Elisa and its type

these slides will let you know about the ELISA and its types and how it is preformed in the laboratory

1.                                     PRESENTATION   ELISA(ENZYME LINKED IMMUNOSORBENT ASSAY )     PRESENTED TO :                       DR. MUHAMMAD ISMAIL     GROUP MEMBERS :                      JONATHAN JAVID (80002)            SHAHRUKH HASSAN (80003)                                                 HAFIZ ABDUL HANNAN (80008)                           BS - BIOINFORMATICS (EVENING)                                      

2.                         Contents Introduction to ELISA History of ELISA Principle of ELISA  Materials Types of ELISA             Competitive             Non-competitive         Direct ASSAY        Indirect ASSAY        Sandwich ASSAY •     Advantage and Disadvantage of ELISA Limitations Application 

3.             Introduction to ELISA   The Enzyme Linked Immunosorbent Assay (ELISA) is a common laboratory technique which is used to measure the concentration of an analyte (usually antibodies or antigens) in solution. The term ELISA was first used by Engvall & Perlma in 1971. The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.

4.                   History of ELISA   Radioimmunoassay was first described in a scientific paper by Rosalyn Yalow and Solomon Berson published in 1960. In 1971, Peter perlmann and Eva Engvall in Sweden, and Anton & Bauke van Weemen in Netherlands independently published papers that synthesized this knowledge into methods to perform EIA/ELISA

5.             Principle of ELISA Based on Immunology Response  Lock and Key concept : Antigen (Key)                                   Antibody (Lock)  Use an enzyme to detect the  binding of antigen Y (Ag)     antibody               (Ab). The enzyme (HRP) converts a colorless substrate (chromogen) to a colored product e.g.TMB(Trimethyl benzidine), indicating the presence of Ag: Ab binding An ELISA can be used to direct either the presence of antigens or antibodies in a sample depending how the test is designed.

6.                       MATERIALS Antibody-coated 96-well microplate  Detection antibody (usually biotinylated) Standard  HRP(Hosre-redish peroxidase)conjugate Diluent buffers Wash buffer Chromogenic substrate (usually TMB (trimethyl benzidine) ) Stop solution  Plate covers 

7.                 Types of ELISA   Competitive ELISA  Non-competitive ELISA            Direct Assay           Indirect Assay           Sandwich Assay   

8.               COMPETITIVE ELISA Antibody coated microwell. Serum antigen & labeled antigen added together. Used to determine small molecules like T3(triodothyroxin), T4(thyroxin), & progesterone. Increased serum antigen result in reduced binding of Ag-enzyme conjugate with the antibody producing less enzyme activity & (yellow) color formation. It is used to detect Ag (Free testosterone)

9.         NON-COMPETITIVE ELISA              DIRECT ASSAY Apply a sample of known antigen to a surface. Enzyme linked primary antibody is applied to the plate. Washed, After this wash, only the antibody-antigen complexes remain attached. Apply a substrate which is converted by the enzyme to dicit a chromogenic signal. It is used to detect Ab (HIV, HCV )

10.                     INDIRECT ASSAY Antigen is added to plate. Adding blocking buffer. Suitable primary antibody is added. TMB substrate is added, is converted to detected (Yellow) form.

11.             SANDWICH ASSAY 1. The plate is coated with suitable antibody       2.  Blocking buffer is added Sample is added to plate so antigen is bounded by capture antibody. A suitable biotin labeled detection antibody is added to plate. Enzyme HRPO is added and bind the biotin labeled detection antibody. TMB substrate is added and converted by HRPO to colored product. It is used to detect Ag (Tumor Markers, Hormones )

12.             ADVANTAGES of ELISA Reagents are relatively cheap & have long shelf life. It is highly specific & sensitive. No radiation hazards occur during labeling or disposal of waste. Easy to perform & quick procedures. Equipment is widely available. It can be used to variety of infections. It can be used on most type of biological samples like plasma, serum, urine, cell extracts.

13.       DISADVANTAGES of ELISA Measurement of enzyme activity can be more complex than the measurement of activity of some type of radioisotopes. Enzyme activity may be affected by plasma constituents. Very specific to particular antigen but won't recognize other antigens. False positive/negative possible, especially with mutated/altered antigen.

14.                   LIMITATIONS Results may not be absolute. Antibody must be available (poor producer, interference). Concentration may  be unclear. False positive (Ab already present). False negative possible.

15.                   APPLICATIONS Screening donated blood for evidence of viral contamination by        HIV-1 and HIV-2 (presence of anti-HIV antibodies)       Hepatitis C (presence of antibodies)       Hepatitis B (testing for both antibodies and a viral antigens) Measuring hormone levels       HCG (as a test for pregnancy)       LH (determining the time of ovulation)        TSH, T3 and T4 (for thyroid function) Detecting infections       Sexually-transmitted agents like HIV, syphilis and chlamydia       Hepatitis B and C       Toxoplasma gondii  Detecting illicit drugs Detecting allergens in food and house dust 

16. REFERENCE WEBSITES AND BOOKS www.Healthline.com/health/elisa https://www.bio-rad-antibodies.com/an-introduction-to-elisa.html www.elisa-antibody.com/ELISA-Introduction www.enzolifesciences.com › Platforms › Immunoassay and Assay Development The ELISA Guidebook-second Edition (John R.Crowther) ELISA: Theory and Practice (John R.Crowther) The Anxiety Book, Blood River, Glasswings,etc