an inhibin coupled column and an activin coupled column and thus an anti alpha subunit antibody and an anti beta_A subunit antibody were obtained. Inhibin A immunofluorometric assay (Inhibin A IFMA) was established using microtiter plates coated with the anti beta_A subunit antibody and the anti alpha subunit antibody labeled with Europium. This inhibin A IFMA is sensitive enough to measure inhibin A concentrations in the circulating blood of non-pregnant women. The study of the change of inhibin A concentrations in various physiological and pathological conditions is continuing. Less