yousif hamed mohamed sharif /ELISA PROCEDURE

1. Enzyme Linked Immunosorbent Assay (ELISA)
by Dr Yousif H.M.Sharif
-------/UOD
14th practical Microbiology
18 April 2017

2. Basic Terms:
• Solid Phase:
Usually a microtiter plate well, having 8 12 well
format.
• Adsorption:
The process of adding an antigen/antibody, diluted in
buffer, so it attaches to the solid phase on incubation.
• Washing:
The simple flooding & emptying of wells with a
buffered solution to separate bound from un-bound
reagents in ELISA.

3. Basic Terms:
• Antigen:
Any molecule that elicits the production of antibodies
when introduced into body.
• Antibodies:
Proteins produced in response to antigenic stimuli.
• Enzyme conjugate:
An enzyme that is attached irreversibly to an antibody.
e.g: Horse-redish peroxidase (HRPO).

4. Basic Terms:
• Chromogen:
A chemical alters color as a result of an enzyme
interaction with substrate (color reaction used as
signal) e.g Trimethyl benzidine (TMB).
• Stopping:
The process of stopping the action of an enzyme on
a substrate.
• Reading:
Spectrophotometric measurement of color developed
in ELISA.

5. ELISA
• Enzyme Linked Immunosorbent Assay (ELISA)
• Biochemical technique used mainly in immunology
• Different Procedures
– Direct ( immunohistochemical staining of cell and tissue )
– Indirect ( to detect Antibody : HIV ,HCV)
– Sandwich(to detect Antigen: Tumor marker ,Hormones)
– Competitive (to detect Antigen :free testosterone )
• Similar To Radioimmunoassay (RIA) , Except No Radiolabel
• Can Be Used To Detect Both Antibody and Antigen
• Very Sensitive, pg/mL
• Relies on Monoclonal Antibodies ?

6. ELISA principle
 Antigen bound to ELISA plate
 Specific antibodies bind to antigen
 Antibodies conjugated with an
enzyme
 Addition of substrate results in a
colored product
 Intensity of color detected by ELISA
reader or spectrophotometer
Both antigen & antibody can be
coated onto polystyrene plates
Both antigen & antibodies can
be conjugated with enzyme
polystyrene plates
spectrophotometer

7. What is it used for?
• Measure antibody levels (allergies, vaccines)
• Detect viruses (hepatitis, HIV, venereal
diseases),Parasite (Toxoplasmosis)
• Detect hormonal changes (HCG in pregnancy),
thyroid hormones
• Detect circulatory inflammatory markers
(cytokines)

8. Different ELISA methods

9. ELISA Equipment's
1.Microwell plate made from
Polystyrene contains 8*12 well
holding 350 µl each
2.Multi channel pipette

10. ELISA equipment cont’d
ELISA washer machine ELISA Plate reader

11. Reagents Used: ELISA Kits
Reagent Composition
Coating Buffer 0.01 M Phosphate Buffer
+ 0.15 M NaCl (PBS)
Diluting/Washing Buffer 0.01 M Phosphate Buffer
+ 0.50 M NaCl + 0.1% Tween 20
Blocking Buffer Bovine Serum Albumin
(BSA)
Enzyme Horse-redish peroxidase
(HRPO)
Chromogenic Substrate Trimethyl benzidine
(TMB)
Stop Solution 0.5 M H₂SO₄

12. • Over view of
ELISA Procedure

13. 13
Types of ELISA
• Qualitative ELISA
– Positive or Negative results
• Quantitative ELISA
– optical density or fluorescent units of the sample is
interpolated into a standard curve, which is
typically a serial dilution of the target.

14. Reading:
• Measure the absorbance at 450nm with the
help of ELISA reader.
• Calculate the absorbance for each sample and
reference.
• Ascent software for the calculation of results
can be used.

15. ELISA TEST RESULTS
ELISA data from three patients.
Numbers are expressed as optical
density at 450 nm. The cutoff value
indicating a positive result is 0.500.
Optical densities of 0.300 to 0.499
are indeterminate and need to be
retested. Values below 0.300 are
considered to be negative. In most
cases, a patient will be retested if
the serum gives a positive result. If
the ELISA retests are positive, the
patient will then be retested by
western blotting analysis.
Positive
Control
Negative
Control Patient A Patient B Patient C CUT-OFF
1.689 0.153 O.055 0.412 1.999 0.500