Presentation elisa ppt

basic concept of ELISA

1. Basic concept of ELISA Digvijay singh IMS, BHU, Varanasi

2. ENZYME LINKED IMMUNOSORBENT ASSAY INTRODUCTION TO ELISA ELISA or enzyme linked immunosorbent assay are quantitative immunological procedures in which the Ag- Ab reaction is monitored by enzyme measurement. or ELISA (enzyme linked immuno sorbent assay ) is a common laboratory technique which is use to measure the concentration of analyte ( usally Ag or Ab ) in solution.

3. Why known as………….? E nzyme L inked I mmuno s orbent A ssay Antigen/Antibody of interest is absorbed on the plastic surface( sorbent ). Antigen is recognised by specific antibody( immuno ). The antibody is recognised by specific antibody( immuno ) which has enzyme attached( enzyme-linked ). substrate reacts with enzyme to produced product usually coloured

4. History of Elisa 1960 Radioimmunoassay 1966 Non radio immunoassay(enzyme linked) 1971 Method to perform EIA/ELISA In 1971 Peter Perlmann and Eva Engvall used term ELISA. 2012 an ultrasensitive enzyme based ELISA test.

5. BASIC PRINCIPLE OF ELISA Use an enzyme to detect the binding of antigen(Ag) antibody ( Ab ). The enzyme converts a colourless substrate ( Chromogen ) to a colour product, incubating the presence of Ag- Ab binding. An ELISA can be used to detect either the presence of Antigen or Antibody in a sample depending how the test is designed.

6. NON COMPETATIVE ELISA 1- DIRECT ELISA * It use a primary labeled antibody that react directly with the antigen. It can be performed with the antigen that is directly immobilized on the assay plate. Not widely used but common for Immuno-histochemical staining of cell & tissue. 2-INDIRECT ELISA * It utilizes a primary un- labeled antibody in conjunction with a labeled secondary antibody. secondry antibody has specific for primary antibody.

7. NON-COMPETATIVE ELISA 3-Sandwich ELISA * Antigen like Tumour markers,hormones,serum proteins may be determined. Antigens in the sample binds with the capture antibody & become immobilized. The antibody of the enzyme conjugate bind with the immobilized antigen to form a sandwich of Ab -Ag- Ab / enzyme bound to microwell .

8. COMPETATIVE ELISA Antibody coated microwell . Serum antigen and labeled antigen added together. Ag- Ab enzyme complex bound is inversely related to the conc. of antigen present in sample. Increased serum antigen results in reduced binding of Ag-enzyme conjugated with the antibody producing less enzyme activity & (yellow colour) formation. Used to determine small molecules like T 3, T 4 etc.

9. REPRESENTATIVE FIGURE OF COMPETITIVE ELISA

10. Multiple & Portable ELISA A new technique use an solid phase made up of an immune-sorbent polystyrene rod with 8-12 protruding ogives pins. The entire device is immersed in a test tube containing the collected sample and the following steps (washing , incubation in the conjugate and incubation in chromogenous ) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.

11. Advantage of ELISA Reagent are relatively cheap & have along self life. ELISA is highly specific and sensitive. No radiation hazard occur during labelling or disposal of waste. Easy to perform and quick procedures. ELISA can be used to variety of infections and research.

12. Disadvantages of ELISA Measurement of enzyme activity can be more complex. Enzyme activity may be affected by plasma constituents. Kits are commercially available but not cheap. very specific to a particular antigen won’t recognize any other antigen. false positives/negatives possible.

13. Limitations Result may not be absolute. Antibody must be available. Concentration may be unclear. False positive possible. False negative possible.

14. Application of ELISA Proteins Hormones Drug Markers Tumor Markers Serum proteins Antibody and Antigen detection vaccine Quality control In clinical research etc.

15. Troubleshooting in ELISA If the negative controls are giving positive result: 1-Contamination of substrate solution, enzyme-labelled antibody, control themselves. 2-Inadequate rinsing of plates. 3-Inadequate blocking of plates. If no colour developed for the positive controls or for the samples: * Check all reagents for dating and storage condition. * Microwell plate not coated properly. *Reagents applied in wrong order or step. *Enzyme conjugate defective or inhibited by contamination

16. If a very little colour has developed for positive controls and the test samples Check the dilution of the enzyme labelled antibody. The concentration of the substrate. Wash buffer not adequately drained after every wash step. Inadequate incubation times. Enzyme conjugate or substrate defective or contaminated. microwell plate poorly coated

17. If colour has developed for the test sample but not positive controls Check the source of positive controls, their expiry date and storage. If the colour can be seen but the absorbance is not high as expected check the wave length.

18. Application of ELISA in viral disease in our VRDL lab Dengue- NS1 -Ag, IgM,IgG Ab. Japanese Encephalitis - IgM,IgG Ab. Influenza - IgM,IgG Ab . Chikungunya - IgM,IgG Ab. Scrub typhus- IgM,IgG Ab. Leptospirosis -IgM Ab. Zika -IgM,IgG Ab. Ebola and other out break of viral infections

19. Instrument used in ELISA- ELISA Reader and Microwell Plate Washer