Elisa: Antibody and Bio-rad Protein Assay Essay

690 Words Nov 29th, 2010 3 Pages
ELISA
(Enzyme-Linked ImmunoSorbant Assay)

ELISA is abbreviated term for Enzyme-Linked ImmunoSorbant Assay. This procedure is one of the most widely used methods in clinical immunology assays to detect the presence and absence of certain antigens or antibodies and also to quantify them when necessary. Quantification can be done in a range of microgram (µg) to nanogram (ng). The ELISA procedure takes advantage of the fact that most proteins will bind firmly to the surface of different kinds of plastic (polystyrene or polyvinyl chloride), usually by hydrophobic interaction.

The steps of the ELISA procedure are simple to perform. There are mainly FOUR phases in ELISA:
1. Coating & Blocking
2. Reaction
3. Labelling
4.
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The absorbance maximum for an acidic solution of Coomassie® Brilliant Blue G-250 dye shifts from 465 nm to 595 nm when binding to protein occurs.2,3,4 The Coomassie blue dye binds to primarily basic and aromatic amino acid residues, especially arginine.5 Spector6 found that the extinction coefficient of a dye-albumin complex solution was constant over a 10-fold concentration range. Thus, Beer’s law may be applied for accurate quantization of protein by selecting an appropriate ratio of dye volume to sample concentration.
Interferences may be caused by chemical-protein and/or chemical-dye interactions. Table 1 lists those chemical reagents not directly affecting the development of dye colour. (Note: Basic buffer conditions and detergents interfere with this assay.) Since every protein-chemical reagent combination has not been assayed, it is possible that some of the listed reagents produce interference in combination with certain proteins.

However, with respect to proteins such as bovine serum albumin and gamma globulin, the listed reagents show little or no interference. The acceptable concentrations of reagents for the Standard Procedure are shown in Table 1. Equivalent concentrations of reagents for the Micro assay Procedure (see Section 2) are 1/40 of those listed in this table, due to the difference of sample-to-dye ratios between the Standard and Micro assay

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