ELISA (ENZYME LINKED IMMUNO SORBENT ESSSAY) NOTES FOR 4 MARKS QUESTIONS
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 makes use of both immunological reaction (the antibody-antigen reaction) to detect the presence of a 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 beforehand 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 performed 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 presence 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 serves 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 colour reaction (in this experiment is the PNPP which produce a yellow colour) to indicate the presence of the specific antibody in the sample. The higher the concentration of the antibody in the test sample, the stronger the colour developed. We can use a spectrometer (an ELISA reader in this experiment) to measure the colour 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 unbound 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 the 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.
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 makes use of both immunological reaction (the antibody-antigen reaction) to detect the presence of a 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 beforehand 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 performed 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 presence 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 serves 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 colour reaction (in this experiment is the PNPP which produce a yellow colour) to indicate the presence of the specific antibody in the sample. The higher the concentration of the antibody in the test sample, the stronger the colour developed. We can use a spectrometer (an ELISA reader in this experiment) to measure the colour 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 unbound 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 the 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.
