Development of an indirect ELISA with artificially synthesized N protein of PPR virus.
The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 μg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Guo-Rui
GR
Zhang
Jiang-Yong
JY
Zeng
Yu-Min
YM
Zhu
Shi-Juan
SJ
Dong
Se
S
Zhu
Rui-Song
RS
Yu
Ciren
C
Duoji
Zhi-Hai
ZH
Lei
Zhen
Z
Li
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006041:Goats; D019251:Nucleocapsid / Q000235:genetics; D029021:Peste-des-Petits-Ruminants / Q000175:diagnosis / Q000662:veterinary*; D018107:Peste-des-petits-ruminants virus / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D012756:Sheep; D018609:Tibet; D014730:Veterinary Medicine / Q000379:methods*; D014773:Virology / Q000379:methods*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins