Using An Elisa To Determine Antigen - Antibody Binding
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Enzyme-Linked ImmunoSorbent Assay, ELISA, is a highly sensitive immunoeassay that is used in place of radioimmunoassay since it does not involve special handling and disposal procedures requiring work with radioactive substances. The ELISA relies on the antibodies to detect the presence of antigens in liquid samples, thus changing from a clear liquid to a colored one. Because they are antibody-based, ELISAs are called immunoassays. ELISAs can detect minute amounts of disease agents in samples in such as body fluids, before the body had had a chance to mount an immune response. When experimenting using an ELISA, the antibodies present in the samples bind to the antigen plate and after removal of excess antibody, bound antibody can be detected by adding enzyme-conjugated second antibody.

The objective of this study is to examine two things: first, are there antigen-antibody binding on a plate that contains two different antibodies specific for two different antigens: Hen Egg Lysozyme (HEL) and Bovine Serum Albumin (BSA), and second, how much of the binding is present in the plate? To examine these questions, using an Enzyme-Linked Immunosorbent Assay to help screen for antibody-secreting hybridomas, which is both highly sensitive and reasonable to perform. The procedure of using an ELISA has increased understandings of bindings of antigen to antibody of such testing as HIV, drug (marijuana), pregnancies, etc.

The purpose of an ELISA is to determine if particular antigen-antibody bindings are present in a sample; if there are antibodies binding to antigens present, an ELISA helps to determine how much. There are two main variations with this method; one is so that we define the amount of antigen-antibody binding and the other, stated previously, is so we can see how much antibody is present in our sample. In this laboratory experiment, we have coated a micro-plate with wells with antigens. The antigen is strongly absorbed by the plastic and remains attached to the well surface throughout the procedure. After coating and addition of the antibodies, we were able to determine the antigen-antibody binding.

In most experiments, researchers use BSA and HEL as unrelated antigens to control for non-specific antigen reactivity. For example, wells coated with HEL antigen should not show positive signs when anti-BSA antibodies are present, and vice versa. This means the amount of enzyme-conjugated antibody bound in each well is able to hydrolyze a colorless substrate to yield a colored product.

In conclusion, the results of using an ELISA should conclude that antigens bind to antibodies, with the provided antigens present. The secondary antibody is attached to an enzyme that chemically changes the enzyme substrate, turning it from a colorless solution to a yellow solution. A side note however, blank wells with no antigens are to be subtracted from the control plate but not form either of the antigen-coated plates. This results in the secondary antibody being covalently bound, conjugated, to an enzyme that catalyzes a chemical reaction when the enzyme substrate is added. This would produce a color change if the serum sample contained antibody to the bacteria, because the enzyme bound secondary antibody would bind to the primary antibody already bound to antigen in the wells.


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