가축사료 중 Zearalenone 분석을 위한 Enzyme Linked Immunosorbent Assay법의 개발

하정기,정덕화,김성영 한국식품위생안전성학회 1991 한국식품위생안전성학회지 Vol.6 No.3

가축사료중 zearalenone 분석을 위한 enzyme linked immunosorbent assay(ELISA) 법의 개발을 위해 우선 zearalenone의 항원성을 증폭시키기 위해 zearalenone oxime 유도체를 합성한 다음 bovine serum albumin(BSA)와 conjugate를 만들고, 이를 항원으로 토끼에 면역시켜 11주에 zearalenone에 특이한 항체를 얻어냈다. 생성된 항체를 zearalenone외에 ${\alpha}-zearalenol$과는 강한 cross reactivity를 나타내었고 ${\beta}-zearalenol,\;{\alpha}-zearalenol\;및\;{\beta}-zearalenol$과는 약간의 반응을 보였으며 확립된 ELISA 조건은 당므과 같다. 먼저 시료를 methanol-phosphate buffered saline-dimethyl formate(70 : 29: 1)을 4배 첨가하여 blending 한 다음 Whatman No. 4를 통한 여액을 ELISA시료로 사용하였다. 효소 반응시간과 발색시간은 각각 $37^{\circ}C$에서 30분과 15분이었고, 흡광도는 410nm에서 ELISA reader로서 측정하였으며, 측정한계는 1~100 ppb로 매우 낮았다. 확립된 ELISA 조건으로 실제시료의 zearalenone오염도는 측정결과 24개 시료 중 4개의 시료가 양성반응을 보였고 그 함량범위는 $3.93~7.43\;\mu\textrm{g}/kg$이었다. 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.

Vinblastine Determination Measured by a Sensitive ELISA Inhibition Assay

Jae-Wha Kim(김재화),Mi-Young Han(한미영),Hee-Gu Lee(이희구),Eun-Young Song(송은영),Tai-Wha Chung(정태화),Kyung-Soo Nam(남경수),In-Seong Choe(최인성),최용경 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.1

Vinblastine을 포함하는 bis-indole alkaloids에 대한 단일클론 항체를 생산하여 Vinca alkaloids의 양을 측정할 수 있는 간편한 immunoassay 체계를 확립하였다. Vinca alkaloids는 periwinkle식물체의 배양된 세포로부터 추출하여 BSA와 접합한 후 Balb/c생쥐에 면역시켜 얻은 비장세포와 골수종양세포의 융합을 유도하여 VBL-BSA에 반응하는 클론을 ELISA 방법으로 분석하였으며 이들 클론 중 bis-indole alkaloids와 특이적으로 반응하는 항체는 inhibition assay를 통하여 분리할 수 있었고 그 결과 두 개의 단일클론 항체를 형성하는 세포주(KN-1과 KN-2)를 확립하였다. KN-1의 경우 dimeric bis-indole alkaloids와는 상당한 교차반응을 나타낸 반면 monomeric bis-indole alkaloids와는 교차반응을 나타내지 않았으며 이 클론의 항체를 이용하여 배양된 세포 추출물에 포함된 Vinca alkaloids의 양을 측정한 결과 0.05 nM정도의 dimeric Vinca alkoloids까지도 측정할 수 있었다. Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA(Enzyme-linked immunosorbent assay). These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.

Enzyme-Linked Immunosorbent Assay of Bovine Tuberculosis by Crude Mycobacterial Protein 70

Cho, Yun Sang,Jung, Suk Chan,Kim, Jong Man,Yoo, Han Sang MARCEL DEKKER 2007 JOURNAL OF IMMUNOASSAY AND IMMUNOCHEMISTRY Vol.28 No.4

<P> MPB70 (mycobacterial protein of BCG 70) as T-cell stimulator has been tried with an intradermal skin test (IST) and enzyme-linked immunosorbent assay (ELISA) of bovine tuberculosis (BTB). In this study, crude mycobacterial protein 70 (CMP70) was prepared by anion exchange chromatography from the culture supernatant of Mycobacterium bovis AN5 and CMP70 ELISA was compared with purified protein derivative (PPD) ELISA. PPD and CMP70 ELISA have shown a positive reaction to the sera of M. bovis infected cattle and IST positive reactors. One of three IST negative cattle showed the nonspecific reaction in PPD ELISA, whereas all of the IST negative cattle (n=3) were did not show the nonspecific reaction in CMP70 ELISA. When each ELISA was applied to sixty-two IST positive cattle, ELISA positive reactors were eighty four per cent to CMP70 antigen and fifty-two per cent to PPD. CMP70 has been shown to be more specific and sensitive than PPD in ELISA.</P>

Vinblastine Determination Measured by a Sensitive ELISA Inhibition Assay

Song,Eun-Young,Chung,Tai-wha,Kim,Jae-Wha,Nam,Kyung-Soo,Han,Mi-Young,Choe,In-Seong,Lee,Hee-Gu THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.1

Vinblastine을 포함하는 bis-indole alkaloids에 대한 단일클론 항체를 생산하여 Vinca alkaloids의 양을 측정할수 있는 간편한 immunoassay 체계를 확립하였다. Vinca alkaloids는 periwinkle식물체의 배양된 세포로 부터 추출하여 BSA와 접합한 후 Balb/c생쥐에 면역시켜 얻은 비장세포와 골수종양세포의 융합을 유도하여 VBL-BSA에 반응하는 클론을 ELISA방법으로 분석하였으며 이들 클론 중 bis-indole alkaloids와 특이적으로 반응하는 항체는 inhibition assay를 통하여 분리할 수 있었고 그 결과 두개의 단일클론 항체를 형성하는 세포주(KN-1과 KN-2)를 확립하였다. KN-1의 경우 dimeric bis-indole alkaloids 와는 상당한 교차반응을 나타낸 반면 monomeric bis-indole alkaloids와는 교차반응을 나타내지 않았으며 이 클론의 항체를 이용하여 배양된 세포 추출물에 포함된 Vinca alkaloids의 양을 측정한 결과 0.05nM정도의 dimeric Vinca alkaloids까지도 측정할 수 있었다. Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA((Enzyme-linked immunosorbent assay). These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.

Comparison of Two Enzyme-Linked Immunosorbent Assays forDetecting Parasitic Diseases

김혜련,이미경,홍성태,채종일 대한임상미생물학회 2008 Annals of clinical microbiology Vol.11 No.1

Background: Serologic tests for specific antibody nowadays are widely employed for the diagnosis of parasitic diseases. Recently, an increasing numbers of kits have adopted enzyme-linked immunosorbent assay (ELISA) for the detection of parasitic antibodies. In this study, we evaluated two ELISA reagents for the diagnosis of parasitic diseases. Methods: A total of 553 serum and 156 CSF samples were assayed using an in-house micro-ELISA and GenediaⓇ Ab ELISA (Green cross PBM, Korea) for Cysticercus, Paragonimus westermani, Clonorchis sinensis, and Sparganum. We reviewed the medical records of all patients. The results from GenediaⓇ Ab ELISA kit were compared with those from the inhouse micro-ELISA method. Results: The overall concordance rate between the two ELISA tests was 95.5%. When compared with the clinical information, the sensitivity, specificity, positive predictive value, and negative predictive value of the in-house micro-ELISA were 100%, 99.0∼99.6%, 82.4∼96.4%, and 100%, and the respective figures for GenediaⓇ Ab ELISA kit were 92.9~100%, 88.0∼ 97.3%, 41.7∼50%, and 99.9~100% with kappa agreement of 0.53-0.63. Comparison of two ELISA methods showed a significant difference (P<0.05). Retesting of 85 discordant samples showed that the concordance rate of the in-house ELISA was 97.7% and that of GenediaⓇ Ab ELISA was 28.2%. Conclusion: GenediaⓇ Ab ELISA kit showed an intermediate level of kappa agreement compared with the in-house ELISA. Further studies are necessary to improve the concordance rate of the two methods, and a careful interpretation of these results is required for a precise diagnosis.

Enzyme-linked immunosorbent assay를 이용한 바이러스성 출혈성 패혈증 바이러스 감염 넙치(Paralichthys olivaceus)의 특이 항체반응 검사

황지연,장진현,김동준,권문경,서정수,황성돈,손맹현,Hwang, Jee Youn,Jang, Jin Hyeon,Kim, Dong Jun,Kwon, Mun Gyeong,Seo, Jung Soo,Hwang, Seong Don,Son, Maeng-Hyun 한국수산과학회 2017 한국수산과학회지 Vol.50 No.5

The viral hemorrhagic septicemia virus (VHSV) has an extensive host range, and infects farmed and wild fish inhabiting both freshwater and marine ecosystems. Enzyme-linked immunosorbent assay (ELISA) is highly useful in diagnosing viral hemorrhagic septicemia. However, ELISA shows high, non-specific background reaction with fish antibodies. In this study, we optimized the antigen and antibody concentrations used for detecting specific antibodies in VHSV-infected olive flounder to reduce non-specific binding, and improve the sensitivity of ELISA. The results suggested that OD (optical Density) values were valid when ELISA was performed with $0.1{\mu}g/well$ of virus, involving blocking with blocking buffer (Roth, Roti-Block), 1:300-1:600 dilution with flounder antisera, and 1:1000 dilution with anti-flounder IgM and HRP-conjugated goat anti-mouse IgG for detecting the VHSV antibody in flounder sera. Furthermore, 11 different VHSV strains isolated in Korea from 2012 to 2016 were used to infect the fish. The results showed no correlation between viral pathogenicity and antibody production. This research is a basic study on the application of antibody detection in the diagnosis of viral hemorrhagic septicemia in the olive flounder.

Serological and epidemiological investigation of Mycobacterium avium subspecies paratuberculosis in bovines in Pakistan

Rehman Aziz Ur,Javed Muhammad Tariq,Ahmed Ishtiaq,Saeed Muhammad Adnan,Ehtisham-ul-Haque Syed,Rafique Muhammad Kamran,Sikandar Arbab,Nasir Amar,Ahmad Latif,Kashif Muhammad,Zeeshan Muhammad Abid 아세아·태평양축산학회 2024 Animal Bioscience Vol.37 No.9

Objective: This study aimed to investigate the prevalence of paratuberculosis in cattle and buffaloes at twelve public dairy farms in Punjab, Pakistan.Methods: A total of 2,181 more than two-year-old animals (1,242 cattle and 939 buffaloes) were tested by avian tuberculin, i.e., killed purified protein derivative of <i>Mycobacterium avium</i> paratuberculosis and indirect enzyme linked immunosorbent assay (ELISA). Blood and fecal samples were collected from tuberculin positive animals. These samples were further processed by indirect ELISA. The data were analyzed using frequency analysis and logistic analysis procedures.Results: The prevalence of paratuberculosis at public dairy farms was 3.8%, as determined by tuberculin+ELISA test. It varied from 0.71% to 13.5% with a 100% herd prevalence. Multivariate logistic regression analysis revealed that species, milk production, total animals, total small ruminants, and total buffaloes were significantly associated with the occurrence of paratuberculosis. Odd ratio analysis revealed that with a one-kilogram increase in body weight, there will be a 0.006% increase in disease occurrence. With the increase in one animal in small ruminants and buffaloes, there will be 0.008% and 0.42% greater chances of developing paratuberculosis, respectively. Bivariate logistic regression analysis of cattle and buffaloes revealed that farm number, age, and total number of cattle were significantly associated with the occurrence of paratuberculosis. A one-month increase in lactation length increases the chance of tuberculosis by 0.004%; similarly, a one-liter increase in milk production increases the chance of disease by 10%. With each additional buffalo in the herd, there will be a 0.007% greater chance for the occurrence of paratuberculosis.Conclusion: This study concluded that tuberculin testing can be used in conjunction with ELISA to screen animals for paratuberculosis in countries with scarce resources, such as Pakistan. The efficacy of disease diagnosis can be improved by combining multiple tests. Objective: This study aimed to investigate the prevalence of paratuberculosis in cattle and buffaloes at twelve public dairy farms in Punjab, Pakistan. Methods: A total of 2,181 more than two-year-old animals (1,242 cattle and 939 buffaloes) were tested by avian tuberculin, i.e., killed purified protein derivative of Mycobacterium avium paratuberculosis and indirect enzyme linked immunosorbent assay (ELISA). Blood and fecal samples were collected from tuberculin positive animals. These samples were further processed by indirect ELISA. The data were analyzed using frequency analysis and logistic analysis procedures. Results: The prevalence of paratuberculosis at public dairy farms was 3.8%, as determined by tuberculin+ELISA test. It varied from 0.71% to 13.5% with a 100% herd prevalence. Multivariate logistic regression analysis revealed that species, milk production, total animals, total small ruminants, and total buffaloes were significantly associated with the occurrence of paratuberculosis. Odd ratio analysis revealed that with a one-kilogram increase in body weight, there will be a 0.006% increase in disease occurrence. With the increase in one animal in small ruminants and buffaloes, there will be 0.008% and 0.42% greater chances of developing paratuberculosis, respectively. Bivariate logistic regression analysis of cattle and buffaloes revealed that farm number, age, and total number of cattle were significantly associated with the occurrence of paratuberculosis. A one-month increase in lactation length increases the chance of tuberculosis by 0.004%; similarly, a one-liter increase in milk production increases the chance of disease by 10%. With each additional buffalo in the herd, there will be a 0.007% greater chance for the occurrence of paratuberculosis. Conclusion: This study concluded that tuberculin testing can be used in conjunction with ELISA to screen animals for paratuberculosis in countries with scarce resources, such as Pakistan. The efficacy of disease diagnosis can be improved by combining multiple tests.

Sequential use of real-time polymerase chain reaction and enzyme-linked immunosorbent assay techniques verifies adulteration of fermented sausages with chicken meat

Benli Hakan,Barutçu Elif 아세아·태평양축산학회 2021 Animal Bioscience Vol.34 No.12

Objective: Detection of adulteration in processed meats is an important issue for some countries due to substitution of beef with a cheaper source of protein like poultry. In this study, the presence of chicken meat was investigated using real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to verify adulteration of fermented sausage samples. Methods: A total of 60 commercial samples were collected from 20 establishments in three replicates including 10 fermented sausage manufacturers and 10 butchers to investigate the presence of chicken meat with the sequential use of real-time PCR and ELISA techniques. In addition, pH, moisture content, water activity and color values of the samples were determined. Results: Both real-time PCR and ELISA showed agreement on the presence or absence of chicken meat in 55 out of 60 fermented sausage samples and chicken meat was identified with both methods in 16 samples. Five samples produced inconsistent results for the presence of chicken meat in the first run. Nevertheless, the presence of chicken meat was verified with both methods when these samples were analyzed for the second time. In addition, the average physico-chemical values of the fermented sausage samples tested positive for chicken meat were not significantly different from some of those fermented sausage samples tested negative for the chicken meat. Conclusion: The sequential use of real-time PCR and ELISA techniques in fermented sausages could be beneficial for the government testing programs to eliminate false negatives for detection of adulteration with chicken meat. Furthermore, consumers should not rely on some of the quality cues including color to predict the adulteration of fermented sausages with chicken meat since there were no statistical differences among some of the samples tested positive and negative for chicken meat. Objective: Detection of adulteration in processed meats is an important issue for some countries due to substitution of beef with a cheaper source of protein like poultry. In this study, the presence of chicken meat was investigated using real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to verify adulteration of fermented sausage samples.Methods: A total of 60 commercial samples were collected from 20 establishments in three replicates including 10 fermented sausage manufacturers and 10 butchers to investigate the presence of chicken meat with the sequential use of real-time PCR and ELISA techniques. In addition, pH, moisture content, water activity and color values of the samples were determined.Results: Both real-time PCR and ELISA showed agreement on the presence or absence of chicken meat in 55 out of 60 fermented sausage samples and chicken meat was identified with both methods in 16 samples. Five samples produced inconsistent results for the presence of chicken meat in the first run. Nevertheless, the presence of chicken meat was verified with both methods when these samples were analyzed for the second time. In addition, the average physico-chemical values of the fermented sausage samples tested positive for chicken meat were not significantly different from some of those fermented sausage samples tested negative for the chicken meat.Conclusion: The sequential use of real-time PCR and ELISA techniques in fermented sausages could be beneficial for the government testing programs to eliminate false negatives for detection of adulteration with chicken meat. Furthermore, consumers should not rely on some of the quality cues including color to predict the adulteration of fermented sausages with chicken meat since there were no statistical differences among some of the samples tested positive and negative for chicken meat.

효소면역검사법에 의한 Yersinia pseudotuberculosis 진단에 대한 연구

백인기,김미애,조종래 대한임상병리학회 1998 대한임상병리학회지 Vol.18 No.3

Enzyme-Linked Immunosorbent Assay for the Detection of Hen`s Egg Proteins in processed Foods

Dong Hwa Shon,Hyun Jung Kim,Soo Ho Kim,Bo Yeon Kwak 한국축산식품학회 2010 한국축산식품학회지 Vol.30 No.1

The Hen`s egg is widely used in many processed foods as an ingredient and is one of the most prevalent food allergens in children. To detect egg proteins in processed foods, we developed a competitive indirect enzyme-linked immunosorbent assay (ciELISA) using an anti-ovomucoid (OM) antibody, which was produced by immunization of rabbits with OM, the most heat-stable component of the egg proteins. The detection limit of this quantitative assay system was 30ng/mL. Cross-reactivity of the anti-OM antibody toward OM, ovalbumin, skim milk, casein, whey protein isolate, and isolated soy protein was 100, 0.4, 0.2, 0.04, 0, and 0%, respectively. In the spike test of egg white powder in milk replacer, commercial sausage, and in-house sausage, the assay recoveries (mean±SD) were 129±13.7%, 73.9±12.5%, and 65.5±13.6%, respectively. When egg white in a commercial crab meat analog and sausage was determined by ciELISA, the assay recovery was found to be 108% and 127%, respectively. The combined results of this study indicate that this novel ciELISA for OM detection could be applied for the quantification of hen`s egg proteins in processed foods.