lab_23_elisa - Miramar College Biology 205 Microbiology Lab...

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Lab Exercise 23: Simulated HIV Diagnosis Using Enzyme-Linked Immunosorbent Assay (ELISA) Page 1 of 3 Miramar College Biology 205 Microbiology Lab Exercise 23: Simulated HIV Diagnosis Using Enzyme-Linked Immunosorbent Assay (ELISA) Background ELISA ( E nzyme- L inked I mmuno S orbent A ssay) is a powerful immunological technique used to detect the presence of an antigen or antibody in a patient sample, based on the specificity of an antibody to its particular antigen. The applications of ELISAs are wide-ranging, although most of the focus is put on their medical importance. ELISAs may be utilized to look for a particular pathogen/antigen in a patient’s serum ( direct ELISA ) or antibodies to a particular antigen/pathogen ( indirect ELISA ). In an indirect ELISA, a patient sample (with an unknown amount of antibody) is added to a well to which antigen has previously been adhered. If antibodies are present in the patient sample, they will bind the antigen, forming an antibody-antigen complex . This complex will be recognized by a second enzyme-linked- antibody forming an antigen-antibody-EL antibody complex . Finally, a chromogenic substrate is added, and because of the EL-antibody, a colorimetric change is noted as a visible marker for both a qualitative and quantitative measure of the patient antigen-antibody complex (Figure 1 and Figure 2). It is important to note that if quantitative results are desired, the signals of the unknown samples must be compared to those of a standard curve. However, even a slight color change is indicative of a positive result. Remember that every antigen-antibody-EL-antibody complex will be capable of converting the substrate to its colored product. Thus, darker color indicates increased incubation and/or more complexes. Washes are used in between additions of antibodies to negate any non-specific binding. In this lab, you will perform an indirect ELISA within the following scenario. The basic indirect ELISA protocol adheres to the following five steps: 1. Coating of the microtiter plate wells with antigen;
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This note was uploaded on 12/23/2009 for the course BIO 205 taught by Professor Murphy during the Fall '09 term at Miramar College.

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lab_23_elisa - Miramar College Biology 205 Microbiology Lab...

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