• Title/Summary/Keyword: ELISA method

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Development of Single-tube Multiplex Immunocapture RT-PCR Assay for Simultaneous Detection of Two Pepper Tobamoviruses

  • Kim, Jae-Hyun;Choi, Gug-Seoun;Kim, Jeong-Soo;Lee, Sin-Ho;Choi, Jang-Kyung;Ryu, Ki-Ryun
    • The Plant Pathology Journal
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    • v.22 no.2
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    • pp.164-167
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    • 2006
  • An immunocapture reverse transcription-polymerase chain reaction (IC/RT-PCR) was developed for simultaneous detection of two pepper-infecting RNA viruses, Pepper mud mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV). The assay could be performed in a single tube for simultaneous and sensitive detection of these tobamoviruses. This detection system revealed thousand-fold increase in detection sensitivity compare to ELISA. This method could save time and reagent cost compare to common RT-PCR which needs several reactions and several procedures of viral RNA extractions for the same number of samples.

Development and evaluation of surface plasmon resonance imaging for the detection of antibodies against classical swine fever virus in swine

  • Cho, Ho-Seong;Lee, Tae-Uk;Park, Nam-Yong
    • Korean Journal of Veterinary Service
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    • v.30 no.2
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    • pp.205-209
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    • 2007
  • A protein chip based on surface plasmon resonance (SPR) imaging was developed for measuring classical swine fever virus (CSFV) antibody using a recombinant gp55 protein as an antigen. The diagnostic potential of SPR imaging for detecting antibodies to the CSFV gp55 protein was compared with that of a enzyme -linked immunosorbent assay (ELISA) using 70 pig sera. There was a strong positive correlation between the SPR imaging and ELISA (n=70, r=0.916, p<0.01). Therefore, the SPR imaging, which is a label-free and high-through put method, is expected to be a valuable tool in the serodiagnosis of CSFV.

Effect of H-Y Antibody on in vitro Development of Mouse Embryos (H-Y항체의 처리가 생쥐수정란의 발달에 미치는 영향)

  • 고정재;심호섭;김종배;박홍양;정길생;이경광
    • Korean Journal of Animal Reproduction
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    • v.10 no.1
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    • pp.42-48
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    • 1986
  • These experiments were carried out to develop new techniques identifying XX-bearing embryos prior to implantation by immunological method. Antiserum to histocompatibility-Y(H-Y) antigen was prepared in adult SD(sprague-dawley) female rat by repeated immunization of newbone testis supernatant from males of the same strain. ELISA test was used to identify the H-Y antibody of antiserum. Total 124 mouse embryos (8-cell stage) were treated with H-Y antiserum and complement in BSA free Ho, pp. and Pitt's medium and cultured under the gas phase of 5% CO2 in air at 37$^{\circ}C$ for 24 to 48 hrs. The morphological characteristics of embryos treated were observed under the phase-contrast micro scope. The results obtained in these experiments were summarized as follows: 1. Optimal Density of H-Y antibody were a, pp.ared to be 0.27-0.47 by ELISA test. 2. Of total 124 embryos treated with H-Y antiserum and complement 69(55.6%) embryos developed to blastocyst and 55(44.4%) destroyed or arrested.

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Cross-Reactivity of Porcine Immunoglobulin A Antibodies with Fecal Immunoglobulins of Wild Boar (Sus scrofa) and Other Animal Species

  • Sang won Seo;Sung J. Yoo;Sunyoung Sunwoo;Bang hun Hyun;Young S. Lyoo
    • IMMUNE NETWORK
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    • v.16 no.3
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    • pp.195-199
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    • 2016
  • Fecal samples obtained from wild boar habitats are useful for the surveillance of diseases in wild boar populations; however, it is difficult to determine the species of origin of feces collected in natural habitats. In this study, a fecal IgA ELISA was evaluated as a method for identifying the porcine species from fecal samples. Both domestic pigs (Sus scrofa domestica) and wild boars (Sus scrofa coreanus) showed significantly higher levels of fecal IgA than other animal species. Additionally, age dependent changes in the level of Ig A in wild boars and domestic pigs were identified; Titers of Ig A were highest in suckling period and lowest in weanling period.

Isolation and purification of chicken egg yolk immunoglobulin against Edwardsiella tarda (Edwardsiella tarda에 대한 계란난황항체의 분리와 정제)

  • Kim, Yeong-Dae;O, Myeong-Ju;Jeong, Tae-Seong;Jeong, Seong-Ju
    • Journal of fish pathology
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    • v.17 no.1
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    • pp.11-20
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    • 2004
  • The present study compared purification methods of hen egg yolk immunoglobulin (IgY) from the hen immunized with Edwardsiella tarda. The purification of anti-E. tarda IgY was performed by four different methods, polyethylene glycol (PEG), chloroform polyethylene glycol (Chloroform-PEG), ammonium sulfate and purification kit. Purified IgY had heavy chain of 64 kDa and light chain of 27 kDa size. IgY purified from the hen immunized with E. tarda showed higher ELISA values and agglutination titers than those with IgY purified from the non-immunized hen as a negative control. In addition, purified IgY recognized similar E. tarda proteins to those with anti-E. tarda rabbit serum by western blotting. Purified IgY had an agglutination titer of 1:512 by PEG method and ammonium sulfate method, and 1:128 by chloroform-PEG method and purification kit. Moreover, PEG method was the most rapid method among the four different IgY purification methods. These results indicate that PEG method is effective purification method maintaining biological activity of the IgY.

An Effective Detection of Potato Virus Y Using RT-PCR Technique (RT-PCR 기법을 이용한 효과적인 감자바이러스 Y의 검정)

  • Joung, Young-Hee;Jeon, Jae-Heung;Choi, Kyung-Hwa;Kim, Hyun-Soon;Yi, Yong-Sub;Joung, Hyouk
    • Korean Journal Plant Pathology
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    • v.13 no.4
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    • pp.219-224
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    • 1997
  • A PT-PCR (reverse transcription-polymerase chain reaction) diagnostic method for potato virus Y (PVY) was developed using primer pair derived from conserved region of coat protein genes of several PVY strains, A 764 bp PCR product was detected from several lines of potato cv. Atlantic. We could prove that the 764 bp DNA fragment was indeed the PVY gene by sequencing analysis. PVY detection method using RT-PCR technique was about tuber tissue.

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Diagnostic Significance of the Serologic Test Using Multiple Antigens of Mycobacterium Tuberculosis by ELISA (다양한 특이결핵항원을 이용한 결핵항체 검사(ELISA)의 진단적 유용성)

  • Kim, Dae-Yun;Choi, In-Hwan;Park, Seung-Kyu;Cho, Shang-Rae;Song, Sun-Dae
    • Tuberculosis and Respiratory Diseases
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    • v.47 no.6
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    • pp.757-767
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    • 1999
  • Background: Diagnosis by smear and/or cultures of the Mycobacterium tuberculosis from body fluid or biopsy specimen is "Gold standard". However the sensitivity of the direct microscopy is relatively low and culture of mycobacteria is time consuming. Despite an explosion in the techniques of rapid identification of mycobacteria by molecular genetic means, it is laborious and expensive and then rapid, inexpensive serodiagnosis is interested in diagnosis of tuberculosis. But sensitivity and specificity of known serologic antigen is not full sufficient level and then new antigen develop and combination cocktails of new developed antigens by ELISA are needed. Method: To compare the efficacy of different mycobacterial specific antigen and to assess the applicability of the combination of several different antigens in the diagnosis of tuberculosis, five ELISA tests derived 14KDa, 16KDa, 19KDa, 23KDa, 38KDa were evaluated in 57 active pulmonary patient and 24 inactive post-therapy follow up patient and 48 normal control. Results: The optical densities of ELISA test with 14KDa, 16KDa, 19KDa, 23KDa, 38KDa were significantly higher in active tuberculosis cases than in normal control(P<0.001, P<0.001, P<0.027, P<0.001, P<0.001) and those with 16KDa, 38KDa were significant higher in active tuberculosis cases than in inactive post-therapy follow up cases(P<0.01. P<0.001) and those of 14KDa, 16KDa, 23KDa, 38KDa were significant higher in inactive post-therapy follow up cases than in normal control(P<0.008. P<0.01. P<0.006. P<0.001). The sensitivity of 14KDa, 16KDa, 19KDa, 23KDa, 38KDa in active pulmonary patient cases was 42.1%, 43.9%, 15.8%, 28.0%, 70.2%, respectively and the specificity of 14KDa, 16KDa, 19KDa, 23KDa, 38KDa in active pulmonary patient cases was 95.8%, 95.8%, 91.7%, 89.6%, 93.8%, respectively. The sensitivity and specificity of combination 38KDa with 16KDa was 87% and 93.7%. Conclusion: The sensitivity and specificity of new antigens for serodiagnosis of the tuberculosis still remains limited at around 70%, which makes its a poor diagnostic tool for disease confirmation. A combination of cocktail antigens provided by cut-off value adjustment for serodiagnosis of tuberculosis some improved diagnostic yield than single antigen serologic test.

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Development of Immunoassay Systems for the Assay of Soy Protein in Meat Products; The Assay of Soy Protein in Meat Blends and Commercial Product by Enzyme-linked Immunosorbent Assay(ELISA) (육제품에 첨가된 대두단백 정량을 위한 면역분석법 개발에 관한 연구 ; ELISA에 의한 고기유화물 및 시판육제품에 첨가된 대두단백 정량)

  • Kim, Cheon-Jei;Kim, Jong-Bae;Kim, Byung-Cheol;Lee, Seoung-Bae;Jung, Sung-Won;Choe, Doo-Young;Ko, Won-Sick
    • Korean Journal of Food Science and Technology
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    • v.24 no.5
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    • pp.397-403
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    • 1992
  • This study was carried out for the development of assay method to quantify the soy protein content in meat homogenate, emulsion-type sausage and commercial meat products by Enzyme-linked Immunosorbent Assay(ELISA). The standard antigen was extracted before and after heat treatment. It was observed that the degree of reaction was not varied significantly according to the heating temperature. The recovery rate in meat homogenate and emulsion-type sausage was not varied significantly according to the heating temperature. The reaction was not interfered with fat and spices of the samples. Samples with 10% soy protein showed lower correlation than those with 2% and 5% soy protein. The recovery rate in commercial meat products showed difference individually. The correlation of some products with raw vegetable and wheat starch was relatively low.

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Methods for Coating the Killed Whole Cell Antigens of Salmonella typhi in Enzyme-linked Immunosorbent Assay (효소면역측정법을 위한 장티푸스 균체항원의 부착방법)

  • Kim, Youn-Won;Hwang, Eung-Soo;Kook, Yoon-Hoh;Choi, Kang-Won;Kim, Ik-Sang;Cha, Chang-Yong;Lee, Seung-Hoon
    • The Journal of the Korean Society for Microbiology
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    • v.20 no.1
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    • pp.91-102
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    • 1985
  • The advantages of enzyme-linked immunosorbent assay(ELISA) are its senstivity and simplicity in detecting IgG, IgM and IgA antibody. To apply ELISA to diagnosis of typhoid fever, antigen such as lipopolysaccharide of Salmonella typhi or killed whole cell must be coated on solid phase. It is easy to coat lipopolysaccharide on ELISA plate but troublesome to purify it. As it is easy to obtain the killed whole cells, the development of the appropriate method by which those antigens of S. typhi are optimally coated on solid phase is needed. To establish the appropriate method, carbonate buffer, methanol or poly-L-lysine was applied as binding substance on polystyrene or polyvinylchloride plate as solid phase when the killed whole cell antigens of S. typhi varided as follows: $10^6$, $10^7$, $10^8$ and $10^9\;cell/ml$. The criteria of the optimal method were determined as follows: 1. The optical density of positive sera is above 1.0(0.6 in IgM) at 1:10 serum dilution and is 0.3(0.2 in IgM) higher than that of negative sera: 2. The O.D. of sera is flat or lowering according to serum dilution: 3. It must be that the O.D. of negative sera is lower than 0.2 at the point of serum dilution where the O.D. of positive sera is higher than 1.0(0.5 in IgM). The results obtained were summarized as follows: 1. The methods which fitted the above criteria were to use poly-L-lysine as binding substance, polyvinylchloride plate as solid phase and $10^7\;cell/ml$ as antigen concentration of S. typhi(poly-L-lysine/polyvinylchloride/$10^7$) and poly-L-lysine/polyvinylchloride/$10^8$ in detecting IgG antibody, methanol/polystyrene/$10^9$, poly-L-lysine/polyvinylchloride/$10^8$ and poly-L-lysine/polyvinylchloride/$10^9$ in IgM and carbonate buffer/polystyrene/$10^8$, carbonate buffer/polystyrene/$10^9$, methanol/polystyrene/$10^8$, methanol/polyvinylchloride/$10^8$, methanol/polyvinylchloride/$10^9$, poly-L-lysine/polyvinylchloride/$10^8$ and poly-L-lysine/polyvinylchloride/$10^9$ in IgA. 2. The coaling method using poly-L-lysine, polyvinylchloride plate and $10^8\;cell/ml$ was best to assay IgG, IgM and IgA antibody all in one. By this method, to assay the each immunoglobulin calss with an appropriate fixed serum dilution, 1:320 dilution was best.

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Effects of Vitamin C on Residual Aflatoxin $B_1$ in Rat Sera Treated with Radiation and Aflatoxin $B_1$ (Vitamin C가 방사선과 Aflatoxin $B_1$을 투여한 흰쥐의 혈청 중 Aflatoxin $B_1$ 잔류량에 미치는 영향)

  • Chung, Do-Young;Kim, Han-Soo;Kang, Jin-Soon
    • Food Science and Preservation
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    • v.18 no.3
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    • pp.374-382
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    • 2011
  • Aflatoxin ($AFB_1$) is a potent hepatotoxic and hepatocarcinogenic mycotoxin in humans. It is also well-known to be accumulated in animal tissues via various metabolic pathways. This study was conducted to determine the effects of vitamin C on the residual $AFB_1$ in rat sera that were treated with radiation and $AFB_1$. Six week-old male Sprague-Dawley rats were randomly divided into five groups: a control group, $AFB_1$-treated group, the group treated with $AFB_1$ and vitamin C, the group treated with X-ray and AFB1, and the group treated with X-ray and $AFB_1$ with vitamin C. On the first day of the experiment, only one dose of X-rays was exposed to the entire liver at 1,500 cGy. Next, vitamin C was injected at 10 mg/kg body weight via intraperitoneal injection, followed 1 hr later by the administration of 0.4 mg/kg of $AFB_1$ via intraperitoneal injection. These treatments were then administered every three days over a period of 15 days. On the 16th day of treatments, the animals were sacrificed. The contents of $AFB_1$ in rat sera were determined via indirect competitive ELISA and HPLC method. In the quantitative analysis of $AFB_1$ in rat sera via ELISA, $5.17{\pm}0.34$ ng/mL of $AFB_1$ was detected in the $AFB_1$-treated groups, but the amount more significantly decreased to $3.23{\pm}0.76$ ng/mL in the groups treated with $AFB_1$ and vitamin C (p<0.01) than in the $AFB_1$-treated groups. The $AFB_1$ contents of the rat sera of the groups treated with X-ray and $AFB_1$ did not significantly decreased with the administration of vitamin C. The $AFB_1$ content of the rat sera that was analyzed via HPLC showed a tendency similar to that of the content that was analyzed via ELISA. With regard to these data, vitamin C was very effective in reducing $AFB_1$ residue in rat sera.