Rapid and efficient preparation of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein by immunizing mice.
OBJECTIVE
To develop a rapid and efficient method for preparing monoclonal antibodies (mAb) against SARS-associated coronavirus (SARS-Cov) nucleocapsid (N) protein.


METHODS
BALB/c mice were injected with the recombinant N protein of SARS-Cov into the foot-pads for the immunization, and the popliteal lymph nodes were isolated 15 d later for mAb-producing hybridomas, from which the mAbs against the N protein of SARS-Cov were screened. The identification of the mAb against the N protein of SARS-Cov was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting.


RESULTS
Four strains of hybridomas were obtained that produced the mAb specific to the N protein without detectable cross-reactivity with other pathogens. Of the 4 strains, 2 were identified as the immunoglobulin G1 (IgG1) isotype, 1 IgG2a, and the other IgG2b, with affinity constants (Ka) of 2 of the strains being 4.14 x 10(-9)M and 3.19 x 10(-9)M respectively.


CONCLUSIONS
This is the first report on the preparation of mAb that is specific to the SARS-Cov, and the high-specificity and high-affinity mAb produced by the 4 strains of hybridomas provide a basis for further researches on the pathogenesis and early diagnosis of SARS.
Che
Xiao-yan
XY
Central Laboratory, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, China. linche@pub.guangzhou.gd.cn
Qiu
Li-wen
LW
Pan
Yu-xian
YX
Xu
Hua
H
Hao
Wei
W
Liao
Zhi-yong
ZY
Mei
Ya-bo
YB
Zhang
Li-ya
LY
Wan
Zhuo-yue
ZY
Yuan
Guo-yong
GY
Huang
Zhen
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D019590:Nucleocapsid Proteins / Q000276:immunology*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins