23 Mar 2015
Enzyme-Linked Immunosorbent Assay (abbreviated as ELISA), is a kind of immunoassay technique to detect the presence of a specific antibody or antigen in the test samples. This method principally make use of both immunological reaction (the antibody-antigen reaction) to detect the present of specific antibody or antigen) and biochemical reaction (the enzyme substrate reaction) to produce a visible signal for both qualitative and quantitative measurement.
The ELISA method used in this experiment is an example of "indirect ELISA" method. The ELISA plate wells are coated with optimized concentration of antigens before hand by means of charge interaction or with the help of a spacer coating (eg. L-lysine). Then the plate wells are washed with buffer solution, and a blocking step is preformed by adding bovine serum albumin or casein, to block any uncoated space in the well before using to detect antibodies in sample serum.
Then the sample serum is added to detect the present of specific antibody, the antibodies will bind to the antigens in the well (in this experiment is the anti-DNA antibody). Then a secondary antibody (usually raised from a species against the antibody of the sample) with enzyme-linked (called conjugate) was added to bind. The enzyme used may be Alkaline phosphate or Horseradish peroxidase (in this experiment is Alkaline phosphate); this also serve as signal amplification step as the enzymes conjugate chose used usually have more than one binding sites for the substrate added subsequently.
Then a substrate is added for the enzyme to produce a color reaction (in this experiment is the PNPP which produce a yellow color) to indicate the present of the specific antibody in the sample. The higher the concentration of the antibody in the test sample, the stronger the color developed. We can use a spectrometer (an ELISA reader in this experiment) to measure the color quantitatively instead of using our eye, which is more objective and accurate.
Washing with buffer (usually a mild detergent) is applied between steps to remove unbind antibodies to avoid non-specific binding of antibodies.
Usually positive and negative controls will be paralleled run with the test sample to validate the result.
The cut-off point between a positive or negative result is usually determined statistically with known standards. In additions, with a serial dilution of a known standard (known concentration of the specific antibody want to detect in the test), we can also find the amount of the specific antibody in the test sample from the graph of absorbance against concentration of the known standard. Thus, the ELISA method can produce both qualitative and quantitative result in detecting the specific antibody in test sample.
ELISA is a relatively high sensitive and specific test for detecting serum protein, the presence of specific antibody or antigen; and also considers as a high-throughput immunoassay. The use of ELISA also includes hormones and infectious antigens (including virus and bacteria). The most common example is detecting HIV in patient samples. In addition, it has the advantage of using non-radioactive substances, is safer than those radio-immunoassays.
"Sandwich ELISA" (or "direct ELISA") is used to detect antigen in sample serum, is less-common. With known quantity of capture antibodies coated to the well, the antigens in the sample will bind to the antibodies to form complex. Then enzyme-linked primary antibodies will be applied to detect the present of the antigens.
"Competition ELISA" is a different method from the indirect and sandwich ELISA, in which the kit contains enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen will compete with the antigen in the sample for the antibody binding sites in the well. Thus, more antigens in the sample will give weaker signal as less labeled antigen can bind to the antibody binding sites in the well. The advantage of this method is can be used to detect antigen in impure samples.
"Reverse ELISA" is a new technology using immunosorbent polystyrene rod with protruding ogives. This ogives will dipped into the sample, thus a higher sample volume can be used to improve the sensitivity. Moreover, the ogives can be sensitized with different reagent to detect different antibodies or antigens simultaneously for multi-target assays.
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