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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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