Studies on Immunological Assay of Urinary Estrogens. II. A Simple and Rapid Hemagglutination Inhibition Reaction Method

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Abstract
Fundamental studies were made on the hemagglutination inhibition reaction (HAIR) method with anti-estriol 16-glucuronide (E3-16-G) serum for a rapid and simplified determination of estrogen in pregnancy urine. The sheep erythrocytes fixed with formalin and treated with tannic acid were sensitized with E3-16-G-rabbit serum albumin (RSA) conjugates. An antiserum to E3-16-G-bovine serum albumin (BSA) was absorbed with BSA and erythrocytes fixed with formalin. The degree of binding of E3-16-G with RSA and the amount of E3-16-RSA sensitized onto erythrocytes, affected the easiness of adjusting sensitivity and the clearcut patterns of HAIR formed at the bottom of the test tubes. A HAIR reagent having sensitivity of 0.2 .mu.g/ml (E3-16-G equivalent in E3) could be prepared by sensitizing an 8% erythrocyte suspension with 0.05-0.08% of E3-16-RSA, in which 20-30 mol of E3-16-G is bound per 1 mol RSA. The sensitivity was adjusted by diluting an antiserum. Cross-reactivity of the HAIR method with estrogens was almost similar to that of radioimmunoassay (RIA) and the reactions found with estrogens other than those conjugated at the C-3 position, i.e., E3-16-G, C-17 conjugated estrogens, free estrogens. Glucose, NaCl, serum protein, urinary protein and urine pH, did not affect the results. E3-16-G added to pregnancy urine was recovered in satisfactory yield, considering the accuracy of the method. Significant correlation (y = 0.78 x + 0.97, r = 0.949) was noted between HAIR and RIA on the measured estrogen values of the same urines from pregnant women. This method, a semi-quantitative determination of urinary estrogen, is evidently a useful tool for feto-placental function in clinical routine.