TROUBLESHOOTING & USAGE TIPS
Envelope (E) Protein: The dominant protein present on a surface of the flavivirus virion and is a major target for neutralizing antibodies. It contains highly conserved regions therefore antibodies directed to the envelope protein tend to be cross-reactive. NS1 Protein: A glycosylated, membrane-associated, secreted glycoprotein with replication and immune evasion functions. NS1 antigens can be detected very early in infection (as early as 1 day post infection). In addition NS1 is serotype-specific, and for viruses such as Dengue where more than one serotype circulates, NS1 antigens enable virus serotyping by ELISA. The Flavivirus genera (e.g. Dengue, Zika, Japanese Encephalitis Virus, West Nile, etc) share epitopes which induce the development of cross-reactive antibodies. This leads to great difficulty in differentially diagnosing flaviviral infections, especially where flaviviruses co-circulate such as in tropical climates. Methods to increase IgG and IgM sensitivity 1. IgM-Capture Assays: Use anti-human IgM Fab fragment antibody as the capture (as opposed to a full-length anti-human IgM antibody). More Fab fragment antibodies are able to bind to the surface area of the solid substrate increasing the number of binding sites available for total IgM antibody that can be captured. 2. Lateral Flow Assays: Employ the bridging method in which colloidal gold-labelled disease specific antibody (e.g. MAb or PAb to Dengue NS1) is pre-mixed with recombinant antigen (e.g. Dengue NS1) and biotinylated anti-human IgM. Conjugating colloidal gold directly to the 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.
Reducing cross-reactivity between flaviviruses in EIA assays 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 successfully been used to increase the specificity and for differentiating flaviviral infections through targeting epitopes on NS1 or E protein. By including low concentrations of unconjugated antigens representing the potential cross-reactive species, it is possible to block their binding to the target antigen.
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