• 센서학회지
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• 제20권2호
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• pp.90-95
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• 2011

In order to protect human lives from disease, various biosensors having the potential to analyze a variety of biomolecules have been utilized. Biosensors constitute one of the most promising ways to monitor and detect various biomolecules corresponding to diseases. In this study, we demonstrate that the reaction of streptavidin molecules with biotin on a gold electrode can be detected using the twoelectrode method with a gold electrode and a platinum reference electrode. We also show the characteristics of the electrical current response. While detecting 2-${\mu}M$ streptavidin molecules dissolved in phosphate buffered saline(PBS) solution, we found that an analytical biosensor can operate on the principle of detecting an antigen-antibody reaction event of protein molecules using the two-electrode method. We think that the "potential step" method might be useful to detect the occurrence of any antigen-antibody reactions and can be combined with other devices or ICs such as BJTs, MOSFETs, and OP-amps for the detection of biomolecules of diseases.

• 대한수의학회지
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• 제45권4호
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• pp.569-574
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• 2005

Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to reticuloendotheliosis virus (REV) at single serum dilution was standardized. REV HI, one of the Korean field isolates, was inoculated into chicken embryo fibroblast (CEF) cells and was harvested from the culture fluids and cells after 10 to 12 days. Viruses were purified by centrifugation at the $107,000{\times}g$ for 12 hours on 20, 30, 45% (W/V) sucrose gradient. Virus specific fraction was collected and used as ELISA antigen. To standardize ELISA, the optimal concentration of coating antigen ($1{\mu}g/well$) and conjugate (1/1000) was determined by corrected OD (OD value of positive serum-OD value of negative serum) and P/N ratio (OD value of positive serum/OD value of negative serum). To calculate ELISA titer by measuring absorbance at 1/400 single serum dilution, serum titrations were carried out for various sample sera together with standard positive and negative sera. The observed titers of serum samples were plotted against sample/positive (s/p) ratios at 1/400 serum dilution. From the above data, the ELISA titers could be calculated by the equation of $log_{10}$ ELISA titer = 2.2763 ($log_{10}$ s/p) + 3.482 (r = 0.93). For evaluating the sensitivity, the standardized method were compared with conventional agar gel immunodiffusion (AGID) test method using serum samples collected from REV infected field chicken flocks. Fifty seven of 60 samples (95%) were positive for REV by ELISA, whereas only 11 (18.3%) samples were positive by AGID test. This results suggested that the ELISA tests developed in this study could be used for detection of antibodies to REV with high sensitivity.

• Clinical and Experimental Pediatrics
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• 제51권10호
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• pp.1071-1076
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• 2008

목적: RSV 호흡기 바이러스 감염은 해마다 영유아에서 심한 호흡기 질환의 중요한 원인이 되고 있다. 이에 따라 최근 검사 방법이 간편하고 결과를 빨리 알 수 있는 면역 크로마토그래피법이 신속 항원 검사법으로서 소개되어 그 정확성과 임상에서의 유용성이 많이 연구되고 있으나 국내에서는 연구례가 드문 실정이다. 이에 저자들은 이 검사법의 정확성과 유용성을 평가하여 실질적인 검사법으로 정착시키고자 하였다. 방법: 2007년 4월부터 2008년 3월까지 발열, 기침, 천명, 호흡곤란, 빈호흡 등의 증상으로 외래 내원 및 입원치료를 받은 112명의 환아를 대상으로 비인두 가검물을 채취하여 RSV Respi-Strip과 효소 면역 측정법, RT-PCR(ASTEC)을 동시에 시행하였다. RT-PCR을 표준으로 하여 RespiStrip 과 EIA의 결과를 비교 분석하여 정확성과 유용성을 평가하였다. 결 과: RSV RespiStrip, RT-PCR, EIA에 양성을 보인 환아는 각각 42명, 45명, 39명 이었다. RespiStrip는 RT-PCR 검사법에 대하여 민감도 88%, 특이도 94%, 양성 예측도 90%, 음성 예측도는 92% 였으며 위양성률과 위음성률 그리고 일치도가 각각 5.9%, 11%, 83% 로 나왔다. EIA는 RT-PCR 검사법에 대하여 민감도 84%, 특이도 94%, 양성 예측도 90%, 음성 예측도 90%, 일치도 79% 로 나왔다. 결론: 신속 항원 검사가 비교적 민감도가 높으므로 RSV 감염의 선별 검사로서 적합하다고 생각한다. 외래 진료실에서 빠르고 경제적이며 간편하게 검사하여 조기에 적절한 치료를 함으로써 합병증을 예방할 수 있고 아울러 불필요한 항생제의 사용을 줄일 수 있다. 앞으로 이러한 신속검사가 실제 임상에서 많이 적용되리라 기대된다.

• 대한수의학회지
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• 제31권4호
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• pp.491-500
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• 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.

• 농촌의학ㆍ지역보건
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• 제16권1호
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• pp.61-69
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• 1991

Serodiagnosis of Clonorchis sinensis infections will probably be a first choice tool for screening of clonorchiasis in a future because of increasing difficulties in collection and examination of stools. The sensitive test such as ELISA can he used effectively. However there are some limitations in serological diagnosis for the detection of serum antibody. One of the major problems is the non-specificity of the antigens which produce cross reaction with other helminthic infection sera. To solve this problem. many investigators have tried to purify the antigens used. In this study, we determined the antigenic profile of the crude saline extract antigen of C. sinensis at early developmental stage based on SDS-PAGE and immunoblotting techniques for the purpose of understanding the nature of C. sinensis worm antigen The following results were obtained : 1) The SDS-PAGE showed many protein hands ranging from 10Kd to 91Kd relative molecular weight. Among them, 66, 46, 40, 33, 27, 24, 16, 14 and 10Kd bands were observed as a principle bands. The protein components of C. sinensis changed chronologically during their early developmental period. 44Kd band was stained unclearly in antigen of 2 weeks worm, but changed to concentrated state in antigen of 5 weeks worm. 35Kd band was found in antigen of 2 weeks worm, however this band was disappeared in antigen of 5 weeks worm. 22Kd band also lost its staining property gradually. 2) In spite of differences in antigenic profile, there was no differences in the data obtained by microplate ELISA using each antigen preparation. Absorbance value began to rise in between 2 to 3 weeks after infection. 3) By EITB. serum antibody recognized major protein bands with molecular weight of 91, 85, 63, 46, 40, 33, 24, 14 and 10Kd hand respectively. Among them 66, 33, 17 and 14Kd bands were observed as non-specific band because they reacted even in normal control sera. Generally, gradual increase of positive reactions were observed as the infection period of C. sinensis was prolonged. In other hand, the reaction of 10Kd hand did not occurred when 26th week sera was tested. 4) The positive reactions using antigens of 2 weeks worm, especially on 40 and 24Kd bands, were most strong and sharply demarcated compared to those of 3~5 weeks worm antigen.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.268-273
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• 2015

The fluorescent-antibody-to-membrane-antigen (FAMA) test is regarded as the "gold standard" to detect protective antibodies to varicella-zoster virus (VZV) because of its high sensitivity and specificity. Because the classic FAMA test uses an infectious virus for detection of antibodies to VZV, it is labor-intensive, and also requires special equipment for handling the virus. For this reason, we attempted to develop a simple and safe FAMA assay. Because VZV glycoprotein E (gE) is one of the major VZV glycoproteins, we used the gE protein for the FAMA test (gE FAMA). Here, we demonstrate that overexpression of gE in HEK293T cells can be used to measure antibodies in human serum, and that gE FAMA titers are closely correlated with gpEIA ELISA data. These results indicate that our gE FAMA test has the potential to measure antibodies to VZV.

• Parasites, Hosts and Diseases
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• 제51권2호
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• pp.177-182
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• 2013

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27- (KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

• 대한수의학회지
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• 제33권2호
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• pp.301-308
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• 1993

Three or 7day old piglets were infected experimentally with different encephalomyocarditis virus isolates to detect the viral antigen by the immunoperoxidase technique and to observe strain difference in their pathogenecity in newborn pigs by comparing clinical signs and pathologic lesions. Clinical signs of the infected pigs were different depending on the virus strain, pig age and infection route. Encephalomyocarditis virus(EMCV) NVSL-PR isolate was more pathogenic than MN-25 and MN-30 isolate. Three day old piglets showed more severe illness than 7 day old piglets. Predominant clinical signs were sudden death without noticeable clinical signs and dyspnea manifested as heavy abdominal breathing. Contact-infection from infected piglets to controls was observed in the oro-nasally infected group but not the intramuscular group. Common necropsy findings of dead piglets in both age groups infected with MN-25 and NVSL-PR were accumulation of excessive fluid in the body cavities and mild to diffuse necrotic areas observed in the hearts and occasionally in the livers. Microscopically, myocarditis with inflammatory cell infiltration, necrosis of the myocardial muscle fibers and occasional mineralization were observed along with interstitial pneumonia and centrolobular necrosis in the liver. Using an immunoperoxidase technique, viral antigen was detected in myocardial muscle fibers of piglets infected with EMCV.

• 한국동물위생학회지
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• 제17권1호
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• pp.37-43
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• 1994

The rate of 58 neonatal calves in infection of Theileria sergenti was investigated in random samples on the farms located in Kyunggi, Chonbuk districts of Korea. 1. The criteria used in veryfying infection with T. sergenti included the detection of parasites by giemsa stain and acridine orange stain in the blood smear slides. 2. Further evidence of current or previous exposure to T. sergenti was based on demonstration of T. sergenti specific antibody and antigen by the western immunoblot and the directed immunofluorescent antibody test in the peripherial blood of the calves. 3. The prevalence rates were 35%, 50% in Kyunggi, Chonbuk provinces respectively and the overall prevalence in all the farms was 43.2％ by means of acridine orange stain. 4. The parasites that were observed in the peripherial blood of calves was showen surely by the western immunoblot to the characteristic 34KD antigen among the proteins of T. sergenti (Korean isolate). 5. And the antigen of the neonatal calves reacted at the very highest titer(1 : 2, 560) 6. These data highlight the significances of T. sergenti in the neonatal calf disease in Korea.

• 한국군사과학기술학회지
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• 제13권3호
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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%.