West Nile IgG, BioAssay(TM) ELISA Kit

Intended Use:\nThe West Nile Detect IgG Test for exposure to West Nile Virus (WNV) is an ELISA assay system for the detection of antibodies in human serum to WNV derived recombinant antigen (WNRA) (1-3). \n\nSummary of the Test:\nExposure to West Nile Virus causes a disease with a number of symptoms including encephalitis (4-7). West Nile Virus is becoming widespread and has been detected in over half of the 50 states. This test has been developed and refined using reagents produced by CDC. The West Nile Detect assay employs a recombinant antigen called WNRA, which can be used as a rapid serological marker for WNV infection. The WNRA protein is a recombinant antigen, which consists of a stretch of peptides from two WNV antigens. \n\nPrinciple of the Test:\nThe West Nile Detect IgG ELISA consists of one enzymatically amplified "two-step" sandwich-type immunoassay. In this assay, the microtitration wells are incubated with standards, controls or unknown serum samples. The serum samples may be directly mixed with sample dilution buffer or sample diluent buffer, and added in the wells. After washing, the wells are treated with an antibody specific for human IgG and labeled with the enzyme horseradish peroxidase (HRP). After a second incubation and washing step, the wells are incubated with the tetramethylbenzidine (TMB) substrate. An acidic stopping solution is then added and the degree of enzymatic turnover of the substrate is determined by absorbance measurement at 450nm. Above a certain threshold, the ratio of the absorbances of the WNRA and the control wells accurately determines whether antibodies to WNV are present. A set of positive and negative samples is provided as internal controls in order to monitor the integrity of the kit components.\n\nBackground:\nWest Nile Virus (WNV) is a member of the Flaviviridae, a plus- stranded virus family that includes St. Louis encephalitis virus, yellow fever virus, and Dengue virus. WNV was initially isolated in 1937 in the West Nile region of Uganda and has become prevalent in Africa, Asia, and Europe. It has rapidly spread across the United States with cases being observed in every continental state. Virus particles consist of a dense core made up of the core/capsid protein encapsulating the RNA genome surrounded by a membrane envelope embedded with envelope and matrix proteins. However, when the viruses are inside of infected cells, the matrix protein exists in its "pre-M" form as a heterodimer with the envelope proteins. Cleavage of the "pre-M" protein to its mature form occurs during release of the virus; this cleavage leads to the dissociation of the heterodimers. The viral core protein is thought to contribute to the WNV-associated inflammation via apoptosis induced though the caspase-9 pathway as delivery of core gene delivery into the striatum of mouse brain and skeletal muscle resulted in cell death and inflammation. The highly glycosylated envelope protein was shown to play a major role for WNV entry into target cells as WNV entry was inhibited by using a recombinant domain III from the envelope glycoprotein. The WNV receptor has recently been identified as alpha v beta 3 integrin.\n\nKit Components:\n1. Microtiter plate, 1x96wells\n2. IgG Sample Dilution Buffer, 1x25ml\n3. WN IgG Positive Control, 1x50ul\n4. WN IgG Negative Control, 1x50ul\n5. Conjugate (HRP), 1x6ml\n6. Wash Buffer (10X), 1x120ml\n7. Wash Solution, 1x20ml\n8. TMP substrate, 1x9ml\n9. Stop solution, 1x6ml\n\nStorage and Stability:\nStore all components at 4 degrees C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial prior to removing the cap.

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