ELISA direct, Haitham ALhakimi- Saadah university..pptx

immunology and serology - ELISA tech. and procedure

1. E L I S A By: Haitham Alhakimi 1

2. Definition: The enzyme-linked immunosorbent assay ( ELISA ) It is a biochemical technique to detect and quantify the presence of specific compound ( antigen ,antibody , hormones) 2

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4. Uses : 1) -ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g. a hormone or virus) in a fluid such as serum or urine 2) The ELISA is a rapid test used for detecting and quantifying antibodies or antigens against viruses, bacteria and other materials. 3) This method can be used to detect many infectious agents affecting poultry and livestock. 4

5. Basic Terms: 12 well Solid Phase: Usually a microtiter plate well, having 8 format. 5

6. Basic Terms: Adsorption: The process of adding an antigen/antibody, diluted in buffer, so it attaches to the solid phase on incubation. Washing: The simple flooding & emptying of wells with a buffered solution to separate bound from un-bound reagents in ELISA. 6

7. Basic Terms: Antigen: Any molecule that elicits the production of antibodies when introduced into body. Antibodies: Proteins produced in response to antigenic stimuli. Enzyme conjugate: An enzyme that is attached irreversibly to an antibody. e.g: Horse-redish peroxidase (HRPO). 7

8. Basic Terms: Chromogen: A chemical alters color as a result of an enzyme interaction with substrate (color reaction used as signal) e.g Trimethyl benzidine (TMB). Stopping: The process of stopping the action of an enzyme on a substrate. Reading: Spectrophotometric measurement of color developed in ELISA. 8

9. Principle of ELISA: Based on Basic Immunology Response Lock and Key Concept: Antigen (key) 2) Antibody (lock): –Key fits into the lock Enzyme conjugate substrates Bound to a secondary antibody that binds with the antibody-antigen complex. 9

10. E q u i p m e nt s : 1) Microwell Plate : Flat bottom polystyrene plate, contains 8 x 12 wells holding 350 μL each. 10

11. E q u i p m e nt s : 2) Multipipette : An 8-channel 100 μL . 11

12. Reagents Used: 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-redish peroxidase (HRPO) Chromogenic Substrate Trimethyl benzidine (TMB) Stop Solution 0.5 M H₂SO₄ 12

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14. General Procedure: 14

15. The main Steps of ELISA 1) Specimen "Analyte“ 2) Substrate 3) Conjugate (Incubation and washing) 15

16. Types of ELISA: On the Basis of Detection: 1) Colorimetric ELISA: Assay to Determine the Antibody Concentration. 16

17. Types of ELISA: 2) Chemiluminescent ELISA: Assay for the Quantitation of an Antigen in a Biological Sample. 17

18. Types of ELISA: T ypes Non- Comp e titi v e (on the basis of procedure) Di r ect Indi r ect S a ndwich Competitive Multiple & Portable 18

19. Non-Competitive: 19

20. Non-Competitive: 20

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22. Non-Competitive: 3) Sandwich ELISA: Antigens like Tumor markers, hormones, serum proteins may be determined. Antigens in the sample bind with the capture antibody & become immobilized. The antibody of the enzyme conjugate bind with the immobili z ed a n ti g en t o f or m a sandwic h o f A b- A g- A b / enzyme bound to microwell. 22

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24. Competitive: 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) color formation. Used to determine small molecules like T₃ , T₄ & Progesterone. 24

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28. R e a d i n g : Measure the absorbance at 450nm with the help of ELISA reader. Calculate the absorbance for each sample and reference. Ascent software for the calculation of results can be used. 28

29. ELISA Reader and Washer washer Reader 29

30. T h a n k You 30