›› 2019, Vol. 46 ›› Issue (6): 1756-1763.doi: 10.16431/j.cnki.1671-7236.2019.06.023

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Establishment of Indirect ELISA Method for Detecting Parabronemosis of Camel

SU Qian, FENG Chenchen, ZHAO Xueliang, WANG Jinling, WANG Wenlong, HUHE Bateer   

  1. Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China
  • Received:2018-11-30 Online:2019-06-20 Published:2019-06-19

Abstract:

In order to establish an indirect ELISA (iELISA) diagnostic method for detection of camel parabronemosis,in this experiment,the CPR gene of cysteine protease was recombinant expression,and the obtained recombinant protein (rCPR) was purified and detected by Western blotting.Using the purified recombinant protein as an antigen,the conditions of coating antigen,coating conditions,antibody dilution,dilution of enzyme-labeled secondary antibody,blocking solution and blocking time were optimized by checkerboard titration test,and the iELISA diagnostic method for detection of Parabronema skrjabini was established.Repeated,sensitive,specific and clinical tests were performed on the established iELISA assay.The results showed that the optimal coating concentration of the antigen was 8 μg/well,the optimal dilution of the serum and the enzyme-labeled secondary antibody were 1:50 and 1:5 000,respectively,and the optimal coating condition was 4℃ overnight.The optimal blocking condition was 3% BSA for 2 h.The cut-off value was 0.235,and the serum D450 nm value>0.235 was determined to be positive.The coefficient of variation in the repeatability test was <10%,and the repeatability was good;The sensitivity of detecting positive serum by this method was 96.3%;This method only specifically reacted with the positive serum of Parabronema skrjabini,and did not cross-react with other parasite-positive sera,the specificity was 100%;140 clinical sera were tested and the positive rate was 86.4%.In summary,this experiment successfully established an iELISA method for rapid and effective diagnosis of camel parabronemosis.

Key words: camel; Parabronema skrjabini; cysteine proteases; iELISA

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