Enzyme Linked Immunosorbent Assay
Enzyme Linked Immunosorbent Assay test procedure prepared to detect antibody or antigen in the blood sample. A lot of tests are done by this method. Following are some of the tests performed by the Elisa method.
- HIV (Human Immunodeficiency Virus)
- HBV ( Hepatitis B Virus
- HCV ( Hepatitis C Virus)
- Typhoid
- MP (Malaria) etc.
Tis test in done in two ways.
- Double antibody method/ Antigen detection
- Indirect method/ Antibody detection
Double Antibody Method / Antigen Detection Principle
Requirement:
Wells – Ab coated, Patient sample, Wash solution, Enzymes conjugate, Substrate, Stop solution, Automatic pipet, (10 to 100 ul – 1000 ul), Analyzer, (Semi automatic chemistry analyzer), or plate reader or strip reader.
Procedure:
Take antibody coated well, Now put the sample of patient in it and incubate for a specific of time. Meanwhile, the antigen present in the sample forms a complex by combining with the antibodies Ab- Ag-attached to the gene wells. Then wash the wells thoroughly three to four time with the wash solution to wash away all the excess antigen and antibody. Now the conjugate or enzymes antibody is add to the wells. Which react with pre-exciting complexes to form a Ab-Ag-Ab* complex. Incubate for specific time and wash the wash with solution three to four time. Now to check the performance of these enzymes, a specific type of substrate is added to it. It changes the color due to the presence of enzymes in the substrate wells. The lack or excess of color is determined with the help of specific type of analyzer.
Indirect method/ Antibody detection
Procedure:
Take antigen coated well, Now put the sample of patient in it and incubate for a specific of time. Meanwhile, the antibody present in the sample forms a complex by combining with the antigen Ag- Ab-attached to the gene wells. Then wash the wells thoroughly three to four time with the wash solution to wash away all the excess antigen and antibody. Now the conjugate or enzymes (Anti Immunoglobulin) is add to the wells. Which react with pre-exciting complexes to form a Ag-Ab-Ig* complex. Incubate for specific time and wash the wash with solution three to four time. Now to check the performance of these enzymes, a specific type of substrate is added to it. It changes the color due to the presence of enzymes in the substrate wells. The lack or excess of color is determined with the help of specific type of analyzer. If the color intensity of the Substrate is greater than the normal color, the sample contain antibody.