ELISA ( Enzyme linked immunosorbent assay)

In this slide contains introduction, principle, types, equipment's and applications of Enzyme linked immunosorbent assay.
Presented by: D.Sudheer Reddy. (Department of pharmacology)
RIPER, anantapur.

1. ELISA(Enzyme Linked ImmunoSorbent Assay) A Seminar as a part of curricular requirement of M-Pharmacy Ι year Ι semester Presented by D.Sudheer Reddy Reg.No:20L81S0113 Under the Guidance/Mentorship of Dr.P.Ramalingam,M.Pharam.,Ph.D.,R&D Director RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

2. CONTENTS Introduction Principle Types Equipments Advantages & Disadvantages Applications conclusion Reference RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

3. INTRODUCTION TO ELISA ELISA,or enzyme-linked immunosorbet assay,are quantitative immunological procedures in which the Ag-Ab reaction is monitored by enzyme measurements. The term ELISA was first used by Engvall & Perlma in 1971. The ELISA test or the enzyme immunoassay(EIA),was the first commonly employed for HIV.It has high sensitivity. It is useful & powerful method to estimating ng/mL to pg/Ml ordered materials in the solution. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

4. Why known as……….? Enzyme Linked Immunosorbent Assay Antigen of interest is absorbed on to plastic surface (‘sorbent’). Antigen is recognised by specific antibody (‘immuno’). This antibody is recognised by second antibody (‘immuno’) which has enzyme attached (‘enzyme-linked’). Substrate reacts with enzyme to produce product,usually coloured. Plate based immunoassay. Detects and quantifies substances such as peptides,poteins,hormones and antibodies. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

5. PRINCIPLE: ELISA use as an enzyme to detect the binding of antigen (Ag) antibody (Ab). The enzyme converts a colorless substrate ( chromogen ) to a coloured product,indicating the presence of Ag:Ab binding. An ELISA can be used to detect either the presence of antigens or antibodies in a sample depending how test is designed. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

6. Types of ELISA : Frequently there are 3 types of ELISA on the basis of binding structure between Basis of binding structure between the Antibody and Antigen. Direct ELISA Indirect ELISA Sandwich ELI S A Specimen samples for ELISA Serum Cerebrospinal fluid Sputum, urine & Supernatant of culture RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

7. Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR

8. Indirect ELISA RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721 The indirect ELISA detects in the presence of Antibody in a sample. Advantage: - Wide variety of enzyme linked secondary antibodies available. -Versatile: many primary antibodies can be made in one species and the same labelled secondary antibody can be used for detection. -Increased sensitivity : each primary antibody contains several epitopes that can be bound by the labelled secondary antibody,allowing for signal amplification. Disadvantages: -Cross-reactivity: might occur with the secondary antibody,resulting in non specific signal. -An extra incubation step is required in the procedure.

9. Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR

10. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721 Direct ELISA The direct ELISA detects the presence of Antigen in a sample. Advantages: Quick methodology since only one antigen is uesd. Cross reactivity of second antibody is eliminated. Disadvantages : Immunoreactivity of primary antibody is reduced because is enzyme linked. Lesser signal amplification than Indirect ELISA.

11. Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR

12. Sandwich ELISA The sandwich ELISA detects the presence of Antigen in a sample. Advantages: High specificity because the antigen is specifically captured and detected. Suitable for crude/impure samples as the antigen does not require purification prior to measurement. Flexible and sensitive, both direct or indirect detection methods can be used. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

13. Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P – 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR

14. Enzymes used in ELISA Horseradish peroxidase (most commonly used) Alkaline Phosphatase β-galactosidase Lactoperoxidase Tetra Methyl benzidine Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P – 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR

15. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721

16. Result Determination: A)IgA and IgG tests : Plot the O.D.result of each reference, except for the negative reference on the vertical. axis (Y-Axis) in relation to the number of corresponding units on the horizontal axis(x-axis). Using the absorbance value for each sample, determine the corresponding concentration of antibodies expressed in units/ml from reference curve. . Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR

17. Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR B)IgM Tests: We can calculate the cut off value A450nm sample/A450nm positive limit reference the normalized value of positive reference is 1. All samples whose value comparised between 0.8 and 1.0 considered dubious and all samples whose normalized value above 1.0 are considered positive for IgM antibodies

18. Applications of ELISA Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu, Anantapuramu A.P - 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Hormones 7. Vaccine Quality Control 2. Proteins 8. For GMO (Genetically modified organism 3. Infections Agent (viral,bacterial,parasitic,fungal) 9. For Rapid test 4. Drug Markers 10. IgG, IgM, IgA 5. Tumor Markers 11. In new born screening 6. Serum Proteins 12. In clinical research

19. Conclusion It is a good techniques and being popular today because of its simple and not involve radiation. It is easy than other test. It is most useful than other test because of their sensitivity. It is not harmful. Besides its disadvantages the technique is widely used in diagnostics and drug screening. Chromogenic detection method used in ELISA. Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu Anantapuramu A.P - 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR

20. Raghavendra Institute of Pharmaceutical Educational and Research- AUTONOMOUS K.R. Palli Cross, Chiyyedu Anantapuramu A.P - 515721 RIPER AUTONOMOUS NAAC & NBA (UG) SIRO - DSIR Reference Engvall E, Perlmann P.Enzyme linked immunosorbent assay.Quantitative assay of immunoglobulin G.Immunochemistry.1971;8(9):871-874. Kuhar M,Yamamura HI.Localization of cholinergic muscarinic receptors in rat brain by light microscopic radioautography.Brain Res.1976;110(2):229-43. Journal of immunoassay and immunochemistry.2012;25(3):241-58.