• 한국식품위생안전성학회지
• /
• 제13권4호
• /
• pp.419-424
• /
• 1998

Zearalenone 검출을 위하여 Z-M-26 hybridoma cell을 mouse의 복강에 투여한 후 생산, 정제한 항체와 합성한 Zearalenone-oxime-OVA conjugate를 이용하여 ELISA법을 확립하였다. Carbonyl buffer로 희석한 Zearalenone-oxime-OVA conjugate를 4$^{\circ}C$에서 하룻밤 coating 하고 1% BSA용액으로 하룻밤 blocking한 다음, 1,000배 희석한 항체를 Zearalenone 또는 시료와 혼합하여 하룻밤 반응시키는 것이 효과적이었다. 또한 2차 항체와 기질용액의 반응시간은 각각 1시간, 30분이 적당하였고, 발색된 반응액 450nm에서 측정하였다. 이 분석법의 결과 0.1~100 ppb의 Zearalenone이 측정 가능하였으며, 본 실험에서 확립한 indirect competitive ELISA법은 농산물 중 Zearalenone 분석에 효과적으로 활용할 수 있으리라 생각된다.

• 한국식품과학회지
• /
• 제32권3호
• /
• pp.538-543
• /
• 2000

가열 처리된 우육, 돈육, 계육, 양육과 염소육과의 종판별을 위한 효소면역측정법(ELISA)을 확립하기 위하여 염소육을 $98^{\circ}C$에서 15분간 열처리해서 추출액을 분리하였다. 열에 안정한 주요 단백질(TS)은 분자량이 36과 38kDa이었고 두 분자량대의 단백질의 pI값은 4.5로 확인되었으며 면역원으로 사용하기 위해 이온교환 (DEAE-Sephadex A-50)과 겔 여과(Sephadex G-75) 크로마토그래피로 정제하였다. 분리, 정제된 TS단백질을 토끼에 면역하여 염소육에 대해 특이성을 갖는 항체를 생산하고 항TS 항체를 이용하여 간접경합 ELISA를 확립하였다. 항TS 항체는 가열 처리된 계육, 우육, 돈육, 양육과는 반응성이 전혀 없었고 동일하게 처리된 염소육과 지표단백질인 TS에만 반응성을 보였다. 따라서, 본 연구에서 확립한 간접경합 효소면역측정법은 가열 처리된 육류 중 염소육 판별에 웅용 가능함을 시사하였다.

• Clinical and Experimental Reproductive Medicine
• /
• 제17권1호
• /
• pp.71-80
• /
• 1990

항정자항체를 검사할 수 있는 면역분석법개발을 위하여 immunoaffinity chromatography로 분리 정제한 정자표현항원을 microtiter plate에 고정화 시킨것과 효소와 화학발광체로 표지된 2차항체를 사용하여 ELISA법과 CIA법을 개발하고 이 방법의 이용 가능성을 검토하기 위하여 기존 방법인 Kibrick test법으로 임상소견이 다른 남성혈청을 분석 비교하여 다음과 같은 결과를 얻었다. 1. 인간정자 표면항원을 분리하기 위한 immunoaffinity column을 제작키 위하여 인간정자를 토끼에 주사하여 정자에 대한 항혈청을 생산하였으며 이를 Protein A-Sepharose column으로 분리 정제하여 CNBr activated Sepharose-4B에 coupling시켜 immunoaffinity column을 제작하였다. 이 column에 균질화된 정자를 반응시키고 SDS를 넣은 Tris-HCI buffer로 용출시켰을때 60KD정도의 분자량을 갖는 분획을 얻었다. 2. 분리 정제된 human IgG를 microtier plate에 농도를 달리하여 고정화 시키고 ELISA용 Goat anti-human IgG-HRP conjugate와 CIA용 Rabbit anti-human IgG-ABEI-H conjugate와 반응시켜 그 활성도를 측정하였던 바 농도에 따라 반응의 정도가 감소하였다. 3. ELISA법으로 양성혈청의 희석곡선을 작성하였을때 경사가 완만한 것과 급한 것의 두 종류의 경향을 띈 곡선으로 대별되었으며 완만한 경사를 나타내는 것에서 1:160 희석치에서 O.D가가 0.1이하를 음성, 0.1이상 0.2이하를 약양성(weak positive) 그리고 0.2이상을 양성으로 판별하였다. 4. ELISA, CIA 그리고 Kibrick test로 동일시료를 분석 비교하였던바 ELISA와 CIA는 거의 동일한 상관관계를 보였으나 Kibrick test와는 50% 수준만 일치함을 보였다.

• Journal of Veterinary Science
• /
• 제23권5호
• /
• pp.32.1-32.11
• /
• 2022

Background: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. Objectives: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. Methods: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. Results: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. Conclusions: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

• 대한수의학회지
• /
• 제31권4호
• /
• pp.491-500
• /
• 1991

This study was conducted to detect the serum antibodies in the experimentally toxoplasma infected dogs and street dogs by use the of an enzyme-linked immunosorbent assay (ELISA). And this test was performed on the polystylene microplate by coating with the tachyzoites soluble antigen of T gondii (RH strain), incubated with sera diluted then, added with HPO-conjugated rabbit anti-dog IgG and o-phenylenediamine used as a substrate. Tachyzoites of T gondii harvested from mouse peritoneal cavity were purified by 30, 40 and 50% Percoll density gradient centrifugation and used as the source of antigen. The results obtained were summarized as follows; 1. The highest ratio of positive to negative (P/N ratio) was obtained at the level of $l{\mu}g/ml$ protein concentration of antigen with the 1/4000 dilution of the conjugate measured by checker-board titration. It was regarded as the optimum concentration of the antigen and conjugate. 2. Cut-off value in this IgG ELISA was 0.375 that was determined by mean absorbance (at 492nm) of IFA negative serum added with the dauble value of the standard deviation $(mean{\pm}2S.D.)$. 3. Serum ELISA IgG antibodies to T gondii in the exyerimentally infected dogs were detected firstly at the Week 3 after inoculation and the highest titer was recognized at the Week 4, 5 and 6 after inoculation. 4. Stability of the antigen absorbed in the microplates that were preserved at $4^{\circ}C$ and $-25^{\circ}C$ separately were prolonged up to 3 weeks and 10 weeks at $4^{\circ}C$ and $-25^{\circ}C$, respectively. However the reproducibility was not reliable after the preservation of 4 weeks and longer. 5. Positive rate of the specific antibodies in 312 test sera was 28.5% and there was no significant differences between the male (27.8%) and female (29.5%), respectively. 6. The IgG ELISA was proved to be a specific procedure for the detection of antibodies to canine toxoplasma infection and also evaluated as a screening test for the large scale of test samples in laboratory.

• 한국동물위생학회지
• /
• 제37권4호
• /
• pp.241-246
• /
• 2014

Q fever is a zoonotic disease caused by the obligate intracellular bacterium Coxiella (C.) burnetii and affects wild and domestic animals worldwide. The aim of this study was to evaluate the seroprevalence of C. burnetii in native Korean goat (Capra hircus coreanae) in Gyeongbuk province, Korea, using ELISA. A total of 256 goat blood samples from 56 farms in Gyeongbuk province were collected between May 2012 and March 2013. Among them, 22 (8.6%) samples from 10 (17.9%) farms were seropositive for C. burnetii by ELISA. According to regional analysis, the seroprevalences among goat farms in eastern, western, southern, and northern areas of Gyeongbuk province were 0%, 18.2%, 36.8%, and 6.3%, respectively, showing the highest seroprevalence in the southern region. Among 22 counties in Gyeongbuk province, 10 (45.5%) counties had one or more farms positive to C. burnetii antibody. Accordingly, the seroprevalence of C. burnetii in high-risk humans and animals are constantly demanded by regional investigation.

• 대한수의학회지
• /
• 제29권4호
• /
• pp.567-575
• /
• 1989

This study was conducted to evaluate the possibility of application of a microenzyme-linked immunosorbent assay(micro-ELISA) for the serodiagnosis of specific toxoplasma antibodies in swine sera and this test was performed as a microplate system by coating the polystyrene plates with toxoplasma soluble antigen, incubated serially diluted sera, then added horse radish peroxidase labelled goat anti-swine IgG(r) conjugate followed by o-phenylenediamine as substrate. The color development by enzyme-substrate reaction was determined by the photometric reading [ELISA reader at 490nm (OD)] and visual reading. The soluble antigen was prepared from the tachyzoites in mouse peritoneal cavity. A total of 1,200 swine sera from pig slaughter-house and a total of 116 swine sera from pig breeding station (S-C farm) were tested for the detection of antibodies to Toxoplasma gondii. The results obtained were summarized as follows: 1. The optimal reactions of indirect ELISA for the test sera were determined by the dilution of antigen 1:256 and 1:3,200 of horse radish peroxidase conjugate [anti-swine IgG(r)]. 2. The specific togoplasma antibody(IgG) in pigs infected with Tp artificially were detected as the serum titers of 1:64 or 1:128 at one week postinfection. 3. Of a total of 1,200 swine sera from pig slaughter-house 505 samples of sera were detected as positive (42.1%) and of a total of 116 swine sera from S-C pig breeding station 68 samples of sera as positive (58.6%). 4. The specific antibody(IgG) detection rates against a total of 1,200 test sera from pig slaughter-house were not significant between male (43.1%) and female (40.7%). 5. The indirect ELISA was proved to be a sensitive and specific procedure for the serodiagnosis of swine toxoplasmosis and also evaluated as an effective screening test for the large scale of test samples in laboratory.

• Parasites, Hosts and Diseases
• /
• 제52권1호
• /
• pp.41-46
• /
• 2014

The mature domain of a cysteine protease of Spirometra erinacei plerocercoid larva (i.e., sparganum) was expressed in Escherichia coli, and its value as an antigen for the serodiagnosis of sparganosis was investigated. The recombinant protein (rSepCp-1) has the molecular weight of 23.4 kDa, and strongly reacted with the sparganum positive human or mice sera but not with negative sera by immunoblotting. ELISA with rSepCp-1 protein or sparganum crude antigen (SeC) was evaluated for the serodiagnosis of sparganosis using patient's sera. The sensitivity and specificity of ELISA using rSepCp-1 protein were 95.0% (19/20) and 99.1% (111/112), respectively. In contrast, the sensitivity and specificity of ELISA with SeC were 100% (20/20) and 96.4% (108/112), respectively. Moreover, in experimentally infected mice, the sensitivity and specificity of both ELISA assays were 100% for the detection of anti-sparganum IgG. It is suggested that the rSepCp-1 protein-based ELISA could provide a highly sensitive and specific assay for the diagnosis of sparganosis.

• 한국환경농학회지
• /
• 제17권1호
• /
• pp.22-25
• /
• 1998

종래에 사용해 오던 HPLC에 의한 quizalofop-ethyl의 분석법을 개선시키고, 또한 ELISA와 GLC를 이용한 새로운 quizalofop-ethyl분석법을 개발하고자 시험을 수행한 결과는 다음과 같다. ELISA 실험에서 적정배양온도는 $37^{\circ}C$였으며 배양시간은 4시간이었다. Coating antigen의 적정수준은 $20{\mu}l/ml$이며 검출 한계는 5ppb이고 회수율은 95%이상이었다. 또 GLC와 HPLC에 의한 검출한계는 5ppb, 100ppb였으며, 토양에서의 회수율은 95.4, 89.5% 이상으로 나타났다. 이상에서 보는 바와 같이 ELISA와 GLC에 의한 quizalofop-ethyl분석법이 HPLC에 의한 것보다 양호한 것으로 나타났다.

• 대한미생물학회지
• /
• 제22권4호
• /
• pp.377-392
• /
• 1987

Monoclonal antibody to the Escherichia coli heat-stable enterotoxin(ST) was produced to develop a rapid and convenient diagnostic method to the ST. The toxin was purified from culture supernatant of enterotoxigenic E. coli O148H28($ST^+/LT^+$) and conjugated to bovine serum albumin(BSA). The ST-BSA conjugate was used to immunize BALB/c mice and the immune spleen cells from these mice were fused with $P3{\times}63$ Ag8.V653 plasmacytoma cells. Hybridomas were screened by ELISA and positive hybridomas were cloned by limiting dilution. Finally, one stable clone (AS36) specific to ST was selected for further growth and characterization. Antibody titers of culture supernatant and ascitic fluid from BALB/c mice were 1:1,024 and 1:20,480 respectively in ELISA. The isotype and subclass of monoclonal antibody was IgG1 in sandwich ELISA. To test the neutralizing effect of monoclonal antibody on toxin activity of ST, mixture of ascitic fluid and ST was assayed by infant mouse assay and this monoclonel antibody was proved to be a neutralizing antibody. The titer of ascitic fluid which completely neutralized biological activity of 4 units of ST was 1:4. Purified ST was quantitatively measured by competitive ELISA and minimum amount of ST detectable by this assay was 250pg, which was an amount six-fold smaller than that detectable by infant mouse assay. Four reference strains of enterotoxigenic E. coli from WHO were detected by competitive ELISA and highly specific, sensitive and reproducible result was obtained.