Download ELISA Lab

ELISA
( Enzyme-Linked Immunosorbent
Assay)
What is an ELISA?
• Enzyme-linked immunosorbent assay
• Name suggests three components
– Antibody
• Allows for specific detection of Ag
– Solid phase (sorbent)
• wash away all the material that is not specifically
captured
– Enzymatic amplification
• Convert a little capture into a visible color change
that can be quantified using an absorbance plate
reader
ELISA PRINCIPLE
The principle of the ELISA is that the target
analyte (the antigen) is recognized with high
specificity by antibodies, which are proteins
produced by the immune system of animals .
These antibodies labeling with enzyme and
after reaction the labeling Ab with Ag the
substrate add and read the color by
spectrophotometer .
What is it used for?
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Measure antibody levels (allergies, vaccines)
Detect viruses (hepatitis, HIV, venereal diseases)
Detect hormonal changes (Exa :pregnancy)
Detect circulatory inflammatory markers (cytokines)
•Infections ( bacterial infection ,parasitic infection )
•Specific disease factors
•Drugs
•Allergens of food
•Residues in food
•Toxins
Advantages
• •Fast—90 samples tested in 2-3 hr
• •Sensitivity (up to 10 pg/mL)
• •Specificity ( even sample with high concentration
contaminants)
• •Many samples can be processed at once
• •Small sample size required (10 μL~ 100μL )
• •Colorimetric results –easily observed and measured
(spectrophotometer)
• •Test for presence of Ag or Ab
• •Flexible usage for research design
• •Easy to learn, simple procedure
The materials for your kit
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1. ELISA plate
2. Positive controls
3. Negative controls
4. Dilution Buffer (already in dilution tubes)
5. Conjugate (secondary antibody labeled with enzyme )
6. Substrate (ex.TMB)
7. Stop solution
8. wash solution
9-procedure leaflet
10- blank solution
11- Standards( may be 2 or more )
Types of ELISA assay
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1.Direct-ELISA
2.Indirect-ELISA
3.Sandwich-ELISA
4.Competition-ELISA
Direct-ELISA
• –Enzyme conjugated Ab is directly bound
to the Ag
• Detect antigen in the sample (e.g., HIV viral
proteins)
Indirect method
Detect antibodies in the sample (e.g., antibodies against
HIV proteins
Sandwich ELISA
Sandwich standard curve
Competitive ELISA
Enzymes used in ELISA
• 1- Horse radish peroxidase .
• 2-Alkaline phosphatase .
• 3- Beta-D-galactosidase
Substrate used in ELISA
• 1- TMB (tetra methyl benzidine) : soluble substrate
yield blue color when react with HRP. Very sensitive,
quick oxidized resulting in faster color development .
• OPO (o-phenylenediamine dihydrochloride yield yelloworenge color when react with HRP.
• PNPP (p-nitrophenyl phosphate ,Disodium salt ) Is a
widely used substrate for detecting alkaline phosphatese
.produce yellow color .
• ABTS ( Azinobis [3-ethelbenzothiazoline-6-sulfonic
acide]-diammonium salt .give green reaction when react
with HRP.
ELISA resultes
• 1-Quantitative :
• The results of patient sample compared to
standard curve (a serial dilution of known
,purified Ag [standards ] )
• 2-Qualitative :
• Give yes or no compared with blank (well not
containing Ag .
• 3- Semi-quantitative
• The result compare with relative levels of Ag or
AD