• 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.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.225-232
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• 1992

효소면역측정법에 의한 aflatoxin $B_1(AFB_1)$의 정량법을 개발하기 위하여, 항체를 생산.정제하여 분석법을 확립하고 직접법과 간접법(direct/indirect competitive ELISA)의 특성 및 문제점을 비교. 검토하였다. Bovine serum albumin(BSA)을 carrier protein으로 한 $AFB_1$-1-(O-carboxymethyl)oxime -BSA를 토끼에 면역하여 항$AFB_1$항혈청을 생산하였다. 정량 침강반응에 의하여 항혈청으로부터 항BSA항체를 제거하고 황산암모늄 침전법 및 EDAE-Sephadex A-50 이온교환 크로마토그래피를 통하여 순도 높은 IgG항체를 정제하여 항$AFB_1$항체를 사용하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제18권10호
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• pp.1498-1504
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• 2005

Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities ($CR_{50}$) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/$B_0$ ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/$B_0$ ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life ($t_{1/2}$) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals.

• 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.

• 대한수의학회지
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• 제38권2호
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• pp.297-303
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• 1998

For the extraction and measurement of zearalenone in the corn, bean, wheat and barley contaminated with Fusarium graminearum, the zearalenone-oxime, zearalenone-oxime BSA and zearalenone monoclonal antibodies were studied to develop and apply the direct competitive enzyme linked immunosorbent assay (ELISA). The extraction range of zearalenone with the monoclonal antibodies produced in this experiment was 10ng to 500ng/g feed and the 50% inhibition value was 50ng/ml. The mean recoveries of zearalenone artificially spiked in the ground corn were 89%. The specificity of F-2 monoclonal antibody for the analogues was favorable for the direct competitive ELISA. The result of the experiment showed the zearalenone in the corn, bean, wheat and barely naturally contaminated with the mold would be suitable for extraction and measurement with the monoclonal antibodies.

• Journal of Microbiology and Biotechnology
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• 제23권1호
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• pp.69-75
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• 2013

Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration ($IC_{50}$) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.

• Applied Biological Chemistry
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• 제36권1호
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• pp.7-10
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• 1993

ELISA법을 zearalenone 생성균주 검색에 응용하였다. 먼저 zearalenone에 대한 항체를 500배 희석한 후 microtiter well에 $125\;{\mu}l$씩 주입하여 $40^{\circ}C$에서 overnight시켜 coating하고, 시료용액과 enzyme을 $37^{\circ}C$에서 30분간 반응시켰다. 배양후 washing buffer로 6회 세척한 다음 plate에 2,2'-azino-di-3-ethyl-benzthiazoline sulfonic acid(ABTS) 용액을 $100\;{\mu}l$씩 첨가하여 15분간 발색시킨 다음 반응 정지액 $100\;{\mu}l$씩을 가해 반응을 정지시키고 ELISA Reader로서 흡광도(410 nm)를 측정하였다. 그 결과 zearalenone 생성이 확인된 19 균주 중 분리균 R-5, C-46 및 S-134가 50 ng/ml 이상의 zearalenone을 생성하였다.

• Toxicological Research
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• 제27권2호
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• pp.125-131
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• 2011

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1-carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with ${\lambda}$-type light chains. The $IC_{50}s$ of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml ($R^2$ > 0.99) and from 1 to 100 ng/ml ($R^2$ > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

• 한국식품영양과학회지
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• 제27권6호
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• pp.1152-1159
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• 1998

In order to develop more rapid and reproducible analysis of biotin known as vitamin H, attempts were made to establish the condition for enzyme linked immunosorbent assay(ELISA) compared with traditional microbiological assay(MBA). Antibiotin and antiserum were obtained from the immunized rabbits injected with emulsion of biotin KLH conjugate and Freund's adjuvant. The antiserum showed cross reactivity on biocytin, a derivative of biotin, which is converted to biotin in intestine, at the rate of 177%(median inhibitory concentration(IC50)=12.58ppb), but not on other derivatives such as desthiobiotin, diaminobiotin and 2 imino biotin. Specific antibody for biotin was purified from the antiserum through protein A column and desalting column. The conditions of competitive direct ELISA (cdELISA) were established. Detection range of biotin concentration by cdELISA was 0.01∼300ng/ ml(ppb). In the spike test with milk, fruit flake and pine carrot juice, the correlation coefficient between two methods of MBA and ELISA was reliably consistent at the value of r=0.992. But detection of biotin by microbiological assay(MBA) was rather restricted in range and nonspecific. Detection range of biotin by MBA was 0.1∼0.5ng/ml(ppb). It showed cross reactivities on biocytin and desthiobiotin at the rate of 80.1% and 66.7%, respectively. In conclusion, ELISA revealed a significant improvement compared with MBA for the biotin detection in terms of sensitivity, detection range and cross reactivity. In addition, a variety of samples could be analyzed rapidly and conveniently at one time by using ELISA. These results strongly suggest that the ELISA is very promising for the practical application to detect biotin contents in a wide range of food stuffs.

• 대한수의학회지
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• 제37권3호
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• pp.613-618
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• 1997

곰팡이에 오염된 저질사료에서 T-2 독소의 존재확인 및 양을 측정하기 위한 Direct Competitive Enzyme Linked Immunosorbent Assay(ELISA)의 Kits를 개발하기 위하여 T-2HS, T-2HS-BSA, T-2HS-HRP 및 T-2 단크론 항체 동을 개발하고저 연구하여 좋은 결과를 얻었다. 분말 옥수수내에 인위적으로 혼합된 T-2 독소의 평균회수율은 83%이었으며, T-2 독소의 추출가능범위는 60ng에서 $6{\mu}g$이었다. 분말옥수수에서 인위적으로 혼합된 T-2 독소의 회수결과에 의하면 이 연구는 T-2 독소의 존재를 확인하기에 적합하고, T-2 독소의 양을 측정하기 위한각종 ELISA Kits 제조의 기틀이 마련되었다.