• Title, Summary, Keyword: ELISA

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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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The Study on the Establishment of Specific ELISA for the Detection of Fish Metallothionein (어류 Metallothionein에 대한 특이적 면역 효소 측정법의 확립에 관한 연구)

  • 황갑수
    • Environmental health and toxicology
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    • v.11 no.1_2
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    • pp.11-17
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    • 1996
  • The purpose of this experiment is to estabilish the sensitive and specific ELISA (enzyme linked immunosorbent assay) system for the detection of fish metallothionein (MT). Silver carp were injected with CA of 1-8mg/kg body wt. 4 times during 10 days. Silver carp was very tolerant species to CA. Cd induced MT in liver was seperated and purified by gel filtration chromatography and ion exchange chromatography and identified by spectrophotometry, native gel electrophoresis and western blot analysis. The rabbit antiserum was produced by immunizing rabbit with lyophilized MT, and the competitive ELISA system was estabilished for the detection of fish MT. In the present ELISA system, the detection limit was about 33 ng/ml. When this ELISA system was employed to determine the MT level in the supernatant sample of fish liver homogenate, the reaction curve showed a good parallel corelationship with the calibration curve over a certain dilution range. The results indicate that the competitive ELISA can be a useful tool for the detection of fish MT in the toxicological study and the evaluation of water pollution.

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ELISA Validation for anti-PA Antibody Titer Measurements (항-보호항원 항체의 역가 측정을 위한 효소면역측정법 밸리데이션)

  • Kim, Yu-Gene
    • Journal of the Korea Institute of Military Science and Technology
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    • v.13 no.3
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    • pp.478-485
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    • 2010
  • The vaccine is biological pretreatment that improves immunity to a particular disease. We can get immunity from producing antibody with injection antigen which has ability to defense against the disease. The ELISA is the most widely used method to measure antibody titer. We have developed and performed validation of ELISA according to the guideline of KFDA and ICH. In this paper, we have verified ELISA method is an excellent method to measure the titer of anti-PA antibody. We have constructed recombinant protective antigen among anthrax toxins and used as antigen of ELISA. In this validation, we have evaluated precision (repeatability, interlaboratory precision), specificity, linearity(range) and LOD, which are validation articles suggested by guideline. Inter-person precision was replaced with inter-laboratory precision. From the results, we have confirmed high precision in all experiments with CV under 20%.

Quantitative Analysis of Phosphinothricin-N-acetyltransferase in Genetically Modified Herbicide Tolerant Pepper by an Enzyme-Linked Immunosorbent Assay

  • Shim, Youn-Young;Shin, Weon-Sun;Moon, Gi-Seong;Kim, Kyung-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.17 no.4
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    • pp.681-684
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    • 2007
  • An immunoassay method was developed to quantitatively detect phosphinothricin-N-acetyltransferase (PAT) encoded by the Bialaphos resistance (bar) gene in genetically modified (GM) pepper. The histidine-tagged PAT was overexpressed in Escherichia coli M15 (pQE3l-bar) and efficiently purified by $Ni^{2+}$ affinity chromatography. A developed sandwich enzyme-linked immunosorbent assay (S-ELISA) method (detection limit: $0.01{\mu}g/ml$) was 100-fold more sensitive than a competitive indirect ELISA (CI-ELISA) method or Western blot analysis in detecting the recombinant PAT. In real sample tests, PAT in genetically modified herbicide-tolerant (GMHT) peppers was successfully quantified [$4.9{\pm}0.4{\mu}g/g$ of sample (n=6)] by the S-ELISA method. The S-ELISA method developed here could be applied to other GMHT crops and vegetables producing PAT.

An Enzyme-Linked Immunosorbent Assay for Detection of Pantothenic Acid (판토텐산의 분석을 위한 효소면역측정법)

  • Shon, Dong-Hwa;Park, Youn-Sick;Bae, Gun-Won
    • Korean Journal of Food Science and Technology
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    • v.32 no.5
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    • pp.1009-1014
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    • 2000
  • In order to detect pantothenic acid (PA), conditions for enzyme-linked immunosorbent assay (ELISA) were established. Anti-PA-BSA antibody was produced from rabbits immunized with PA-bovine serum albumin (BSA) conjugates which were prepared by the bromoacetyl chloride [Bc] method (PA-BSA[Bc]) and by the periodate oxidation [Po] method (PA-BSA[Po]). PA-BSA[Bc] and PA-BSA[Po] was used as a coating antigen for competitive indirect(ci)ELISA. The Anti-PA-BSA[Po] antibody on ciELISA showed no competitive reaction. The detection limit of PA by ciELISA using Anti-PA-BSA[Bc] antibody was 1 ppm. The Anti-PA-BSA[Bc] antibody showed little cross-reactivity to PA derivatives such as pantoyllactone, pantetheine, pantothenyl alcohol, and acetyl CoA. The detection limit of PA by microbiological assay (MBA) was 10 ppb. Assay recoveries of PA in egg, cow's liver, and lettuce by ciELISA were 109, 64, and 344%, respectively, comparing with the MBA results.

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Microplate Enzyme-Linked Immunosorbent Assay for Bovine Virus Antibody (우백혈병(牛白血病) Virus 항체측정(抗体測定)을 위한 효소면역법(酵素免疫法))

  • Choi, Won Pil
    • Current Research on Agriculture and Life Sciences
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    • v.1
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    • pp.195-199
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    • 1983
  • A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to bovine leukemia virus(BLV) is described and compared its sensitivity with that of the agar gel immunodiffusion test (ID) with BLV glycoprotein (gp) antigen using 263 sera collected in Korea and Japan. There was 98.5 per cent agreement between ELISA and ID when ELISA value, the value of tested serum(T) was devided with that of standard negative seurm(N) after the value of control was eliminated from T and N (T-C/N-C), of 1.5 or greater was considered positive. One hundred and forty four (99.6%) of 145 sera which were positive by ID were greater than 1.5 by ELISA, and 115 (97.5%) of 118 sera which were negative by ID were less than 1.5 by ELISA. As a result, it suggest that the ELISA test using BLV-gp antigen provides a useful serological tool for the diagnosis of BLV infection.

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Studies on avian infectious bronchitis: II. Standardization of an indirect enzyme-linked immunosorbent assay (ELISA) for antibody measurement (닭 전염성 기관지염에 관한 연구: II. 간접 enzyme-linked immunosorbent assay(ELISA)에 의한 항체가 측정)

  • Chang, Chong-ho;Kim, Sun-joong
    • Korean Journal of Veterinary Research
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    • v.29 no.4
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    • pp.503-515
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    • 1989
  • Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to avian infections bronchitis virus(IBV) were standardized. By adopting the optimized conditions an equation calculating ELISA antibody titers from the observations at single serum dilution was formulated. The purified antigen of IBV Mass-41 strain was dispensed into polystyrene microplate wells at a concentration of 300ng per well($100{\mu}l$) and the plates were coated by completey drying at $37^{\circ}C$. Diluted chicken serum and horseradish peroxidase conjugated goat anti-chicken IgG were added in order in $100{\mu}l$ volumes per well and allowed to react for 30 minutes each at room temperature. Just before use and after each reaction the plates were washed three times with distilled water. Finally o-phenylenediamine solution was added as an enzyme substrate. After incubation for another 15 minutes at room temperature absorbances were read at 492nm. Hyperimmune serum against Mass-41 strain was used as internal reference positive(IRP) serum. After repeated titration of IRP and negative serum, a constant titer of IRP was determined. Serum titrations were carried out for various sample sera together with IRP and negative sera and the observed titers of sample sera were corrected by reflecting the ratio between observed and constant titers of IRP serum. These corrected titers of the sample sera were plotted against sample/positive(S/P) OD ratios. All the OD's measured in the serum titrations were also corrected by substracting negative serum OD. The following equation was formulated from the above data; $Log_{10}$ ELISA titer=$5.568({\log}_{10}S/P)+4.161$ Thus it was possible to calculate ELISA titer by measuring absorbance at 1/400 single serum dilution. Titer measured by cross ELISA tests employing Mass-41 strain and three local IBV isolates were similar. These results suggest that the ELISA tests standardized in this study can be used for evaluating not only vaccinal immunity but also for infection status against fields IBV's.

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Development of Enzyme Linked Immunosorbent Assay for Determination of Zearalenone in Animal Feeds (가축사료 중 Zearalenone 분석을 위한 Enzyme Linked Immunosorbent Assay법의 개발)

  • 하정기;정덕화;김성영
    • Journal of Food Hygiene and Safety
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    • v.6 no.3
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    • pp.111-117
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    • 1991
  • We examined to develop the enzyme linked immunosorbent assay (ELISA) for determination of zearalenone in animal feeds. Zearalenone was first converted to 6'-(carboxymethyl) zearalenone oxime(zearalenone oxime) to get a coupling site and then conjugated to bovine serum albumin(BSA) for use as immunogen and to horseradish peroxidase(HRP) for use as enzyme marker. Antibody against zearalenone was obtained after 11 weeks of immunization of rabbit with zearalenone oxime-BSA. Cross reactivity of the antibody with ${\alpha}-zearalanol,\;{\beta}-zearalenol,\;{\alpha}-zearalanol\;and\;{\beta}-zearalanol$ were 168, 46, 26 and 20% respectiviely. A simple procedure was devised for the screening of zearalenone in feeds using ELISA. Feeds samples(5g) were extracted by blending with 25 ml of methanol-phospate butTered saline-dimethylformate(70 : 29 : 1) and the extract was filtered and aqueous filterate analyzed. It took only 1 hours to do whole procedure for the analysis of zearalenone in feeds by the direct competitive ELISA, and detectable limit was 1-100 ppb. Using this procedure, only 4 of 24 feed samples showed positive results with 3.93-7.43 ppb levels.

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Changes of maternal antibodies in broilers vaccinated with infectious bronchitis, infectious bursal disease and Newcastle disease viruses detected by ELISA (육계에서 전염성기관지염, 전염성 F 낭병, 뉴캣슬병 백신투여에 따른 혈중항체가의 변동)

  • 고원석;백귀정;이정원;서이원;김태중;송희종;오언평
    • Korean Journal of Veterinary Service
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    • v.21 no.3
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    • pp.277-284
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    • 1998
  • Serum samples were collected from 100 breeders and their progeny 600 broilers. The breeders and broilers were vaccinated against infectious bronchitis(IB), infectious bursal disease(IBD) and Newcastle disease(ND) viruses according to general vaccination program. The antibodies in serum samples against IB, IBD and ND viruses were detected by ELISA using commercial ELISA kit. Geometric mean titer(GMT) of ELISA was monitored from 1-day-old to 35-day-old broilers and compared to that of breeder chickens. The GMT of ELISA to IB, IBD and ND was declined half level of the day old broiler's antibody titers at about 4, 9 and 4 days of age. The GMT of ELISA to IB, IBD and ND was declined than that of protective antibody titer at about 12, 11, and 15 days of age. Thereafter, the GMT of ELISA to IB, ND were declined and disappeared according to age of broilers. The GMT of ELISA to IBD was declined according to age of broilers, but at 25 days of age increased and 31 days of age increased than that of protective antibody titer. Taken together, these studies led to conclusion that time-course of antibody titers of broilers from vaccinated breeders and that of progeny broliers which vaccinated according to vaccine program. Those are very important data to design vaccine program to breeders and broilers.

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Western blot analysis of stray cat sera against Toxoplasma gondii and the diagnostic availability of monoclonal antibodies in sandwich-ELISA

  • Sohn, Woon-Mok;Nam, Ho-Woo
    • The Korean Journal of Parasitology
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    • v.37 no.4
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    • pp.249-256
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    • 1999
  • A total of 198 sera from stray cats was assayed against Toxoplasma gondii antigen by western blot. Out of 198 sera assayed, 26 sera (13.1%) showed typical blot patterns against T gondii. When spotted by ELISA absorbance and indirect latex agglutination lest (ILAT) titer, all 26 cases were distributed over the cut-off value of ELISA whereas 24 cases (92.3%) were in the positive range of 1:32 or higher and 2 cases in negative range by ILAT. Among western blot negative 172 sera, 162 cases were negative in both ILAT and ELISA while 10 cases were reactive falsely such that three cases were ILAT positive with 1:32 titer and 9 cases were ELISA positive (2 cases overlapped). These 10 cases reacted peculiarly without typical binding pattern in Western blot. Sandwich-ELISA was performed with monoclonal antibodies (mAbs) of Tg563 (30 kDa, SAG 1), Tg505 (22 kDa, SAG2), Tg605 (43 kDa, SAG3), Tg556 (28 kDa, GRA2), Tg737 (32 kDa, GRA6). Tg695 (66 kDa, ROP2), Tg786 (42 kDa, ROP6), and Tg621 (32 kDa, anonymous but cytosolic) clone, respectively. All western blot-positive cases were in the positive range and negative cases in the negative range clearly. Among the 10 false reactive cases, 3 cases were in the positive range with one or more mAbs. All mAbs used in this study were confirmed to be specific to T. gondii infection as a standardized sandwich-ELISA to differentiate it from other pathogens.

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