[Development and application of triple antibodies-based sandwich ELISA for detecting nucleocapsid protein of SARS-associated coronavirus].
OBJECTIVE
To prepare and characterize monoclonal antibodies (mAb) and polyclonal antibodies against nucleocapsid (N) protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and to establish antibodies-based sandwich ELISA for detecting N protein of SARS-CoV, which might apply to early diagnosis of patients with SARS-CoV infection.


METHODS
BALB/c mice were immunized with purified recombinant N protein of SARS-CoV for producing mAbs, and New Zealand white rabbits were immunized for producing polyclonal antibodies. The identification of antibodies was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting. Capturing and detecting antibodies were selected by pairing the mAbs and polyclonal antibodies one by one and an antibodies-based sandwich antigen capture ELISA was used for detecting N antigen of SARS-CoV.


RESULTS
Nine mAbs and hyperimmune rabbit polyclonal antibodies, specifically against SARS-CoV nucleocapsid protein were obtained. Using paired ELISA assay, three mAbs N1E8, N8E1 and N10E4 were selected as capturing antibody and rabbit polyclonal antibodies as detecting antibody then triple antibodies-based sandwich ELISA was established following horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G. The recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml with this assay. When tested with 420 serum specimens from serologically confirmed SARS patients, the positive rates of serum N protein were 90.1%, 23% and 0%, in which sera collected from 1 to 10 days, 11 to 20 days and beyond 21 days respectively after the onset of symptoms. The specificity of the assay was 99.86% in 715 control serum specimens. There was no cross-reaction with other respiratory viruses and coronaviruses.


CONCLUSIONS
Specific and high affinity mAbs and rabbit polyclonal antibodies were obtained. By paired and optimized sandwich ELISA, a sensitive and specific antigen capture ELISA was established for detecting N antigen of SARS-CoV, which might apply to early diagnosis, source tracing and epidemiological studies of SARS.
Qiu
Li-wen
LW
Center of Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Tang
Han-wen
HW
Wang
Ya-di
YD
Liao
Jin-e
JE
Hao
Wei
W
Wen
Kun
K
He
Xiu-min
XM
Che
Xiao-yan
XY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000914:Antibodies, Viral / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D019251:Nucleocapsid / Q000276:immunology*; D011817:Rabbits; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000821:virology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral