China Animal Husbandry and Veterinary Medicine ›› 2020, Vol. 47 ›› Issue (4): 1191-1198.doi: 10.16431/j.cnki.1671-7236.2020.04.025

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Development of a Time-resolved Fluorescence Immunochromatography Assay Kit for Detection of Bovine Serum Amyloid A

WU Meng1, WEI Zhijing1, FAN Yiming2, CUI Chenguang3, LI Shuang4, DONG Weiya5   

  1. 1. Biology Institute, Hebei Academy of Sciences, Shijiazhuang 050081, China;
    2. College of Veterinary Medicine(Traditional Chinese Veterinary Medicine), Hebei Agricultural University, Baoding 071000, China;
    3. Animal Products Quality Monitoring Station of Xingtang County, Hebei Province, Shijiazhuang 050600, China;
    4. Shijiazhuang Junlebao Dairy Co., Ltd., Shijiazhuang 050221, China;
    5. Hebei Animal Epidemic Prevention and Control Center, Shijiazhuang 050000, China
  • Received:2019-09-27 Online:2020-04-20 Published:2020-04-17

Abstract: The purpose of this study was to develop a time-resolved fluorescence immunochromatography kit for clinical rapid detection of bovine serum amyloid A (SAA) content in milk.By using double antibody sandwich method and fluorescence immunochromatography detection technology,the immunochromatography detection kit was manufactured.The SAA monoclonal antibody used for labeling was labeled with fluorescent microspheres,and chicken IgY was labeled with fluorescent microspheres,too.Then the above two kinds of fluorescent microspheres labeled antibody mixture was fixed on the binding pad.The SAA monoclonal antibody used for coating was coated on the detection area of nitrocellulose membrane,and goat anti-chicken IgY was coated on the quality control area of nitrocellulose membrane.The standard curve of the kit was drawn after screening of antibody pairing materials and optimization of the process of fluorescent microsphere labeled antibody.Besides,the blank limit,precision,stability and sample testing performance of the kit were preliminarily evaluated.The results showed that,the Medix SAA-2 monoclonal antibody as coating antibody and YBX SAA-3 monoclonal antibody as labeling antibody was the optimum antibody pairing materials.In the process of labeling antibody with fluorescent microsphere,the optimal results were that the mass ratio of fluorescent microspheres to antibodies was 40∶1,and the carboxyl molar ratio of coupling agent to fluorescent microspheres was 2∶1.The four-parameter fitting curve equation of the standard curve of the kit was y=(1.03947-0.00182)/[1+(x/12.08222)×(-0.84692)]+0.00182,the linear correlation coefficient was R2=0.9997.The blank limit of the developed bovine SAA detection kit was 0.052 mg/L.Precision test result showed that,the intra-assay coefficient of variation was < 15%,and the inter-assay coefficient of variation was < 20%.The room temperature stability test showed that,the fluorescence T/C value of the sealed kit stored at room temperature for 6 months decreased by about 15%.The correlation coefficient R2 of self-made kit and Shanghai Blue Gene kit was 0.97.In summary,the developed kit had the advantages of simple operation,high sensitivity and low cost,which could meet the needs of clinical determination,and provided a new type of fast and accurate detection method for bovine SAA detection.

Key words: bovine serum amyloid A; mastitis; fluorescence immunochromatography; quantitative detection

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