ELISA

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By: ad871368 (20 month(s) ago)

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Slide 1: 

بسم الله الرحمن الرحيم

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Enzyme Linked Immunosorbent Assay ELISA By: Dr.Hossam Eldin Mahmoud

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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).

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Historical background

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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.

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Principle of EIA

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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.

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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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Criteria for choice of a marker enzyme

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High specific activity, not reduced by conjugation. Stability & Solubility. Simple, sensitive. Activity constant under test condition.

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Types of ELISA

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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

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Applications of EIA

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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.

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Advantages of EIA

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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

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Disadvantages of EIA

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Enzyme activity may be affected by plasma constituents. Homogenous ELISA is not as sensitive as radioimmunoassay.

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Troubleshooting in ELISA assays

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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.

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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.

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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.

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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

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Thanks