ASSAYS AND APPLICATIONS OF IMMUNE RESPONSE PRESENTED BYBILAL ANJUM BUTT 50-E 11 May 2002 2
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) 11 May 2002 3
ELISA(Enzyme-linked immunosorbent assay) 11 May 2002 4
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. 11 May 2002 5
HISTORY It is first described by Peter Perlmannand Eva Engval , and Anton SchuursandBauke van Weemen. The technique was subsequently developed by Voller and col. using microplates, that permitted the development of highly sensitive and accurate kits. 11 May 2002 6
COMPONENTS OF AN ELISA Antibody: IgG fraction of serum Enzyme:Peroxidase from horseradish,Alkalinephosphatase from E. coli,β-galactosidase from E coli.,Glucoseoxidase. Substrate: TMB (3,3',5,5', tetramethylbenzidine). 11 May 2002 7
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. 11 May 2002 8
8. Observe colour development 7. Add substrate for enzyme 1. Add antigen 2. Wash with PBST 6. Wash with PBST 4. Wash with PBST 3. Add primary antibody 5. Add secondary antibody 11 May 2002 9
ELISA Step 1 inactivated HIV antigens Step 2 serum antibodies Step 3 Anti-human Ig coupled to enzyme Step 4 Chromogen or substrate Step 5 ( Measurement )
CRITERIA FOR CHOICE OF A MARKER ENZYME Should 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. 11 May 2002 11
TYPES OF ELISA INDIRECT ELISA SANDWICH ELISA COMPETETIVE ELISA 11 May 2002 12
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. 11 May 2002 13
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. 11 May 2002 14
SANDWICH ELISA Plate is coated with a antibody. Sample is added, and any antigen present binds to antibody. Detecting antibody is added, and binds to antigen. Enzyme-linked secondary antibody is added, and binds to detecting antibody. Substrate is added, and is converted by enzyme to detectable form. 11 May 2002 15
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. 11 May 2002 16
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. 11 May 2002 17
Immunologic tests: Types of ELISA’s… 11 May 2002 18
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) 11 May 2002 19
Detecting infections sexually-transmitted agents like HIV, syphilis and chlamydia hepatitis B and C Toxoplasmagondii Detecting allergens in food and house dust Measuring toxins in contaminated food Detecting illicit drugs, e.g., cocaine opiates 11 May 2002 20
Enzyme activity may be affected by plasma constituents.
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APPLICATIONS OF IMMUNE RESPONSE(NEUTRILIZATION AND PRECIPITATION) 11 May 2002 24
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 of Clostridium botulinumin food. 11 May 2002 25
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 11 May 2002 27
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. 11 May 2002 28
IMMUNODIFFUSION In immunodiffusion, antigens and antibodies diffuse through a semisolid gel until they reach the zone of equivalence. TWO METHODS 1.Single diffusion(oudin) 2.Double diffusion(Ouchterlony plate technique)
Both reactants diffuse.
Antigen and antibody solutions are placed in wells cut into agar in petridishes.The plates are incubated and precipitation lines form at zone of equivalence.
Used to detect fungal antigens of histoplasma,blastomyces,coccidioides.