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Edit Comment Close Premium member Presentation Transcript Slide 1: بسم الله الرحمن الرحيم Slide 2: Enzyme Linked Immunosorbent Assay ELISA By: Dr.Hossam Eldin Mahmoud Slide 3: 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. EIA are the developmental successor to radioimmunoassay. The most widely used names are ELISA; EIA (enzyme immunoassay); EMIT (enzyme multiplied immunoassay technique). Slide 4: Historical background Slide 5: The use of enzymes as immunological labels instead of radionucides was reported in 1971. The EIA was 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. Slide 6: Principle of EIA Slide 7: The sample with an unknown amount of antigen is immobilized on a solid support either: Non-specifically or Specifically The detection antibody is added, forming a complex with the antigen. Slide 8: 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. Slide 9: Criteria for choice of a marker enzyme Slide 10: High specific activity, not reduced by conjugation. Stability & Solubility. Simple, sensitive. Activity constant under test condition. Slide 11: Types of ELISA Slide 12: Homogeneous vs. Heterogeneous Competitive vs. Noncompetitive Indirect vs. Direct Homogeneous EIA : Homogeneous EIA Advantages: These assays don’t require the separation and washing steps. So it is easy, simple and adaptable for automation. Disadvantage: It is difficult to use for detection of high molecular weight substances with sufficient sensitivity. Used in determination low molecular weight analytes such as hormones and drugs. Heterogeneous EIA: : Heterogeneous EIA: The free reactant is always separated from the bound labeled reactant after immunological incubation. Competitive ELISA : Competitive ELISA Non competitive ELISA : Non competitive ELISA Indirect EIA : Indirect EIA Indirect EIA : Step 1 inactivated HIV antigens Step 2 serum antibodies Step 3 Anti-human Ig coupled to enzyme Step 5 ( Measurement ) Step 4 Chromogen or substrate Indirect EIA Direct EIA : Direct EIA Slide 20: Applications of EIA Slide 21: Evaluate the presence of antigen or antibody in a sample. In food industry in detecting potential food allergens such as peanuts and eggs. ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs. Slide 22: Advantages of EIA Slide 23: 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. Reproducible. ELISA can be used on most types of biological samples, such as plasma, serum, urine, and cell extracts Slide 24: Disadvantages of EIA Slide 25: Enzyme activity may be affected by plasma constituents. Homogenous ELISA is not as sensitive as radioimmunoassay. Slide 26: Troubleshooting in ELISA assays Slide 27: If the negative controls are giving positive results: Contamination of the substrate solution, enzyme-labelled antibody, control themselves. Inadequate rinsing of plates. Inadequate blocking of plates. Slide 28: If no colour has developed for the positive controls or for the samples: Check all reagents for dating and storage conditions. Microwell plates not coated properly. Reagents applied in wrong order or step omitted. Enzyme conjugate defective or inhibited by contaminant. Slide 29: If very little colour has developed for positive controls and the test samples: Check the dilution of the enzyme labelled antibody. The concentration of the substrate. Wash buffer not adequately drained after every wash step. Inadequate incubation times. Enzyme conjugate defective or inhibited by contaminant, Substrate defective or contaminated, Micro well plates poorly coated. Slide 30: 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. Preparation of ELISA : Preparation of ELISA 2 1 3 4 ELISA Reader : ELISA Reader Slide 34: Thanks You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.