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2. ELISA (Enzyme linked ImmunoSorbant Assay)
DEPARTMENT OF MICROBIOLOGY
GOVERNMENT COLLEGE UNIVERSITY FAISALABAD
Iqbal Danish
m.Phil 2nd semester

3. Introduction
 ELISA, are quantitative immunological procedure in
which Ag-Ab reaction is monitored by enzyme
measurements.
 It is used to detect the antibodies in the blood.
 The ELISA test was the first screening test commonly
for HIV.
 It is highly sensitive.

4. Principle
 Use an enzyme to detect the binding of Ag –Ab.
 The enzyme converts a colorless substrate to colored
product. That indicate the presence of Ag.
 It can be used to detect the presence of both Ag and
Ab.

6. Antigen (Ag)
 Any molecule that induces production of antibodies
when introduced in the body is called antigen.
OR
 Any thing that is foreign to immune system.
E.g Bacteria, Virus, etc.

7. Antibody (Ab)
 These are proteins that produced by the immune
system which help defend against antigens.

8. Antigen –Antibody Interaction

9. Material Required
 Test Sample
 Micro titeration plate
 ELISA Reader
 Washing Buffer
 Stop Solution
 Substrate
 Incubator

10. Material Required
 Test Sample
 Micro titeration plate
 ELISA Reader
 Washing Buffer
 Stop Solution
 Substrate
 Incubator

11. Procedure
 Take 96 wells plates coated with antigen.
 Use blocking agend.
 Take serum and then add into the wells.
 Add Ab with enzyme .
 Add substrate to make color.
 Add stop solution.
 Use spectrophotometer to read color.

13. Types
Frequently there are 3 types of ELISA on the
basis of binding structure between the Antibody
and Antigen.
 Direct ELISA
 Indirect ELISA
 Sandwich ELISA
 Competitive ELISA

14. Specimen Samples for ELISA
 Serum
 Cerebrospinal fluid
 Sputum
 Urine
 Supernatant of Culture
 etc

15. Direct ELISA
 Is one where there is only one set of antigens and one
set of antibodies to react. In this ELISA
method, antigens from the patient sample fixed to the
Elisa plates are made to react with an antibodies
sample which is tagged to a marker enzyme I.e.
directly to the antigen in the test an enzyme-linked
antibody is added to produce a color reaction with
externally added substrate i.e. Elisa reagent.
 Ag or Ab + Ab or Ag-(e) −−−−−→ Reaction color.

16. Procedure
 Antigen is immobilized onto the wells of a 96-well
polystyrene plate via passive adsorption.
 An enzyme-labeled primary antibody specific for the
target antigen is added to the wells and directly binds
to the antigen.
 A respective enzyme substrate is added, which upon
reaction with the enzyme, produces a visible
colorimetric output that can be measured by a
spectrophotometer or absorbance microplate reader.

17. Direct ELISA

18. Advantages
 It is used to detect antibodies.
 It is simple test.
 It is quick and easy than other.
 It is used to detect antigen .

19. Indirect ELISA
 This has a difference to the Direct Elisa in that one more
additional antibody is added in the reaction. To the antigen
(fixed to Elisa plate) an antibody is added. Again secondary
antigen is added which is enzyme-linked. This requirement
is due to the reason that sometimes in patients antigen of
the disease-causing agent may not be present but a
corresponding antibody is available in the patient sample.
which can be traced. These are of two types like normal
Indirect Elisa and other is sandwich, Elisa.
 Ag or Ab + Ab or Ag +Ag or Ab-(e) −−−−−→Reaction color.

20. Procedure
 Coat the micro titer plate wells with antigen.
 Block all unbound sites to prevent false positive
results.
 Add sample containing antibody (e.g. rabbit
monoclonal antibody) to the wells and incubate the
plate at 37°c.
 Wash the plate, so that unbound antibody is removed.
 Add secondary antibody conjugated to an enzyme (e.g.
anti- mouse IgG).

21.  Wash the plate, so that unbound enzyme-linked
antibodies are removed.
 Add substrate which is converted by the enzyme to
produce a colored product.
 Reaction of a substrate with the enzyme to produce a
colored product.

23. Advantages
 A wide variety of labeled secondary antibodies are
available commercially.
 Cost Effective.
 Different visualization markers can be used with the
same primary antibody.
Disadvantages
 Cross-reactivity might occur with the secondary
antibody, resulting in nonspecific signal.
 An extra incubation step is required in the procedure.

24. Sandwich ELISA
 Antigen can be detected by sandwich ELISA
 Antibody is coated on the microtiter well.
 Sample containing antigen is added to the well and
allowed to react with the antibody attached to the well,
forming antigen-antibody complex.
 Washing can be done.

25.  Enzyme-linked antibody specific for a different epitope
on the antigen is added and allowed to react with the
bound antigen.
 Washing can be done.
 Substrate is added to the plate which is hydrolyzed by
enzyme to form colored products.

27. Advantages
 High specificity, since two antibodies are used the
antigen 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.

28. Competitive ELISA.
 This test is used to measure the concentration of an
antigen in a sample.
 Antibody is first incubated in solution with a sample
containing antigen.
 The antigen-antibody mixture is then added to the
microtitre well which is coated with antigen.

29.  The more the antigen present in the sample, the less
free antibody will be available to bind to the antigen-
coated well.
 Washed wells.
 enzyme conjugated secondary antibody specific for
iso-type of the primary antibody is added to determine
the amount of primary antibody bound to the well.
 The higher the concentration of antigen in the sample,
the lower the absorbance.

31. Advantages.
 High specificity, since two antibodies are used.
 High sensitivity, since both direct and indirect
detection methods can be used.
 Suitable for complex samples, since the antigen does
not require purification prior to measurement.

32. Application of ELISA.
 Presence of antigen or the presence of antibody in a
sample can be evaluated.
 Determination of serum antibody concentrations in a
virus test.
 Used in food industry when detecting potential food
allergens.
 Applied in disease outbreaks- tracking the spread of
disease e.g. HIV, bird flu, common, colds, cholera,
STD etc.

33. Qualitative and Quantitative Test
 Qualitative: Determine the present of antigen and
antibody .
 Posive or negative result.
 Quantitative: Determine the quantity of antibody.
 No of positive and negative result.

35. Conclusion
 It is a good techniques and being popular today
because of its simple and not involve radiation .
 It is easy than other test .
 It is most useful than other test because of their
sensitivity.
 It is not harmful.