its a ppt based on elisas

1. BY :
MT MAINAK GHOSH
M.SC IN MLT
WBUHS,VU
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2. INTRODUCTION TO
ELISA
• 1. ELISA, or enzyme-linked immunosorbent assay, is an
immunoassay technique involving the reaction of antigen &
Antibody
• 2. Elisa is sensitive are usually performed in microwell Plate
based assay designed for detecting and quantifying peptides,
protein, antibodies & hormonese
• 3. certain enzyme react with appropriate substarte, they can
result in changes in color, which can be used as a signal
• 4. ELISA a powerful tool for measuring specific analytes
within a crude preparation.
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ELISA USED FOR PLATE MICROLITER WELL, HAVING 8*12 WELL FORMAT.

4. PRINCIPLE
It is a plate based assay technique. Along with the enzyme labeling of antigen or antibodies
1. Immune reaction
2. Enzymatic chemical reaction : enzyme catalyses from the color product from colorless substrate
3. Signal detection & quantification : detection & measurement of color intensity of the colored products
generated by enzyme linked antibody & added substarte.
BASIC TERMS :
Antigen : Any molecule that elicts the production of antibodies when introduced into body
Antibody : protein produced in response to antigenic stimuli
Enzyme conjugate : An enzyme that is attached irreversibly to an antibody.
Chromogen : A chemical alters color as a result of an enzyme interaction with substrate
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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.
Stopping: The process of stopping the action of an enzyme on a substrate
Reading: Spectrophotometric measurement of color developed in ELISA
Equipment :
1. Microwell plate 2.multipipette
3. microplate washer

6. REAGENT USED
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7. TYPES OF ELISA
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• Direct Elisa
• Indirect Elisa
• Sandwich Elisa
• Competitive Elisa

8. DIRECT ELISA
• It uses a primary labeled anti-body that reacts directly with the antigen
• It can be performed with the antigen that is directly immobilized on assay plate
• The direct ELISA test is used in various applications, including the detection of antigens in viral
infections, such as HIV
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9. INDIRECT ELISA 9
• It utilizes a primary un-labeled antibody in conjunction with a labeled secondary
antibody
• Secondary antibody has specificity for primary antibody
• A wide variety of secondary antibody are available commercialy
• The higher the concentration of the primary antibody that was present in the serum,
the stronger the color change. A spectrometer is used to give quantitative values for
color strength

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11. SANDWICH ELISA
• Antigen like tumor marker hormones serum
protein are use to determine through this
• A sample containing antigen is added and
allowed to react with immobilized antibody
• The antibody of the enzyme conjugate bind
with the immobilized antigen to form a
sandwich of Ab-Ag-Ab/ enzyme bound to
microwell
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12. COMPETITIVE ELISA 12
• Another variation for measuring amounts of antigen is competitive ELISA
• In this technique antibody is first incubated with a simple containing antigen
• The antigen-antibody mixtures is then added to an antigen coated microtiter well.
• The more antigen present in the sample the less free antibody will be available to bind to the
antigen-coated well.

13. READING :
• Measure the absorbance at 450nm with the help of ELISA reader
• Calculate the absorbance for each sample & reference
• Ascent software for the calculation of result can be used
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14. RESULTS :
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15. APPLICATION
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16. THANK YOU
MAINAK GHOSH
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