• 동의생리병리학회지
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• 제18권3호
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• pp.874-879
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• 2004

Alakaline phosphatase (ALP)- or horseradish peroxidase (HRP)-antibody conjugate was used frequently on the immunological detection methods such as enzyme-linked immunosobent assay (ELISA), immunobolt, immunohistochemistry. The classical enzyme-antibody coupling method by one-step (direction) injection of glutaraldehyde bring into being disadvantage such as low sensitivity of antigen detection because of homopolymers. This study was modified with the dialysis glutaraldehyde method to provide simple coupling through E-amino residues present in most protein. The dialysis glutaraldehyde coupling effects were better than the classical one-step glutaraldehyde injection in antigen detection of ELISA and immunobolt. Optimal dose of the dialysis glutaraldehyde solution was 0.10-0.25 %. This results suggest that the dialysis glutaraldehyde coupling method can readily applied to antigen detection of in vitro and in vivo.

• 대한수의학회지
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• 제33권4호
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• pp.611-615
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• 1993

젖소에서 progesterone과 testosterone의 측정을 위하여 solid phase 효소면역분석법(酵素免疫分析法)를 개발하였다. 1차 항체로서 $11{\alpha}$-hemisuccinate-progesterone bovine serum albumin과 4-androsten-$17\;{\beta}$-ol-3-one-carboxymethyloxime bovine serum albumin에 대한 토끼 혈청(血淸)을 각각 항혈청(抗血淸)으로 사용하였고, 2차 항체로서 면약 IgG를 사용하였다. Conjugates로서는 각각 progesterone-$11\;{\alpha}$-hydroxy-hemisuccinate-horseradish peroxidase와 4-androsten-$17\;{\beta}$-ol-3-hemisuccinate-horseradish peroxidase를 사용하였다. Progesterone과 testosterone에 대한 최저측정치(最低測定値)는 각각 6.7 pg/well과 1.0 pg/well이었다.

• Bulletin of the Korean Chemical Society
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• 제18권5호
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• pp.515-519
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• 1997

In a competitive enzyme immunoassay, the performance was tested with different analyte-enzyme conjugates (signal generators) in their binding constants to antibody. Analyte (progesterone)-enzyme (glucose oxidase; GO) conjugates were chemically synthesized and purified by using a gel column with an immobilized antibody to progesterone. In an elution range from the column, four peaks were detected by measuring total enzyme activities. Results from further analysis indicated that the first peak contained mainly unreacted GO while the next three peaks conjugated GO with progesterone. These three conjugate preparations were compared in dose-response curves along with the unpurified mixture. The purified conjugates showed higher detection capabilities than did the mixture. Especially, the preparation in the second peak next to the free GO peak improved the detection limit five times. This performance was comparable to that of a progesterone-horseradish peroxidase conjugate that has been identified to have one progesterone ligand.

• KSBB Journal
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• 제14권1호
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• pp.82-90
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• 1999

병원성 미생물을 측정할 수 있는 분석시스템을 구성하기 위해 면역학적 성분들을 합성하였고, 이를 이용하여 모델 시스템을 구성함으로써 균 세포 분석원리가 연구되었다. 구성성분을 준비하기 위해 Salmonella thompson에 대한 복합 클론항체를 면역 친화 크로마토그래피를 이용하여 정제하였고, 이렇게 정제된 항체를 Streptavidin과 horseradush peroxdase에 화학결합시켰다. 항체와 Streptavidinfdm은 각각 SMCC와 SPDP에 의해 활성화 되있고 두 물질을 반응시킴으로써 중합체가 합성되었다. 중합체는diaminobiotion 젤과 sephades G-100젤을 이중 층으로 쌓은 칼럼을 이용하여 정제되었다. 항체- HRP 중합체의 합성을 위해, HRP를 $NAIO_4$ 처리에 의해 안정화된 중합체는 Biohel A5M을 이용한size exchusion크로토그래피로 정제되었다. 이렇게 준비된 중합체들과 dot-bloner 그리고 biotim이 고정화된 nitrocellulose membrane($12\mum$ pore size)을 이용하여 모델시스템을 구성하였다. 분석물질(S.Thormpson cells)을 먼적 액상에서 두 중합체와 반응되었고 반응먹을 membrane이 정착된 blotter에 옮긴 후 하부에 진공을 걸어 면역복합체를 biotin-streptavidin 반응에 의해 membrane 표면에 포획하였다.최적조건 하에서 시스템의 균 세포 분석원리를 확인하였으며 측정하한농도는 약 $1{\mu}g/m{\ell}(10^5 {\cdot} 10^6\;cells/m{\ell}$인 것으로 나타났다. 이러한 측정성능의 주요조절인자는 두항체 종합체 농도의 증가는 항원-항체 응집반응을 초래하는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.527-533
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• 2013

Polyclonal antibodies labeled with a tracer have been commonly used as secondary antibodies in immunochemical assays to quantify the concentration of antibody-antigen complexes. The majority of these antibodies conjugated with a tracer are commercially available, with the exception of few untouched targets. This study focused on the production and application of mouse anti-quail IgY as an intermediate antibody to link between quail egg yolk IgY and goat anti-mouse IgG-HRP as primary and secondary antibodies, respectively. Subsequently, the produced mouse anti-quail IgY was labeled with horseradish peroxidase (HRP) and its efficiency on enzyme linked immunosorbent assay (ELISA) was compared with that of commercial rabbit anti-chicken IgY-HRP. As an intermediate antibody, mouse anti-quail IgY was successfully produced with good affinity and sensitivity (1:10,000) to the primary and secondary antibodies. Subsequently, mouse anti-quail IgY was effectively conjugated with HRP enzyme, resulting in a secondary antibody with good sensitivity (1:10,000) to quail anti-V. parahaemolyticus and V. vulnificus IgY. The detection limit was $10^5$ CFU/ml for both V. parahaemolyticus and V. vulnificus. The efficiency of the produced conjugate to detect quail IgY on ELISA was comparable to that of the commercial rabbit anti-chicken IgY-HRP, and hence the produced and labeled mouse anti-quail IgY-HRP can be used as a secondary antibody to detect any antibody produced in quail.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.612-619
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• 2004

A competitive direct enzyme-linked immunosorbent assay (cdELISA) was developed for selective and rapid detection of a glycopeptide antibiotic, teicoplanin (TP). TP was conjugated to bovine serum albumin (BSA) for use as an immunogen. Repeated subcutaneous injections of 0.5 mg of the conjugate was effective in generating specific polyclonal antibody (PAb) toward TP in rabbits, as determined by cdELISA. TP-horseradish peroxidase conjugate (TP-HRP) was used as an enzyme marker. The cdELISA was developed based on a competition reaction between TP-BSA PAb and TP-HRP conjugate. The TP-BSA PAb was highly sensitive (detection limit, 0.3 ng/ml and specific toward teicoplanin, showing no cross-reactivity to other glycopeptide antibiotics including vancomycin. There were good correlations ($r^2$=0.84 and 0.76, respectively) between cdELISA and microbiological assay, and high-performance liquid chromatography. The cdELISA system developed in this work is expected to be useful not only for selective and rapid monitoring of TP but also for study of TP pharmacokinetics.

• 한국식품위생안전성학회지
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• 제9권3호
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• pp.123-131
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• 1994

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of residual gentamicin(GM) in edible animal products. The immunogen(GM-KLH conjugate) and coating antigen(GM-BSA conjugate) were prepared by coupling GM sulfate to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, respectively. Polyclonal antibody to GM was produced in rabbits(New Zealand White, female) by using the immunogen and the antibody titer was measured by indirect ELISA. A competitive ELISA was developed using GM-bovine serum albumin conjugate as a coating antigen, GM(as standards or sample), polyclonal antibody to GM, secondary antibody conjugated with horseradish peroxidase as an enzyme, and H2O2 and o-phenylenediamine dihydrochloride as a substrate and a chromophore, respectively. The detection limit of GM was 10 ng/ml and the standard curve of GM(n=26) was linear up to 10 $\mu\textrm{g}$/ml in this competitive ELISA system. There were no cross-reactivities of the partially purified antibody between GM and the various antibiotice such as amikacin, benzyl-penicillin, chloramphenicol, erythromycin, furazlidone, kanamycin, neomycin, oleandomycin, streptomycin, sulfathiazole and thiamphenicol(CR50<0.05%)

• 대한의생명과학회지
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• 제4권1호
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• pp.57-63
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• 1998

본 연구는 돼지 지방세포 원형질막 단백질에 대한 항체를 면양에서 생산하고 생산된 항체의 역가 및 조직특이성을 조사하기 위하여 실시되었다. 지방세포, 뇌, 심장, 신장, 간장 및 비장으로부터 원형질막 단백질을 추출하였으며, 그중 지방세포로부터 분리한 원형질막 단백질을 면양(체중 40kg)에 3주 간격으로 3회 면역 접종시켰다. 면역접종 전, 3차 면역접종 후 10일 (AS-1), 12일 (AS-2)및 14일 (AS-3)째에 각각 면양의 경정맥으로부터 혈액을 채취하여 혈청을 분리하였다. 항체의 역가 및 기타 조직과의 교차반응성은 enzyme-linked immunosorbent assay (ELISA)로 측정하였다. 면양에서 생산된 돼지 지방세포 원형질막 단백질에 대한 항혈청은 지방세포 원형질막 단백질과 강한 항원-항체 반응을 나타내었다. 항혈청의 교차반응성을 조사한 결과, 기타 조직의 원형질막 단백질과는 매우 미약한 반응을 나타낸 반면 지방세포 원형질막 단백질과는 강한 반응을 나타내었다. 이러한 항혈청의 지방세포 원형질막 단백질과의 조직특이적인 반응은 anti-sheep immunoglobulin G-horseradish peroxidase conjugate를 2차 항체로 이용한 immunoblot에 의해서도 재확인되었다. 이상의 결과, 면양으로부터 생산된 돼지 지방세포 원형질막 단백질에 대한 항체는 높은 역가를 지니고 있었으며, 지방세포 원형질막 단백질에 특이적으로 작용함을 알 수 있었다.

• 대한의생명과학회지
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• 제5권1호
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• pp.11-15
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• 1999

갑상선 질환의 진단에 필요한 갑상선 호르몬의 측정시 thyroxine (T$_4$)농도 측정방법이 가장 흔히 이용되고 있다. 과거에 많이 활용되어온 방사면역측정법 (RH)이 폐기물 처리 및 관리인원 등의 인적, 물적인 문제가 크게 대두되고 있는 점을 감안하면 효소결합면역측정법의 확립은 큰 의미가 있다고 하겠다. 본 연구에서는 thyroxine (T$_4$)에 대한 단클론 항체 (monoclonal antibody)를 이용하여 T$_4$에 horseradish-peroxidase를 화학적으로 결합시킨 microtiter plate에 HRP-conjugate T$_4$와 혈청내의 T$_4$간의 서로 경쟁적인 반응을 이용한 표준곡선을 얻었다. 그리고 이렇게 확립한 기법을 임상적인 갑상선질환 환자의 혈청을 기존의 chemiluminescence 방법을 이용하여 얻은 결과와 비교하여 본 실험에서 확립한 ELISA검사 수치의 유용성을 검토하였으며 이 방법의 임상이용 가능성을 검토하였다.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.877-880
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• 2004

In this study, we determined the content of chitooligosaccharides (COS) in Korean commercial salt-fermented shrimps by competitive direct enzyme-linked immunosorbent assays (cdELISAs), using anti-COS mixture (COSM) antibody and COSM horseradish peroxidase (HRP) conjugate. When COS6 was spiked into salt-fermented shrimps at the level of $10-300\mu{g/g,}$ the average recovery was $120\pm19%$ ($mean\pmS.D.$). The COS contents of the 92 samples of Korean commercial salt-fermented shrimps collected during February 2000 and August 2002 were $36.3\pm20.7\mug$ COS6 equivalent/g (expressed as "$\mug/g$" hereafter). Among the samples, the COS contents of yuk-jeot ( $40.3 \pm 22.5 \mug/g, n=27$) and buksaewoojeot ($40.2 \pm 21.6 \mug/g, n=5$) were higher than the others. The COS contents of salt-fermented shrimps produced at Gwangcheon ($47.1 \pm 20.7 \mug/g, n=18$) and Gomso ($44.1 \pm 21.8 \mug/g, n=6$) areas were higher than those produced at the other areas. This is the first report to determine COS of salt-fermented shrimps by cdELISA.