A Rift Valley Fever in vitro diagnostic lateral flow test
Publication Date: 2007-Feb-14
The IP.com Prior Art Database
A Rift Valley Fever Virus recombinant protein was prepared (Onderstepoort Veterinary Institute )and incorporated into a lateral flow diagnostic assay (Vision Biotech).
Preparation of recombinant antigen
The gene encoding the nucleoprotein of RVFV was amplified with RT-PCR from viral RNA extracted from Onderstepoort RVFV isolate 35/74. After cloning and sequencing of the PCR product, the gene was sub-cloned into a bacterial expression vector and the protein was expressed in bacteria.
The recombinant protein containing a 6X Histidine fusion at the N-terminal was purified using commercial Nickel chelate columns
Stability and empirical PI estimation: 25µg stock RVF-nc-3574 was prepared in 5mM Borate pH 8 (no initial centrifugation of antigen to pellet cell debris). 20nm and 40nm AU colloid were used for conjugation. Different coating buffers and molarities were used and stability points were tested against analyte.
Conjugate Functionality was tested using a 50mM TRIS buffer system at pH 10 and concentrations of 0.4 µg/ml up to 4 µg/ml RVF-nc-3574, blocked with 1% BSA pH 9.2. RVF rNC (batch 4) recombinant antigen was spotted on 8µm Whatman nitrocellulose membrane and glass-fibre conjugation pad was used as the release pad. The best specificity and functionality was observed at 0.8 µg/ml pH 10 using standard c++ (3763) and –ve blood as analytes. Consequently a candidate conjugate was prepared using 100ml of 40nm AU colloid pH 10 (OD 1.125, Lmax 525nm), 50 ml of 50mM TRIS pH 10, 0.8 µg/ml RVF-nc-3574, 1% BSA pH 9.2. ...