Seven hybridoma cell lines, 051-01 (common name : 15A, $IgG_1$), 051-02 (common name : 28A, $IgG_{2b}$), 051-03 ($IgG_1$), 051-04(IgM), 051-05 ($IgG_{2a}$), 051-06(IgM), and 051-07($IgG_{2a}$) producing monoclonal antibodies to progesteron were established. Progesterone 11α -hemi-succinate-bovine serum albumin conjugate, prepared using carbodiimide, was used to immunize BALB/c mice. The spleen cells from the immunized mice were taken to fuse with mouse myeloma (P3-X63-Ag8.654) cells. The hybridoma cells thus obtained were screened by radioimmunoassay using [$^3H$]-progesterone. The clones 051-01, 051-03, and 051-05 were purified using protein A-Sepharose 4B affinity chromatography utilizing their subtype characteristics. The purity of the isolated IgG was identified by SDS-PAGE and capillary electophoresis. The affinity constant ranged from 5$\times$10$^7$M$^{-1}$ to 4$\times$10$^8$M$^{-1}$ and the progesterone binding capacity of the monoclonal antibodies from 7 to 72 pmole/mg IgG by the Scatchard analysis method. The cross-reactivity of the monoclonal antibodies, 15A and 28A with other steroid hormones has shown that these antibody reactions were highly specific and sensitive in a competitive immunoassay.
To develope EIA system, the influence of the conjugation site on the specificity of monoclonal antibodies to progesterone and on the performance of enzyme immunoassay was assessed. Monoclonal antibodies against progesterone conjugated to carrier protein through substituent on the A-ring (C-3 position) or the C-ring (C-11 position) of progesterone were used in the enzyme immunoassay. Antibody specificities were determined by testing the ability of 11 representative steroids to displace labelled progesterone in a competitive enzyme immunoassay and a radioimmunoassay (RIA). Immunization with progesterone conjugated to BSA through substituent on the A-ring (C-3) resulted in the formation of monoclonal antibody (mAb) which fairly specific for progesterone. While immunization with progesterone at the 11-position (C-11) resulted in the mAbs which were very specific for progesterone. To assess the influence of structure of immunogens and tracers on the competitive enzyme immunoassay (EIA), two kinds of the horseradish peroxidase (HRP) tracers were employed; one of the HRP tracers is synthesized from progesterone-11α-hemisuccinate (P-11HS) and the other from progesterone-3(O-carboxymethyloxime) (P-3CMO). The best EIA system was developed by mAb against progesterone-11α-hemisuccinyl BSA (P-11HS-BSA) and tracer of progesterone-3(O-carboxymethyloxime)-HRP (P-3CMO-HRP). The approach is to confirm the overall orientation of the steroid at the binding site of monoclonal antibody. Immunobiochemical epitope analysis of mAb suggested that the D-ring is substantially more buried in the binding pocket than the A-ring. The assay was designed to use the reagent inactivating cholesterol binding globulin in serum so that the extraction of the hormone into organic solvent was unnecessary. Therefore, this EIA system can directly and rapidly detect the progesterone level in serum within one hour.
By using the best combination of mAb and tracer of P- 3CMO-HRP, a visual membrane EIA was also developed based on utilization of reagents immobilized on membranes or porous carriers for the semiquantitative determination of progesterone.
A quantitative measurement of progesterone was studied using fluorescein isothiocyanate (FITC)-labelled progesterone-3(O-carboxy methyloxim)-BSA, antibody-bound Sepharose 4B particles, and a FACScan flow cytometer (Becton Dickinson, U.S.A.). The FACScan flow cytometer was an alternative reader for the FITC-labelled fluoro-immunoassay using the microparticle solid phase.
Early embryo development and implantation were arrested in ICR mice which were passively immunized with a monoclonal anti-progesterone antibody given as a single intraperitoneal injection at 12 hrs or 60 hrs post coitum (p.c.). Unimplanted embryos were recovered from the reproductive tract of the antibody-treated mice and none of these progressed to the blastocyst stage. The most pronounced effect was an arrest of embryonic development at a stage prior to cavitation. The plasma progesterone concentration in the blood taken by cardiac puncture increased greatly after the treatment by virtue of high affinity binding by the antibody in circulation. The results showed that passive immunization against progesterone shortly after mating interfered with early hormone dependent steps which were essential for normal embryonic development.
Other study was carried out to find if the anti-idiotypic (Anti-Id) antibodies to progesterone could mimic the antigenicity of the progesterone hormone and whether these were biologically active. The relationships between the antifertility effects and anti-progesterone antibody level were studied by the active immunization of anti-Id and progesterone-BSA. The result showed that a higher antifertility effect was observed with progesterone-BSA immunization (70%) than with anti-Id immunization (55%). The plasma level of anti-progesterone was as high as 3,500 μg/ml with progesterone-BSA immunization. The comparative study of the antibody levels between pregnant (10μg/ml) and nonpregnant mice (44μg/ml) after immunization with the anti-Id indicates that antibody formation at a certain level prevents pregnancy.
Progesterone-11α -hemisuccinyl BSA (P-11HS-BSA)를 immunogen으로 사용하여 여러가지 subtype의 immunoglobulin을 분비하는 7개의 hybridoma cell line을 얻었다. 이들 단일클론항체들을 각각의 subtype의 특성에 따라 분리하여 SDS-PAGE와 capillary electrophoresis로 순수정제 되었슴을 확인하였다. 단일클론항체들을 scatchard analysis한 결과 affinity constant는 $5\times10^7\M^{-1}$ $-4\times10^8\M^{-1}$, progesterone binding capacity는 7-72 pmole/mg IgG 정도였다. progesterone의 A-ring(C-3)이나 C-ring(C-11)에 BSA를 결합시킨 immunogen에 의해 생성된 단일클론항체들을 효소면역측정법 (enzymeimmunoassay ; EIA)에 이용하였다. 이들 단일클론항체와 progesterone-3(O carboxy methyloxime)-HRP(P-3CMO-HRP)나 progesterone-11α hemisuccinyl HRP (P-11HS-HRP)를 tracer로 이용하여 항체의 항원결합부위(Ag binding site)에 대한 경쟁적 반응을 응용한 최적의 효소면역측정법을 개발하였다. 효소면역측정법에 사용되는 tracer로는 P-11HS-HRP보다 P-3CMO-HRP가 free progesterone과의 경쟁반응을 더욱 잘 하였으므로 tracer로는 P-3CMO-HRP를 이용하였다.
P-3CMO-HRP를 이용하여 11개의 유사한 steroid hormone 에 대한 단일클론 항체들의 특이반응성을 조사하였다. P-3CMO-BSA를 주사하여 만든 단일클론항체 2B7는 progesterone에 대한 특이성이 대체로 낮았으나, P-11HS-BSA를 면역 주사하여 얻은 단일클론항체 15A와 28A는 특이성이 매우 높았다. progesterone의 epitope 분석결과, progesterone의 D-ring이 A-ring보다 항체 (15A, 28A)의 hydrophobic binding pocket에 훨씬 깊이 들어감을 나타내었다.
혈청 중의 steroid를 측정하려면 대체로 유기용매로 steroik를 추출하는 과정이 필요한데 본 EIA에서는 steroid-binding protein을 불활성화 시키는 reagent로 ANS를 사용하여 혈청중의 progesterone농도를 직접 1시간 내에 빠르게 측정할 수 있었다.
보통의 EIA에서는 항체에 결합된 항원과 결합되지 않은 항원을 분리하는 과정이 필요한데, 이러한 분리과정이 필요없는 homogeneous polarization fluoroimmunoassay(PFIA)를 이용하여 progesterone농도를 측정하였다. 시료중에 함유되어 있는 progesterone이 항체에 대하여, P-11HS-fluorescein ethylenediamine (P-11HS-FED)이나 p-3CMO-FED와 같은 tracer와 경쟁적인 반응을 하게되어 항체에 FED tracer가 적게 결합하면, polarization signal이 감소하게된다. 이들 두 종류의 tracer와 두 종류의 항체(P-3CMO-BSA에 의해 유도된 2B7과 P-11HS-BSA에 의해 유도된 15A)를 이용하여 PFIA를 수행한 결과 2B7과 P-3CMO-FED, 15A와 P-11HS-FED의 조합을 사용하여 10μl의 시료중에서 10 ng/ml 까지 측정할 수 있었다.
이외에도 progesterone측정을 위한 다른 방법으로 nitrocellulose membrane을 이용한 visual membrane EIA을 수행하였다. 여러가지 membrane의 기질에 대한 비특이성을 조사한 결과 Ultrabind US-450(0.45 μm)의 비특이성이 제일 적게 나타나 이 membrane을 이용하였다. 또한 FITClabelled immunoassay를 위한 측정기기로 Flow Cytometer를 사용 하였으며, microparticle solid phase로는 antibody-bound Sepharose 4B, tracer로는 FITC-labelled P-3CMO-BSA를 이용하여 progesterone을 측정하였다.
ICR mice는 mating후, 12시간 또는 60 시간후에 progesterone에 대한 단일클론항체를 복강에 한번 수동면역함으로써, 초기의 embryo의 발육과 착상이 억제되었다. 착상되지 않은 embryo를 항체처리군의 수란관에서 회수해 보니 이들이 모두 blastocyst stage로 전환되지 못했다. 항체 처리후 혈청내의 progesterone농도는 매우 증가했다.
15A의 idiotype (Id)에 대한 anti-15A-Id가 progesterone의 항원성 (antigenicity) 효과를 대신 나타내는지를 알아보기위해 anti-15A-Id를 3가지의 affinity-chromatography를 수행하여 분리정제한 후 mice에 능동면역하였다. Anti-15A-Id를 능동면역 주사한 결과 anti-progesterone antibody를 44 μg/ml 정도까지 생성한 mice는 임신이 억제되었으나, 10 μg/ml정도 밖에 생성되지 않은 mice는 임신이 계속 유지되었다. progesterone-BSA를 능동 면역시킨 mice는 3,500 μg/ml 정도의 많은 양의 anti-progesterone이 생성되어 임신억제율이 70% 정도로 나타났으며, anti-Id를 면역주사한 경우는 progesterone-BSA를 면역시킨 경우에 비해 1% 정도의 anti-progesterone antibody 생성율을 보여주었으나 임신은 55% 나 억제되었다. 이점으로 미루어 보아 Anti-15A-Id는 progesterone의 internal image를 지녀 anti-progesterone antibody를 생성케 하고 생성된 항체는 생체 내에서 progesterone에 결합하여 progesterone의 기능을 active하게 억제한다고 볼 수 있다.
