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Preparation of Anti-SEA Chicken Immunoglobulin Y and Detection of Circulating Schistosoma Japonica Antigen

Author: ZhangZuoMing
Tutor: GuanXiaoHong
School: Nanjing Medical University
Course: Pathology
Keywords: Schistosoma japonicum Immunodiagnostic Circulating antigen Yolk antibody Soluble Egg Hang original Enzyme - linked immunosorbent assay
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 69
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Abstract

The schistosomiasis immunodiagnostic including the detection of antibodies and circulating antigen detection. Detection antibody specificity and sensitivity are good, but the antibodies after cure still present in the serum longer time, and is difficult to distinguish the current infection and past infection, i.e. efficacy assessment value is not high; circulating antigen detection can reflect activity infection, better pathogen detection significance and efficacy assessment of the value of the schistosome circulating antigen detection specificity and high sensitivity to chronic infection. Therefore, the establishment of a sensitive and specific efficacy appraisal value of circulating antigen detection method is a hot topic in the current schistosomiasis basic research. Yolk antibody (immunoglobulin Y, IgY) is the the specific antigen hen corresponding antibody accumulated and transferred to the yolk, and the yolk immunoglobulin class. Compared to mammalian IgG, IgY has a high affinity to prepare a simple economic stability, does not activate complement, not combined with rheumatoid factor, nor the Fc receptor binding of mammalian immunoglobulin molecules, etc., in addition to distance with the mammalian germline, IgY does not mammalian immune globulin serological cross-reactivity occurs, as immunodiagnostic reagents can reduce false positives and improve the detection sensitivity and specificity. Objective: The application of anti-schistosome soluble egg antigen (soluble egg antigen, SEA) polyclonal IgY and enzyme-labeled anti-SEA monoclonal antibody NP28-5B combination to establish a double-antibody sandwich assay (sandwich ELISA, the S-ELISA) detection of circulating antigen to explore IgY value for the diagnosis of schistosomiasis diagnosis. Method: 1. Preparation and identification of anti-Schistosoma japonicum SEA chicken egg yolk antibodies: Schistosoma japonicum SEA via intravenous and subcutaneous immune under the wings of 25-week-old Highland hen 4 times (the first dose of 60μg / booster dose of 30μg / only), at intervals of 10d. Taken before immunization and yolk the 35d of eggs after the first immunization, purified IgY was diluted with water, extracted respectively. After purification of IgY concentration was measured by BCA method (2.2 '-Bicinohoninic acid.BCA) and detection by polyacrylamide gel electrophoresis (SDS-PAGE) analysis of its relative molecular weight and purity, indirect enzyme-linked immunosorbent assay (indirect-ELIsA) IgY activity and specificity. Anti-SEA monoclonal antibody NP28-5B preparation and enzyme markers: take Schistosoma japonicum infection BALB / c mouse spleen cells with syngeneic mouse myeloma cells SP2 / 0 fusion. Positive clones were screened by indirect ELISA positive wells cells were repeatedly subcloned to culture supernatants of hybridoma cell growth holes can with the SEA positive reaction so far, this time to confirm the anti-SEA monoclonal antibody NP28-5B cell lines to establish . After subculturing the hybridoma cell supernatants were collected, with 50% ammonium sulfate precipitation, and dissolved in 10 mM PBS (pH 7.4), to adequate dialysis, frozen at -70 ℃ standby. Horseradish peroxidase (HRP) labeled NP28-5B (Simple sodium iodide method). 3.IgY detect Schistosoma japonicum circulating antigen-antibody sandwich establishment: purified chicken IgY as a capture antibody, HRP-labeled NP28-5B double antibody sandwich enzyme-linked immunosorbent assay (S-ELISA) for the detection of antibodies to establish. Created from the package, antibody concentration, serum incubation time, the enzyme-labeled antibody concentration and enzyme-labeled antibody incubation time in four aspects to optimize the use of orthogonal test. SEA-ELISA and conventional detection of antibodies to detect the number of copies of acute and chronic schistosomiasis and healthy human serum. Sera from infected sources: of 9 acute schistosomiasis in human serum and 45 copies of chronic schistosomiasis human serum were collected from the the Jiangxi Jiujiang DUCHANG schistosomiasis control the Dali Bai Autonomous Prefecture, Yunnan Weishan County hamster parastic mouse Jiecun. Healthy serum 50 were taken from non-endemic areas, Binzhou City, Shandong Province. Results: 1. Highland hens after SEA after the first immunization 10d that specific IgY strengthen immune aftereffect price gradually increased to 35d Each egg to about 61mg antibodies can be obtained after purification by non-reduced SDS -PAGE analysis, purified IgY major protein band of a relative molecular weight of 130,000. Also, the reduced SDS-PAGE analysis showed that after cleavage of IgY of band, which contains a Mr of 66,000 and 35,000 two main belt (7). 2.NP28-5B hybridoma culture supernatant was precipitated by 50% ammonium sulfate solution, the concentration of antibodies obtained after 61.75mg/ml adequate dialysis, measured by indirect ELISA antibody activity; simple marker enzyme sodium iodide method after The working concentration of 1:600 ??~ 1:1000. 3.S-ELISA method to measure SEA concentration (y) and the absorbance (A450) of the value (x) in the linear relationship between 2.25ng/mL ~ 900 ng / mL (r = 0.948,1, y = 459.22x-108.14), the lowest detection limit was 2.2 5ng. Detect the acute schistosomiasis people circulating antigen positive rate of 100.00% (9/9), chronic schistosomiasis circulating antigen positive rate was 84.40% (38/45), healthy specificity of 96.00% (48/50). SEA-ELISA detection rate of schistosomiasis was not statistically significant (P> 0.05). Conclusion: 1.SEA immune hens can produce anti-SEA IgY. 2 S-ELISA method established by detection of circulating antigen of Schistosoma japonicum better sensitivity and specificity, and can be used for the diagnosis of schistosomiasis immune.

Preparation of Anti-SEA Chicken Immunoglobulin Y and Detection of Circulating Schistosoma Japonica Antigen

Abstract

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