Dr. Shataha S. Jumaah/Lecturer _____________________________________________ Virology – 4th /1st Semester

shatha.saadi@tiu.edu.iq https://tiu.edu.iq/

2020 - 2021

TIU - Faculty of Science Medical Analysis Department

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ELISA

Introduction ELISA, are quantitative immunological procedure in

which Ag-Ab reaction is monitored by enzyme

measurements.

It is used to detect the antibodies in the blood. The ELISA test was the first screening test commonly

for HIV.

It is highly sensitive.

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Principle Use an enzyme to detect the binding of Ag -Ab. The enzyme converts a colorless substrate to colored

product. That indicate the presence of Ag. It can be used to detect the presence of both Ag and

Ab.

Antigen (Ag) Any molecule that induces production of antibodies

when introduced in the body is called antigen. OR

Any thing that is foreign to immune system.

E.g Bacteria, Virus, etc.

Antibody (Ab) These are proteins that produced by the immune

system which help defend against antigens.

Antigen -Antibody Interaction

Material Required Test Sample Micro titeration plate

ELISA Reader

Washing Buffer

Stop Solution

Substrate

Incubator

Material Required Test Sample Micro titeration plate

ELISA Reader

Washing Buffer

Stop Solution

Substrate

Incubator

Procedure Take 96 wells plates coated with antigen.

Use blocking agend.

Take serum and then add into the wells.

Add Ab with enzyme .

Add substrate to make color.

Add stop solution.

Use spectrophotometer to read color.

Types Frequently there are 3 types of ELISA on the basis of binding structure between the Antibody

and Antigen.

Direct ELISA

Indirect ELISA

Sandwich ELISA

Competitive ELISA

Specimen Samples for ELISA Serum

Cerebrospinal fluid

Sputum

Urine

Supernatant of Culture

etc

Direct ELISA Is one where there is only one set of antigens and one

set of antibodies to react. In this ELISA method, antigens from the patient sample fixed to the

Elisa plates are made to react with an antibodies

sample which is tagged to a marker enzyme I.e. directly to the antigen in the test an enzyme-linked

antibody is added to produce a color reaction with

externally added substrate i.e. Elisa reagent. Ag or Ab + Ab or Ag-(e) −−−−−→ Reaction color.

Procedure Antigen is immobilized onto the wells of a 96-well polystyrene plate via passive adsorption. An enzyme-labeled primary antibody specific for the

target antigen is added to the wells and directly binds to the antigen.

A respective enzyme substrate is added, which upon

reaction with the enzyme, produces a visible colorimetric output that can be measured by a

spectrophotometer or absorbance microplate reader.

Direct ELISA

Advantages It is used to detect antibodies.

It is simple test.

It is quick and easy than other.

It is used to detect antigen .

Indirect ELISA This has a difference to the Direct Elisa in that one more additional antibody is added in the reaction. To the antigen

(fixed to Elisa plate) an antibody is added. Again secondary

antigen is added which is enzyme-linked. This requirement is due to the reason that sometimes in patients antigen of the disease-causing agent may not be present but a corresponding antibody is available in the patient sample. which can be traced. These are of two types like normal Indirect Elisa and other is sandwich, Elisa. Ag or Ab + Ab or Ag +Ag or Ab-(e) −−−−−→Reaction color.

Procedure Coat the micro titer plate wells with antigen. Block all unbound sites to prevent false positive

results. Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and incubate the

plate at 37°c. Wash the plate, so that unbound antibody is removed.

Add secondary antibody conjugated to an enzyme (e.g.

anti- mouse IgG).

Wash the plate, so that unbound enzyme-linked

antibodies are removed. Add substrate which is converted by the enzyme to

produce a colored product. Reaction of a substrate with the enzyme to produce a

colored product.

Advantages A wide variety of labeled secondary antibodies are

available commercially.

Cost Effective. Different visualization markers can be used with the

same primary antibody.

Disadvantages Cross-reactivity might occur with the secondary

antibody, resulting in nonspecific signal. An extra incubation step is required in the procedure.

Sandwich ELISA Antigen can be detected by sandwich ELISA Antibody is coated on the microtiter well. Sample containing antigen is added to the well and allowed to react with the antibody attached to the well,

forming antigen-antibody complex. Washing can be done.

Enzyme-linked antibody specific for a different epitope

on the antigen is added and allowed to react with the bound antigen.

Washing can be done. Substrate is added to the plate which is hydrolyzed by

enzyme to form colored products.

Advantages High specificity, since two antibodies are used the

antigen is specifically captured and detected. Suitable for complex samples, since the antigen does

not require purification prior to measurement. Flexibility and sensitivity, since both direct and

indirect detection methods can be used.

Competitive ELISA. This test is used to measure the concentration of an

antigen in a sample. Antibody is first incubated in solution with a sample

containing antigen. The antigen-antibody mixture is then added to the

microtitre well which is coated with antigen.

The more the antigen present in the sample, the less

free antibody will be available to bind to the antigen-

coated well. Washed wells.

enzyme conjugated secondary antibody specific for iso-type of the primary antibody is added to determine

the amount of primary antibody bound to the well.

The higher the concentration of antigen in the sample,

the lower the absorbance.

Advantages. High specificity, since two antibodies are used.

High sensitivity, since both direct and indirect

detection methods can be used.

Suitable for complex samples, since the antigen does

not require purification prior to measurement.

Application of ELISA. Presence of antigen or the presence of antibody in a

sample can be evaluated. Determination of serum antibody concentrations in a

virus test. Used in food industry when detecting potential food

allergens. Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc.

Qualitative and Quantitative Test Qualitative: Determine the present of antigen and

antibody . Posive or negative result. Quantitative: Determine the quantity of antibody.

No of positive and negative result.

Thanks for your

attention

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