• 한국식품위생안전성학회지
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• 제33권3호
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• pp.185-192
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• 2018

본 연구에서는 수산가공품 중 고등어 어육을 신속하게 검출하기 위하여 고등어 어육 중 열 안정-수용성 단백질에 특이한 단클론성 항체(3A5-2)를 이용하여 indirect ELISA 법을 개발하였다. Indirect ELISA을 개발하기에 앞서 먼저 시료 전처리는 이전 연구의 결과를 바탕으로 진행되었다. 이전 연구에서는 3A5-2 항체가 37 kDa 부근의 열 안정-수용성 단백질과 반응하였고, 0.05 M carbonate buffer로 추출하였을 때 흡광도가 가장 두드러지게 증가한 것을 확인하였다. 따라서 indirect ELISA의 시료 전처리는 0.05 M carbonate buffer를 이용한 열처리 추출법으로 추출한 후 0.05 M PBS로 희석하는 것으로 확립하였고, indirect ELISA 법을 최적화하였다. 개발된 indirect ELISA법에 실험실에서 임의로 열처리한 고등어 샘플을 적용한 결과 indirect ELISA법은 0.001% (0% 흡광도 표준편차 양의 값: $0.0003{\times}3$)의 검출한계를 확인하였으며, 꽁치 중 고등어는 0.002%(0% 흡광도 표준편차 양의 값: $0.0006{\times}3$)까지 검출이 가능하였다. 또한 조리($100^{\circ}C$, 30분)와 멸균($121^{\circ}C$, 30분) 처리된 고등어의 검출이 가능한 것으로 확인되었고, 멸균된 제품에서도 고등어에 특이적으로 반응하여 고등어의 혼입여부 판별도 가능한 것으로 판단되었다. 시판되는 고등어 가공품에 대해서는 양성결과를 나타내었고 다른 수산물에서는 음성으로 판단되어 개발된 분석법은 가공품에 혼입될 수 있는 알레르겐인 고등어를 보다 신속하고 민감하게 분석할 수 있고, 점차 가격이 증가하고 있는 고등어의 혼입여부를 확인할 수 있는 분석 도구로서 활용이 가능할 것으로 판단된다.

• 한국축산식품학회지
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• 제24권2호
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• pp.182-188
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• 2004

Lactoperoxidase(LPO)를 농도를 측정하기 위한 ELISA을 개발하기 위해 LPO로 면역시킨 갈색 산란계의 계란에서 형성된 anti-LPO IgY 항체를 분리 정제하고, 분리된 anti-LPO IgY 항체의 특이성을 ELISA 와 double immunodiffusion 방법으로 조사한 후 indirect ELISA 방법을 이용한 표준곡선을 만들었다. 분리 정제된 anti-LPO IgY항체의 titer는 1:520,000이며, ELISA와 double immunodiffusion 방법 모두에서 $\alpha$-lactalbumin, $\beta$-lactoglobulin, casein 및 lysozyme하고는 교차반응을 하지 않고 LPO만 높은 특이성을 갖는 항체로 나타났다. Indirect ELISA방법에서 LPO의 coating 농도는 0.25 $\mu\textrm{g}$/mL이며 anti-LPO IgY 최적 희석배수는 1:8,000으로 나타났다. Indirect ELISA 방법으로 LPO를 측정할 수 있는 표준곡선에서 민감도의 범위는 0.0l-l $\mu\textrm{g}$/mL로 나타났다.

• 대한수의학회지
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• 제52권1호
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• pp.25-31
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• 2012

Brucella (B.) canis is mainly transmitted by direct or indirect contact with aborted fetuses and placenta. It's also known to be able to infect human, which likely results in providing veterinarians and companion animal owners for infectious risk. To develop diagnostic ELISA, we cloned and expressed rp1L gene of B. canis, which encodes the ribosomal protein L7/L12. Using this purified recombinant protein, indirect-ELISA (iELISA) was evaluated using 78 positive and 44 negative sera. The sensitivity and the specificity of iELISA were 94% and 89%, respectively. The results indicated that indirect-ELISA using recombinant ribosomal protein L7/L12 may be useful for diagnosis of canine brucellosis.

• 대한수의학회지
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• 제30권2호
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• pp.187-192
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• 1990

돼지로부터 분리한 Staphylococcus hyicus subsp hyicus 489주의 protein A 존재여부와 함량을 indirect hemagglutination 및 enzyme-linked immunosorbent assay(ELISA)법으로 조사하였다. Indirect hemagglutination text에 의하여 cell-bound protein A 및 extracellular protein A 보유균은 489주 중 각각 87.7% 및 36.0%로 나타났다. ELISA법에 의한 이들 균의 protein A함량 측정에서 전균주의 extracellular protein A는 1ng/ml미만이었으며, cell-bound protein A함량은 대부분의 균주에서 1ng/ml 미만이었고 11주가 25~108ng/ml 수준이었다.

• 한국축산식품학회지
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• 제43권6호
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• pp.989-1001
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• 2023

Processed foods containing pork fat tissue to improve flavor and gain economic benefit may cause severe issues for Muslims, Jews, and vegetarians. This study aimed to develop an indirect enzyme-linked immunosorbent assay (iELISA) based on a monoclonal antibody specific to thermal stable-soluble protein in pork fat tissue and apply it to detect pork fat tissue in heat-processed (autoclave, steam, roast, and fry) beef meatballs. To develop a sensitive iELISA, the optimal sample pre-cooking time, coating conditions, primary and secondary dilution time, and various buffer systems were tested. The change in the iELISA sensitivity with different 96-well microtiter microplates was confirmed. The detection limit of iELISA performed with an appropriate microplate was 0.015% (w/w) pork fat in raw and heat-treated beef. No cross-reactions to other meats or fats were shown. These results mean that the iELISA can be used as an analytical method to detect trace amounts of pork fat mixed in beef.

• Journal of Microbiology and Biotechnology
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• 제33권9호
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• pp.1170-1178
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• 2023

Food allergy represents a severe problem for many societies, including sensitive populations, academies, health authorities, and the food industry. Peanut allergy occupies a special place in the food allergy spectrum. To prevent consumption by consumers suffering from a peanut allergy, a rapid and sensitive detection method is essential to identify unintended peanut adulteration in processed foods. In this study, we produced four monoclonal antibodies (MAbs; RO 3A1-12, PB 4C12-10, PB 5F9-23, and PB 6G4-30) specific to thermo-stable and soluble proteins (TSSPs) of peanut and developed an enzyme-linked immunosorbent assay (ELISA) based on the MAbs. Among them, PB 5F9-23 MAb was firmly bound to Ara h 1, and other MAbs strongly reacted to Ara h 3 in the Western blot analysis. An antibody cocktail solution of the MAbs was used to enhance the sensitivity of an indirect ELISA, and the limit of detection of the indirect ELISA based on the antibody cocktail solution was 1 ng/ml and improved compared to the indirect ELISA based on the single MAb (11 ng/ml). The cross-reaction analysis revealed the high specificity of developed MAbs to peanut TSSPs without cross-reaction to other food allergens, including nuts. Subsequently, analyzing processed foods by indirect ELISA, all foods labeled as containing peanuts in the product description were confirmed to be positive. The results indicate that the developed antibodies exhibit high specificity and sensitivity to peanuts and can be used as bio-receptors in immunoassays or biosensors to detect intentional or unintentional adulteration of peanuts in processed foods, particularly heat-processed foods.

• 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.

• 한국가축번식학회지
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• 제14권2호
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• pp.107-114
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• 1990

These experiments were undertaken as a basic study to develop immunocontraceptive vaccine and to understand the role of zona pellucidae in early fertilization process by identifying the monoclonal and polyclonal antibody to porcine zona pellucidae and polyclonal antibody to mouse zona pellucidae by indirect ELISA and indirect immunofluorescence test. The results obtained in these experiments were summarized as follows : 1. The titer of the antibodies to zona was determined by indirect ELISA using solubilized porcine zona coated plates. Both monoclonal and rabbit polyclonal antibodies showed very high titers ; O.D at 1 : 12,800 dilution of antibodies was still significantly higher than that of non-immunized control serum. Rabbit anti-mouse zona pellucidae sera also reacted with porcine zona pellucidae. 2. By indirect immunofluorescence test strong fluorescences were observed on the egg treated with homologous and heterologous rabbit polyclonal antibodies and FITC lablled 2nd antibodies and found to crossreact strongly with the eggs from the pig and mouse. While weaken fluorescences were observed on the eggs treated with monoclonal antibodies.

• 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.

• The Plant Pathology Journal
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• 제20권4호
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• pp.252-257
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• 2004

We developed a polyclonal antibody based-ELISA system to monitor inocula accurately and rapidly before onset of anthracnose on soybean sprouts. Titer of mouse antisera against conidia of Colletotrichum gloeosporioides, determined by indirect ELISA, was high enough to be detectable up to ${\times}$25,600 dilutions. Both PAb1 and PAb2 had the highest level of reactivity to Colletotrichum gloeosporioides. Absorbance readings exceeded 0.15. Sensitivity of PAb to C. gloeosporioides was precise enough to detect spore concentration as low as 500 conidia/well by indirect ELISA. Both antibodies are very sensitive and highly specific to the target pathogen Colletotrichum gloeosporioides, apparently discriminating other unrelated pathogen, or epiphytes. This kit fulfills the requirements far detecting inocula before infection and onset of anthracnose. Our ELISA system should also be feasible to detect C. acutatum (Mungbean sprouts rot) and G. cingulata (C. gleosporioides), (apple, pepper). It was remarkable that absorbance value was not reduced even after 4 consecutive washings (Fig.4), suggesting that antigenic determinants are on the surface of conidia. Antigenic determinant was characterized by heating and enzyme treatment: Both PAb1 and PAb2 bind to protein epitope that does not contain residue of amino acid, arginine, and Iysine, even though more work needs to be done.