ELISA Protocol - Types of ELISA
The Enzyme-Linked Immunosorbent Assay (ELISA) / (EIA) involves coating (binding) of an antigen (Protein) to a solid support such as a membrane (as used in immunoblotting/western blotting) or a 96-well micro plate (ELISA Plate). The coating is done using bicarbonate / carbonate coating buffer. Proteins which have not been separated by electrophoresis can be bound to membranes and analyzed with primary and secondary antibodies as in the immunoblotting procedure. Such analysis are often called dot blots. The more common format is to absorb the antigen to the wells of a 96-well microplate and to use substrates that produce a colored product. The ELISA is suitable for the analysis of large numbers of samples and most of the procedure can be automated.
The first step is to bind the antigen to the micro well plate. The antigen used can be a purified protein , peptide or a crude protein extract. The choice of antigen will depend on the application and the nature of the antibodies being tested. Binding to 96-well micro plates is achieved by incubating the wells with a solution containing the antigen. Protein binding to the plate is due to hydrophobic interactions between the protein and the plastic. coating incubation may vary from 2hrs at room temperature or overnight at 4oC.
Once antigen is bound to the plates, it is important to block the remaining surface of the plate / membrane to prevent nonspecific binding and the detection antibodies during subsequent steps. Many Blocking buffers are available to block the free sites on the plate, BSA, non fat dry milk powder etc in PBS(phosphate buffered saline) or TBS (Tris buffered saline) with minute percentage of tween 20 or Triton X-100 can be used as blocking buffer. The protein in the blocking solution will attach to the membrane in all places where the target proteins have not attached. thus when antibody is added which will bind only to the target protein so the background interference will be reduced.
Incubation with Primary & Secondary Antibodies
After blocking the, excess blocking agent is removed by washing the plate / membrane with PBS and tween 20 (wash buffer) for sometime Then the antigen-containing wells are incubated with the primary antibody. Appropriate antibody dilutions need to be made which will better results, Serial dilutions are often carried out and the minimum titer which is giving a good response can be used. Optimization of the antibody concentration need to be done for getting better results. Antibody dilution are made in PBS or TBS. ELISAs are typically carried out in duplicate or triplicate. A positive and a negative control is also included along with the test samples. Primary antibody incubation may vary from 2 hours or even more incubation periods are required to get good results. After incubation with the primary antibody the wells are washed extensively to remove any unbound antibody. Similarly membrane / plate is incubated with 2o antibody with suitable dilutions. incubation period varies from 2 hrs or sometimes even more, secondary antibody will bind to the primary antibody, secondary antibodies are conjugated with enzyme which on reaction with substrate in the developing solution will yield colour. excess antibodies are washed off with wash buffer. there are variety of tags or enzyme labels can be conjugated to the secondary antibody. Most widely used enzymes are Horse Radish Peroxidase(HRP) and Alkaline Phosphatase (AP).
A developing solution having chromogenic substrate is added to the wells, the enzyme reacts with the substrate and gives colour. The intensity of the color will be proportional to the amount of secondary antibody which proportional to the amount of specific primary antibody in the sample. Alternatively a constant amount of a defined antibody can be used to quantify the amount of a specific protein in the sample. ELISA plate readers is basically a spectrophotometer designed to read the individual wells in a 96-well microplate. ELISA plate readers are interfaced with a computer to assist in data management.
most widely used substrates are pNPP (p-nitrophenyl phosphate); BCIP (5-bromo-4-chloro-3-indolyl phosphate); NBT (nitro blue tetrazolium); TMB (3,3',5,5'-tetramethyl-benzidine); DAB (3,3-diaminobenzidine)
Types of ELISA
The method described above is for the indirect ELISA.
Competitive ELISAAntigen (Sample) is incubated with an unlabelled antibody, these bind each other to form antigen-antibody complex. These bound antibody/antigen complexes are then added to an antigen-coated well. The plate is washed, so that unbound antibody is removed, there will be competition for binding of antigen in the well, then a secondary antibody specific to the primary antibody is added. This secondary antibody is coupled to an enzyme. When a substrate is added, enzyme acts on the chromogenic substrate and emits colour. ELISA reader is used to measure the intensity.
In sandwich ELISA first the Plate is coated with a capture antibody; to the antibody coated well sample is added and if any antigen present in the sample will binds to capture antibody; an antibody which detects the antigen is added, and this antibody binds to antigen. Finally an enzyme-linked secondary antibody is added, and this secondary antibody binds to detecting antibody when a substrate is added, the secondary antibody having enzyme converts the substrate and colour is developed which can be read in ELISA reader.
Advantages and Applications of ELISA
- ELISA can be used to quantify Antibody in the sample
- ELISA can be used to quantify Antigen in the sample
- Large no of samples can be processed at a time.
- Highly sensitive.
- Detection of HIV antibodies in serum
- Diagnosing Lyme Disease
- Diagnosing Lyme Disease, etc
- Detecting plant diseases.
- Application in toxicology for screening of Drugs.
- Detecting Food Allergens.
Methods in Cell Biology, Mark F. Wiser