ELISA (ENZYME LINKED IMMUNO SORBENT ENZYME

ELISA

1. Noida Institute of Engineering and Technology (Pharmacy Institute) Greater Noida Enzyme-linked immunosorbent assay (ELISA) Submitted to: Chandana Majee (Assistance Professor ) 1 Unit: 5 (MPC201T )Advanced Spectral Analysis Course Details M.PHARM 2 nd Semester Submitted by: Rachit Sharma (0231PCH006)

2. Content ELISA Principle of ELISA Types of ELISA Advantages Application 2

3. ENZYME LINKED IMMUNOSORBANT ASSAY ELISA Add a footer 3

4. Principle of ELISA “Wet lab" analytic biochemistry assay, ELISA involves detection of an "analyte“ in a liquid sample by a method that continues to use liquid reagents during the "analysis“. Lock and Key Concept: Antigen (key) & Antibody (lock) key fits into the lock The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding. The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag: Ab binding. 4

5. Principle of ELISA

6. Types of ELISA On the Basis of Detection Colorimetric ELISA Chemiluminescent ELISA Competitive Fluorescence ELISA On the basis of procedure Non competitive ELISA Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA Direct ELISA Indirect ELISA Sandwich ELISA Add a footer 6

7. Colorimetric ELISA These type of assays are used to determine the concentration of antibody present in the sample provided by the change in colour . On the basis of detection 7

8. Chemiluminescent ELISA Chemiluminescent ELISA is the technique which work on the emission of light occur due to reaction of antigen and antibodies. It is used for the quantitation of an antigen in a biological sample. On the basis of detection 8

9. Competitive Fluorescence ELISA Competitive fluorescence ELISA based on the principle of change in the direction of emitted fluorescent light from the fluorescent labelled tracer due to the reaction of Ag – Ab. It can define both antigen and antibodies. On the basis of detection 9

10. Add a footer 10 Colorimetric ELISA Chemiluminescence ELISA Competitive Fluorescence ELISA

11. On the basis of procedure Direct ELISA Add a footer 11

12. On the basis of procedure Indirect ELISA Add a footer 12

13. On the basis of procedure Sandwich ELISA Add a footer 13

14. Antibody coated microwell. Serum antigen & labeled antigen added together .... Competition Ab-Ag enzyme complex bound is inversely related to the conc. Of antigen present in sample. Increased serum antigen results in reduced binding of Ag enzyme conjugate with the antibody producing less enzyme activity & (yellow) colour formation. It is used to determine small molecules like T₃ , T₄ & Progesterone. On the basis of procedure Competitive ELISA Add a footer 14

15. Requirement for ELISA Microwell Plate: Flat bottom made up of polystyrene plate, containing 8 x 12 wells holding 350 μ L volume in each. Multi – pipette: An 8-channel 100 μ L pipette is a good help for even small-scale work. Microplate washer : These are very efficient with unusually low carry-over contamination. Recorder: Measure the absorbance at 450nm with the help of ELISA reader. Ascent software for the calculation of results can be used. Add a footer 15

16. Requirement for ELISA Add a footer 16 REAGENT COMPOSITION Coating Buffer 0.01 M Phosphate Buffer + 0.15 M NaCl (PBS) Diluting/Washing Buffer 0.01 M Phosphate Buffer + 0.50 M NaCl + 0.1% Tween 20 Blocking Buffer Bovine Serum Albumin (BSA) Enzyme Horse-reddish peroxidase (HRPO) Chromogenic Substrate Trimethyl benzidine (TMB) Stop Solution 0.5 M H₂SO₄

17. ADVANTAGES of ELISA Reagents are relatively cheap & ‘ve long shelf life. It is highly specific & sensitive (<1pg/ml). No radiation hazards occur during labeling or disposal of waste. Easy to perform & quick procedures. Equipment is widely available. It can be used to a variety of infections. It can be used on most type of biological samples like plasma, serum, urine, cell extracts. 17

18. APPLICATION of ELISA 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 antigen) 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 18

19. Thank You. ELISA IMMUNO ASSAY

20. REFERENCE https://www.slideshare.net/ParasuramanParasuraman/principles-and-applications-of-elisa