ELISA TECHNIQUE

Newer techniques in Antigen Antibody reactions including ELISA and its different types.

1. ANTIGEN ANTIBODY REACTION ARPITA CHANDRA MICROBIOLOGIST

2. NEWER TECHNIQUES ELISA ( Enzyme linked Immunosorbent Assay ) IFA (Immunofluorescence assay ) RIA ( Radioimmunoassay) CLIA ( Chemiluminescence -linked immunoassay) Immunohistochemistry Rapid test – Lateral flow assay ( Immunochromatographic test ) Flow through assay Western blot Immunoassay using electron microscope.

3. ELISA ENZYME – Used to label antigen or antibody. IMMUNOSORBENT – An absorbing material is used (ex- polystyrene, polyvinyl) Absorb Ag and Ab present in serum

4. ENZYMES USED IN ELISA AND THEIR SUBSTRATE CHROMOGEN SYSTEM ENZYME SUBSTRATE CHROMOGEN Horseradish Peroxidase Hydrogen peroxide Tetramethyl benzidine (TMB) Urease Urea Bromocresol B- Galactosidase ONPG ONPG Alkaline Phosphatase pNPP ( para-Nitrophenyl phosphate chromogenic substrate, Converted to para - Nitrophenol (color product) pNPP

5. PRINCIPLE The enzyme reacts with substrate, which in turn activates the chromogen to produce a color The color change is detected by spectrophotometry in an ELISA reader.

6. Intensity of the color is Directly propotional to The amount of detection molecules (Ag or Ab ) present in test serum .

7. (Ag- Ab complex)-enzyme + substrate Activates the chromogen Color change Detected by spectrophotometry

8. Necessary reagents Enzyme conjugate, dilution buffer, substrate/ Chromogen etc . Steps At each step, a reagent is being added, and then incubated followed by washing of the wells(manually or by an automated ELISA washer )

9. Types of elisa

10. Principles of different ELISA types ELISA type Used for the detection of Enzyme is labeled with Direct ELISA Antigen Primary antibody Indirect ELISA Antigen or antibody Secondary antibody Sandwich ELISA Antigen primary antibody in direct sandwich ELISA Secondary antibody in indirect sandwich ELISA Competitive ELISA Antigen or antibody Secondary antibody ELISPOT Cells producing antibody or cytokine Primary antibody

11. DIRECT ELISA Used for detection of antigen in test serum Primary antibody (targeted against the serum antigen) is labeled with the enzyme .

12. Wells(not precoated with Ag or Ab ) + Ag (test serum) + primary Ab -Enzyme + substrate- chromogen Color change

13. Indirect ELJSA It is used for detection of antibody. It differs from direct ELISA in that the the secondary antibody is labeled with enzyme instead of primary antibody.

14. Wells (coated with Ag) + primary ( test serum) + secondary Ab - Enzyme + substrate- chromogen Color change

15. Sandwich ELJSA It detects the Ag in test serum. The antigen gets sandwiched between a capture antibody and a detector antibody.

16. There are two types of sandwich ELISA- direct and indirect Detector antibody Primary antibody (direct ) Secondary antibody (indirect)

17. Direct sandwich elisa Wells coated with capture Ab + Ag (test serum)+ primary Ab enzyme + substrate- chromogen Color

18. In Indirect Sandwich ELISA Wells coated with captureAb + Ag (test serum) + primary Ab +sec o ndary Ab -enzyme+ substrate , chromcgen Color

19. Competitive ELISA Competitive ELISA is so named because, antigen in test serum competes with another antigen of same type coated on well to bind to die primary antibody.

20. Elispot test Modification of ELISA that allows the quantitative detection of cells producing antibodies (plasma cells) or cytokines (e.g. macrophage)

21. Advantage of elisa large number of samples can be tested together using the 96 well microtiter plate . It is economical, takes 2-3 hours for performing the assay.

22. ELISA is the most sensitive immunoassay, it is commonly used for performing screening test at blood banks and tertiary care sites.

23. Disadvantages In small laboratories having less sample load, ELISA is less preferred than rapid rests later can be done on individual samples. It needs expensive equipments such as ELISA washer and reader.

24. Applications of ELISA ELISA can be used both for antigen and antibody derection . Antigen detection: HBsAg , HBeAg , NS1 antigen for dengue, etc.

25. ELISA can also be used for antibody detection against hepatitis B, hepatitis C, HIV, dengue, EBV, HSY, toxoplasmosis, leishmaniasis , etc.