Zika Virus

PRODUCT USAGE NOTES

Protein Confirmation & Molecular Weight The Zika antigens are expressed in insect cells and accordingly are expected to have the necessary post-translational modifications (e.g. glycosylation) required for proper folding. The native conformation of Zika NS1 is proposed to be hexameric, similar to other NS1 proteins, so molecular weight testing may produce varying results depending on the technique used (e.g. higher molecular weights may be seen using analytical SEC techniques due to oligomer formation). The specification for molecular weight provided on the COAs for the antigens were determined by reducing SDS-PAGE. There is an urgent medical need for rapid ZIKV diagnostic assays for use in the field to screen large numbers of samples. Existing tests include PCR assays and antibody (IgM/ IgG) detection assays, however differentiation between flaviviruses remains challenging. Assays that directly test for the presence of ZIKV (e.g. PCR) are only able to detect infections up to 5 days after onset of symptoms – after this, the virus is no longer detectable. To overcome flavivirus cross-reactivity in diagnostics, recombinant antigens to envelope and NS1 proteins are commonly used. IgG and IgM antibodies typically show a high sensitivity and specificity to these epitopes, especially NS1 which is thought to contain more species specific epitopes than the envelope proteins. The biological safety level assigned to live Zika virus is BSL-2.

Reducing cross-reactivity with Dengue To improve assay specificity, it is necessary to remove any cross-reacting antibodies that could bind to the antigen and cause a false result. Defined epitope blocking ELISAs have also been used to increase the specificity and have been useful for differentiating flaviviral infections through targeting epitopes on NS1 or E protein. By including low concentrations of unconjugated Dengue NS1 and Chikungunya NS1 antigens, it is possible to block antibodies that are highly cross reactive between the three flaviviruses.

RECOMMENDED DENGUE RECOMBINANTS: R01656 (NS1, Type 1) R01659 (Envelope, Type 1) R01657 (NS1, Type 2) R01660 (Envelope, Type 2) R01658 (NS1, Type 3) R01661 (Envelope, Type 3) R01663 (NS1, Type 4) R01662 (Envelope, Type 4)

Methods to increase IgM sensitivity To improve assay sensitivity to IgM, we recommended the following:

1. IgM-Capture Assays: Use anti-human IgM Fab fragment antibody as the capture as opposed to a full-length anti-human IgM antibody. This allows more Fab fragment antibodies to bind to the surface area on the solid substrate increasing the

number of sites available for total IgM antibody to be captured. 2. Lateral Flow Assays: Employ the bridging method in which colloidal gold-labelled disease specific antibody (e.g. MAb or PAb to Zika NS1) is pre-mixed with recombinant antigen (e.g. Zika NS1). Conjugating colloidal gold directly to the Zika antigen can inhibit its ability to bind to captured IgM. Furthermore using a gold-conjugated PAb, which has a broad reactivity, can further increase assay sensitivity. A PAb can bind to different antigen epitopes therefore enabling more than one PAb to bind to the same antigen simultaneously to generate a stronger signal.

RECOMMENDED ZIKA POLYCLONALS: C01885G (Total IgG) C01886G (Affinity Purified) * C01886G Purified using Zika Virus NS1 to minimize cross-reactivity to other flaviviruses such as Dengue NS1