• 한국미생물·생명공학회지
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• 제27권4호
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• pp.344-348
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• 1999

A competitive enzyme-linked immunosorbent assay(ELISA) for the detection of antibodies to Treponema pallidum(T.pallidum) was developed and evaluated. T.apllidum lysate was immobilized on the surface of microplate wells and horseradish peroxidase labeled human anti-T.pallidum lysate was immobilized on the surface of microplate wells and horseradish peroxidase labeled human anti-T.pallidum was prepared and used as a tracer. The performance of the competitive ELISA was evaluated by using different specimens. The competitive ELISA showed a sensitivity of 100% in a performance panel consisting of serum and plasma with anti-T.pallidum reactivity ranging from negative to strong positive by FTA-ABS test system and 120 plasma samples positive by TPHA. The specificity of the competitive ELISA was 100% in 1,200 plasma samples collected from healthy seronegative blood donors. These results suggest that the competitive ELISA provides an excellent assay method for the detection of antibodies to T.pallidum, and may be particularly useful for serological blood screening of syphilis.

• BMB Reports
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• 제31권5호
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• pp.519-523
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• 1998

We have previously reported that anti-glucose-6-phosphate dehydrogenase (G6PD) serum raised against native G6PD (nG6PD) enzyme recognized nG6PD antigen poorly in competitive enzyme-linked immunosorbent assay (ELISA) (Kim, 1997). In the present study, we investigated whether anti-G6PD serum raised against nG6PD can react with denatured G6PD effectively in competitive ELISA. We used partially active G6PD (paG6PD) by repeated freeze-thawing or SDS-denatured G6PD (SDS-G6PD) as both immobilized and soluble antigens, and anti-G6PD serum raised against nG6PD for competitive ELISA. The polystyrene cuvettes coated with either paG6PD or SDS-G6PD were challenged with a mixture of a limiting amount of anti-G6PD serum and various doses of paG6PD or SDS-G6PD as competitors, followed by incubation with alkaline phosphatase-anti-IgG conjugate. The competitive ELISA with paG6PD or SDS-G6PD antigen exhibited the sigmoidal dose-response curve characteristic of competition immunoassays. Furthermore, Triton-denatured G6PD (Triton-G6PD) was used in competitive ELISA. The paG6PD, SDS-G6PD, or Triton-G6PD used as competitors increased the inhibition of antibody binding to immobilized either of nG6PD or denatured G6PD compared with nG6PD competitor. The inhibition by denatured G6PD competitors was more pronounced at high competitor concentrations than at low counterparts. We conclude that anti-G6PD serum raised against nG6PD can effectively react with denatured G6PD in competitive ELISA and that our anti-G6PD serum recognizes denatured enzymes better than active enzymes.

• 생명과학회지
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• 제23권2호
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• pp.306-310
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• 2013

본 연구는 요중 아플라톡신 M1 (AFM1)의 농도를 측정할 수 있는 competitive ELISA 방법의 특성을 HPLC-fluorescence detector (HPLC-FLD) 방법과 비교하여 평가하였다. ELISA 방법에서의 AFM1의 회수율은 105% (73-124%)였고 측정의 변이계수는 6.85%로 나타났다. ELISA 방법에서의 검출한계와 정량한계는 각각 0.20 pg/ml과 0.62 pg/ml로 조사되었으며, 두 방법을 이용하여 측정한 요중 AFM1 농도는 상관계수 0.96의 매우 높은 상관성이 있는 것으로 확인되었다(p<0.01). 그러나, 이렇게 높은 상관성에도 불구하고, ELISA 방법을 이용한 요중 AFM1의 농도는 HPLC-FLD 방법으로 측정한 값에 비해 상대적으로 높게 나타나는 경향을 보여 ELISA를 이용한 방법이 단시간에 많은 시료를 분석할 수 있는 장점은 있으나 그 결과는 HPLC-FLD 방법을 이용해서 얻은 회귀식을 이용하여 보정을 한 후 제시할 필요가 있는 것으로 판단된다.

• 한국식품과학회지
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• 제38권2호
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• pp.176-182
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• 2006

본 연구는 축산농가에서 가축의 세균성 질병의 예방과 치료 및 성장촉진을 목적으로 주로 사용되고 있으며, 축산물과 유가공품에 잔류 가능성이 높은 sulfamethazine(SMZ)을 검출할 수 있는 직접경쟁 효소면역분석법(direct competitive ELISA)의 개발과 이를원유 및 시판우유 분석에 이용하는 것을 목적으로 하였다. 면역항원의 합성은 hemiglutarate와 hemisuccinate를 이용하여 SMZ-hemiglutarate(SMZ-HG)와 SMZ-hemisuccinate(SMZ-HS) hapten을 유도한 후 단백질 KLH와 STI를 결합시켜 마우스 면역에 사용하였고, SMZ-HG-KLH를 이용하여 면역한 마우스에서 가장 높은항체 생성정도를 나타내었다. 세포융합과 cloning을 실시하여 총15종의 hybridoma cell line을 확보하였고 그 중 가장 경쟁성이 뛰어나고 민감도가 높은 1H11-5 hybridoma를 선택하고 대량 생산하였다. 생산된 항체는 sulfamethazine에만 54%의 교차 반응성을 나타내었고, 다른 설파계 항생제와는 반응을 하지 않는 특이성이 높은 항체로 확인되었고 이를 이용하여 확립된 direct competitive ELISA법은 검출범위가 0.1-100 ppb 수준으로 기존에 보고된 ELISA보다 민감도가 높았다. 민감도와 특이성이 높은 direct competitive ELISA를 이용하여 원유와 시판우유를 분석하기 위한 전처리법을 확립하여 회수율을 확인한 결과 원유의 경우 82-121%까지 회수가 되는 것으로 나타났으며, 시판유의 경우는 82-97%까지 회수가 되는 것으로 나타나 우유 샘플 중에 미량 잔류하는 SMZ를 신속하고 정확하게 분석할 수 있을 것으로 사료되었다. 이러한 연구결과를 종합해 볼 때 확립된 direct competitive ELISA법은 우유 시료뿐만 아니라 모든 축산물에 잔류 가능성이 높은 SMZ의 분석을 신속하고 정확하게 분석이 가능할 것으로 사료되었고 확립된 direct competitive ELISA법의 안정화 조건을 확립하고 응용한다면 외국으로부터 생산 및 수입되는 ELISA kit을 대체할 수 있는 저가형 국산화 ELISA kit의 상용화가 가능할것으로 사료되었다.

• 한국환경보건학회지
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• 제22권1호
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• pp.51-56
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• 1996

In order to detect the T-2 toxin accumulation in the animal tissues, T-2 toxin, produced by Fusarium sporotrichioides M-1-1, was injected to mouse by 0, 1 and 2 mg per kilogram of body weight, respectively, and T-2 toxin extracted from serum and organs were analyzed by the indirected competitive ELISA. The indirect competitive ELISA established in the laboratory can be check less than 0.1 ppb level of T-2 toxin and average recovery of T-2 toxin spiked was 80~113% in animal samples such as serum, liver and kidney. After 6 weeks of treatment with 2 mg of T-2 toxin per kg body weight, T-2 toxin was accumulated in serum (133.0 ng/ml), liver(1.4 ng/g) and kidney(14.3 ng/g) of mouse injected with 2 mg of toxin per kg body weight.

• 한국식품위생안전성학회지
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• 제13권4호
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• pp.419-424
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• 1998

Zearalenone 검출을 위하여 Z-M-26 hybridoma cell을 mouse의 복강에 투여한 후 생산, 정제한 항체와 합성한 Zearalenone-oxime-OVA conjugate를 이용하여 ELISA법을 확립하였다. Carbonyl buffer로 희석한 Zearalenone-oxime-OVA conjugate를 4$^{\circ}C$에서 하룻밤 coating 하고 1% BSA용액으로 하룻밤 blocking한 다음, 1,000배 희석한 항체를 Zearalenone 또는 시료와 혼합하여 하룻밤 반응시키는 것이 효과적이었다. 또한 2차 항체와 기질용액의 반응시간은 각각 1시간, 30분이 적당하였고, 발색된 반응액 450nm에서 측정하였다. 이 분석법의 결과 0.1~100 ppb의 Zearalenone이 측정 가능하였으며, 본 실험에서 확립한 indirect competitive ELISA법은 농산물 중 Zearalenone 분석에 효과적으로 활용할 수 있으리라 생각된다.

• 대한수의학회지
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• 제40권3호
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• pp.601-609
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• 2000

An enzyme linked immunosorbent assay (ELISA) was developed to screen residues of sulfadimethoxine (SDM) in edible animal products. An indirect competitive ELISA was allowed to compete with rabbit anti-SDM for binding to a limited amount of SDM-gelatin conjugate and SDM in serum samples. Sera was diluted 20 times with phosphate buffered saline (PBS) and boiled for 5 minutes to destruct immunoglobulins of serum. Detection limit of this competitive ELISA for SDM was 0.1 ppb or less. Among eight sulfonamide analogues tested for specifity, only sulfamonomethoxine showed significant cross-reaction in the assay. The EC-50 value for sulfamonomethoxine was 3.5 ppm. Recovery of SDM in spiked serum samples between 100 ppb and 500 ppb ranged from 110.7% to 128.9%.

• Environmental Analysis Health and Toxicology
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• 제11권1_2호
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• pp.11-17
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• 1996

The purpose of this experiment is to estabilish the sensitive and specific ELISA (enzyme linked immunosorbent assay) system for the detection of fish metallothionein (MT). Silver carp were injected with CA of 1-8mg/kg body wt. 4 times during 10 days. Silver carp was very tolerant species to CA. Cd induced MT in liver was seperated and purified by gel filtration chromatography and ion exchange chromatography and identified by spectrophotometry, native gel electrophoresis and western blot analysis. The rabbit antiserum was produced by immunizing rabbit with lyophilized MT, and the competitive ELISA system was estabilished for the detection of fish MT. In the present ELISA system, the detection limit was about 33 ng/ml. When this ELISA system was employed to determine the MT level in the supernatant sample of fish liver homogenate, the reaction curve showed a good parallel corelationship with the calibration curve over a certain dilution range. The results indicate that the competitive ELISA can be a useful tool for the detection of fish MT in the toxicological study and the evaluation of water pollution.

• 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.

• 한국동물위생학회지
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• 제34권2호
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• pp.153-157
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• 2011

In order to investigate serum antibodies for detection of brucellosis in pigs, a total of 1208 sera were tested by Rose Bengal test (RBT), the standard tube agglutination test (STAT) and competitive ELISA (cELISA). The sera were collected from pigs of Gyeonggi, Chungnam, Chungbuk, Jeonnam and Jeonbuk, provinces during the period 2002 to 2004. All the sera were screened by RBT, and were confirmed by STAT and cELISA. Among 1208 sera, 26 sera (2.2%) were positive in screening test. All the 26 positive sera were positive by STAT, while all the sera were negative by cELISA. On the basis of this study, farmed pigs may be exposed to Brucella species. Furthermore, these results suggest that establishment of diagnoses for detection of porcine brucellosis is necessary.