Application and Development of Double Antibody Sandwich ELISA for Detection Haemophilus parasuis

doi:10.7668/hbnxb.2016.S1.079

Abstract

Abstract: In order to develop a double antibody sandwich ELISA to detect Haemophilus parasuis quickly and accurately,which using specific monoclonal antibody 1E2 to outer membrane protein P5 of Haemophilus parasuisas the capture antibody, and the rabbit anti-Haemophilus parasuis polyclonal antibody as the detection antibody, then determined the optimal working conditions of ELISA by square matrix titrimetry,and verified for specificity, sensitivity and detected the clinical samples. The results showed that the optimal enveloped concentration of monoclonal antibody 1E2 was 3.434 μg/mL, and the best diluted concentration of polyclonal antibody was 3.350 μg/mL,and the 10 mg/mL gelatin which was used as confining liquid was better than other closed liquid, and the optimal diluted concentration of goat-anti-rabbit IgG which labeled by HRP was 1:4 000.The developed ELISA had good specificity and the results were positive to detect the 15 serovars of Haemophilus parasuis, but no cross reactions with other bacteria.The sensitivity result of the ELISA showed that the minimum detection limit was 1×10⁶ cfu/mL.When the ELISA was used to detect the suspect Haemophilus parasuis 115 clinical samples, the results showed that the 83 samples of which were positive to Haemophilus parasuis, and the number of positive samples was higher than the results of the bacteria isolation and PCR identification.The above results showed that the developed sandwich ELISA was specific and sensitive, and can be applied to the detection of clinical samples.

Key words: Haemophilus parasuis, Monoclonal antibody, Sandwich ELISA

CLC Number:

S432.4

SONG Shuai, LI Chunling, ZANG Yingan, GUO Haixiang, LI Miao, YANG Dongxia, XU Zhihong. Application and Development of Double Antibody Sandwich ELISA for Detection Haemophilus parasuis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(S1): 463-469. doi: 10.7668/hbnxb.2016.S1.079.

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http://www.hbnxb.net/EN/Y2016/V31/IS1/463

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