Abstract
Rift Valley fever (RVF) is a mosquito-borne zoonotic disease that is caused by the Rift
Valley fever virus (RVFV); Bunyaviridae: Phlebovirus. RVF disease can infect a number
of different species, including ruminants, camels and humans. In livestock, the RVFV
infection is characterised by an acute hepatitis, abortion with high mortality rates in
newborn animals. The current RVF diagnostic techniques have shown good sensitivity;
however, they require extensive sample processing and complex instrumentation.
The first part of the study aimed to validate the binding of the RVFV antibodies to the
developed antigen for use in the development of an RVFV diagnostic assay. Recombinant
RVFV nucleocapsid protein (rNp) was used as a biorecognition molecule (antigen) to
detect pure anti-RVFV nucleocapsid antibodies. This was achieved by expressing a spliced
Np coding sequence into an E coli expression system that translated the complete Np gene
into the rNp. Western Blot (WB) analysis and Enzyme-Linked Immunosorbent Assay
(ELISA) were used to validate the interaction between the rNp and anti-RVFV antibodies.
The next phase of the study was to develop a Protein-A (SpA)/Gold nanoparticles (AuNP)
detection conjugate for the application in RVF lateral flow strip using the rNp antigen. The
RVF half strip lateral flow strip was designed to detect RVF positive, negative antibodies
and serum using the wet test. The SpA/AuNP conjugate was used as a chromatographic
probe to visualise the test and control line on the prepared lateral flow strip. The results
obtained in this part of the study showed detection of RVFV antibodies by the immobilised
5 mg/ml rNp antigen on the test tine and SpA on the control line of the prepared lateral
flow strip. The developed RVFV half-strip is capable of detecting as low as 0.125 mg/ml
of the anti-RVFV nucleocapsid antibody.
The Surface Enhanced Raman Spectroscopy (SERS) has shown potential in disease
detection without the prerequisites presented by the current RVF diagnostic assays. This
study reports on the potential use of SERS platform in the development of bio-sensing
probes for the detection of the Rift Valley Fever virus. The RVF rNp was immobilised on a
SERS substrate and used to capture RVF antibodies. Subsequently, a detection conjugate,
consisting of AuNPs labelled with SpA and a Raman reporter 4-mercaptobenzoic acid
(MBA), was allowed to bind to the tested RVF antibodies forming AuNP/Protein A/MBA hybrids. The hybrids were interpreted using vibrational Raman Spectroscopy, which
indirectly detects the binding interaction between the rNp antigen and RVF antibodies via
the Raman Reporter labelled conjugate. The ability of the SERS probes to detect an RVFV
infection was tested using varying concentrations of pure RVFV antibodies. The probes
exhibited sensitivity towards RVFV antibodies, with a detection limit of 1μg/ml showing
potential for a quantitative diagnostic assay for detecting the RVF virus.
The SERS based assay is able to detected a lower concentration range of 1 to 8 μg/ml and
is thus recommended for use as a confirmatory quantitative test after screening samples
with a lateral flow immunoassay.