![Commonly used enzymatic markers
• Alkaline Phosphatase
– PNPP (p-Nitrophenyl Phosphate, Disodium Salt) yellow
• HRP (Horseradish Peroxidase)
– OPD (o-phenylenediamine dihydrochloride) Amber
– ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic
acid]-diammonium salt) Green
– TMB (3,3',5,5'-tetramethylbenzidine) Blue
• turns yellow after the addition of sulfuric or phosphoric acid.](https://image.slidesharecdn.com/enzymelinkedimmunosorbantassay-200203092828/85/Enzyme-linked-immunosorbant-assay-4-320.jpg)
Enzyme linked immunosorbant assay
- 1. Enzyme Linked Immunosorbant Assay ELISA -Kavya Rojesh (19HGMA13) I M.Sc Human Genetics And Molecular Biology
- 2. History and Development of ELISA • Before the development of the ELISA, the only option for conducting an immunoassay was radioimmunoassay. • As radioactivity poses a potential health threat, a safer alternative was sought. • In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands independently published papers that synthesized this knowledge into methods to perform EIA/ELISA
- 3. • ELISA is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. ELISA Competitive Non- Competitive Direct Indirect Sandwich
- 4. Commonly used enzymatic markers • Alkaline Phosphatase – PNPP (p-Nitrophenyl Phosphate, Disodium Salt) yellow • HRP (Horseradish Peroxidase) – OPD (o-phenylenediamine dihydrochloride) Amber – ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) Green – TMB (3,3',5,5'-tetramethylbenzidine) Blue • turns yellow after the addition of sulfuric or phosphoric acid.
- 5. Direct ELISA • A direct ELISA is intended for the detection and quantification of a specific analyte (e.g. antigens, antibodies, proteins, hormones, peptides, etc.) from within a complex biological sample. • Of the four different ELISA formats, direct ELISA is the simplest and quickest to perform
- 6. Procedure • Antigen is immobilized onto the wells of a 96-well polystyrene plate via passive adsorption. • An enzyme-labeled primary antibody specific for the target antigen is added to the wells and directly binds to the antigen. • A respective enzyme substrate a suitable substrate is added, which upon reaction with the enzyme, produces a visible colorimetric output that can be measured by a spectrophotometer or absorbance microplate reader.
- 7. Advantages • Less reagents and fewer steps are required making this ELISA format simple and quick while minimizing potential user error • Cross-reactivity of secondary antibody is eliminated Disadvantages • Antigen immobilization is not specific resulting in potentially high background interference • Primary antibody must be labeled individually, which is time- consuming and expensive • No signal amplification • Low flexibility – a specific conjugated primary antibody is needed for each target protein • Immunoreactivity of the primary antibody may be adversely affected by labeling with enzymes
- 8. Indirect ELISA • Antibody can be detected or quantitatively determined by indirect ELISA. • In this technique, Antigen is coated on the microtiter well. • Serum or some other sample containing primary antibody is allowed to react with the coated antigen. (Any free primary antibody is washed away) • Ag-1’Ab Complex is detected by adding an enzyme conjugated secondary antibody that binds to the primary antibody. (Unbound secondary antibody is then washed away ) • A specific substrate for the enzyme is added to form colored products. • The amount of colored end product is measured by absorbance of all the wells of 96-well plate.
- 9. Procedure of Indirect ELISA • Coat the micro titer plate wells with antigen. • Block all unbound sites to prevent false positive results. • Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and incubate the plate at 37°c. • Wash the plate, so that unbound antibody is removed. • Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG). • Wash the plate, so that unbound enzyme-linked antibodies are removed. • Add substrate which is converted by the enzyme to produce a colored product. • Reaction of a substrate with the enzyme to produce a colored product.
- 10. Advantages: • Increased sensitivity • A wide variety of commercially available secondary antibodies. • Maximum immunoreactivity of the primary antibody is retained because it is not labeled. • Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. • Flexibility, Different primary detection antibodies can be used with a single labeled secondary antibody. • Cost savings, since fewer labeled antibodies are required. • Different visualization markers can be used with the same primary antibody. Disadvantages: • Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal. • An extra incubation step is required in the procedure.
- 11. Sandwich ELISA • Antigen can be detected by sandwich ELISA. • In this technique, antibody is coated on the microtiter well. • A sample containing antigen is added to the well with the antibody attached to the well, forming antigen-antibody complex. (the well is washed) • A 2’Ab specific for a different epitope on the antigen is added and allowed to react with the bound antigen. (unbound secondary antibody is removed) • Finally substrate is added to the plate which is hydrolyzed by enzyme to form colored products.
- 12. Procedure of sandwich ELISA • Prepare a surface to which a known quantity of antibody is bound. • Add the antigen-containing sample to the plate and incubate the plate at 37°c. • Wash the plate, so that unbound antigen is removed. • Add the enzyme-linked antibodies which are also specific to the antigen and then incubate at 37°c. • Wash the plate, so that unbound enzyme-linked antibodies are removed. • Add substrate which is converted by the enzyme to produce a colored product. • Reaction of a substrate with the enzyme to produce a colored product.
- 13. Advantages • High specificity, since two antibodies are used the antigen is specifically captured and detected. • Suitable for complex samples, since the antigen does not require purification prior to measurement. • Flexibility and sensitivity, since both direct and indirect detection methods can be used. Disadvantages • Optimization in terms of antibody becomes problematic due to cross-reactivity issues. • For recognition of a specific epitope, only monoclonal antibodies can be applied as matched pairs. • To procure monoclonal antibodies it is a tedious process in case of matched pairs and are more expensive than polyclonal antibodies.
- 14. 3. Competitive ELISA • This test is used to measure the concentration of an antigen in a sample. • Antibody is first incubated in solution with a sample containing antigen. • The ag-ab mixture is then added to the microtitre well which is coated with antigen. • The more the antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. • After the well is washed, enzyme conjugated secondary antibody specific for isotype of the primary antibody is added to determine the amount of primary antibody bound to the well. • The higher the concentration of antigen in the sample, the lower the absorbance.
- 15. Procedure • Antibody is incubated with sample containing antigen. • Antigen-antibody complex are added to the microtitre well which are pre-coated with the antigen. • Wash the plate to remove unbound antibody. • Enzyme linked secondary antibody which is specific to the primary antibody is added. • Wash the plate, so that unbound enzyme-linked antibodies are removed. • Add substrate which is converted by the enzyme into a fluorescent signal.
- 16. Advantages • High specificity, since two antibodies are used. • High sensitivity, since both direct and indirect detection methods can be used. • Suitable for complex samples, since the antigen does not require purification prior to measurement.
- 17. Applications Of ELISA • Screening of Donated Blood for Viral contaminations – HIV-1 & HIV-2 (Ab) – Hepatitis –C (Ab) – Hepatitis-B (Both Ag & Ab) • Measuring hormone Levels – hCG (Pregnancy Test) – LH (Ovulation) – TSH, T3 & T4 (Thyroid Function) Other Disease Diagnosis • HIV, which causes AIDS • Lyme disease • pernicious anemia • Rocky Mountain spotted fever • rotavirus • squamous cell carcinoma • syphilis • toxoplasmosis • varicella-zoster virus, which causes chickenpox and shingles • Zika virus
- 19. Thank You