An indirect enzyme immunoassay for the measurement of total 17alpha-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17alpha-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.
Hybridomas secreting monoclonal antibodies (MAbs) against 17alpha-hydroxyprogesterone (17OHP) have been generated. These MAbs are highly specific and have an affinity of 7-12 x 10(7) M(1). The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma P3X63 Ag8.653 cells. The antigen used for immunization was 17OHP conjugated to bovine serum albumin (17OHP:BSA). Fused cells were plated and cloned in 96-well microtiter plates. Wells containing hybridomas were screened simultaneously for specific gamma globulin (IgG) and anti-17OHP activity using an enzyme-linked immunosorbent assay (ELISA)-based method, which is faster than the conventional radioimmunoassay (RIA) screening procedure. Limiting dilution methods were used to obtain single hybridoma clones producing MAb. The stable hybridomas secreting anti-17OHP MAbs were expanded into bioreactors or ascites fluid for large-scale production of the required antibodies. These MAbs will be used in the formulation of a 17OHP assay kit to screen for congenital adrenal hyperplasia (CAH) in local newborn human population.
The effects of exogenously introduced oestradiol-17 beta (E) and relaxin (RLX) on cervical extensibility and collagen organisation were tested in rats ovariectomised in late pregnancy. When the cervices were stretched in vitro by 1 mm increments, it was found that those from rats given E alone generated significantly higher tensions than those from control rats, while cervices from rats given both E and RLX had tensions similar to controls. Examination of cervical sections under the light microscope and ultra-thin sections under the electron microscope showed that the collagen fibres in the cervices from E-treated rats were highly organised, whereas those from animals given E+RLX and control animals were disorganised and dispersed. It was concluded that E decreased cervical extensibility, while RLX counteracted the effect of E to maintain a soft and easily extensible cervix.