ELISA

(Enzyme-linked immunosorbent assay )

ANITA SINGHMEDICAL ONCOLOGY

HISTORY

Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively- labeled antigens or antibodies.

Avramais (1966, 1969) and Pierce (1967) developed methods to chemically link antibodies to biological enzymes whose activities produce a measurable signal with solutions containing appropriate substrates.

This signal has to be associated with the presence of antibody or antigen .

COMPONENTS OF ELISA Antibody: IgG fraction of serum.

Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4 lysine residues.

Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color. Reaction stopped with dilute acid to cause complex to turn yellow.

INTRODUCTION TO ELISA

A 96 - well microtiter plate 

being used for ELISA.

A test that uses antibodies and color change to identify a substance.

ELISA is a popular format of a "wet-lab" type analytic biochemistry assay.

ELISA involves at least one antibody with specificity for a particular antigen.

ELISA can perform other forms of  ligand binding assays instead of strictly "immuno" assays.

“Wet lab" analytic biochemistry assay, ELISA involves detection of an "analyte" in a liquid sample by a method that continues to use liquid reagents during the "analysis“.

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.

PRINCIPLE

Different antigen in sample

substrate

Enzyme

Colored

product

Primary antibody

Secondary antibody

There are variations of the ELISA test but the most basic type

consist of an antibody attached to the solid surface. This antibody

has affinity for (will latch onto) the substance of interest.

For example… Human Chorionic Gonadotropin (HCG), the

commonly measured protein which indicates pregnancy.

A mixture of purified HCG linked to an enzyme and sample (blood,

urine, etc.) under test are added to the test system. If no HCG is

present in the test sample the only HCG with linked enzyme will

bind. The more HCG which is present in the test sample the less

enzyme linked HCG will bind.

HOW DOES ELISA WORK ?

TYPES OF ELISA

INDIRECT ELISA

DIRECT ELISA

SANDWICH ELISA

COMPETETIVE ELISA

NON -COMPETETIVE ELISA

INDIRECT ELISA

Antigen is added to plate.

Added Blocking buffer.

Suitable primary antibody is added.

Secondary antibody- HRPO is then added which recognizes and binds to primary antibody.

TMB substrate is added, is converted to detectable form.

Under standard condition ,the enzyme activity measured is proportional to amount of specific antibody in the original serum.

ADVANTAGES OF INDIRECT DETECTION

Wide variety of labeled secondary antibodies are available commercially.

Versatile, since many primary antibodies can be made in one species and the

Same labeled secondary antibody can be used for detection.

Immunoreactivity of the primary antibody is not affected by labeling.

Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.DISADVANTAGES OF INDIRECT

DETECTION Cross-reactivity may occur with the secondary antibody, resulting

in nonspecific signal.

An extra incubation step is required in the procedure.

DIRECT ELISA

1. Apply a sample of known antigen to a surface.

2. Enzyme linked primary antibody is applied to the plate.

3. Washed, After this wash, only the antibody-antigen complexes remain attached.

4. Apply a substrate which is converted by the enzyme to elicit a chromogenic signal.

Under standard condition ,the enzyme activity measured is proportional to amount of specific antibody in the original serum.

ADVANTAGES OF DIRECT DETECTION

Quick methodology since only one antibody is used.

Cross-reactivity of secondary antibody is eliminated.

DISADVANTAGES OF DIRECT DETECTION

Immunoreactivity of the primary antibody may be reduced as a result of labeling.

Labeling of every primary antibody is time-consuming and expensive.

No flexibility in choice of primary antibody label from one experiment to another.

Little signal amplification.

SANDWICH ELISA

1. a. Plate is coated with suitable antibody.b. Blocking buffer is added.

2. Sample is added to plate so antigen is bounded by capture antibody.3. A suitable biotin labeled detection antibody is added to plate.4. Enzyme HRPO is added and binds the biotin labeled detection

antibody.5. TMB substrate is added and converted by HRPO to colored product.

Under standard condition ,the enzyme activity measured is proportional to the Amount of specific antigen in the original serum.

MATERIALS NEEDED FOR ELISA KIT

ELISA Plate

Positive control

Negative control

Dilution Buffer

Conjugate

TMB Substrate

Stop Solution

Wash

3X

Wash

4X

Wash

4X

Wash

4X

A EDCB

Alkaline phosphatase

substrate is added and developed

colour is read at 405 nm

wavelength to measure plasma

cencentration

Wells are coated with 0.2

μg primary antibody

Diluted plasma

is added to coated wells,

which bind to

antibodies

0.1 μg of biotinylated (biotin =

– ) antihuman secondary antibody

Incubated overnight at

4˚C

Incubated at room temperature (24˚C)

2h 2h 1h

PROCEDURE OF ELISA

Add 1.2000 dilution of streptavidi

n conjugate to alkaline phosphatas

e ( E)

FINAL PLATE OF ELISA

COMPETETIVE ELISACOMPETETIVE ELISA

COMPETETIVE ELISA

Solid phase coated with antibody

Add unknown amount of unlabeled antigen and known amount of labeled antigen

Free and labeled antigen are captured

Color formation by oxidation of substrate into a colored compound

Under standard condition ,the enzyme activity measured is proportional to the proportion of labeled antigen in the mixture of labeled and unlabled antigen.

ADVANTAGES:

Suitable for complex (crude or impure) samples, since the antigen does not require purification prior to measurement.

DISADVANTAGES

Each antigen may require a different method to couple it to the enzyme.

COMPARISON BETWEEN VARIOUS TYPES OF ELISA

Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA

The ELISA assay yields three different types of data output:

Quantitative: ELISA data can be interpreted in comparison to a standard curve in order to precisely calculate the concentrations of antigen in various samples.

Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is

present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen.

Semi-quantitative: ELISAs can be used to compare the relative levels of antigen

in assay samples, since the intensity of signal will vary directly with antigen concentration.

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.

SENSITIVITY

ELISAs are one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody – antigen interaction.

In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results.

As mentioned earlier, indirect detection will produce higher levels of signal and should therefore be more sensitive. However, it can also cause higher background signal thus reducing net specific signal levels.

PRECAUTIONSNegative control with strong signal

The excessive background signal can be caused by inadequate rinsing of plates, reagents not sufficiently diluted, inadequate blocking of plates or non-specific binding of enzyme conjugate. The appearance of color in negative control wells may also indicate cross-reactivity of secondary antibody with components in the antigen sample.

Positive control with no signal

Microwell plates not coated properly.Reagents applied in wrong order or step omitted.Secondary antibody not matched to the species of primary antibody.Enzyme conjugate defective or inhibited by contaminant.

ELISA with weak signal

Wash buffer not adequately drained after every wash step.Inadequate incubation times.Enzyme conjugate defective or inhibited by contaminant.Substrate defective or contaminated.Microwell plates poorly coated.Loss of capture antibody during blocking/washing.

PRACTICAL ASPECTS

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).

APPLICATIONS Screening donated blood for evidence of viral

contamination 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)

Detecting infections

Sexually-transmitted agents like HIV, syphilis and chlamydia

Hepatitis B and C Toxoplasma gondii

Detecting illicit drugs.

Detecting allergens in food and house dust

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.

A NEW TECHNIQUE:- REVERSE ELISA

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