• 한국식품위생안전성학회지
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• 제13권2호
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• pp.129-134
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• 1998

Nivalenol(NIV)의 검출을 위한 효소면역측정법(ELISA)을 개발하기 위하여 tetraacetyl nivalenol(Ac4-NIV)에 대한 다클론항체를 생산하고 그 조건을 확립하였다. Ac4-NIV-hemisuccinate를 bovine serum albumin에 공유결합 시킨 Ac4-NIV-HS-BSA를 Freund's adjuvant와 함께 수차례 토끼에 피하면역하였다. 가장 높은 항체가를 나타낸 항혈청으로부터 정제한 항체와 Ac4-NIV-HS-HRP conjugate를 이용하여 직접 경합 ELISA(cdELISA)를 확립하였다. 그 표준 곡선으로부터 Ac4-NIV의 검출 범위는 10~5,000 ng/ml(ppb)임을 알 수 있었다. 특이항체의 Ac4-NIV과 acetyl T-2에 대한 반응성은 각각 100, 70%였으나, NIV, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, triacetyl deoxynivalenol, fusarenon-X, T-2에 대한 반응성은 0.1% 이하로 극히 미약하였다. NIV를 인위적으로 오염시킨 옥수수 시료를 70% acetonitrile 추출하고 acetylation 한 다음 cdELISA를 행하였을 때, 분석의 회수율은 100, 300, 1,000 ng/g(ppb)에서 각각 108, 143, and 70%(평균, 107%)로 나타났다.

• 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.

• 한국미생물·생명공학회지
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• 제25권4호
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• pp.414-419
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• 1997

In order to develop an enzyme-linked immunosorbent assay (ELISA) for deoxynivalenol (DON) in com, we produced a specific monocl- onal antibody and established ELISA conditions. After the spleen cells from mice immunized with DON-bovine serum albumin conjugate were fused with S$_{p}$2/0 myeloma cells, a hybridoma cell 3G7 producing anti-DON antibody was screened by ELISA. From the standard curve of competitive direct ELISA (cdELISA) using 3G7 monoclonal antibody and DON-HRP conjugate, the detection range of DON showed 3-3,000 ng/ml (ppb). The monoclonal antibody showed some cross-reactivities against DON analogues such as 15 acetyl-DON (110%), nivalenol (5.0%), 3 acetyl-DON (1.7%), fusarenon-x (0.72%), and T-2 (0.59%). When the cdELISA was applied to the spiked coms after extracting with 60% methanol and diluting 5- fold with washing buffer, the assay recoveries of DON were 313, 163, 106, and 88.9% (av., 168%) in the levels of 200, 600, 2,000, and 6,000ng/g, respectively. For the quantitation of DON in coms, 30 samples kept under two different storage conditions of cold and room temperature were assayed by cdELISA. The mean detection concentrations were 595 (detection range, 0-2,750) and 2,448 (detection range, 0-4,500) ppb, respectively.

• 한국식품과학회지
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• 제41권2호
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• pp.215-219
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• 2009

$AFB_1$의 대사산물 중 하나인 $AFM_1$을 사람과 돼지의 뇨에서 cdELISA를 통해 분석함으로써 간접적으로 $AFB_1$의 위해성 평가를 수행하고자 하였다. 항 $AFM_1$ 항체와 ${AFB_1}-HRP$를 이용한 cdELISA에서 $AFM_1$의 검출한계는 10 pg/mL로 나타났다. 사람의 요에 $AFM_1$을 3-100 pg/mL 농도가 되게 첨가한 후 cdELISA로 분석하였을 때 그 회수율은 117-167%(평균 139${\pm}$19.1)의 범위로 나타났다. 사람의 뇨 중 $AFM_1$의 오염량을 cdELISA로 분석한 결과 전체 172개의 시료 중 165개의 시료에서 평균 2.74${\pm}$1.89 pg/mL 농도로 검출되었다. 뇨 중 $AFM_1$의 일일배출량은 평균 3.97 ng/day으로 나타났고, $AFB_1$의 섭취량은 체내전환율 5%로 가정하였을 때, 79.4 ng/day로 추정되었다. 따라서 사람의 일일섭취추정량 (PDI)은 1.28 ng/kg bw/day로 나타났으며 돼지의 PDI는 9.2 ng/kg bw/day로 나타났다. 한편 $AFB_1$ 섭취의 위해성 평가에서 사람의 PDI는, 일일섭취감내량(TDI, 0.15 ng/kg bw/day)보다 8.5배나 높은 값을 보였지만 외국의 연구결과와 비교하였을 때 대체로 비슷하게 나타났다.

• 한국식품위생안전성학회지
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• 제18권3호
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• pp.95-100
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• 2003

된장과 고추장에 대해 cdELISA에 의한 $AFB_1$의 분석방법을 확립하고 국내에서 생산되는 재래식 및 개량식 된장과 고추자을 수거하여 $AFB_1$의 오염도를 조사하였다. 표준곡선을 작성하였을 때, 이의 검출 한계는 0.2 ng/m(ppb)이었다. 된장의 Spike test 후 회수율은 1~100 ng/g 범위에서 평균 71.5%로 나타나 이 범위에서 cdELISA에 의한 $AFB_1$의 측정이 가능함을 보여주었다. 이로부터 된장 및 고추장에서 cdELISA를 통한 $AFB_1$의 분석 방법을 확립하였다. 된장 및 고추장 시료의 $AFB_1$의 오염도는 총 30종의 고추장 시료에서는 $AFB_1$이 검출되지 않았고 총 30종의 된장 시료 중 6종에서 1.0~6.0 ng/g 수준으로 검출되었다. 이는 우리 나라의 허용 기준인 10 ng/g(ppb) 이하였다.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.686-692
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• 2004

To develop the enzyme-linked immunosorbent assay (ELISA) for the analysis of N-acetylehitooligosaccharides (NACOS) and chitooligosaccharides (COS), specific antibodies (Abs) were produced, and their properties were compared. N-acetylehitohexaose (NACOS6), chitohexaose (COS6), and COS mixture (COSM) conjugated to bovine serum albumin (BSA) were used to immunize rabbits. By the use of specific Abs and NACOS6-horseradish peroxidase (HRP), COS6-HRP, and COSM-HRP conjugates, competitive direct ELISA (cdELISA) was developed. The detection limits of NACOS6 by the anti-NACOS6 Ab and COS6 by the anti-COS6 and the anti-COSM Abs in the cdELISAs were about 0.2, 2, ana 2 ng/ml (ppb), respectively. In the cdELISA, the anti-NACOS6 Ab was found to recognize NACOS3-NACOS6, but not N-acetyl-D-glucosamine (GlcNAc), NACOS2, and COSs; the anti-COS6 Ab recognized COS2-COS6 and COSM, but not glucosamine (GlcN) and NACOSs. The recognition pattern of the anti-COSM Ab was almost the same as that of the anti- COS6 Ab, except that the former recognized COS2 and COS3 slightly better than the latter.

• 한국미생물·생명공학회지
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• 제24권5호
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• pp.630-635
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• 1996

For a survey of the occurrence of aflatoxin M$_{1}$ (AFM$_{1}$) in domestic cow's milk, we developed an enzyme-linked immunosorbent assay (ELISA) system, and quantitated the toxin in cow's milk. In order to produce specific antibodies AFM, conjugated to bovine serum albumin (AFM$_{1}$-BSA) and Freund's adjuvant were immunized subcutaneously to rabbits. By use of the antiserum showing the highest titer and AFB$_{1}$-HRP conjugate, we established a competitive direct ELISA (cdELISA) for AFM$_{1}$, whose detection limit was 0.003 ppb. The cross-reactivities of the antiserum against aflatoxin M$_{1}$ M$_{2}$, B$_{1}$, B$_{2}$, G$_{1}$, G$_{2}$, B$_{2a}$, and G$_{2a}$, were 100, 29.9, 25.0, 2.7, 13.0, 0.65, 0, and 0%, respectively. When the cdELISA was applied to the cow's milk spiked with AFM$_{1}$ and followed by cleanup with C$_{18}$ cartridge, the mean recovery of the assay was 104% (mean of CV, 6.4%) in the final concentration of 0.01-1 ppb (10-1, 000 ppt). When cow's milk samples gathered from markets and farms were assayed by the cdELISA, the mean concentration and SD of AFM$_{1}$ was 80.4 $\pm$ 55.0 ppt (n=64; range, 5.6-280 ppt).

• 대한본초학회지
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• 제24권3호
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• pp.29-37
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• 2009

Objectives : The present study was carried out to investigate the effect of Trichosanthis Radix extract (TRE) on the proliferation and activation of eosinophils which were prepared from lung cells of asthma-induced mice by ovalbumin (OVA) treatment. Methods : C57BL/6 mouse was exposed to OVA three times a week for 6 weeks. The mouse lung tissues were dissected out, chopped and dossiciated with collagenase (1 $\mu$g/ml). Eosinophils were activated by rmIL-3/rmIL-5 co-treatments. The lung cells were treated with TRE, incubated for 48 hr at 37$^{\circ}C$, and analyzed by flow cytometer, ELISA and RT-PCR methods Results : To measure cytotoxicity, mouse lung fibroblast cells (mLFCs) were pretreated with various concentrations of TRE. TRE at 100 $\mu$g/ml, the highest concentration, examined did not have any cytotoxic effects on mLFCs. In FACS analysis, number of granulocyte/lymphocyte, CD3e-/CCR3+, CD3e+/CD69+, CD4+/CD8+ T cells in asthma-induced lung cells were significantly decreased by TRE treatment compared to the control group. But CD4+/CD25+ T cells were not examined significant change in lung cells treated with TRE. In ELISA analysis, production levels of IL-3, IL-5, IL-13 and histamine in asthma-induced lung cells, which were induced by rIL-3 plus rmIL-5 co-treatment, were significantly decreased by TRE treatment. Conclusions : The present data suggested that Trichosanthis Radix on the inhibition of parameters associated with asthma responses in eosinpophils, and thus implicate the possibility for the clinical application of Trichosanthis Radix.

• 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.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.