Enzyme Linked Immunosorbent Assay

ELISA

Enzyme-linked Immunosorbent Assay

ELISA Kits for Antibody Detection Commercial ELISA test kits are

available to detect Avian influenza virus antibody in chicken

serum

Newcastle disease virus antibody in chicken serum

Newcastle disease virus antibody in turkey serum

ELISA Kits for avian Ab Detection

SYNBIOTICS

IDEXX

Definition of ELISA

A sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein, especially an antigen or antibody. It is often used as a diagnostic test to determine exposure to a particular infectious agent (technique involving the reaction of the antigen or antibodies in vitro )

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.

Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.

ELISA objective

To detect the presence of an antigen or antibodies in a sample and to use it as a diagnostic tool in medicine.

To detect potential food allergens.

To be used in toxicology as a rapid presumptive screen for certain classes of drugs

Principle of ELISA

Principle of ELISA

Antibody is immobilized on micro-plate wells

Competition between in sample and labeled enzyme for antibody binding sites

The unbound material is washed out

Chromogenic substrate added to develop color

Resulting color is read

Component of ELISA

Antigen

Primary antibodies

Secondary antibodies

Enzyme

Substrate

Stop solution

• Equipments are widely available.

• No radiation hazards.

• Reagents are cheap with long shelf life.

• Adaptable to automation and high speed.

• Qualitative and quantitative.

• Reproducible.

• ELISA can be used on most types of

biological samples, such as plasma,

serum, urine, and cell extracts

• Sensitive assay

Types of ELISA

Direct method

In direct method

Sandwich method

Competitive method

Direct ELISA

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Direct ELISA

The direct Enzyme-Linked Immunoabsorbent Assay (ELISA) is a method for detecting and measuring antigen concentration in a sample. Using a capture monoclonal antibody, the presence of a particular antigen in a sample is detected.

Advantage of direct ELISA

This type of ELISA has two main advantages:

It is faster, since fewer steps are required

It is less prone to error, since there are fewer steps and reagents

Indirect ELISA

Indirect ELISA is a two-step method that uses a primary antibody and labeled secondary antibody. The primary antibody is incubated with the antigen followed by the incubation of the secondary antibody. But, this may give a nonspecific signal result because cross-reaction with the secondary antibody may occur

Antigen coated to a polystyrene multiwell plate is

detected in two stages or layers. First an unlabeled

primary antibody, which is specific for the

antigen, is applied. Next, an enzyme-labeled

secondary antibody is bound to the first antibody.

The secondary antibody is usually an anti-species antibody and is often polyclonal.

Advantage of indirect method

This method has several advantages: Increased sensitivity, since more than

one labeled antibody is bound per primary antibody

Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody

Cost savings, since fewer labeled antibodies are required

ELISA Results

Results should be recorded by reading the optical densities of the plates in a plate reader at the correct absorbance:

ELISA Results

The status of a sample are evaluated by the sample to positive ratio (S/P ratio):

Sample mean - negative control mean positive control mean - negative control mean

(mean of optical absorbance)

With the IDEXX kit S/P ratios of greater than 0.5 are considered positive

With the IDEXX kit S/P ratios of greater than 0.5 are considered positive

ELISA Results

Example:

Sample mean= 0.820

Negative control mean=0.053

Positive control mean=0.563

ELISA titer =(1.642xlog10 SP)+3.568

Values are relatively quantitative: a higher value indicates more antibody.

ELISA Laboratory

Materials Needed

ELISA plate

Record Sheet

Test samples

Dilution Tubes

Pipets and tips

Materials Needed

The materials for your kit

ELISA plate

Positive control

Negative control

Dilution Buffer (already in dilution tubes)

Conjugate (secondary antibody)

TMB Substrate

Stop solution

ELISA Laboratory 1

Label dilution tubes

Add 1ml of diluent to dilution tubes (done)

Add 2μl of test serum to a dilution tube

Do NOT dilute controls

ELISA Laboratory 2

1 2 3 4 5 6 7 8 9 10 11 12

A + - + 1 2 3 4 5 6 7 8 9

B - + -

C

D

E

F

G

H

Add 100μl of diluted test serum to the plate according to your record sheet

Incubate for 30 minutes

1 2 3 4 5 6 7 8 9 10 11 12

A + - + 1 2 3 4 5 6 7 8 9

B - + -

C

D

E

F

G

H

ELISA Laboratory 3

Wash with 350 μl distilled water (three times)

Add 100 μl of conjugate to test wells on your plate

Incubate for 30 minutes

ELISA Laboratory 4

Wash with distilled water (3 times)

Add 100 μl of TMB substrate to each well

Incubate for 15 minutes

Add 100 μl of stop solution to each well

Read results

Interpretation of Results

Negative control = 0.150 or less

The difference between the positive and negative control means must be greater than 0.075

Example: if negative control mean = 0.100, the positive control mean must be 0.176 or greater

Calculation of Results

Average the 2 negative control wells

Average the 2 positive control wells

Average 2 wells for each sample

Calculation of Results

Example:

Sample mean= 0.820

Negative control mean=0.053

Positive control mean=0.563

S/P ratios of greater than 0.5 are considered positive

(Positive values will be different for each kit)

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.

are negative controlsIf the results: positivegiving

Contamination of the substrate solution, enzyme-labelled antibody, control themselves.

Inadequate rinsing of plates.

Inadequate blocking of plates.

If no colour has developed for the positive controls or for the samples:

a. Check all reagents for dating and storage conditions.

b. Microwell plates not coated properly.

c. Reagents applied in wrong order or step omitted.

d. Enzyme conjugate defective or inhibited by contaminant.

If very little colour has developed for positive controls and the test samples:

a. Check the dilution of the enzyme labelled antibody.

b. The concentration of the substrate.

c. Wash buffer not adequately drained after every wash step.

d. Inadequate incubation times.

e. Enzyme conjugate defective or inhibited by contaminant, Substrate defective or contaminated,

f. Micro well plates poorly coated.

If colour has developed for the test samples but

not the positive controls:

Check the source of positive controls, their

expiry date and storage.

If the colour can be seen, but the absorbance is

not high as expected, check the wave length.