AIDS KIL Simulation of v- Detection y ELISA udent Eperimental Procedu 12. Wash e

Question

AIDS KIL Simulation of v- Detection y ELISA udent Eperimental Procedu 12. Wash eac well once with PBS bstter (as doscrbed rtes Washe 13. Add 100 of the sulstrate to all 12 wels 14 Incubate at 37 C for s minutes 15. Remove the p late for analysis It color is not fully developed after 5 min utes, incubat timo Qulck Reference: te at 37-C for a longer period of The ponitive contreA, whhich cortains g3 directed agairnt HIV nntgens, serum snmples wil niso contain anti-HIV IgG, wile negative serum samplos will not contain arw-Hrv gG udy Questions laboratory notebook or on a separate Answer the following study questions in your worksheet LISA. Why is ELISA so sensitive? Why is it necessary to Describe the mechanism of E block unoccupied binding sites in the microtiter wells? Why is it important to have a positive control? 1. 2. Why can the onset of AIDS take several years? 3. Why is anti-HIV-1 1gG screened instead of the virus itself? 4. Why does the destruction of T, cells compromise the entire immune system? How does HIV target T, cells? 5. Why are there so many immunological variants of HIV? The elimination of several steps in the ELISA could be accomplished if the primary an- tibody was made into an enzyme conjugate. Why is this generally not done? What can cause a false positive in an ELISA? 6. Duplication of any part of this document is permited for non proft educational purposes onk Coprieht 1992-2013 EDNOTE Inc all rights reserved 271.130318 1.800 EDVOTEK www.edvotek.com- The Biotechnology Education Company

Explanation / Answer

(1) Performing an ELISA test includes minimum one particular antigen specific antibody. The sample with an obscure measure of antigen is immobilized on a strong support, generally a polystyrene microtiter plate, either specifically or non-specifically. After immobilization of the antigen, the detection antibody or secondary antibody is added, which forms a complex with the antigen. The secondary antibody can be linked to an enzyme covalently, or would itself be able to be recognized by a secondary antibody that is connected to an enzyme through bioconjugation. The antibody incubation of ELISA is comparative with western blot. Between each progression, the plate is washed with a detergent in order to expel out any antibodies or proteins that are not particularly bound. After the last wash step, an enzymatic substrate is added to the plate, to produce a visible signal, which shows the amount of antigen in the sample.

ELISA is very sensitive because antigen-antibody complexes are very specific, and enzymes used in ELISA convert substrate quickly.

In order to prevent nonspecific binding of antibody with the plastic of the microliter plate, it is necessary to block unoccupied, and nonspecific binding sites in the microtiter wells.

A positive control is a gathering in an analysis that gets a treatment with a known outcome, and hence should demonstrate a specific change during the experiment. It is utilized to control for unknown factors during the experiment and to give the researcher something to compare with the test gathering.

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