How to Perform an ELISA Assay

By Dmitriy Richter, eHow Contributor An ELISA, or enzyme-linked immunoabsorbent assay, is a biochemical lab technique done to detect an antigen (such as a protein) or antibody in an experimental sample. The assay takes advantage of antibody-antigen binding and can be used to assay other cell and molecular binding mechanisms as well. In a protein-based ELISA assay, a "capture" antibody or protein is laid down in an assay dish and allowed to adhere to the dish, after which the "probe" agent (such as one's protein of interest) is laid down and incubated with the capture agent. After several washes, a "detection" antibody is added in order to visualize the presence or absence of antibody-antigen or protein-protein binding. Instructions 1 .Dilute capture antibody to 5 ug/mL in 50 mM NaHCO2 (pH 9.6). Add 50 uL to each well of the sample dish used for testing. Cover with plastic or aluminum and incubate overnight at 4 degrees C on a shaker. 2 . Dump out capture antibody and wash two times with 200 uL PT buffer. Add 200 uL of blocking buffer per well (PB or 5 percent skim milk). Incubate anywhere from two hours to overnight while shaking at 4 degrees C. 3. Dump out blocking buffer and wash 4 times with 200 uL PT buffer. Add "probe" protein samples at various concentrations in PB or 5 percent skim milk, adding 100 uL per well. Incubate one hour shaking at 4 degrees C. Dump out probe solutions and wash six times with 200 uL PT buffer. 4 . Add detection antibody (HRP-conjugated antibody) diluted 1:4000 in PB buffer or 5 percent skim milk (either one containing 0.05 percent Tween-20), adding 50 uL per sample well. Incubate for 30 minutes to one hour shaking at 4 degrees C. 5. Dump out detection antibody solution and wash 6 times with 200 uL PT buffer per well, followed by washing two times with 200 uL of 1x PBS (phosphate-buffered saline). Dump out PBS and add 100 uL of detection reagent per well(for HRP, use freshly-mixed TMB reagent by mixing TMB substrate and peroxide at 1:1). Incubate for 15 minutes at room temperature, shaking occasionally. When using TMB and HRP: Finally, add 100 uL of 1M H3PO4 (phosphoric acid) per well in order to quench the TBM reaction. Read plate using sample analysis reader, such as a 96-well plate reader. (For HRP and TMB, use the "ELISA End-Point Assay" template available on most readers.

How to Design an ELISA By Kerstin Cunningham, eHow Contributor | updated May 08, 2011 ELISA is a technique often used to detect proteins, peptides in research or in medical diagnosis. ELISA is the acronym for enzyme linked immunosorbent assay, a method to study the presence of proteins, peptides, or infectious agents, for example. It is a biochemical method used in research and medicine often for diagnosis. There are several different versions of ELISAs: indirect, direct, sandwich and competitive, though all versions use 96-well plates, antibodies against the molecule under investigation and a colorimetric reaction determining the presence of the molecule under study. This color change can be the equivalent to the quantity of the studied molecule in the test samples.

Instructions 1 Determine the molecule under investigation and the samples required to investigate this molecule. Assess the ELISA technique required, this will depend on whether the studied molecule will stick to the plastic of the 96 well plate, direct, or requires an antibody to capture the molecule of interest, indirect. Determine the sensitivity needed, sandwich ELISA is the most sensitive using the molecule under study to be sandwiched between two antibodies. Assess the structure of the molecule, if the molecule under study has one binding site for antibodies a competitive ELISA may be more appropriate. 2 Select the appropriate antibodies, either monoclonal, derived from one cell type and binding to one site on the investigated molecule; or polyclonal, derived from different cell types and binding to different sites on the studied molecule. Ensure the antibodies can be used in ELISA and will work in the selected ELISA. 3 Determine the method of detection, either colorimetric or fluorescence. Colorimetric assays require an enzyme tagged to an antibody, while fluorescence require a fluorescene tagged to an antibody. Colorimetric assays utilize an enzyme and a substrate which produce a change in color. The color change is equal to the molecule under study as the enzyme-antibody complex is bound to the molecule under study. Assessing the investigated samples as human tissues can contain the enzymes used in colorimetric assays and interfere with the results. 4 Optimize all dilutions including the samples and antibodies in a blocking buffer. The samples being tested must contain over three percent of the target protein. This ensures that the target of interest is at a level that can be detected. Optimize incubation of the antibodies. Ensure controls are available for the standard curve.

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