elisa (1) enzyme linked immunosorbant assay.pptx

enzyme linked immuosorbant assay

1. ELISA

2. ELISA ELISA (Enzyme-linked immuno-sorbent assay) is one of immunoassay method used to detection and quantification of 1-Antibodies 2-Proteins 3-Peptides 4-Biomolecules

3. 1. Antigen/antibody of interest is absorbed on to plastic surface (‘sorbent’). 2. Antigen is recognised by specific antibody (‘ immuno ’). 3. This antibody is recognised by second antibody (‘ immuno ’) which has enzyme attached (‘enzyme-linked’). 4. Substrate reacts with enzyme to produce product, usually coloured.

4. Antibody (antiserum) Antibody: proteins produced by the immune system which help defend against antigens The variable regions are though to be the place for recognition and binding with the antigen.

5. Antigen Any molecule that induces production of antibodies when introduced in the body is called antigen. OR Any “thing”, foreign to the immune system. e.g. bacteria, viruses, (or their parts), pollen, etc.

6. Specimen Sample For ELISA SERUM (Ag/ Ab ) CSF SPUTUM URINE SEMEN SUPERNATANT OF CULUTRE STOOL

7. 1-Direct ELISA 2-Indirect ELISA 3-Sandwich ELISA Types Of Elisa According to the coating substance and no of antibodies used:

8. Direct ELISA

9. Indirect ELISA

10. Sandwich ELISA The ELISA plate is coated with Antibody to detect specific antigen

11. Steps of ELISA Basic steps: Coating Blocking Sample load Washing 2ry Ab Detection

12. Materials Needed Testing sample Antibody (1st, 2nd) / Antigen Polystyrene microtiter plate Blocking buffer Washing buffer Substrate Enzyme 8

13. ELISA Procedure

14. STEPS INVOLVED in indirect ELISA Binding is achieved by incubating the wells with a solution containing the antigen for 1-2hrs at RT or overnight at 4°C. Antigen Binding The sample antigen is binded or immobilized to the micro plate via adsorption to the surface. The protein adheres due to hydrophobic interactions between the protein & the plastic. Coating is done using carbonate/bicarbonate buffer at a pH 9. 6.

15. Blocking  All unbound sites on the solid support are blocked to prevent nonspecific binding. Blocking buffers like BSA( bovine serum albumin ) , Non fat dry milk powder (casein) in PBS ( Phosphate buffered saline) or TBS ( Tris buffered saline) at a pH 7.4 with of ( 0.01% to 0.10%) Tween 20 are used to block the free sites. STEPS INVOLVED in ELISA Blocking volume should be double coating volume

16. The benefit from the addition of a surfactant such as Tween™ 20 (a gentle non-ionic detergent) to the blocking solution to minimize hydrophobic interactions between the blocking protein and the antigen or antibodies

17. Protein in the blocking solution will attach to membranes in places where the target proteins have not attached. Excess blocking agent is removed by washing the plate membrane with washing buffer. Washing is performed by buffers such as Tris -buffered saline (TBS) or phosphate-buffered saline (PBS) with detergent such as 0.05% Tween-20 .

18. Primary Antibody The 1° antibody is added and will be bound only if there is a recognized epitope within sample antigen. The dilution of Ab added depends on titration experiment. The solvent used in dilution is called diluting buffer, in some cases the diluting buffer is the same like blocking buffer . After incubation of Primary Ab with samples, washing is required. 3-5 times washing to remove unbound antibodies.

19. Optimizing the antibody dilution: titration experiments The optimal antibody concentration, which gives the best result with minimum background, must be determined experimentally for each assay and is usually determined by using a series of dilutions in a titration experiment. For example, it is recommended to make dilutions of 1:50, 1:100, 1:200, 1:400 and 1:500.

20. Secondary Antibody -An enzyme-linked 2° antibody is added with suitable dilutions which will bind to any available 1° antibody (i.e. it is bound to the antigen). -2° antibodies are linked to the enzyme through biconjugation . - Titration experiment is also done to determine the optimal dilution of antibody -Plate is washed again with buffer to remove any unbound secondary antibodies .

21. Detection/ Developing After the final wash step, the plate is developed by adding an enzymatic chromogenic substrate  to give color, which also give indication on the amount of Ag/Ab present. The entire plate is placed into a plate reader and the OD is determined for each well. Intensity of color reflects the amount of specific 2° antibody bound to the target. STEPS INVOLVED in ELISA

22. ELISA reader

23. Enzymes Used in Elisa - Horseradish peroxidase (most commonly used) -Alkaline Phosphatase - β- galactosidase - Lactoperoxidase

24. enzyme substrate Color produced absorbance Stop soln AP(alkaline phosphatase) PNPP(colorless) p- nitro phenyl phosphate yellow 405nm NaoH HRP(Horseradish Peroxidase) TMB ( blue ) Tetramethyl Benzidine yellow 450nm sulfuric or phosphoric acid HRP OPD  colorless ( o- phenylenediamine dihydrochloride) yellow-orange 492nm HCL

25. ELISA PLATE READY FOR READING Measures the absorbance at certain λ according to the color produced. For example measure at 450nm if TMB is used The final color is yellow after addition of stopping soln. The color before stopping is blue. Calculate the absorbance for each sample and reference.

26. Standard curve Of ELISA Standard curve of ELISA prepared by plotting standard concentration on X-axis and absorbance on Y-axis Limit of detection Limit of quantification

27. APPLICATIONS OF ELISA -detection of Mycobacterium antibodies in tuberculosis. -detection of rotavirus in feces. -detection of hepatitis B markers in serum. -detection of HIV antibodies in blood samples.

28. Advantages of ELISA Reagents are relatively cheap & have a long shelf life  ELISA is highly specific and sensitive  No radiation hazards occur during labelling or disposal of waste.  Easy to perform and quick procedures  Equipment can be inexpensive and widely available.  ELISA can be used to a variety of infections. Not require highly qualified experienced researcher .

29. Disadvantages of ELISA Enzyme activity may be affected by plasma constituents. Kits are commercially available, but not cheap False positives/negatives possible, especially with mutated/altered antigen Variation of the result

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