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PRESENTED BY BILAL ANJUM BUTT 50-E

ASSAYS AND APPLICATIONS OF IMMUNE RESPONSE

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IMMUNE ASSAYS

Immunoassays are a group of sensitive analytical tests that utilize very specific antibody/antigen complexes to produce a signal that can be measured and related to the concentration of a compound in solution.

TYPES Radioimmunoassays (RIAs) Fluorescent Polarized Immunoassay Enzyme Multiplied Immunoassay (EMIT) Enzyme linked immunosorbant assay (ELISA)

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ELISA(Enzyme-linked immunosorbent assay)

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INTRODUCTION TO ELISA ELISA, or enzyme-linked immunosorbent

assay, is an immunoassay technique involving the reaction of antigen and antibody in vitro. ELISA is a sensitive and specific assay for the detection and quantitation of antigens or antibodies. ELISA tests are usually performed in microwell plates.

It is a sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein.

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HISTORY It is first described by Peter

Perlmann and Eva Engval , and Anton Schuurs and Bauke van Weemen.

The technique was subsequently developed by Voller and col. using microplates, that permitted the development of highly sensitive and accurate kits.

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COMPONENTS OF AN ELISA

Antibody: IgG fraction of serum

Enzyme:Peroxidase from horseradish,Alkaline phosphatase from E. coli,β-galactosidase from E coli.,Glucose oxidase.

Substrate: TMB (3,3',5,5', tetramethylbenzidine).

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PRINCIPLE OF ELISA

The sample with an unknown amount of antigen is immobilized on a solid support.

The detection antibody is added ,forming a complex with antigen.

The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme.

Between each step the plate washed with a mild detergent solution.

After the final wash step, adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

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1. Add antigen7. Add substrate for enzyme

2. Wash with PBST

4. Wash with PBST

3. Add primary antibody

6. Wash with PBST

5. Add secondary antibody

8. Observe colour development

Step 1

inactivated HIV antigens Step 2

serum antibodies

Step 3Anti-human Ig

coupled to enzyme

Step 5 ( Measurement )

Step 4

Chromogen or substrate

ELISA

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CRITERIA FOR CHOICE OF A MARKER ENZYMEShould be easily coupled to ligands & the labelled complex must be stable.

The reactivity should be retained after linking of the enzyme to the antigen/antibody.

The chosen enzymes should not be normally present in the patient samples.

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TYPES OF ELISA

INDIRECT ELISA SANDWICH ELISA COMPETETIVE ELISA

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INDIRECT ELISA

The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since first the antigen is coated and specific antibody is added which forms complex.Then add enzyme-conjugated secondry antibody and add substrate and measure colour.

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ADVANTAGES AND DISDVANTAGES

Advantages of indirect detection Wide variety of labeled secondary antibodies are

available commercially. Sensitivity is increased because each primary

antibody contains several sites that can be bound by the labeled secondary antibody, allowing for signal amplification.

Secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect

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.

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SANDWICH ELISA1. Plate is coated with a antibody.2. Sample is added, and any antigen

present binds to antibody.3. Detecting antibody is added, and binds to

antigen.4. Enzyme-linked secondary antibody is

added, and binds to detecting antibody.5. Substrate is added, and is converted by

enzyme to detectable form.

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Competitive binding assay

Unlabeled antibody is incubated in the presence of its antigen.

These bound antibody/antigen complexes are then added to an antigen coated well.

The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")

The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.

A substrate is added and check the colour change.

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Advantages

The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.

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Immunologic tests: Types of ELISA’s…

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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)

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Detecting infections sexually-transmitted agents like HIV,

syphilis and chlamydia hepatitis B and C Toxoplasma gondii

Detecting allergens in food and house dust

Measuring toxins in contaminated food Detecting illicit drugs, e.g.,

cocaine opiates

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ELISA Reader

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ADVANTAGES OF ELISA

Sensitive assay Equipments are widely available.No radiation hazards.Reagents are cheap with long shelf life.Adaptable to automation and high speed.Qualitative and quantitative.ELISA can be used on most types of

biological samples, such as plasma, serum, urine.

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DISADVANTAGES OF ELISA

Enzyme activity may be affected by plasma constituents.

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APPLICATIONS OF IMMUNE RESPONSE(NEUTRILIZATION AND PRECIPITATION)

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NEUTRALIZATION

It is a serological test used to identify used to identify toxins and antitoxins as well as viruses and viral antibodies.

It involves antigen-antibody reaction. Laboratory animals are used as

“indicator systems” in these tests. For example it is used to detect

exotoxin ofClostridium botulinum in food.

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NEUTRALIZATION

Antitoxin is mixed with the food that contains toxins. Thus neutralization of toxin takes place.

If toxin is produced by some other organism no neutralization occurs and mixture is lethal to

animal.Schick test The Schick test is used to determine if a person is

immune to diphtheria (intradermal test) In this test diphtheria toxin is injected

intradermally.No skin reactions occurs if person has neutralizing antibodies. A local edema occurs if no neutralizing antibodies are present , indicating person is susceptible to diphtheria.

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Virus neutralization by specific antibodies

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PRECIPITATION

The immunoprecipitation technique detects soluble antigens that react with antibodies called precipitins.

The antibodies link the antigen to form a large antibody-antigen network or lattice that settles out of solution at the equivalence zone when it becomes sufficiently large.

In fluids,antigen and antibody are layered over each other in a thin tube.The molecules then diffuse through the fluid untill they reach equivalence zone.A visible mass of particles is now formed at interface or at bottom.

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IMMUNODIFFUSION

In immunodiffusion, antigens and antibodies diffuse through a semisolid gel until they reach the zone of equivalence.

TWO METHODS1.Single diffusion(oudin) 2.Double diffusion(Ouchterlony plate technique)

Both reactants diffuse. Antigen and antibody solutions are placed in wells

cut into agar in petri dishes.The plates are incubated and precipitation lines form at zone of equivalence.

Used to detect fungal antigens of histoplasma,blastomyces,coccidioides.

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IMMUNO-ELECTROPHORESIS In this technique gel electrophoresis

and diffusion are combined.

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