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. 2007 Jun;35(3):203-10.
doi: 10.1016/j.biologicals.2006.11.001. Epub 2007 Jan 29.

Establishment of a reference panel for the detection of anti-SARS-CoV antibodies

Chunyu Liu  1 , Xiuhua Li, Chuntao Zhang, Sihong Xu, Yiming Shao, Hui Zhuang, Xiaoyan Che, Yan Qiu, Hongzhang Yin, Defu Li, Youchun Wang
Affiliations

Establishment of a reference panel for the detection of anti-SARS-CoV antibodies

Chunyu Liu et al. Biologicals. 2007 Jun.

Abstract

The immunological assays for detection of antibodies against SARS-CoV were developed in-house and some of them are available commercially. However, the antigens used in these assays differed. In order to validate the reliability of these assays, the standard panel should be established. In this study, we have expressed and purified severe acute respiratory syndrome (SARS) structural proteins and their fragments and developed indirect enzyme-linked immunosorbent assays (ELISAs) that detect antibodies against the SARS N, N(1), N(2), S(1), S(C), S(2), and M proteins as well as the human coronavirus OC43 and 229E N proteins. These assays were used to screen 58 samples from SARS convalescent patients, 40 serial serum specimens from patients at different phases of SARS infection, and 88 plasma specimens from normal blood donors. The samples from normal blood donors were also tested for antibodies against other respiratory virus. The representative samples were chosen to comprise a reference panel of SARS antibodies that may be used for the detection of SARS. The panel is composed of 25 positive samples, 25 negative samples, 7 diluted samples for anti-N antibody, 6 diluted samples for anti-S antibody, and one sample for validating precision. Comparison of detection results with different SARS antibody assays indicated that our panel should differentiate the specificity and sensitivity of different assays.

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Figures

Fig. 1
Fig. 1
The expressed fusion proteins analyzed by SDS-PAGE. Lane 1, protein marker. Lane 2, supernate of BL-21 with pET-30a as negative control. Lane 3–5, SARS-CoV N, N1 and N2 protein expressed in supernate. Lane 6–8, SARS-CoV S1,SC and S2 protein expressed in inclusion bodies. Lane 9, pellet of BL-21 with pET-30a as negative control. Lane 10, HcoV OC43 N protein expressed in M1 supernate. Lane 11, HcoV 229E N protein expressed in M1 supernate. Lane 12, supernate of M1 with pQE30-OC43N as negative control.
Fig. 2
Fig. 2
The purified proteins analyzed by SDS-PAGE. Lane 1, protein marker. Lane 2–7, purified SARS-CoV N protein (48.3 kD), SARS-CoV N1 protein (29.5 kD), N2 protein (32.3 kD), S1 protein (76.5 kD), SC protein (54.8 kD) and S2 protein (53.6 kD). Lane 8, purified HcoV OC43 N protein (50.8 kD). Lane 9, purified HcoV 229E N protein (44.7 kD).
Fig. 3
Fig. 3
Identification of recombinant proteins by Western blot. Lane 1, protein marker. Lane 2, BL-21 with pET-30a as negative control. Lane 3–8, SARS-CoV N,N1,N2,S1,SC and S2 protein performed.
Fig. 4
Fig. 4
Change in detection rate of different antibodies in serial sera over the course of infection.
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