The document outlines the principles and procedures of the Enzyme-Linked Immunosorbent Assay (ELISA), detailing its components, types (direct, indirect, sandwich, and others), and applications in diagnosing various infections like HIV and dengue. It discusses the mechanics of each ELISA type, their advantages, disadvantages, and the nature of the antigens and antibodies involved. Overall, it emphasizes ELISA's high sensitivity and capacity for large sample testing despite its longer processing time and equipment costs.

1. ELISA

2. Principle of ELISA
• 2 Components
• Immunosorbent: Absorbs antigen or antibody
present in serum
• Enzyme: used to label one of the components of
immunoassay
• Substrate-chromogen system : added at the final
step of ELISA
(Ag-Ab complex) – enzyme + substrate activates the chromogen
Color change detected by spectrophotometry

3. Procedure of ELISA
• Microtitre plate- 96 wells- polyvinyl, polystyrene or
polycarbonate material
• ELISA kits- commercially available- enzyme
conjugate, dilution buffer, substrate/chromogen
• At each step- reagent added – incubated- washing

4. Types
• Direct ELISA
• Indirect ELISA
• Sandwich ELISA
• IgM antibody capture (MAC ELISA)
• Competitive ELISA
• ELISPOT TEST
• IgG avidity ELISA

5. Direct ELISA
• Detection of antigen in test serum
Step
1. Wells of microtitre plate is empty not precoated
with Ag or Ab
2. Test serum (antigen) – added to well
3. After washing- enyme labeled primary antibodies
added
4. Substrate-chromogen added
5. Colour measured

6. Indirect ELISA
• Detection of antibody in serum

7. • Uses - determination of serum antibodies for
diagnosis of HIV
• Japanese encephalitis
• Dengue, many other viral infections

8. SANDWICH ELISA
• Detects antigen in the serum
• Antigen gets sandwiched between capture Ab
& detector antibody

9. • Used to detect rotavirus & enterotoxin of
E.coli in feces.

10. IgM ANTIBODY CAPTURE ELISA(MAC)
• Enzymatically amplifies sandwich type
immunoassay
• Based on capturing primary IgM Ab
• Microtitre plate precoated with antihuman
IgM antibody (capture IgM)
• Followed by addition of Recombinant Ag
eg.Dengue antigen

11. • Enzyme labelled secondary antibody specific
for antigen – added
• Addition of substrate chromogen system

12. COMPETITIVE ELISA
• Antigen in the test serum competes with
another antigen of same type coated on the
well – to bind to primary antibody.

13. • Most commonly used – detection of HIV
antibodies in serum

14. ELISPOT TEST
• Modification of ELISA
• Allows quantitative detection of cells
producing antibodies (plasma cells) or cytokines
(lymphocytes or macrophages)
•Used in IGRA – diagnosis of latent TB
•Sensitized T cells capable of producing IFN- γ
are measured

15. IgG AVIDITY ELISA
• Differentiating recent from past infection
Principle
• Avidity of an antibody- indicates how firmly it is
bound with its antigen
• Avidity reflects maturity of antibodies- which usually
increases with time
• Detection of low avidity IgG – recent infection
• High avidity IgG- past infection

16. Uses
Diagnosis of following infections:
• Rubella, CMV, VZV, toxoplasmosis, EBV, HIV,
viral hepatitis & west nile virus infection.

17. ADVANTAGES 0F ELISA
• Large sample can be tested together
• Economical
• High sensitivity

18. DISADVANTAGES OF ELISA
• Small laboratories- ELISA less preferred
• Takes more time (2-3hrs) compared to rapid
test (10-20min)
• Needs expensive equipments- ELISA washer,
reader

19. APPLICATIONS OF ELISA
• Antigen detection: HBsAg, HBeAg, NS1 antigen
for dengue
• Antibody detection: Hepatitis B, hepatitis C,
HIV, dengue, EBV, HSV, toxoplasmosis,
leishmaniasis

20. Consists of two antigen–antibody complement systems:
• (a) an indicator system
• (b) a test system
• Indicator system:
 Consists - RBCs that have been preincubated with a
specific anti-RBC antibody, in concentrations that do
not cause agglutination, and no hemolysis of RBCs
occurs in the absence of complement.
 Such RBCs -“sensitized” red cells