Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

Abstract

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.

Fig. 1

ELISA reactivity of mAbs with whole, inactivated SARS-CoV. Hybridoma supernatants were tested at a 1:4 dilution in PBS+0.2% BSA on pre-blocked plates, coated with 18 ng per well of inactivated, highly purified SARS-CoV. Positive clones were identified as having positive binding (color) in wells that were at least four-fold higher than the background level reactivity on BSA. Antigen legend: black bars—native, highly purified SARS-CoV; white bars—heat denatured, highly purified SARS-CoV; grey bars—BSA control.

Fig. 2

Western immunoblot reactivity of mAbs with highly purified SARS-CoV. The positive and preimmune control sera were from the corresponding immune mouse and tested at 1:2000 dilution in TBS+0.2% BSA. Lanes marked 1 were loaded with highly purified SARS-CoV, lanes marked 2 with infected Vero cell lysate. The molecular weights of the pre-stained kaleidoscope markers (BioRAD) are indicated on the left of the figure.

Fig. 3

Competition ELISA measuring the binding of murine mAbs to highly purified SARS-CoV in the presence of human patient serum. Dilutions (as indicated) of a normal human serum control (white bar), or serum from convalescent SARS patient S3 were applied to wells coated with highly purified whole SARS-CoV. mAb F26G6 (Spike specific; black bars) or F26G15 (nucleoprotein specific; grey bars) were then added to the reactions. The results depicted for the pooled normal human serum (NHS) represent the mean of three replicate tests performed in the presence of mAb F26G6 combined with three replicate tests performed in the presence of mAb F26G15. Results are representative of identical assays performed in duplicate with gamma-irradiated patient sera (2 Mrad) (*P=0.01, **P=0.004, Student’s t-test).

Fig. 4

Immunofluorescence staining of SARS-CoV-infected Vero cells with Western immunoblot negative, neutralising mAbs to SARS-CoV. (A) F26G6, non-neutralising mAb specific for the Spike protein. (B) F26G3, neutralising mAb. (C) F26G7, neutralising mAb. (D) F26G9, neutralising mAb. (E) F26G10, neutralising mAb. (F) Anthrax toxin mAb, F25G1. (G) Anthrax toxin mAb F25G1 in bright field.