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ELISA (ENZYME LINKED IMMUNO SORBENT ENZYME
- 1. Noida Institute ofEngineering 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 2nd Semester Submitted by: Rachit Sharma (0231PCH006)
- 2. FR Content • ELISA • Principleof ELISA • Types of ELISA • Advantages • Application 2
- 3. The ELISA isa common laboratory technique which is used to measure the concentration of an analyte (usually antibodies or antigens) in solution. Detection by secondary antibodies conjugated to enzymes (alkaline phosphatase, horse radish peroxidase, β- galactosidase). It is "wet-lab" type analytic biochemistry assay. A 96 - well microtiter plate being used for ELISA. ENZYME LINKED IMMUNOSORBANT ASSAY ELISA Add a footer 3
- 4. FR • “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 4
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- 6. FR 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. Colorimet ric ELISA These typeof 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. Chemiluminescen t ELISA Chemiluminescent ELISA isthe 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 On the basis of detection 8
- 9. Competitive Fluorescence ELISA Competitive fluorescence ELISAbased 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 footer10 Colorimetric ELISA Chemiluminescen ce ELISA Competitive Fluorescence ELISA
- 11. It uses aprimary labeled anti-body that react directly with the antigen. It can be performed with the antigen that is directly immobilized on assay plate. The antigen is bound by passive adsorption to the solid phase, washed to remove any unbound molecules and then directly incubated with conjugated antibody. Substrate is added to produce signal that is allowed to develop. After certain time, the substrate reaction is stopped and the resulting signal quantified. On the basis of procedure Direct ELISA Add a footer 11
- 12. It utilizes aprimary un-labeled antibody in conjunction with a labeled secondary antibody. In this system, initial antigen binding and washing steps are the same as the direct method. In this case, unconjugated antibody to bind the immobilized antigen upon incubation at optimal temperature (usually 37⁰C). Following a washing, the remaining antigen-bound antibodies are targeted by a conjugated secondary antibody that will generate the readout signal as described for direct ELISA. This system has been widely applied in diagnostics because it allows large number of samples to be screened with a single conjugated secondary antibody. On the basis of procedure Indirect ELISA Add a footer 12
- 13. Antigens in thesample bind 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 HRPO is added and binds the biotin labeled detection antibody. On the basis of procedure Sandwich ELISA Add a footer 13
- 14. • Antibody coatedmicrowell. • 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. FR Requirement for ELISA • MicrowellPlate: Flat bottom made up of polystyrene plate, containing 8 x 12 wells holding 350 μL volume in each. • Multi – pipette: An 8-channel 100 μL 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. FR Requireme nt for ELISA Add afooter 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. FR ADVANTAGE S of ELISA • Reagentsare 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. FR APPLICATI ON of ELISA • Screeningdonated 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
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