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. 2013 Jan;187(1):72-8.
doi: 10.1016/j.jviromet.2012.09.006. Epub 2012 Sep 17.

Quantitative and sensitive detection of the SARS-CoV spike protein using bispecific monoclonal antibody-based enzyme-linked immunoassay

Hoon H Sunwoo  1 , Arivazhagan Palaniyappan, Advaita Ganguly, Pravin K Bhatnagar, Dipankar Das, Ayman O S El-Kadi, Mavanur R Suresh
Affiliations

Quantitative and sensitive detection of the SARS-CoV spike protein using bispecific monoclonal antibody-based enzyme-linked immunoassay

Hoon H Sunwoo et al. J Virol Methods. 2013 Jan.

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein is known to mediate receptor interaction and immune recognition and thus it is considered as a major target for vaccine design. The spike protein plays an important role in virus entry, virus receptor interactions, and virus tropism. Sensitive diagnosis of SARS is essential for the control of the disease in humans. Recombinant SARS-CoV S1 antigen was produced and purified for the development of monoclonal and bi-specific monoclonal antibodies. The hybridomas secreting anti-S1 antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. The sandwich ELISA was formed by using F26G18 as a coating antibody and biotinylated F26G18 as a detection antibody with a detection limit of 0.037μg/ml (p<0.02). The same detection limit was found with P136.8D12 as a coating antibody and biotinylated F26G18 as a detection antibody. The sensitivity was improved (detection limit of 0.019μg/ml), however, when using bi-specific monoclonal antibody (F157) as the detection antibody. In conclusion, the method described in this study allows sensitive detection of a recombinant SARS spike protein by sandwich ELISA with bi-specific monoclonal antibody and could be used for the diagnosis of patients suspected with SARS.

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Figures

Fig. 1
Fig. 1
Reactivity of anti-SARS-CoV S-protein mAb (P136.812) to SARS-CoV S1. (A) SDS-PAGE analysis. Lane M: standard protein molecular weight markers. Lane 1: SARS-CoV S1. (B) Western blot analysis of S-protein probed with anti-SARS-Cov S1 mAb (P136.812) in lane 1.
Fig. 2
Fig. 2
Titer of purified bsmAb-HRPO complex (P157 and F157 at a concentration of 20 ng/ml) by ELISA.
Fig. 3
Fig. 3
ELISA for detection of SARS-CoV S1 antigen using biotinylated mAb (F26G18). (A) Sandwich ELISA with anti-SARS-CoV S1 mAb (F26G18) as a coating antibody and same mAb labeled with biotin as a detection antibody. The biotinylated mAb (F26G18) was detected using Streptavidin-HRPO. (B) Sandwich ELISA with anti-SARS-CoV S1 mAb (P136.8D12) as a coating antibody and another mAb labeled with biotin (F26G18). The biotinylated mAb was detected using Streptavidin–HRPO. The results are expressed as absorbance reading at 650 nm wavelength. Arrows indicate the limit of detection.
Fig. 4
Fig. 4
ELISA for detection of SARS-CoV S1 antigen using bsmAb (F157). (A) Sandwich ELISA with anti-SARS-CoV S1 mAb (F26G18) as a coating antibody and bsmAb-HRPO complex (F157) as a detection antibody. (B) Sandwich ELISA with anti-SARS-CoV S1 mAb (P136.8D12) as a coating antibody and bsmAb-HRPO complex (F157) as a detection antibody. The color was developed using TMB substrate. The results are expressed as absorbance reading at 650 nm wavelength. Arrows indicate the limit of detection.
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