Characterization of monoclonal antibody against SARS coronavirus nucleocapsid antigen and development of an antigen capture ELISA.
This report describes the production of several MAbs against N195 protein, a major immunodomain of SARS CoV nucleocapsid protein [He, Q., Chong, K.H., Chang, H.H., Leung, B., Ling, A.E., Wei, T., Chan, S.W., Ooi, E.E., Kwang, J., 2004. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11 (2) 417-422.]. One representative IgG1 monoclonal antibody (MAb), S-A5D5, was selected and characterized. S-A5D5 reacted specifically react with both recombinant and native nucleocapsid protein of SARS CoV. The reactivity of S-A5D5 with purified N195 protein and utilization of the MAb as a detector antibody to develop an antigen capture ELISA was assessed. As little as 37.5 pg of purified N protein and 50 TCID(50) of SARS CoV could be detected by the antigen capture ELISA. Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The N protein in N195-spike recombinant baculovirus-infected Sf-9 cells could also be identified. N protein was detected in 18 IFA IgM-positive serum samples collected from SARS confirmed patients, but not in nine samples collected from SARS recovery patient. No false positive results were given when 60 samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (IBV), chicken coronavirus, was tested. This monoclonal antibody-based antigen capture ELISA is thus a powerful tool for early diagnosis of SARS CoV infection.
He
Qigai
Q
Animal Health Biotechnology, Temasek Life Science Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore.
Du
Qingyun
Q
Lau
Suelyn
S
Manopo
Ivanus
I
Lu
Liqun
L
Chan
Shzu-Wei
SW
Fenner
Beau J
BJ
Kwang
Jimmy
J
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000032:analysis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D019590:Nucleocapsid Proteins / Q000032:analysis / Q000276:immunology; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology* / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D019590:Nucleocapsid Proteins