• Title, Summary, Keyword: enzyme immunoassay

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Studies on enzyme immunoassay for determining progesterone of bovine plasma and its clinical application I. Optimizing double antibody for progesterone in enzyme immunoassay (Enzyme immunoassay(EIA)에 의한 소의 혈중 progesterone 측정과 이의 응용에 관한 연구 I. 이항체(二項體)의 최적조건에 관한 연구)

  • Kang, Chung-boo;Shin, Jong-uk;Choe, Sang-yong
    • Korean Journal of Veterinary Research
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    • v.28 no.2
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    • pp.321-325
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    • 1988
  • This experiment was carried out to determine the progesterone concentration of bovine plasma by liquid phase double antibody enzyme immunoassay. The optimum conditions of assay-system, double (first and second) antibody and carrier (normal rabbit serum) were investigated. The optimum dilution rate of first antibody, second antibody and normal rabbit serum was $10{\times}10^3$ to $15{\times}10^3$, 20 and $1{\times}10^3$ times, respectively.

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Studies on enzyme immunoassay for determining progesterone of bovine plasma and its clinical application. II. Establishment of enzyme immunoassay for progesterone (Enzyme immunoassay(EIA)에 의한 소의 progesterone 측정과 이의 응용에 관한 연구 II. Progesterone 측정에 대한 효소면역측정방법(酵素免疫測定方法)의 확립)

  • Kang, Chung-boo;Shin, Jong-uk;Choe, Sang-yong
    • Korean Journal of Veterinary Research
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    • v.29 no.1
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    • pp.21-25
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    • 1989
  • This experiment was carried out to determine the progesterone concentration of bovine plasma by liquid phase double antibody enzyme immunoassay. The optimum conditions of assay-system, enzyme conjugate and gelatin were invested. The sensitivity, recovery rate and reproducibility by this assay were also analyzed. The results obtained were as follows: 1. The suitable supplementation level of gelatin was 0.2%. As the gelatin level increased to 1%, coefficient variation of interassay was shown to be irregular. 2. The optimum dilution rate of enzyme conjugate was 30 times. Enzyme activity was greatly fluctuated depending on the minor condition of enzyme conjugate technique. Therefore, it was considered to be checked when determined. 3. The sensitivity of the assay was 12 pg/tube. 4. Recovery rate was decreased when the amount of sample was too little or too much, but the recovery rate was high as 97.8% when the amount of sample between 50 and $200{\mu}l$. 5. Mean intra-assay and inter-assay coefficient of variation was 4.5% and 5.9%, respectively. By using liquid phase double antibody enzyme immunoassay, progesterone in plasma can be detected, and also this assay system is applicable to study on physiological function of progesterone and to diagnosis of reproductive disorders.

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Role of enzyme immunoassay for the Detection of Helicobacter pylori Stool Antigen in Confirming Eradication After Quadruple Therapy in Children (소아에서 4제요법 후 enzyme immunoassay에 의한 Helicobacter pylori 대변 항원 검출법의 유용성에 대한 연구)

  • Yang, Hye Ran;Seo, Jeong Kee
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.7 no.2
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    • pp.153-162
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    • 2004
  • Purpose: The Helicobacter pylori stool antigen (HpSA) enzyme immunoassay is a non-invasive test for the diagnosis and monitoring of H. pylori infection. But, there are few validation studies on the HpSA test after eradication in children. The aim of this study was to assess the diagnostic accuracy of HpSA enzyme immunoassay for the detection of H. pylori to confirm eradication in children. Methods: From January 2001 to October 2003, 164 tests were performed in 146 children aged 1 to 17.5 years (mean $9.3{\pm}4.3$ years). H. pylori infection was confirmed by endoscopy-based tests (rapid urease test, histology, and culture). All H. pylori infected children were treated with quadruple regimens (Omeprazole, amoxicillin, metronidazole and bismuth subcitrate for 7 days). Stool specimens were collected from all patients for the HpSA enzyme immunoassay (Primier platinum HpSA). The results of HpSA tests were interpreted as positive for $OD{\geq}0.160$, unresolved for $$0.140{\leq_-}OD$$<0.160, and negative for OD<0.140 at 450 nm on spectrophotometer. Results: 1) One hundred thirty-one HpSA tests were performed before treatment. The result of HpSA enzyme immunoassay showed three false positive cases and one false negative case. The sensitivity, specificity, positive predictive value, and negative predictive value of HpSA enzyme immunoassay before treatment were 96.4%, 97.1%, 90%, and 99%, respectively. 2) Thirty-three HpSA enzyme immunoassay were performed at least 4 weeks after eradication therapy. The results of HpSA enzyme immunoassay showed two false positive cases and one false negative case. The sensitivity, specificity, positive predictive value, and negative predictive value after treatment were 88.9%, 91.7%, 80%, and 95.7%, respectively. Conclusion: Diagnostic accuracy of the HpSA enzyme immunoassay after eradication therapy was as high as that of the HpSA test before eradication therapy. The HpSA enzyme immunoassay was found to be a useful non-invasive method to confirm H. pylori eradication in children.

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An Improved Method for Detection of Salmonella Typhi O Antigen with Staphylococcal Protein A Using Enzyme Immunoassay (포도구균의 A단백질을 이용한 효소면역법으로 살모넬라 O항원 검출)

  • Rhyu, Mun-Gan;Kim, Gum-Ryong;Lee, Choong-Ki
    • The Journal of the Korean Society for Microbiology
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    • v.22 no.4
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    • pp.445-451
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    • 1987
  • Coagglutination method is widely used for the diagnosis of Salmonella infection. This test, however, has a disadvantage of false positive reaction due to the coagglutination of staphylococci with non-specific immune complexes or anti-staphylococci antibody in serum. Salmonell O antigen was detected by enzyme immunoassay with protein A-bearing Staphylococcus aureus as in the solid phase. Horse radish peroxidase was labeled to IgG specific against Salmonella O antigen. This enzyme immunoassay was much more sensitive than conventional coagglutination method without false poitive agglutination. To improve the sensitivity for detection of Salmonella O antigen in samples, we tried to determine the optimal concentration of normal IgG that inhibits non-specific binding of horse radish peroxidase labeled IgG to staphylococci, and to establish the optimal condition of reaction between antigen-antibody complex and staphylococci. Non-specific binding of horse radish peroxidase labeled specific IgG to staphylococci was almost blocked when the enzyme labeled IgG was 500-fold diluted with phosphate buffered saline containing 2mg/ml of normal IgG. When staphylococci coated with antibody to Salmonella O antigen were mixed with antigen-antibody complex and then incubated for 1 hour at room temperature, the minimal detectable concentration of Salmonella O antigen was 1ng/ml. The sensitivity of enzyme immunoassay was 100-fold greater than a conventional coagglutination method. This enzyme immunoassay could be expected as an improved method for detection of other infectious agents.

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Thermal Inactivation Kinetics of Tyichoderma viride Cellobiohydrolase Determined by Enzyme Linked Immunosorbent Assay and Residual Enzyme Assay (면역학적 방법에 의한 Cellobiohydrolase의 열역학적 특성)

  • 오태광;박관화
    • Microbiology and Biotechnology Letters
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    • v.17 no.4
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    • pp.365-369
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    • 1989
  • Thermal inactivation of Tyichoderma viride cellobiohydrolase was investigated by immunoassay and residual enzyme assay such as carboxymethyl cellulase (CMCase) and filter paper degradation activity (FPase). Arrhenius plots of cellobiohydrolase were appeared as straight line. The Z-values of cellobiohydrolase calculated by CMCase, FPase and immunoassay were 5.2$^{\circ}C$, 6.4$^{\circ}C$ and 5.8$^{\circ}C$, respectively. The thermodynamic parameters obtained from FPase were better agreement with those of immunoassay than CMCase assay.

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Development of a One-step Two-site Enzyme Immunoassay for Measuring Human Alpha-fetoprotein by Eliminating Hook-effect

  • Kim, Se-Ho
    • BMB Reports
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    • v.34 no.1
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    • pp.47-50
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    • 2001
  • A one-step, two-site enzyme immunoassay was developed for measuring human alpha-fetoprotein (AFP) in serum and amniotic fluid using monoclonal antibodies (McAb) by eliminating the high-dose hook effect. Three McAbs that recognize different epitopes were selected among 16 different clones on the basis of epitope mapping, two for immobilization and one for horseradish peroxidase conjugation. This one-step immunoassay system is more convenient and rapid compared to a conventional two-step sandwich immunoassay system. It did not exhibit the hook effect to around 2.7 mg/ml of AFP, which is probably one of the highest concentrations of AFP in the serum. The dose-response curve of the system was linear to 500 mg/ml of AFP and the system could differentiate as low as 1 mg/ml of AFP The intra- and inter-assay variations were in an acceptable range; 95~104% and 97~105% respectively Its correlation with other commercial systems was around 95%.

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Screening of Deoxynivalenol Producing Fungi from Greenhouse Horticulture by Enzyme Amplification System Immunoassay (Enzyme Amplification System Immunoassay에 의한 시설원예산물의 Deoxynivalenol 생성곰팡이의 검색)

  • Park, Mi-Ja;Park, Jung-Hyun;Chung, Duck-Hwa
    • Korean Journal of Food Science and Technology
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    • v.32 no.2
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    • pp.439-443
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    • 2000
  • In order to evaluate the safety of greenhouse horticulture products in Korea, we carried out this work by screening of Fusarium species, which produce deoxynivalenol (DON) from greenhouse horticulture in Western Gyeongnam and Northern Gyeongbuk, Korea. For this study, high sensitive enzyme-linked immunosorbent assay, ALP/NADP method, was applied to detection of DON by enzyme amplification system. From 192 samples of greenhouse horticulture soil and its products, 103 isolates of Fusarium species were obtained. The isolates were cultured at 28C for 15 days and the cultured mediums were extracted by ethyl acetate. The production of DON was verified by thin layer chromatography (TLC). As the results of TLC, 8 strains were identified as DON producing strain. We screened potential producers of DON by ALP/NADP. The levels of DON production were shown from 0.007 to 1.21 g/ml of YES medium. The maximum DON producing strain No. 32-D-3 was isolated from soil in Namhae, Korea. In conclusion, the above results indicate that DON producing fungi contaminated greenhouse horticulture products in Korea. Therefore, further studies are required to accumulate more detailed data about the contamination of DON in various cereals.

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Optimization of liquid phase enzyme immunoassay for determining of progesterone (Progesterone 측정을 위한 액상(液相) 효소면역측정법(酵素免疫測定法)의 최적조건에 관한 연구)

  • Kang, Chung-boo;Choi, Il-kwan;Son, Min-soo;Hur, Ju-hyeong;Kim, Chur-ho
    • Korean Journal of Veterinary Research
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    • v.32 no.3
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    • pp.429-434
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    • 1992
  • This study was carried out to develop an effective liquid-phase double antibody enzyme immunoassay for determining of progesterone. The optimum conditions of assay system, 1st and 2nd antibodies, enzyme conjugate, and time reaction were invested. The bovine plasma progesterone level in dairy cattle and korean native bulls were also analyzed. The results obtained were as follows; 1. The reproducibility of petroleum ether was superior to that of ethyl ether as extract solvent of progesterone in plasma. 2. The optimum dilution rate of 1st and 2nd antibody was 30,000 and 10 times, respectively. Affer the reaction of enzyme conjugate to progesterone 1st antibody, and then 2nd antibody competition reaction was enough for over 1hr. 3. Average plasma progesterone level in 4 pregnant and 9 nonpregnant Holstein was $2.5{\pm}0.5$ and $0.7{\pm}0.2ng/m{\ell}$, respectively. Average plasma progesterone level of 10 Korean native bulls was $0.1{\pm}0.001ng/m{\ell}$ From these results, by using liquid phase double antibody enzyme immunoassay for progesterone is applicable to detect of early pregnancy diagnosis, factorial analysis of reproductive disorder, and also reproductive physiological function such as monitoring of cyclicity during the post-partum period.

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Development of Homogeneous Enzyme Immunoassay for the Organophosphorus Insecticide Fenthion

  • Kim, Bok-Hee;Park, Eun-Yong;Lee, Yong-Tae;Lee, Jung-Hun;Lee, Sang-Hee
    • Journal of Microbiology and Biotechnology
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    • v.17 no.6
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    • pp.1002-1009
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    • 2007
  • A rapid, convenient homogeneous competitive enzyme immunoassay for estimating the amount of fenthion is described. The assay utilizes glucose-6-phosphate dehydrogenase-hapten conjugates that are inhibited in solution by antibodies obtained from bovine serum albumin-hapten conjugates. In order to investigate the effects of bridging group recognition on the sensitivity of dose response characteristics, the bridging groups of varying alkyl chain length were attached at the phosphate position of fenthion. Among the antibodies used, the one obtained from the use of hapten (fenthion analog) with the same bridging group structure that was used in preparing the enzyme-fenthion conjugates showed maximum inhibition (up to 51.8%) in the absence of fenthion. In the presence of fenthion, the activity of the enzyme-hapten conjugate is regained in an amount proportional to the fenthion concentration. Under the optimized condition, the $ED_{50}$ value for fenthion was $0.809{\mu}g/ml$. The assay developed in this study is a rapid effective screening method for fenthion prior to precise analysis.

Novel Liposome Immunoassay for Detection of Ultratrace Amount of Bioactive Substances : an Assay for Insulin

  • Lim, Soo-Jeong;Kim, Chong-Kook
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.281-281
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    • 1996
  • The immunoassay method is frequently used for the identification and quantitation of ultratrace amount of bioactive substances. Homogeneous liposome immunoassays, which can avoid the use of radioisotopes and separation steps, have recently been reported in many publications. Cytolysin-mediated liposome immunoassay using melittin ever been studied but showed limited applications. Here, we designed a homogeneous liposome immunoassay using Clostridium perfringens phospholipase C (PLC), an enzyme which catalyzes the hydrolysis of phosphatidylcholine in biological membranes, as a cytolysin.

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