Measurement of luteinizing hormone (LH) in the urine has much significance in the diagnosis of menstrual disorders. Urinary LH assay has been previously carried out by troublesome, time-consuming bioassay. Recently, immunological method has become available for that purpose, since Wide and Gemzell (1961) reported LH-immunoassay using HCG antigen-antibody system which was based upon the cross-reaction between HCG and LH. It is the purpose of this report to demonstrate the cross-reaction between HCG and LH, to establish the immunological assay method of urinary LH and to investigate the relationship between immunological and biological activites of LH.
Anti-HCG sera were obtained from rabbits received 2 injections each of 1,000 IU of crude preparation of HCG (Primogonyl, HCG-P), freshly emulsified with complete Freund's adjuvant at two weeks interval. The injections were made in the footpads and i.d. After 3 weeks of rest, a series of booster injections with 1,000 IU of HCG-P without adjuvant were given i.p. nine times at every other day. Rabbits were bled 7 days after the last injection and the sera were kept. The antibody titers were in the range from 6,400 to 51,200 by tanned hemagglutination. Antiserum which had the highest antibody titer was used in this study.
This anti-HCG serum contained several antibodies which reacted not only with HCG but also with normal human serum (NHS) and child urine protein (CUP) by immunoelectrophoresis. In order to remove antibodies against proteins other than HCG, 1 ml of anti-HCG serum was absorbed with 30mg of lyophilized NHS and 20mg of CUP, thus the absorbed anti-HCG serum showed one precipitin band only to HCG. This absorbed serum also showed one precipitin band to human menopausal gonadotropin (Pergonal, HMG-P) which contained LH and FSH by agar gel diffusion and immunoelectrophoresis, and this band completely coalosced to the band wnich appeared against HCG.
This absorbed anti-HCG serum was examined for neutralization of LH and FSH. An assay method based on the rate of increase in the weight of the ventral prostates of hypophysectomized male rats (Greep) was used for LH assay and Steelman-Pohley's augmentation method was used for FSH assay. When 2 IU or 4 IU of LH and 2 IU or 4 IU of FSH were incubated with 0.002 ml of absorbed anti-HCG serum at 37°C for 60 min., biological activity of LH was completely neutralized but the FSH activity was not. From the above immunological and biological results, it could be concluded that LH had a similar antigen to HCG and LH was completely neutralized with anti-HCG serum.
The amount of LH in the urine is so small for immunoassay that concentration of urine using pervaporation, carbowax 6,000 and alcohol precipitation to 1 : 20 is necessary. The recovery of LH was almost complete. The components of medium to dissolve the standard HCG influenced the sensitivity of tanned hemagglutination inhibition reaction. The concentration from 1 to 1 : 20 of the child urine as the medium of HCG, showed 4 fold as sensitive as phosphate buffered saline, thus 1 : 2 concentrated child urine was used for the medium to dissolve the standard HCG or to dilute the test materials.
Ten women from 23 to 34 years old who had the normal menstrual cycle, were examined every day for BBT, and LH value in the first morning urine by immunoassay. Seven women who showed clearly biphasic BBT gave the peak of LH level, in the range from 80 IU to 320 IU HCG equivalent/L, from 15 to 13 days before next menstruation. These peaks coincided with the rise of BBT.
Three women among them were examined LH level in the urine by using immunological and biological methods simultaneously. The urine for each 48 hours during the cycle war collected, and COml of that was used for immunoassay and the rest was used for bioassay. The kaolin adsorption method by Bradbury-Matsushima was used to extract gonadotropin from the urine.
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