Elisa

ELISA techniques use in ANIMAL DISEASE DIAGNOSIS.

1. Enzyme Linked I mmunosorbent Assay (ELISA) Submitted by Dinesh Patel M.V.Sc scholar(AGB) Reg no:04-3211-2017 Submitted to Dr. P. G. Koringa Assistant professor Department of animal biotechnology 1

2. Contents Introduction Basic principle of ELISA Antigen Antibody Requirements of ELISA test Equipments Steps of ELISA test Types of ELISA Troubleshooting in ELISA Precautions Advantages Disadvantages Applications Limitations 2

3. Introduction Enzyme Linked Immunosorbent Assay (ELISA). Term Was Coined By Engvall and Pearlmann in 1971. Similar To RIA, Except No Radiolabel. Can Be Used To Detect Both Antibody and Antigen. ELISA may be run in a Qualitative or Quantitative format. 3

4. Basic principle of ELISA Lock and key concept. Use an enzyme to detect the binding of antigen (Ag) antibody (Ab). The enzyme converts a colorless substrate ( chromogen ) to a colored product, indicating the presence of Ag : Ab binding. An ELISA can be used to detect either the presence of Antigens or antibodies in a sample depending how the test is designed. 4

5. Substrate Primary antibody Enzyme Secondary antibody Different antigens in sample Coloured product 5

6. ANTIGEN (Ag) Any molecule that induces production of antibodies when introduced in the body of an animal is called antigen . OR Any “thing”, foreign to the immune system. e.g. bacteria, viruses, (or their parts), pollen, etc. Protein molecule. Carbohydrate molecule. Microorganisms. Allergens. Viruses Etc. SYMBOL FOR ANTIGEN 6

7. ANTIBODY (Ab) Antibody : proteins produced by the immune system which help defend against antigens. SYMBOL FOR ANTIBODY Y 7

8. Requirements for ELISA test Purified antigen (if you want to detect or quantify antibody). Purified antibody (if you want to detect or quantify antigen). Standard solutions (positive and negative controls). Sample to be tested. Micro-titer plates: plastic trays with small wells in which the assay is done. Wash fluid (buffer). Enzyme-labeled antibody and enzyme substrate. ELISA reader (spectrophotometer) for quantitative measurements. 8

9. Specimen Sample For ELISA SERUM CSF SPUTUM URINE SEMEN SUPERNATANT OF CULUTRE STOOL 9

10. Equipments 1 ) Microwell Plate: Flat bottom polystyrene plate, contains 8 x 12 wells holding , which will passively bind antibodies and proteins . 10

11. Equipments 2) Multipipette : An 8-channel 100 μL pipette is a good help for even small-scale work. 11

12. Equipments 3) Washing Device: manually operated washing devices. may be of use particularly when there is a risk that the samples tested in ELISA contain infectious material, so must be collected for subsequent disinfection. 12

13. Equipments 4) Microplate washer : These are very efficient with unusually low carry-over contamination. 13

14. Equipments 5) ELISA reader : Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical  or physical events of samples in microtiter plates. 14

15. Enzyme labels Enzyme labels are used to detect the binding of antigen-antibody complex. It should have high specific reactivity. It should be easily coupled to antigen-antibody complex and must stable. Enzymes used in labelling should not be normally present in the patient samples. Examples of enzyme labels are Horse radish peroxidase , Alkaline phosphatase, and Glucose oxidase. 15

16. 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 16

17. General Procedure 17

18. Basic Steps Of Enzyme-Linked Immunosorbant Assay 18

19. Types of ELISA Based on detection: Colorimetric ELISA Chemiluminescent ELISA Competitive Fluorescence ELISA Based on procedure: Non competitive ELISA Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA Modified ELISA( avidin -biotin ELISA) 19

20. Types of ELISA On the Basis of Detection: 1) Colorimetric ELISA: Assay to Determine the Antibody Concentration. 20

21. Types of ELISA 2) Chemiluminescent ELISA: Assay for the Quantification of an Antigen in a Biological Sample. 21

22. Types of ELISA 3) Competitive Fluorescence ELISA: 22

23. Types of ELISA: (on the basis of procedure )

24. Direct ELISA Antigen can be detected or quantitated by this method. The direct ELISA uses the method of directly labeling the antibody itself. Microwell plates are coated with a sample containing the target antigen. Not widely used but common for immuno-histochemical staining of cells & tissues. 24

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26. Advantages and Disadvantages Advantages of Direct ELISA Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated. Disadvantages of Direct ELISA Immunoreactivity of the primary antibody may be reduced as a result of labeling. Labeling of every primary antibody is time-consuming and expensive. No flexibility in choice of primary antibody label from one experiment to another. Little signal amplification. 26

27. Indirect ELISA In this unknown antibody can be detected or quantitated. The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody . Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies). 27

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30. Advantages and Disadvantages Advantages of indirect ELISA Wide variety of labeled secondary antibodies are available commercially. Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. Immunoreactivity of the primary antibody is not affected by labeling. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Disadvantages of indirect ELISA Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal. An extra incubation step is required in the procedure. 30

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32. Sandwich ELISA It is done for the antigen detection and quantification. 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 immobilized antigen to form a sandwich of Ab -Ag-Ab . 32

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36. Advantages of sandwich ELISA High specificity, since two antibodies are used the antigen/ analyte is specifically captured and detected. Suitable for complex samples, since the antigen does not require purification prior to measurement. Flexibility and sensitivity, since both direct and indirect detection methods can be used. Sandwich ELISA is a common tool to diagnose  Influenza , e.g.  H5N1 (Avian Flu) Hemagglutinin ELISA kit. 36

37. Competitive ELISA 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. 37

38. Competitive ELISA 38 Quick methodology.

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40. Modified ELISA ( Avidin -biotin ELISA ) Enzymes may inhibit antibody activity or lose enzymatic activity in the process of conjugating them to antiglobulin . One alternative is to use biotin and its specific binding protein avidin . Biotin can bind to protein (antibody) without affecting their biological activity . Avidin binds very strongly and specifically to biotin and may be conjugated with enzymes. 40

41. Modified ELISA 41

42. Dot ELISA Similar to the plate ELISAs, but the solid phase to which antigen is bound is the nitrocellulose membrane (usually) and the chromogenic substrate used is DAB which forms an insoluble product . Hence in positive cases a brown dot is obtained at place of antigen deposition . The advantage of dot ELISA over other ELISAs is that the color reaction can be read visually without sophisticated ELISA readers . The test is also easy to perform and can be done in the field .  The results  i.e.  dots can be stored for retrospective analysis. 42

43. Reading 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. 43

44. Results 44

45. Troubleshooting in ELISA Negative control with strong signal The excessive background signal can be caused by inadequate rinsing of plates, reagents not sufficiently diluted, inadequate blocking of plates or non-specific binding of enzyme conjugate. The appearance of color in negative control wells may also indicate cross-reactivity of secondary antibody with components in the antigen sample. 45

46. Positive control with no signal Microwell plates not coated properly. Reagents applied in wrong order or step omitted. Secondary antibody not matched to the species of primary antibody. Enzyme conjugate defective or inhibited by contaminant. Detector antibody not compatible with capture antibody (for sandwich assays). 46

47. ELISA with weak signal Wash buffer not adequately drained after every wash step. Inadequate incubation times. Detection reagents too dilute. Enzyme conjugate defective or inhibited by contaminant. Substrate defective or contaminated. Microwell plates poorly coated. Loss of capture antibody during blocking/washing. Decrease or eliminate use of Tween-20 ( nonionic detergent ) 47

48. Precautions 1) Use of Exchange type pipette: (always use new tip) 48

49. Precautions 2) Washing: 49

50. Precautions 3) Reagents: 50

51. Precautions 3) Reagents: 51

52. Precautions 4) Plate cover: During incubation, well plate should be covered using the plate cover. Plate cover is effective only under suitable conditions i.e room temp. H umidity > 50%. 52

53. Precautions 5) Coating of wells: Coating of wells should be proper with the addition of Blocking solution. Improper coating False positive results. 53

54. Advantages of ELISA Less costly and safest. Easy visualization of results with high level of accuracy. Specific and highly sensitive assay that can detect protein at the picomolar to nanomolar range. Easily automated for performance of large numbers of tests. Require minimal reagents. Qualitative detection or Quantitative measurement of either antigen or antibody. Wells can be coated with antigens or antibodies. Can be done by personnel with only minimal training. 54

55. 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. Kits are not cheap. Very specific to particular antigen but won’t recognize other antigens. False positive/ negative possible, especially with mutated/ altered antigen. 55

56. Applications of ELISA Analysis of hormones, vitamins, metabolites, and diagnostic markers. Therapeutic drug monitoring. Diagnostic procedures for detecting infection. Detection of potential allergens in food. 56

57. Limitations Results may not be absolute. Antibody must be available (poor producer, interference). Concentration may be unclear. False positive possible (Ab already present). False negative possible. 57

58. THANK YOU 58