Enzyme Linked Immunosorbent Assay Video Lecture | Crash Course for CSIR NET Life Sciences

Ans. Indirect ELISA is based on the principle of antigen-antibody interaction. In this assay, an antigen is coated onto a microplate. When a sample containing antibodies is added, these antibodies bind to the antigen. After washing, a secondary antibody, which is linked to an enzyme, is introduced. This secondary antibody binds to the primary antibodies. A substrate is then added, which the enzyme converts into a detectable signal, usually a color change, indicating the presence and quantity of antibodies in the sample.

Ans. The main components of an Indirect ELISA include: 1. <b>Coated Antigen</b>: The specific antigen that is immobilized on the plate. 2. <b>Sample</b>: The test serum or fluid that may contain antibodies against the antigen. 3. <b>Primary Antibody</b>: An antibody that binds specifically to the antigen. 4. <b>Secondary Antibody</b>: An enzyme-linked antibody that binds to the primary antibody. 5. <b>Substrate</b>: A chemical that the enzyme acts upon to produce a measurable signal, usually a color change.

Ans. Indirect ELISA has several advantages over Direct ELISA, including: 1. <b>Increased Sensitivity</b>: The use of a secondary antibody that can bind multiple primary antibodies increases signal amplification. 2. <b>Flexibility</b>: Multiple primary antibodies can be detected using the same secondary antibody, allowing for the simultaneous analysis of different targets. 3. <b>Cost-Effectiveness</b>: Fewer labeled antibodies are required, as one labeled secondary antibody can be used for various primary antibodies.

Ans. Indirect ELISA is widely used in various fields, including: 1. <b>Diagnostic Testing</b>: For the detection of antibodies against pathogens such as viruses and bacteria. 2. <b>Vaccine Development</b>: To evaluate immune responses in clinical trials. 3. <b>Research</b>: For quantifying specific antibodies in serum or other biological samples. 4. <b>Quality Control</b>: In the manufacturing of vaccines and therapeutic antibodies to ensure consistency and efficacy.

Ans. The steps of performing an Indirect ELISA include: 1. <b>Coating the Plate</b>: Add the antigen to the wells of a microplate and incubate to allow binding. 2. <b>Blocking</b>: Add a blocking solution to prevent non-specific binding. 3. <b>Sample Addition</b>: Introduce the test sample containing antibodies and incubate. 4. <b>Washing</b>: Remove unbound antibodies through washing steps. 5. <b>Secondary Antibody Addition</b>: Add an enzyme-linked secondary antibody and incubate. 6. <b>Washing Again</b>: Wash to remove unbound secondary antibodies. 7. <b>Substrate Addition</b>: Add substrate to allow the enzyme to react and generate a measurable signal. 8. <b>Signal Measurement</b>: Measure the signal using a spectrophotometer to determine the concentration of antibodies in the sample.