Enzyme immunoassays

Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.

1. Enzyme Immunoassays Presented by: MOSTAFA ISMAIL Faculty of Science Helwan University 1

2. Out lines Introduction What ELISA Does ELISA Procedures Types of ELISA: 1.Direct ELISA 2.Indirect ELISA 3.Sandwish ELISA 4.Competitive ELISA 5. Avidin-Biotin systems 2

3. Introduction The first immunoassay applications in the clinical laboratories often involved detection with radioactive compounds. These radioimmunoassays ( RIAs ) have now been replaced by enzyme immunoassays ( EIA ), which offer easy and nonhazardous detection of antigens or antibodies in clinical samples. Most commercially available EIA systems require separation of specific antigens from nonspecific complexes. Such systems are called solid-phase immunosorbent assays ( SPIA ) or enzyme-linked immunosorbent assays ( ELISA ). 3

4. What (ELISA)Does ELISA is a biochemical assay that uses antibodies and an enzyme -mediated color change to detect the presence of either antigen (proteins, peptides, hormones, etc.) or antibody in a given sample. the most commonly used enzymes being alkaline phosphatase and horseradish peroxidase (HRP) . http://www.bbisolutions.com/productshub/enzymes/immunoassays/ 4

5. 1. Coating : Polystyrene plate is treated with a solution of either antigen or antibody 2. Blocking : An unrelated protein-based solution is used to cover all unbound sites on the plate 3. Detection : Enzyme-conjugated antibody or antigen binds specifically to the target antigen or antibody 4. Read result : Substrate is added and the signal produced by the enzyme- substrate reaction is measured ELISA Procedures Remove liquid and wash plate Remove liquid and wash plate 5

6. Types of ELISA Direct ELISA: An antigen coated to a multiwell plate is detected by an antibody that has been directly conjugated to an enzyme . This can also be reversed , with an antibody coated to the plate and a labeled antigen used for detection, but the second option is less common. 6

7. 2. Indirect ELISA: Antigen coated to a polystyrene multiwell plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next , an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal. Types of ELISA 7

8. 3.Sandwich ELISA: Require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule. The first antibody , termed the capture antibody , is coated to the polystyrene plate. Next, the analyte or sample solution is added to the well. A second antibody layer, the detection antibody , follows this step in order to measure the concentration of the analyte . Types of ELISA 8

9. 3.Sandwich ELISA cont : If the detection antibody is conjugated to an enzyme , then the assay is called a direct sandwich ELISA . If the detection antibody is unlabeled , then a second detection antibody will be needed resulting in an indirect sandwich ELISA . Types of ELISA 9

10. 4. Competitive ELISA 1. Primary antibody ( unlabeled ) is incubated with sample antigen. 2.Antibody-antigen complexes are then added to 96-well plates which are pre-coated with the same antigen. 3.Unbound antibody is removed by washing the plate. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence " competition .") 4.The secondary antibody that is specific to the primary antibody and conjugated with an enzyme is added. 5.A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. Types of ELISA 10

11. 4. Competitive ELISA cont. For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. (Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample the less labeled antigen is retained in the well and the weaker the signal). 11 Types of ELISA

12. Types of ELISA 4. Competitive ELISA 12

13. 13 Types of ELISA

14. Avidin is a protein derived from both avians and amphibians that shows considerable affinity for biotin , a co-factor that plays a role in multiple eukaryotic biological processes. Avidin and other biotin-binding proteins, including streptavidin and NeutrAvidin Protein, have the ability to bind up to four biotin molecules. 14 Avidin-Biotin systems

15. The avidin-biotin system can be incorporated into virtually every immunoassay, whereby an antibody is conjugated to biotin and then detected with avidin or streptavidin conjugated to variety of fluorochromes and enzymes. This makes biotinylated antibodies advantageous to signal amplification and increased sensitivity. 15 Avidin-Biotin systems

16. REFERENCES Koivunen ME, Krogsrud RL. Principles of immunochemical techniques used in clinical laboratories. Laboratory Medicine. 2006 Aug 1;37(8):490-7. Gan SD, Patel KR. Enzyme immunoassay and enzyme-linked immunosorbent assay. Journal of Investigative Dermatology. 2013 Sep 30;133(9):1-3. https://www.bio-rad-antibodies.com/elisa-types-direct-indirect-sandwich-competition-elisa-formats.html http://www.rockland-inc.com/streptavidin-biotin-tips.aspx 16