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[Development and application of triple antibodies-based sandwich ELISA for detecting nucleocapsid protein of SARS-associated coronavirus].
Zhonghua Liu Xing Bing Xue Za Zhi. 2005 Apr; 26(4):277-81.ZL

Abstract

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.

CONCLUSION

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.

Authors+Show Affiliations

Center of Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article
Research Support, Non-U.S. Gov't

Language

chi

PubMed ID

15941537

Citation

Qiu, Li-wen, et al. "[Development and Application of Triple Antibodies-based Sandwich ELISA for Detecting Nucleocapsid Protein of SARS-associated Coronavirus]." Zhonghua Liu Xing Bing Xue Za Zhi = Zhonghua Liuxingbingxue Zazhi, vol. 26, no. 4, 2005, pp. 277-81.
Qiu LW, Tang HW, Wang YD, et al. [Development and application of triple antibodies-based sandwich ELISA for detecting nucleocapsid protein of SARS-associated coronavirus]. Zhonghua Liu Xing Bing Xue Za Zhi. 2005;26(4):277-81.
Qiu, L. W., Tang, H. W., Wang, Y. D., Liao, J. E., Hao, W., Wen, K., He, X. M., & Che, X. Y. (2005). [Development and application of triple antibodies-based sandwich ELISA for detecting nucleocapsid protein of SARS-associated coronavirus]. Zhonghua Liu Xing Bing Xue Za Zhi = Zhonghua Liuxingbingxue Zazhi, 26(4), 277-81.
Qiu LW, et al. [Development and Application of Triple Antibodies-based Sandwich ELISA for Detecting Nucleocapsid Protein of SARS-associated Coronavirus]. Zhonghua Liu Xing Bing Xue Za Zhi. 2005;26(4):277-81. PubMed PMID: 15941537.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Development and application of triple antibodies-based sandwich ELISA for detecting nucleocapsid protein of SARS-associated coronavirus]. AU - Qiu,Li-wen, AU - Tang,Han-wen, AU - Wang,Ya-di, AU - Liao,Jin-e, AU - Hao,Wei, AU - Wen,Kun, AU - He,Xiu-min, AU - Che,Xiao-yan, PY - 2005/6/9/pubmed PY - 2007/7/11/medline PY - 2005/6/9/entrez SP - 277 EP - 81 JF - Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi JO - Zhonghua Liu Xing Bing Xue Za Zhi VL - 26 IS - 4 N2 - 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. CONCLUSION: 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. SN - 0254-6450 UR - https://www.unboundmedicine.com/medline/citation/15941537/[Development_and_application_of_triple_antibodies_based_sandwich_ELISA_for_detecting_nucleocapsid_protein_of_SARS_associated_coronavirus]_ L2 - http://journal.yiigle.com/LinkIn.do?linkin_type=pubmed&issn=0254-6450&year=2005&vol=26&issue=4&fpage=277 DB - PRIME DP - Unbound Medicine ER -