Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens

Abstract

Background: Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection.

Methods: RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006-2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems.

Results: rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%.

Conclusions: Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

Keywords: Expression; IgG sandwich ELISA; IgM capture ELISA; Nucleocapsid protein; RVFV.

Fig. 1

Expression and purification of recombinant RVFV-N protein. Recombinant plasmid containing the full length RVFV N gene was transformed into E. coli XL1-blue strain and induced with IPTG. E.coli cells were collected and dissolved in 10 mM PBS (pH 7.5) with 500 mM NaCl. After sonication, the E. coli cell lysate was centrifuged and the recombinant protein was purified from the supernatant by Talon™ IMAC affinity column. The E. coli cell lysate and purified recombinant protein were analyzed in a 10% SDS-PAGE gel and revealed with Coomassie brilliant blue staining. Lane 1: protein marker (Precision plus protein standards, Bio-Rad); Lane 2: supernatant of sonicated E. coli cell lysate after centrifugation; Lane 3: pellet of sonicated E.coli cell lysate; Lane 4: purified recombinant protein

Fig. 2

Western-blot analysis of purified RVFV-N protein. Pre-stained protein marker and purified recombinant RVFV-N protein were separated by SDS-PAGE and transferred to a PVDF membrane. Each membrane was incubated with diluted rabbit RVF hyper-immune sera or mouse anti-Histidine serum followed by horseradish peroxidase(HRP)-conjugated anti-rabbit IgG or anti-mouse IgG (1:1000 dilution) and detected by DAB staining. a Reactivity of recombinant protein to rabbit RVF hyper-immune sera. b Reactivity of recombinant protein to mouse anti-Histidine antibody. Lane 1: protein marker (Precision plus protein standards, Bio-Rad); Lane 2: purified RVFV-N protein

Fig. 3

Comparison of rRVFV-N protein with inactivated RVFV antigen in inactivated virus based-IgG sandwich ELISA system. Inactivated virus-based IgG sandwich ELISA system used in the RVF diagnosis reference laboratory was used to evaluate the rRVFV-N protein by replacing the inactivated virus antigen (RVFV culture fluid) with the rRVFV-N protein. Twelve human serum samples collected during the RVF surveillance were tested by using the virus culture fluid (X axis) or rRVFV-N protein as antigen (Y axis)

Fig. 4

Comparison of rRVFV-N protein with inactivated virus antigen in inactivated virus based-IgM capture ELISA system. Inactivated virus based IgM capture system used in the RVF diagnosis reference laboratory was used to evaluate the RVFV-N protein by replacing the inactivated virus antigen (RVFV culture fluid) with the rRVFV-N protein. Twelve human serum samples collected from an RVF outbreak were tested by using the virus culture fluid (X axis) or rRVFV-N protein as antigen (Y axis)