Microbiology

1. Presented by
Daxa Rathwa
4th year B.Sc Nursing

2. INTRODUCTION
DEFINITION
TYPES
PRINCIPLES
Uses

3.  An enzyme-linked immunosorbent assay, also called
ELISA or EIA, is a test that detects and measures
antibodies in your blood. This test can be used to
determine if you have antibodies related to certain
infectious conditions.
 IMMUNO- refers to immune response that couses the
body to generate antibodies.
 ASSAY - refers to test or check amount substant
present in sample

4.  ANTIGEN
Antigens are large molecules of proteins, present on
the surface of the pathogen- such as bacteria, fungi
viruses, and other foreign particles. When these
harmful agents enter the body, it induces an immune
response in tHe body for the production of
antibodies.
 ANTIBODY
Antibodies are proteins that your body produces in
response to harmful substances called antigens.

5.  ELISA works on the principle that specific
antibodies bind the target antigen and detect
the presence and quantity of antigens
binding. In order to increase the sensitivity
and precision of the assay, the plate must be
coated with antibodies with high affinity.
ELISA can provide a useful measurement of
antigen-antibody concentration.

6.  Microtiter plate
 Antibody
 Antigen
 Washing buffer
 Enzyme[ horseradish,
alkline phosphate]
 Substrate
 speectrophotometer

7. Washing
buffer
Spectro
photometer
Substrate

8.  ELISA tests can be classified into three types
depending upon the different methods used for
binding between antigen and antibodies,
namely:
 Direct Elisa
 Indirect Elisa
 Sandwich elisa
 Competitive elisa

9.  Take a empty microtiter plate. And add patient
sample containing antigen.
 Do washing to remove unbound antigen.
 An enzyme-labeled primary antibody (e.g. HRP-
labeled primary antibody) specific for the target
antigen is added to the wells and directly binds to
the antigen.

10. Do washing to remove remove
unbound antibody.
 respective enzyme substrate
is added, which upon
reaction with the enzyme,
produces a visible
colorimetric output that can
be measured by a
spectrophotometer

11.  Indirect ELISA detects the presence of an antibody in a
sample.
 The antigen is attached to the wells of the microtitre
plate[readymade kit]
 A sample containing the antibodies is added to the antigen-
coated wells for binding with the antigen.
 The free primary antibodies [sample] are washed away and
the antigen-antibody complex is detected by adding a
secondary antibody [enzyme linked] conjugated with an
enzyme that can bind with the primary antibody.

12. All the free secondary
antibodies are washed
away. A specific substrate
is added which gives a
colourd product. The
absorbance of the coloured
product is measured by
spectrophotometry.

13.  Sandwich elisa helps to detect the
presence of antigen in a sample.
 coat the well with capture antibody
and washed to remove free antibody.
 The sample containing the antigen is
added to the well and washed to remove
free antigen.

14.  Then an enzyme-linked
secondary antibody (detection
antibody), which binds to another
epitope on the antigen is added.
The well is washed to remove
any free secondary antibodies.
The enzyme-specific substrate is added to the plate
to form a coloured product, which can be
measured. 2-5 time More sensitive than direct
elisa.

15.  Competitive ELISA helps to detect antigen
concentration in a sample.
 The microtitre wells are coated with the antibody.
 Mixed patient sample containing antigen and
secondary antigen that linked with enzyme.
 The mixed solution of both antigen is added into
the microtitre well. The well is then washed to
remove any unbound antigen.

16.  The enzyme specifi substrate add in well and
measure colour product to find out the
concentration of antigen
 The both specific antigen compitte with each other
to bind with antibody
 If enzyme linked antigen more bind with antibody
that indicate more concentration of enzyme linked
antigen and produced more colour and least
concentration of antigen in patient sample

17.  If the sample antigen is
more bind with
antibody that produced
less colour that indicate
more concentration of
antigen in patient
sample.

18. 1) It can be used to detect the presence of antigen
or antibody in a sample.
2) It can be used to determine antigen and
antibody concentrations in a sample.
3) It can be used in food industry to detect
potential food allergens.
4) It can be used in disease outbreaks to track the
spreading of diseases

19. Screening donated blood for evidence of viral contaminatio
by
• HIV-1 and HIV-2 (presence of anti-HIV antibodies)
Hepatitis C (presence of antibodies)
Hepatitis B (testing for both antibodies and a viral antigen)
Measuring hormone levels
HCG (as a test for pregnancy)
LH (determining the time of ovulation)
TSH, T3 and T4 (for thyroid function)