• Clinical and Experimental Reproductive Medicine
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• v.17 no.1
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• pp.87-91
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• 1990

Development of a solid-phase chemiluminescence immunoassay for the detection of progesterone in serum extract was described. The chemiluminescence immunoassay was establised utilizing anti-progesterone monoclonal antibody that coated on polystyrene tubes and progesterone-ABEI conjugate as tracer. The light yield generated from antibody bound conjugate was counted on clinilumat luminometer by oxidation with microperoxidase and peroxide. The chemiluminescence immunoassay was high specific and accurate and detects as little as 3.9ng/ml of progesterone. The intra-assay CV ranged from 6% to 11.5% and inter-assay CV ranged from 13.6% to 18.7%. This assay system was good correlated with conventional kit radioimmunoassay system (r=0.98).

• Korean Journal of Animal Reproduction
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• v.11 no.1
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• pp.16-21
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• 1987

We describe a simple, solid-phase chemiluminescence immunoassay for the meausrement of serum T4. An immunoglobulin G fraction of antibody to thyroxine was passively absorbed onto the walls of polystyrene tubes. The labeled antigen was thyroxine-aminobutylethylisoluminol. After the bindings reaction (37$^{\circ}C$ for 1 hour), the solution is removed by aspiration and the antibody-bound fraction was washed once with buffer. Sodium hydroxide (5mol/1,200${mu}ell$) was added and the mixture incubated for 30 minutes at 6$0^{\circ}C$. Luminescence was initiated by oxidation of the label with micropeeroxidase-hydrogen peroxide and the signal of light emission was intergrated for 10 sec. The light yield was inversely proportional to the concentration of T4 in the standard or sample. An evaluation of the method gave the following values sensitivity of calibration curve 7.5$\pm$2.8 nmol/l (mean$\pm$SD). The intra-assay precision (CV%) was 8.9, 7.3 and 5.4. The inter-assay precision (CV%) was 10.2, 8.1 and 7.1. When seum samples were assayed for T4, the results obtained by solid-phase CIA and the conventional RIA agreed well(n=3.5, r=0.954).

• Korean Journal of Animal Reproduction
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• v.15 no.2
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• pp.149-155
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• 1991

본 실험은 H-Y 항원 유전자 크로닝을 위한 기초연구로서 H-Y 항원의 특성을 규명하기 위하여 친화성 크로마토그래피에 의하여 H-Y 항원을 분리·정제하였다. 정소 추출액을 항체가 결합된 column에 결합시킨 뒤 10% acetic acid로 용출시켰다. 용출된 분획을 모아 농축한 후 HPLC와 SDS-PAGE를 실시하여 H-Y 항원의 분자량은 약 6,7000달톤 임을 알 수 있었으며 isoelectric focusing에 의하여 등전점(pI)은 5.0인 것으로 측정되었다. H-Y 항원에 대한 단일클론항체와 표지항원으로는 H-Y 항원-ABEI(aminobutylethyl isoluminol)를 사용하여 H-Y 항원 정량을 위한 화학발광면역분석법을 개발하였다. 항원항체 반응후 빛의 측정은 NaOH 존재하에서 microperoxidase/H2O2를 이용한 산화반응으로 실시하여 10초간 측정한 빛의 양을 적분하였다. H-Y 항원의 농도와 빛의 양과는 역비례하였으며 감도는 11.8ng/tube 정도이었다.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.215-221
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• 1996

This study was carried out to develop an immunoassay for the diagnosis of the pregnancy and ovarian function of domestic animals. Using 2E92C10 monoclonal antibody(McAb) generated against estrone-3-glucuronide(E1-3-G) and appeared a high cross-reactivity with estrone-3-sulfate(E1-3-S), chemiluminesence immunoassay (CIA) to detect E1-3-S was developed. 2E92C10 McAb cross-reacted with E1-3-S (30%) was purified from ascites fluid using protein G sepharose gel column. The purity of purified antibody fraction was monitored by SDS-PAGE and was better compared to that of crude ascite fluid. The soild and liquid phase CIA for E1-3-S were established utilizing 2E92C10 antibody and E1-3-G-ABEI conjugate used as a tracer. As the results, the titer of 2E92C10 antibody was 5g/ml in soild phase and 1:2000 in liquid phase. The sensitivity on soild and solid phase CIA were about 200 pg/ml. These results indicate that CIA for measurement of E1-3-S was successfully developed by using ant-E1-3-G McAb cross-reacted with E1-3-S and could be usefully used to research this area.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.1
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• pp.71-80
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• 1990

New immunoassay systems for the detection of anti-sperm antibodies were developed. For this, sperm surface protein was purified by the immunoaffinity column prepared by the coupling of rabbit anti-human IgG antibodies to Sepharose-4B. Fraction eluted by tris-HCI buffer containing SDS showed a single band having molecular weight of about 60KD on electrophoresis. Enzyme HRP labelled goat anti-human IgG and chemiluminescence aminobutylethyl-isoluminol(ABEI) labelled rabbit anti-human IgG were used for ELISA and CIA, respectively. These two labelled conjugate bound well with human IgG. When serum dilution curves were made to titrate positive serums, two kinds of curves with steep and sluggish slopes were obtained Serum samples were categorized into 3 groups: positive, weak positive and negative based on slope of curve and O.D. values at 1:160 dilution of serum. When ELISA and CIA were compared to conventional method Kibrick test by the determinations of 62 male serums with different diagnosis, the results of ELISA and CIA agreed well, but both disagreed with that of Kibrick test. This study showed that purified sperm surface antigen can be used to develope solid-phase immunoassay systems such as ELISA and CIA which may eliminate the problems encounted the immobilization of living sperm in other tests.

• Clinical and Experimental Reproductive Medicine
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• v.9 no.1_2
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• pp.43-53
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• 1982

여성의 난소기능은 뇨중 Oestrone-3-glucuronide를 간편한 solid-phase 의 화학발광성 면역학적 측정법 (Chemiluminescence Imrnunoassay(CIA) 에 의하여 그 기능이 탐지될 수 있다. Oestrone-3-g1ucuronyl-6-bovine serum albumine에 대한 antiserum의 IgG fraction은 polystyrene 실험관벽에 흡착시켰으며, 항원으로서는 est r one-3- gl ucuronyI-6-aminoethyl-ethyl-isoluminol 을 항원 (antigen) 에 labeI 시킨 것이다. 시험 대상물인 뇨는 매일아침뇨(early morning urine) 을 희석 (1:1000 V/V)한 후 100mcl 를 취하여 이를 각기 이중분석액으로 택하였다. 시험관 내에서 결합반응 (1 hour at $4^{\circ}C)이 일어난 후에는 시험관내의 액체를 전부 흡입 폐기시켰으며, 항체반응이 일어난 후 ( antibody-bound fraction )에는 완충액 (400mcl)으로 한번 세척시켰다. 그후 염화수산화물(2N , 200mcl)을 가지고 $22^{\circ}C$에 60 분간 방치 혼합케 한 후 효소(microperoxidase) 와 과산화수소를 가하면서 산화작용에서 발생되는 발광양을 10초동안 측정하여 그 결과를 분석하였다. 위에 기술한 분석방법을 평가하면 다음과 같은 결론을 얻었다. Calibration curve sensitivity$3.12{\pm}0.75$ PG/tube ($mean{\pm}SD$)였고, lntra-assay precision(CV%) 9.52 (20 replicates;$38.4{\pm}3.66$nmol/1) 와 8.81 (15 replicates; $102.4{\pm}8.82$nmol/1)였다. Inter-assay precision(CV%) 은 11.9 (mean of 4 pools-7.03, 23.16, 52.11 과 117.53 nmol/1)로 2개월 동안에 걸쳐 시행되었고, 평균 비이어스(mean bias)는 -0.78 로 28에서 448 nmol 범위로서 매일아침 "뇨"의 차이분(different aliquots)은 좋은 결과를 얻었다. 건강한 여성으로부터 채취된 뇨중 Oestrone-3-glucuronide 의 농도(nmol/1)를 보면 월경주기의 여포기와 배난기 및 황체기에 있어서 각기 $40.2{\pm}9.9$ , $102.3{\pm}39.4$와 $84.3{\pm}13.3$nmol/1였다. 이와같은 결과는 동일한 검사뇨를 방사면역학적 방법(RIA)으로 측정 (6 menstrual cycle)한 결과와 유사한 측정치를 얻으므로서 간편하고 진보된 좋은 방법중의 하나라고 사료되는바이다.