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ELISA

BIOCHEMISTRY

mprasadnaidu
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ELISA

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  1. Enzyme-Linked Immunosorbent Assay M.Prasad NaiduMSc Medical Biochemistry, Ph.D,.

  2. INTRODUCTION • Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. • ELISA is so named because the technique involves the use of an immunosorbent, an absorbing material specific for one of the components of the reaction, the antigen or antibody. • ELISA is usually done using 96-well microtitre plate suitable for automation

  3. What is ELISA? Technique used to detect (assay) specific molecules (e.g. proteins & carbohydrates) in samples.  Immunological technique: uses antibodies.  Quantitative.  Very sensitive.  Commonly used in medicine and scientific research.

  4. Secondary antibody Enzyme Coloured product Primary antibody PRINCIPLE Substrate Different antigens in sample

  5. The technique is divided into : 1- Competitive ELISA 2- Sandwich ELISA (also called direct ELISA) 3- Indirect ELISA

  6. COMPETITIVE ELISA • The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). • The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal. • Advantage: • The ability to use crude or impure samples and still selectively bind any antigen that may be present. • Application: • Used for the detection of human T-cell leukemia-lymphoma virus type III (HTLV-III). 

  7. INDIRECT ELISA

  8. COLOR OF SOLUTION & COLOR OF SOLUTION ARE COMPLEMENTARY

  9. INDIRECT METHOD • This method is largely used to measure antibodies in almost all human infections • Eg : HIV antibody detection • In patients with AIDS, the human immuno deficiency virus(HIV) produces specific antibody. • To detect the HIV antibody, indirect ELISA method is used

  10. SANDWICH METHOD

  11. Eg : Assay of thyroid hormone (T4)

  12. Reaction : HRP • H2O2 H2O + [O] Diamino oxidized Benzidine DAB (colorless) (brown color)

  13. MULTIPLE & PORTABLE ELISA • This new technique uses a solid phase made up of an immunosorbent polystyrene rod with 8-12 protruding ogives. • The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.

  14. The advantages of this technique are as follows: • The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and/or different antigens for multi-target assays • The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples • One ogive is left unsensitized to measure the non-specific reactions of the sample • The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required

  15. APPLICATIONS OF ELISA • Serum Antibody Concentrations • Detecting potential food allergens (milk, peanuts, walnuts, almonds and eggs) • Disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc

  16. Detections of antigens e.g. pregnancy hormones, drug allergen, , mad cow disease • human chorionic gonadotropin (HCG), the commonly measured protein which indicates pregnancy. • A mixture of purified HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are added to the test system. • If no HCG is present in the test sample, then only HCG with linked enzyme will bind.

  17. The more HCG which is present in the test sample, the less enzyme linked HCG will bind. • The substrate the enzyme acts on is then added, and the amount of product measured, such as a change in color of the solution.

  18. Detection of antibodies in blood sample for past exposure to disease e.g. Lyme Disease, trichinosis, HIV, bird flu • ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.

  19. To monitor diabetes, glucose concentrations will be checked. • Bacterial left-over in milk can be determined with an ELISA test. • ELISA assays are as well employed in foodstuff protection through signifying the occurrence of salmonella • ELISA assays can also be utilised to diagnose several cancers (eg: bladder cancer)

  20. ADVANTAGES OF ELISA • ELISA tests are generally relatively accurate tests. • Highly sensitive and specific • Antigens of very low or unknown concentration can be detected since capture antibody only grabs specific antigen • Generally safe: do not require radioactive substances, contains diluted sulfuric acid • Used in wide variety of tests

  21. DISADVANTAGES • Only monoclonal antibodies can be used as matched pairs • Monoclonal antibodies can cost more than polyclonal antibodies • Monoclonal antibodies are more difficult to find • Negative controls may indicate positive results if blocking solution is ineffective [secondary Ab or antigen can bind to open sites in well] • Enzyme/substrate reaction is short term so microwells must be read as soon as possible

  22. THANK YOU

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