Radioimmunoassay of Human Chorionic Gonadotropin (HCG) and Luteinizing Hormone (LH) in the Urine

Abstract

In 1965, Wilde et al tried to use radioimmunoassay for estimating human chorionic gonadotropin (HCG) and Bagshawe et al, Odell and Midgley et al. also used this method for assaying HCG and luteinizing hormone (LH). One of our aims in this paper was to determine whether even partially purified HCG (5,000 I.U./mg) could be used for radioimmunoassay or not, and the other was to determine the proper experimental condition for each step of the reactions, in order to get the best results.
Anti-HCG sera were prepared by giving two injections to four rabbits at 2-week intervals of 1,000 I.U. of crude preparation of HCG (1,000 I.U./mg) in 1 ml of phosphate saline buffer, freshly emulsified with 1 ml of complete Freiund's adjuvant (Difco) each time. The injections were made in the footpads and i.d. After 3 weeks of rest, a series of booster injections with 1,000 I.U. of HCG without adjuvant were given i.p. 5 times every other day. Rabbist were bled 7 days after the last injection and the sera were obtained. The antibody titers were from 1 : 6,400 to 1 : 25,600 by hemagglutination.
For preparing absorbed anti-HCG serum, one ml of antiserum was incubated with 30 mg of an alcohol precipitate of urine from a child and 30 mg of human serum protein at 37°C for 60 min., and was kept at 4°C over-night. After centrifuging the mixture, the supernatant, the absorbed anti-HCG serum, showed only one precipitin band to HCG antigen on immuno-electrophoresis. Radioiodination of HCG by ¹³¹I was carried out by the modified chloramine T method of Greenwood.
All materials were diluted with 0.5% bovine serum albumin (BSA) in borate buffered saline (BBS) -diluent-, and the 2nd IRP-HMG and Pergonal 500^TM were used as standard LH.
In radioiodination, the ratio of chloramine T to HCG was 0.4-1.3 : 1. The high specific activity of ¹³¹I-HCG which was more than 200 mc/mg, was obtained, and around 60% of the ¹³¹I-HCG preparation was precipitable with absorbed anti-HCG serum.
The procedure of radioimmunoassay was as follows : The diluted anti-HCG serum was mixed with sample (HCG) and ¹³¹I-HCG. After incubation, the inactivated diluted rabbit serum (as carrier protein) and horse anti (rabbit serum r-globulin) serum globulin were added and then incubated. Th resulting precipitate was washed once with diluent and hydrolyzed with 1 ml of 2N NaOH and then counted with Scintillation Counter.
Th proper experimental conditions for each step of radioimmunoassay were determined. 1. The combination of ¹³¹I-HCG and anti-HCG needed 48 hrs. for the maximum reaction (1 st reaction). 2. Cold HCG (sample) should be added to anti-HCG serum at least 4 hrs. before the addition of ¹³¹I-HCG in the 1st reaction. 3. The proper temperature during incubation in the 1st and 2nd reaction was 4°C. 4. As diluent, 0.5% bovine serum albumin in BBS was necessary and enough. 5. In the 2nd reaction, 0.1 ml of diluted (1 : 2,000) rabbit serum as carrier protein, gave the maximum precipitate with 0.2 mg of our horse anti (rabbit serum r-globulin) serum globulin. 6. The 2nd reaction needed more than 6 hrs. at 4°C.
The standard curve of HCG shows that the amount of HCG from 0.0025 I.U./ml to 0.3125 I.U./ml was sufficient to be estimated quantitatively and the amount as much as 0.001 I.U./ml of HCG was also qualitatively detectable by this method. The amount of HCG in the urine was measured simultaneously by radioimmunoassay, hemagglutination inhibition rection and bioassay. The results showed that the radioimniunoassay could be clinically used. This method was also very useful to detect the low titer of HCG in the urine which could not be detected by other methods.
The amount of LH in the urine of adult women having normal ovarian cycle, was radioimmunologically assayed, and it was shown that the peak of LH appeared one to three days before ovulation assumed by B.B.T.