ELISA is an immunological technique used to detect the presence of antigens or antibodies in samples. It works by immobilizing an antigen or antibody on a plate and detecting it using an enzyme-linked antibody or antigen. This allows quantification of the analyte being tested for. ELISA has advantages like being sensitive, specific, easy to perform, and having a long shelf life for reagents. It is commonly used to detect proteins, hormones, drugs, tumor markers, antibodies, and antigens in clinical and research applications.

1. Basic concept of ELISA
Digvijay singh
IMS, BHU, Varanasi

2. ENZYME LINKED
IMMUNOSORBENT ASSAY
INTRODUCTION TO ELISA
• ELISA or enzyme linked immunosorbent assay are quantitative
immunological procedures in which the Ag-Ab reaction is monitored by
enzyme measurement.
or
• ELISA (enzyme linked immuno sorbent assay) is a common laboratory
technique which is use to measure the concentration of analyte (usally Ag
or Ab) in solution.

3. Why known as………….?
Enzyme Linked Immunosorbent Assay
• Antigen/Antibody of interest is absorbed on
the plastic surface(sorbent).
• Antigen is recognised by specific
antibody(immuno).
• The antibody is recognised by specific
antibody(immuno) which has enzyme
attached(enzyme-linked).
• substrate reacts with enzyme to produced
product usually coloured

5. History of Elisa
• 1960 Radioimmunoassay
• 1966 Non radio immunoassay(enzyme linked)
• 1971 Method to perform EIA/ELISA
• In 1971 Peter Perlmann and Eva Engvall used
term ELISA.
• 2012 an ultrasensitive enzyme based ELISA
test.

6. BASIC PRINCIPLE OF ELISA
• Use an enzyme to detect the binding of
antigen(Ag) antibody (Ab).
• The enzyme converts a colourless substrate
(Chromogen) to a colour product, incubating
the presence of Ag-Ab binding.
• An ELISA can be used to detect either the
presence of Antigen or Antibody in a sample
depending how the test is designed.

9. NON COMPETATIVE ELISA
• 1- DIRECT ELISA *It use a primary labeled
antibody that react directly with the antigen.
• It can be performed with the antigen that is
directly immobilized on the assay plate.
• Not widely used but common for Immuno-
histochemical staining of cell & tissue.
• 2-INDIRECT ELISA * It utilizes a primary un-
labeled antibody in conjunction with a labeled
secondary antibody.
• secondry antibody has specific for primary
antibody.

10. NON-COMPETATIVE ELISA
• 3-Sandwich ELISA * Antigen like Tumour
markers,hormones,serum proteins may be
determined.
• Antigens in the sample binds 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/ enzyme bound to
microwell.

12. COMPETATIVE ELISA
• Antibody coated microwell.
• Serum antigen and labeled antigen added
together.
• Ag-Ab enzyme complex bound is inversely
related to the conc. of antigen present in sample.
• Increased serum antigen results in reduced
binding of Ag-enzyme conjugated with the
antibody producing less enzyme activity &
(yellow colour) formation.
• Used to determine small molecules like T3,T4 etc.

13. REPRESENTATIVE FIGURE OF
COMPETITIVE ELISA

14. Multiple & Portable ELISA
• A new technique use an solid phase made up of
an immune-sorbent polystyrene rod with 8-12
protruding ogives pins.
• The entire device is immersed in a test tube
containing the collected sample and the following
steps (washing , incubation in the conjugate and
incubation in chromogenous) are carried out by
dipping the ogives in microwells of standard
microplates pre-filled with reagents.

19. Advantage of ELISA
• Reagent are relatively cheap & have along self
life.
• ELISA is highly specific and sensitive.
• No radiation hazard occur during labelling or
disposal of waste.
• Easy to perform and quick procedures.
• ELISA can be used to variety of infections and
research.

20. Disadvantages of ELISA
• Measurement of enzyme activity can be more
complex.
• Enzyme activity may be affected by plasma
constituents.
• Kits are commercially available but not cheap.
• very specific to a particular antigen won’t
recognize any other antigen.
• false positives/negatives possible.

21. Limitations
• Result may not be absolute.
• Antibody must be available.
• Concentration may be unclear.
• False positive possible.
• False negative possible.

22. Application of ELISA
• Proteins
• Hormones
• Drug Markers
• Tumor Markers
• Serum proteins
• Antibody and Antigen detection
• vaccine Quality control
• In clinical research etc.

23. Troubleshooting in ELISA
If the negative controls are giving positive result:
• 1-Contamination of substrate solution, enzyme-labelled
antibody, control themselves.
• 2-Inadequate rinsing of plates.
• 3-Inadequate blocking of plates.
If no colour developed for the positive controls or for the
samples:
* Check all reagents for dating and storage condition.
*Microwell plate not coated properly.
*Reagents applied in wrong order or step.
*Enzyme conjugate defective or inhibited by contamination

24. If a very little colour has developed for
positive controls and the test samples
• Check the dilution of the enzyme labelled
antibody.
• The concentration of the substrate.
• Wash buffer not adequately drained after every
wash step.
• Inadequate incubation times.
• Enzyme conjugate or substrate defective or
contaminated.
• microwell plate poorly coated

25. If colour has developed for the test
sample but not positive controls
• Check the source of positive controls, their
expiry date and storage.
• If the colour can be seen but the absorbance
is not high as expected check the wave
length.

26. Application of ELISA in viral
disease in our VRDL lab
• Dengue-NS1 -Ag, IgM,IgG Ab.
• Japanese Encephalitis-IgM,IgG Ab.
• Influenza-IgM,IgG Ab .
• Chikungunya- IgM,IgG Ab.
• Scrub typhus- IgM,IgG Ab.
• Leptospirosis-IgM Ab.
• Zika-IgM,IgG Ab.
• Ebola and other out break of viral infections

27. Instrument used in ELISA- ELISA
Reader and Microwell Plate Washer