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Elisa P
Elisa P
YESRA ARSHAD
INTRODUCTION
The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique which is used to measure the
concentration of an analyte (usually antibodies or antigens) in solution.
INTRODUCTION TO ELISA
The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding (antigen- antibody binding). The
enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab
binding.
It combines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or
antigens coupled to an easily assayed enzyme.
It involves the usage of an enzyme to detect the binding of an Antigen (Ag)and an Antibody (Ab).
The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag : Ab
binding.
Therefore, ELISA can be used to detect either the presence of antigens or antibodies in a sample depending upon
how the test is designed.
STEPS INVOLVED IN ELISA
1. Antigen Binding
The sample antigen is binded or immobilized to the micro plate via adsorption to the surface.
Binding is achieved by incubating the wells with a solution containing the antigen for 2hrs at RT or overnight at
4°C.
The protein adheres due to hydrophobic interactions between the protein& the plastic.
Coating is done using carbonate/bicarbonate buffer at a pH 9.6.
STEPS INVOLVED IN ELISA
2. Blocking
All unbound sites on the solid support are blocked to prevent nonspecific binding of the antibodies.
Blocking buffers like BSA, Non fat dry milk powder (casein) in PBS ( Phosphate buffered saline) or TBS ( Tris
buffered saline) at a pH 7.4 with minute percentage of Tween 20 are used to block the free sites.
Protein in the blocking solution will attach to membranes in places where the target proteins have not attached.
Excess blocking agent is removed by washing the plate membrane with washing buffer.
STEPS INVOLVED IN ELISA
3. Primary Antibody
The 1° antibody is added and will be bound only if there is a recognized epitope within sample antigen.
4. Secondary Antibody
An enzyme-linked 2° antibody is added with suitable dilutions which will bind to any available 1° antibody (i.e. it
is bound to the antigen).
2° antibodies are linked to the enzyme through biconjugation.
Enzymes generally used are Horse Radish Peroxidase (HRP) & Alkaline Phosphatase (AP).
Plate is washed with buffer or a mild detergent to remove any unbound antibodies or proteins
STEPS INVOLVED IN ELISA
5. Detection/ Developing
After the final wash step, the plate is developed by adding an enzymatic chromogenic substrate to give color.
The entire plate is placed into a plate reader and the OD is determined for each well.
Intensity of color reflects the amount of specific 2° antibody bound to the target.
A spectrophotometer is designed to read each well in the 96 well plate. It is interfaced with a computer for data
management.
Substrates used are pNPP ( p- nitro phenyl phosphate), BCIP (5-bromo,4- chloro,3-indoyl phosphate), NBT (nitro
blue tetrazolium), TMB (3,3´,5,5´- tetra methyl benzidine), DAB (3,3-diamino benzidine).
COMPONENTS OF ELISA
Reagent Composition
Coating Buffer » » 0.01M 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
Solid phases used in ELISA include cross-linked dextran or polyacrylamide beeds, filter paper(cellulose) disc or
polypropylene tubes and disposable polystyrene microtitre plates.
Antigen or antibodies attached to solid phase by passive absorption.
Conjugate used must contain a highly reactive antibody coupled to an enzyme with a highly turnover
number(alkaline phosphatase and horseradish peroxide are commonly used enzymes).
TYPES OF ELISA
Competitive ELISA
Reverse/Multiple/portable ELISA
DIRECT ELISA
It uses a primary labeled anti-body that react directly with the antigen.
It can be performed with the antigen that is directly immobilized on assay plate.
Not widely used but common for immuno-histochemical staining of cells & tissues.
DIRECT ELISA
DIRECT ELISA
ADVANTAGES
A major disadvantage of the indirect ELISA is the method of antigen immobilization is not specific.
Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may
competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.
Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen
from complicated mixtures before the measurement.
Simplifies the assay.
Increases the specificity and the sensitivity of the assay.
COMPETITIVE ELISA
A new technique uses a solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives.
The entire device is immersed in a test tube containing the collected sample and the following steps (washing,
incubation in conjugate and incubation in chromigeneous) are carried out by dipping the ogives in microwells of
standard microplated prefilled with reagents.
Advantages of this technique:
The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different
antibodies and different antigens for multi-target assays.
The sample volume can be increased to improve the test sensitivity in clinical, food and environmental samples.
The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not
required, facilitating ready-to-use lab kits and on-site kits
ELISA RESULTS
ELISA data is typically graphed with Optical density Vs Log concentration to produce a sigmoidal curve.
Known concentrations of antigen are used to produce a standard curve and then this data is used to measure the
concentration of unknown samples by comparison to the linear portion of the standard curve.
This can be done directly on the graph or with curve fitting software which is typically found on ELISA plate
readers.
PRECAUTIONS