• Title/Summary/Keyword: indirect competitive ELISA

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Establishment of Indirect Competitive ELISA for the Detection of Zearalenone Produced by Fusarium sp. (Fusarium속이 생성하는 zearalenone 측정을 위한 Indirect Competitive ELISA의 확립)

  • 강성조;정덕화
    • Journal of Food Hygiene and Safety
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    • v.13 no.4
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    • pp.419-424
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    • 1998
  • An enzyme-linked immunosorbent assay (ELISA) was established for the detection of zearalenone by using monoclonal antibodies produced by Z-M-26 hybridoma cells when injected into a mouse and zearalenone-oxime-OV A conjugate. Zearalenone-oxime-OV A conjugates were diluted with carbonyl buffer, coated to 96 well microtiter plates at $4^{\circ}C$ overnight and blocked with 1% BSA overnight. One thousand times diluted antibody solution together with standard zearalenone or sample was added to 96-well microtiter plates and stood overnight. A secondary antibody conjugated with HRP was added and an hour later, enzyme substrate (TMBZ) solution was added for color develpment. Mter 30 minutes, coloring reaction was terminated by adding 2 N $H_2S0_4$ and the O.D. was measured at 450 nm. Detection range of this method was about 0.1~100 ppb. The established indirect competitive ELISA method was suitable for a rapid and effective analysis of zearalenone in agricultural products.

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The Detection of T-2 toxin in Serum and Organ of Mouse by ELISA (ELISA법에 의한 mouse의 혈청 및 조직중의 T-2 toxin의 검색)

  • 김동술;송재영;정덕화
    • Journal of Environmental Health Sciences
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    • v.22 no.1
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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.

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Development of an Enzyme-Linked Immunosorbent Assay for the Iletection of Aflatoxin $B_1$ (Aflatoxin $B_1$의 검출을 위한 효소면역측정법의 개발)

  • 손동화;박애란;서병철;김진철;이인원;남영중;허우덕
    • Microbiology and Biotechnology Letters
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    • v.20 no.2
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    • pp.225-232
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    • 1992
  • In order to develop an enzyme-linked immunosorbent assay(ELISA) for detecting aflatoxin $B_1(AFB_1)$, we produced and purified antibodies, thereafter established and evaluated methods of direct and indirect competitive ELISA. Anti-AFB, antisera, produced by immunizing rabbits with $AFB_1$-1-(0-carboxymethy1)oxime-bovine serum albumin conjugate ($AFB_1$-BSA), were removed of anti-BSA antibodies by quantitative precipitation reaction and further purified by ammonium sulfate precipitation and DEAE-Sephadex A-50 ion exchange chromatography. Purified IgG fractions were used as anti-$AFB_1$ antibodies. The antibodies, whose titer was deterrnined extremely high above $2 \times 10^6$, showed low cross-reactivity of 3~34% against $AFB_1$ analogues such as G2, B2, and GI. From the standard curves of direct and indirect competitive ELISA for AFBI, the detection ranges were found 0.2~20 and 1~10, 000 ng/ml(ppb) respectively. In their sensitivity, stability, simplicity, and rapidity, the direct method was more suitable than the indirect method for practical use.

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Screening of Ochratoxin A Producing Fungi from Greenhouse Horticulture (시설원예산물로부터 Ochratoxin A 생성 곰팡이의 검색)

  • Kang, Sung-Jo;Park, Bong-Jung;Lee, Jong-Ok;Kang, Jin-Soon;Chung, Duck-Hwa
    • Korean Journal of Food Science and Technology
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    • v.30 no.6
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    • pp.1415-1419
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    • 1998
  • In order to evaluate the safety of greenhouse horticultures, a large number sample sources were collected, and the fungi of Aspergillus sp. and Penicillium sp. were isolated from them. Indirect competitive ELISA method and high performance liquid chromatography (HPLC) were applied to confirm the ochratoxin A producing abilities of isolated strains. One hundred ninety two sample sources including soil, pepper, strawberry and water mellon were collected for fungi isolation from western Gyeongnam, Andong and Gyeongbok. One hundred forty two strains of Aspergillus sp. and one hundred fifty three strains of Penicillium sp. were isolated respectively from them. The isolated fungi were tested for the production of ochratoxin A by ELISA. After culture of them on the modified sucrose low salt medium at $28^{\circ}C$ for 15 days, we found that five strains of Penicillium sp. produced ochratoxin A at the levels of $0.084{\sim}2.128\;{\mu}g/mL$. Among them, #129-2 strain isolated from water melon, showed the highest level of ochratoxin A as $2.128\;{\mu}g/mL$ broth. However, all of isolated Aspergillus sp. didn't produce ochratoxin A. When we compared the results of ELISA method with HPLC method, ochratoxin A production of each isolated strains showed very similar levels.

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Development of an ELISA kit for the detection of residual sulfadimethoxine in edible animal products (축산물 잔류 sulfadimethoxine 검출용 ELISA kit 개발)

  • Kim, Woo-taek;Kim, Seong-hee;Yoon, Byoung-su;Lim, Yoon-kyu
    • Korean Journal of Veterinary Research
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    • v.40 no.3
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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%.

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Development of Competitive Indirect ELISA for the Detection of Buckwheat in Processed Foods (가공식품 중 메밀 검출을 위한 경합 ELISA의 개발)

  • Back, Su-Yeon;Do, Jeong-Ryong;Shon, Dong-Hwa
    • Korean Journal of Food Science and Technology
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    • v.46 no.3
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    • pp.269-275
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    • 2014
  • We developed a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for determining the buckwheat content in processed foods by using rabbit polyclonal antibodies against buckwheat proteins (BWP). The detection limit of this assay was $0.05-100{\mu}g/mL$. The cross-reactivities of the anti-BWP antibodies toward BWP, buckwheat flour, whole buckwheat, and cereals (wheat flour, whole wheat, black bean, mung bean, red bean, brack rice, brown rice, glutinous rice, white rice, millet, African millet, nonglutinous millet, adlay, and rye) were 100, 17.9, 11.8, and 0%, respectively. Thus, the antibodies were found to be specific for buckwheat only. When buckwheat flour was heated for 30 min, the mean assay recoveries of BWP were 83.0% at $60-90^{\circ}C$ and 44.5% at $100^{\circ}C$. The spike test showed that the mean assay recoveries of buckwheat from raw noodle, boiled noodle, starch gel, and cereal flour were 99.1, 98.6, 81.1, and 104%, respectively. For the 22 commercial items tested, the qualitative coincidence ratio of assay result and the corresponding value indicated on the item's package label was 100%. However, the average quantitative coincidence ratios from 12 commercial items were 31.6%. Thus, the results suggest that ciELISA is an efficient tool to detect buckwheat in processed foods.

Development of Enzyme-Linked Immunosorbent Assay for Glyphosate-Tolerant Soybeans (제초제내성 유전자재조합 콩의 검출을 위한 면역분석법 개발)

  • Kwak, Bo-Yeon;Ko, Seung-Hee;Park, Chun-Wuk;Son, Dae-Yeul;Shon, Dong-Hwa
    • Korean Journal of Food Science and Technology
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    • v.35 no.3
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    • pp.366-372
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    • 2003
  • Enzyme-linked immunosorbent assay (ELISA) for assaying the 5-enolpyruvyshikimate-3-phosphate synthase from Agribacterium sp. CP4 (CP4 EPSPS) in genetically modified soybeans was developed. Polyclonal and monoclonal antibodies (Pab, Mab) specific to the CP4 EPSPS were produced. When using the Pab, the detection limit of sandwich ELISA toward CP4 EPSPS (0.03 ${\mu}g/mL$) was better than that of competitive indirect ELISA(ciELISA) (1 ${\mu}g/mL$). It was found that 2 of 3 monoclonal antibodies, Mab1 and Mab2, recognized the same antigenic determinant on CP4 EPSPS, but Mab3 recognized different antigenic determinant when competitive ELISA was performed using the Mabs. On the other hand, when the sensitivity of sandwich ELISA using combination of Pab and/or Mabs was determined, the sandiwich ELISA using Mab2 as a capture antibody and Pab-HRP as a secondary antibody showed the lowest detection limit of CP4 EPSPS (0.02 ${\mu}g/mL$). The sandwich ELISA developed in this study could be applied to detect glyphosate-tolerant soybeans.

Assessment of Allergenicity of Fermented Dairy Products by Immunoassay (면역 분석법을 이용한 발효 유제품의 알레르기원성 평가)

  • Kang, Kun-Og
    • The Korean Journal of Food And Nutrition
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    • v.19 no.3
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    • pp.296-300
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    • 2006
  • Immunoblotting and competitive indirect enzyme-liked immunosorbent assay(Ci-ELISA) was used for detection of ${\beta}$-lactoglobulin(BLG) in dairy products, such as milk, dried milk and fermented milk. In immunoblotting, human IgE weakly recognized proteins of fermented milk, but still responded to dried milk even though become weak. Rabbit polyclonal antibody to BLG, used as a model of antigen, and milk allergic patients' IgE was used in the ELISA. Reactivities of Abs were the highest in market milk. BLG in fermented milk was detected in a low content. This result indicates the fermented milk have the lowest BLG content and could be used as hypo-allergenic food for milk-allergic individual.

Analysis of Microtoxins in the Nakdong River Watershed

  • Park, Jung-Min;Lee, Jae-Jung;Hwang, Dong-Jin;Yang, Sang-Yong
    • Proceedings of the Korean Environmental Sciences Society Conference
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    • 2003.11a
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    • pp.99-102
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    • 2003
  • The different methods such as HPLC, indirect- and direct-ELISA were employed for the analysis of microtoxins and the results of each method were compared in terms of the detection limit and accurary. Three toxins, microcystin-RR, -LR and -YR were clearly separated by HPLC using 0.05 M methanol and phosphate buffer used as a solvent system. The calibration curves for the toxins were linear in the range of 5 ng to 50 ng. The standard curves for the immunoassay of microcystin obtained by direct and indirect ELISA are compared. The linear responses of inhibitions of binding by microcystin in the direct and indirect competitive ELISA were in the range of 10 ng to 1000 ng and 50 pg to 160 pg, respectively. Distribution of microtoxins at 11 sites in the Nakdong river and several lakes in Korea was also studied. The most dominant microcystin variant in the test sites was found to be microcystin-RR.

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Development of Immunoassay Systems for the Assay of Soy Protein in Meat Products; Antibody Production and Properties for the Assay of Soy Protein (육제품에 첨가된 대두단백 정량을 위한 면역분석법 개발에 관한 연구: 대두단백 정량을 위한 항체생산 및 특성조사)

  • Kim, Cheon-Jei;Kim, Jong-Bae;Kim, Byung-Cheol;Lee, Seoung-Bae;Jung, Sung-Won;Shin, Hyun-Kil;Ko, Won-Sick
    • Korean Journal of Food Science and Technology
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    • v.24 no.3
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    • pp.204-208
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    • 1992
  • This study was carried out to develop a practical enzyme-linked immunosorbent assay(ELISA) for the determination of soy protein in processed meat products as a preliminary study. The titer of antiserum raised in rabbit by injection of SDS-treated whole buffer extract(WBE) from isolates soy protein(ISP) was above 1:10,000 in indirect ELISA. When the SDS concentration was higher than 0.03% the antibody-antigen reaction was inhibited significantly. However, the antibody-antigen reaction inhibition was not observed when the SDS concentration was less than 0.02%. The antibodies used in this experiment also reacted with renatured antigen after removing SDS by dialysis, though not better than with SDS-denatured antigen(immunogen). The calibration curve with $100\;{\mu}g/100\;ml$ of sensitivity was obtained in indirect competitive ELISA.

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