• 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.

• Applied Biological Chemistry
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• 제42권4호
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• pp.310-316
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• 1999

대두가공제품 중에 존재하는 Bowman-Birk protease inhibitor(BBPI) 함량을 protease 저해활성 측정 및 경합 Enzyme-Linked Immunosorbent Assay(ELISA)로 살펴보았다. 항체제조를 위한 BBPI는 ion exchange chromatography와 전기영동 후 gel slicing 방법으로 시판 soybean trypsin-chymotrypsin inhibitor로부터 순수, 분리하였다. 순수분리한 BBPI를 면역원으로 rabbit anti-BBPI antibody를 조제하였으며 단백질 농도별 titration방법으로 BBP에 비교적 선택적으로 결합하는 항체임을 확인하였다. 이를 이용한 경합 ELISA 방법으로 BBPI를 정량하기 위한 표준 정량곡선을 작성하였으며 시료용액중의 BBPI 함량이 $0.03{\sim}30\;{\mu}g/ml$ 범위일 경우에 정량적인 분석이 가능하였다. 대두품종별 chymotrypsin 저해활성은 $8,462{\sim}12,428\;U/g$이었으며 BBPI 함량은 $482{\sim}692\;mg%$ 이었다. 시판 대두 가공제품 중에서 5종의 콩나물은 건물량 기준으로 $10,695{\sim}13,249\;U/g$의 chymotrypsin 저해활성과 $529{\sim}803\;mg%$의 BBPI 함량을 나타내었으며 일부 두부제품에서도 68.9 mg%정도의 BBPI가 검출되었다. 그 밖의 두유, 된장, 고추장 및 간장 등의 대두발효식품, 탈지대두박 등에서는 chymotrypsin 저해활성 및 BBPI가 거의 검출되지 않았다.

• 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%.

• 한국식품영양학회지
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• 제19권3호
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• pp.296-300
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• 2006

시유, 분유, 발효유에 존재하는 우유알레르겐인 ${\beta}$-Lactoglobulin (BLG)를 측정하기 위해 면역 blotting 및 간접결합면역 분석법(Competitive-indirect Enzyme linked Immunosorbent Assay; Ci-ELISA)으로 분석하였다. Immunoblotting 결과, 발효유에서는 우유 알레르기 환자의 IgE와의 반응이 나타나지 않았고 분유 및 시유에서는 약하지만 반응이 나타났다. 다클론 항체 및 우유 알레르기 환자의 IgE로 이들의 BLG 함량을 정량한 결과, BLG 함량은 시유가 가장 높았으며 발효유는 매우 낮았다. 이상의 결과에서 발효유에서는 가장 낮은 BLG 함량이 측정되어 우유 알레르기 환자에 대한 저알레르기원성 식품으로 사용될 수 있을 것으로 사료된다.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.185-185
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• 2002

A murine local lymph node assay (LLNA) has been developed as an alternative test to guinea pig maximization test. The disadvantage of LLNA is the need for the use of radioactive material. In this study, we aimed to investigate the development of non-radio isotopic endpoint for local lymph node assay in Balb/c mice using Enzyme-linked immunosorbent assay (ELISA) based on Bromodeoxyuridine (BrdU) incorporation.(omitted)

• 대한수의학회지
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• 제43권1호
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• pp.129-137
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• 2003

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failures in sows and respiratory problems in piglets. The nucleocapsid(N) protein, encoded by the open reading frame 7 (ORF7) gene, is known to be the most abundant and antigenic protein in PRRS virus. Therefore, it was suggested that the N protein could be a suitable candidate for the detection of PRRS virus-specific antibodies and diagnosis of PRRS. In the present study, the ORF7 gene encoding the N protein was cloned and expressed as a fusion protein with the glutathione S-transferase (GST) in Escherichia coli. The resulting GST-N recombinant protein was used as an antigen for an indirect sandwich enzyme-linked immunosorbent assay (i-ELISA). Expressed GST-N recombinant protein was migrated at 41 kDa and reacted with ORF7-specific monoclonal antibody by Western blotting. In order to increase the specificity of the ELISA for the detection of PRRS virus-specific antibodes, an i-ELISA was developed using an anti-GST antibody as a capture antibody. The sensitivity and specificity of developed i-ELISA were 92% and 96%, respectively. Based on these results, it was suggested that the i-ELISA is a simple and rapid test for screening a large number of swine sera for the anti-PRRS virus 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.

• 한국임상수의학회지
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• 제27권4호
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• pp.402-406
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• 2010

Bovine leukemia virus (BLV) is a delta-retrovirus which causes chronic lymphocytosis in cattle. BLV infections have been divided into two groups such as enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL) according to the clinical symptoms in infected cattle. The conventional detection method of BLV was hematological procedure which is determining lymphocytosis in the suspected animals. Recently several sensitive methods were developed to detect antibody to BLV and nucleic acid of the BLV from infected cattle. In this study we have compared the difference of positive rates between agar gel immunodiffusion (AGID) and enzyme linked immunosorbent assay (ELISA) which are using for BLV antibody detection methods. The positive detection rate of ELISA test was 7.4% greater than the positive rate of AGID. The discrepancy of the positive rate between ELISA and AGID were showed in the group of age over one year old to under three year old group. The result from each test agreed very well in the group of over 5 year old cattles. The serological test is very useful method to select the infected cattle for the eradication or control of the disease in the infected herd. But it has a limit by interference of the maternal antibody from the cow of under 6 month old. This study shows that 16.2% of these ages group showed BLV gene positive by polymerase chain reaction (PCR) method. The result suggests that ELISA test need to be used with PCR to clarify misinterpretation of positive animals by antibody response due to the natural infection from maternally derived antibody in calves of under 6 months old.

• 한국동물위생학회지
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• 제43권4호
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• pp.237-244
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• 2020

Porcine transmissible gastroenteritis (TGE) has been a significant cause of economic losses in pig farming industry since 1950s. Although transmissible gastroenteritis virus (TGEV) has declined in recent years, it should not be excluded because of its characteristics; the frequency of gene mutation, the mortality in piglets, and the possibility for sudden incidence. Therefore, the herd-level monitoring of the virus is important to prevent further circulation of TGE. The aim of this study is to develop a large-scale sandwich enzyme-linked immunosorbent assay (ELISA) with high specificity to rapidly detect TGEV in feces by using monoclonal antibodies (Mabs). The TGEV specific Mabs were produced in hybridoma cells. Among the Mabs belonged to the IgG class developed by this study, the final selected 8H6, 1B7, 4G3, and 1F8 were identified to have the neutralization ability against TGEV. The sandwich ELISA was established using 8H6 as a reporter antibody and 1B7 and the reported 5C8 as a capture antibody. The developed sandwich ELISA was able to distinguish TGEV from other pathogenic diarrheal agents (porcine rotavirus, porcine reovirus, porcine epidemic diarrhea virus (PEDV), E. coli, and C. perfringens) in tissue culture as well as fecal samples. And the detection rate of TGEV in feces was 80% compared with RT-PCR. The results suggested that the developed sandwich ELISA may be useful in the herd-level monitoring for effective preventive measures due to the early diagnosis of TGEV using a large amount of samples.

• KSBB Journal
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• 제21권3호
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• pp.212-219
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• 2006

재조합 생물의약품 생산시, 오염물질의 유래는 외인성(외래성 바이러스, 마이코플라즈마, 미생물) 및 내인성(생산세포주 유래 단백질) 두 가지의 일반적인 가능성으로 존재한다. 이 중 생산세포주 유래의 불순 단백질은 환자에게 면역이상반응을 일으킬 수 있어 제품의 안정성과 사용자의 안전상의 문제가 될 수 있다. 따라서 이들 단백질의 오염 여부를 검출하는 방법의 개발이 요구된다. 상용화된 분석제품들은 일반적인 생산세포주 유래 단백질에 대한 분석은 가능하지만, 생산 방법이나 재조합 된 세포의 특성에 따라 달라질 수 있는 고유한 생산세포주 유래 단백질에 대한 분석은 적합하지 못하다. 본 연구에서는 재조합 항체의약품 생산과 정제과정에서 목적단백질의 생산 유전자를 지니지 않는 null cell mock 배양에 의해 생산세포주 유래 모든 단백질을 얻어 이에 대한 다클론항체를 제작하여 면역학적 반응을 이용하여 생산세포주 유래 단백질을 정량 할 수 있는 ELISA 방법을 개발하였다. 생산세포주 유래 단백질에 대한 다클론항체는 rabbit에서 얻었으며, 이 중 생산세포주 유래 단백질에 특이적인 항체만을 정제하였다. 또한 direct sandwich ELISA 방법 개발을 위해 항체에 발색효소(HRP)를 표지하였다. 이렇게 만들어진 항체를 이용하여 정성분석을 위한 Western blot과 정량분석을 위한 ELISA 방법을 개발하여 목적단백질의 정제과정 내에서 생산세포주 유래 단백질이 제거되는 것을 확인하였다. 또한 개발된 ELISA 방법은 ICH 규정에 따라 validation을 실시한 결과 되어 객관적 검증을 받은 결과, 특이성, 직선성, 정확성, 정밀성의 기준을 만족하며 검출한도가 10.8 ppm인 방법임을 확인하였다.