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Mouse anti-Tat IgG Immunoassay

Enzyme linked immunosorbent assay (ELISA) for the quantitative determination of anti- Tat IgG concentrations in mouse serum and plasma as an aid in the diagnosis of antibody production in mouse immunized with (HIV) Tat protein.


The HIV-1 regulatory proteins Tat are considered attractive targets for the development of a multicomponent vaccine against HIV-1 infection. The protein is well conserved among different isolates and thus may be less susceptible to mutation leading to the production of escape virus variants. Tat is produced early after infection and is essential for virus replication and infectivity. Tat protein is also immunogenic and antibodies (Ab) against Tat have been found to correlate with delayed disease progression and may exert protective effects by inhibiting both HIV replication and the effects of extracellular Tat. Moreover Tat is efficiently taken up by monocite-derived dendritic cells, promotes their maturation and antigen presenting functions directing Th-1 and CTL responses against itself and other Ags since it enters the major histocompatibility complex class I pathway. Finally, vaccination of mice with a biologically active Tat protein has been shown to be safe, immunogenic and elicits anti-Tat neutralizing Ab and CTL.(..more..)


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(..more..) The Tat protein was expressed in Escherichia Coli and purified by heparin sepharose affinity chromatography. The purified Tat protein is 95% pure and fully monomeric, as tested by sodium dodedecyl sulphate polyacrylamide gel elettroforesis (SDS-PAGE) and posses full biological activity as tested by the rescue assay and by MDDCs uptake. Measurement of IgG anti-Tat is especially valuable in sera and plasma mice immunized with Tat protein.

Tat protein has been coated onto the wells of the microplate. Revelation step includes a polyclonal anti-IgG HRP-conjugated specific for mouse and ABTS as chromogen. Sample or standard are pipetted into wells of a microplate pre-coated with Tat protein and pre-blocked; the protein can then capture anti-Tat IgG and, thereby, immobilizing it to the well.


Limitation of the Procedure:


• No drugs have been investigated for assay interference
• Do not use reagents beyond expiration date printed on the kit label
• Any variation in specimen diluent, operator, pipetting technique, washing technique, incubation time or temperature or kit age can cause variation in binding.

 

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