The McGill Physiology Virtual Lab

Immunology Laboratory

Elisa assay
 

The Enzyme-Linked ImmunoSorbent Assay is a serological test that can be used to detect particular antigens or antibodies.

An ELISA assay such as the one you will do in this lab would be used to diagnose HIV infection by detecting the presence of antibodies to HIV.  

A few Elisa methods exist:  their difference resides in the sequence in which antigens and antibodies are added to a solid substrate (96-well plate or a micro-well strip).
 
 
  • In an antibody capture assay, antigen is first bound in the plastic wells.
  • Then, the primary antibody  binds to (or is captured by) the immobilized antigen.
  • A secondary antibody (conjugated to the enzyme horseradish peroxidase (HRP)) is added, and binds to the first antibody.
  • The substrate 3,3’,5,5’-tetramethylbenzidine (TMB) is added. HRP reacts with TMB (chromogenic reagent) and gives a blue colour which can be quantified.


 

  • In an antigen capture assay, primary antibody is fixed onto the plastic wells,
  • antigen is captured by the immobilized primary antibody,
  • and the captured antigen is detected by a secondary antibody, also conjugated to HRP,
  • that turns the assay solution blue upon reaction with TMB.
 
Protocol  

 
Antigen is bound    
Label the 12-well strip. On each strip label the first 3 wells with a “+” for the positive controls and the next 3 wells with a “–” for the negative controls. Label the remaining wells to identify the samples being tested (3 wells each). Use a fresh pipet tip to transfer 50 μl with purified antigen (Ag) into all 12 wells of the microplate strip. Wait 5 minutes for the antigen to bind to the plastic wells.

The appropriate antigen is first absorbed to the walls of a microplate strip: HIV capsid protein p24 could serve as the antigen.

  Wash    
Tip the microplate strip upside down onto the paper towels, and gently tap the strip a few times upside down. Make sure to avoid splashing sample back into wells. Discard the top paper towel. Use your pipet to fill each well with wash buffer, taking care not to spill over into neighbouring wells. Note: the same pipet tip is used for all washing steps. Tip the microplate strip upside down onto the paper towels and tap. Discard the top 2–3 paper towels. Repeat wash.

 

Washing removes what is not bound to the plastic.

  Antibody and controls    
Use a fresh pipet tip to transfer 50 μl of the positive control (+) into the three “+” wells. Use a fresh pipet tip to transfer 50 μl of the negative control (–) into the three “–” wells. Transfer 50 μl of each of your team’s serum samples into the appropriately initialed three wells, using a fresh pipet tip for each serum sample.

Wait 5 minutes for the antibodies to bind to their targets. Wash the unbound primary antibody out of the wells two times.

 

Serum that may contain antibodies against the antigen is added to the well (human IgG). If the antibodies are present in the sample, they will bind to the antigens that are absorbed to the wall of the well. If the sample contains only non-specific antibodies, they will not bind to the antigen.
Washing removes any antibodies which do not specifically attach to the antigen absorbed to the well.


Control samples (samples which will give known results) must be run side by side with actual samples to ensure that the assay is working correctly.

  Secondary antibody    
Use a fresh pipet tip to transfer 50 μl of secondary antibody (HPRO-2ndAb) into all 12 wells of the microplate strip.
Wait 5 minutes for the antibodies to bind to their targets.
Wash the unbound secondary antibody out of the wells 3 times.

If antibodies in the previous step have attached to the antigen, these antibodies can be detected by the addition of an anti-human IgG antibody-enzyme conjugate. The wash step gets rid of the antibody-enzyme conjugate which did not find a specific antibody and could not form a complex.

  Enzyme substrate    
Use a fresh pipet tip to transfer 50 μl of enzyme substrate (TMB) into all 12 wells of the microplate strip. Wait 5 minutes.

A colour change indicates that the serum contained specific antibodies which reacted against the original antigen.

Click here to continue with the topic of hemagglutination