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RIA and ELISA-3 (1).pptx
- 1. Presented to : DrSidra Yaseen Presented by : Momna Faisal
- 2. Definition : It isan immunological assay commonly used to measure antibodies, antigens, proteins,harmones and glycoproteins in biological samples ELISA is a distinguished analysis compared to other antibody-assays as it yields quantitative results
- 3. ELISA workson the principle that specific antibodies bind the target antigen and detect the presence and quantity of antigens binding. In order to increase the sensitivity and precision of the assay, the plate must be coated with antibodies/antigen with high affinity. ELISA can provide a useful measurement of antigen-antibody concentration.
- 4. Generally thefollowing apparatus is used for ELISA : ELISA Kits Pipettes Pipette tips Microplates Spectrophotometer Well wash microplate washer
- 5. ELISA assaysare generally carried out in 96 well plates, allowing multiple samples to be measured in a single experiment. These plates need to be special absorbent plates (e.g. NUNC Immuno- plates) to ensure the antibody or antigen sticks to the surface. Each ELISA measures a specific antigen, and kits for a variety of antigens are widely available.
- 8. • An antibodyis attached to a polystyrene plate which is a solid surface and is attracted or has an affinity towards bacteria, other antibodies and hormones. • A microtiter coated with antigen is filled with this antigen-antibody mixture after which free antibodies are removed by washing. • A second antibody specific to primary antibody is added which is usually conjugated with an enzyme. • Free enzyme-linked secondary antibodies are removed by washing the plate. • Finally, the substrate is added. The substrate is converted by the enzyme to form a coloured product, which can be measured by spectrophotometry.
- 10. EXAMPLE: HCG protein whichindicates pregnancy is detected by ELISA. A combination of blood or urine sample and purified HCG linked to an enzyme is added to the system. If HCG is absent in the test sample, then only the linked enzyme binds to the solid surface
- 11. ELISA testscan be classified into three types depending upon the different methods used for binding between antigen and antibodies, namely: • Indirect ELISA – Antigen is coated to the microtiter well • Sandwich ELISA – Antibody is coated on the microtiter well • Competitive ELISA – Microtiter well which is antigen-coated is filled with the antigen-antibody mixture
- 12. Indirect ELISA : •It detects the presence of antibody in sample. The antigen is attached to the wells of the microtitre plate. • A sample containing the antibodies is added to the antigen-coated wells for binding with the antigen. • The free primary antibodies are washed away and the antigen-antibody complex is detected by adding a secondary antibody conjugated with an enzyme that can bind with the primary antibody. • All the free secondary antibodies are washed away. A specific substrate is added which gives a coloured product. • The absorbance of the coloured product is measured by spectrophotometry
- 13. Sandwich ELISA • SandwichELISA helps to detect the presence of antigen in a sample. • The microtitre well is coated by the antibody. • The sample containing the antigen is added to the well and washed to remove free antigens. • Then an enzyme-linked secondary antibody, which binds to another epitope on the antigen is added. The well is washed to remove any free secondary antibodies. • The enzyme-specific substrate is added to the plate to form a coloured product, which can be measured
- 15. Competitive ELISA • CompetitiveELISA helps to detect antigen concentration in a sample. • The microtitre wells are coated with the antigen. • Antibodies are incubated in a solution having the antigen. • The solution of the antigen-antibody complex is added to the microtitre wells. The well is then washed to remove any unbound antibodies. • More the concentration of antigen in the sample, lesser the free antibodies available to interact with the antigen, which is coated in the well. • The enzyme-linked secondary antibody is added to detect the number of primary antibodies present in the well. • The concentration is then determined by spectrophotometry
- 16. Ebola Perniciousanemia AIDS Rotavirus Lyme disease Syphilis Toxoplasmosis Zika virus Carcinoma of the epithelial cells
- 17. The presenceof antibodies and antigens in a sample can be determined. It is used in the food industry to detect any food allergens present. To determine the concentration of serum antibody in a virus test. During a disease outbreak, to evaluate the spread of the disease For Example, during recent COVID-19 outbreak, rapid testing kits are being used to determine presence of antibodies in the blood sample.
- 18.
- 19. INTRODUCTION Radioimmunoassayis one of the sensitive immunoassay techniques which helps in the determination of antigens or antibodies in a sample with the use of radioisotopes. A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. RIA was first described in 1960 for the measurement of endogenous plasma insulin by Solomon Berson and Rosalyn Yalow of the Veterans Administration Hospital in New York.
- 20. It isan in vitro type of antigen-antibody interaction. When radioisotopes instead of enzymes are used as labels to be conjugated with antigens or antibodies, the technique of detection of the antigen- antibody complex is called radioimmunoassay (RIA). Radioimmunoassay (RIA) is an in vitro assay that measures the presence of an antigen with very high sensitivity.
- 21. RIA issubdivided into four sections (primarily because there are four basic steps in setting up an RIA procedure): a) sample or standard b) label or labeled antigen c) primary binder d) separation step.
- 22. Pipettors and/orpipets that accurately and precisely deliver the required volumes Polypropylene test tubes Test tube rack Vortex mixer Beakers or flasks Centrifuge (refrigerated, with swinging bucket rotor) Liquid scintillation counter.
- 23. There aretwo different methods of RIA that are commonly employed for drug detection. Double-antibody RIA Coated-tube RIA.
- 24. Types ofassays Enzyme Immunoassay (EIA) or Enzyme-linked immunosorbent assay (ELISA) Radioimmunoassay (RIA) Fluoroimmnoassay (FIA) Chemiluminescence immunoassay (CLIA) Counting assay
- 25. A radioimmunoassay(RIA) uses radiolabeled antigens to measure concentrations of substances in body fluids such as blood and saliva. A radioisotope is attached to an antigen of interest and bound with its complementary antibody. A sample with the target antigen is then added, which competes with the radioactive antigen, kicks it out of the binding spot and replaces it.
- 26. After washingaway unbound antigens the radioactivity of the sample is measured. The smaller the radioactive signal, the more target antigen is present. Compared to other immunoassay techniques, extra precautions are needed due to the radioactive substances involved, but RIA’s high sensitivity and specificity means that it remains in use today.
- 27. Fluoroimmunoassay Fluorescentimmunoassays (FIA) use a fluorescent compound as the detection reagent to detect and quantify a variety of compounds. FIA is widely used in the in vitro diagnostics (IVD) industry and has the advantage of being fast and highly sensitive compared to other methods. In FIA, antibodies are labeled with fluorescent probes. FIA fluorescent dyes illuminate in UV light and are used to detect a specific antigen-antibody binding. After incubation with the antigen, the antibody-antigen complexes are isolated, and the fluorescent intensity is measured.
- 28. Chemiluminescent immunoassay isa variation of the standard enzyme immunoassay (EIA), which is a biochemical technique used in immunology. Applications Chemiluminescent immunoassay (CLIA) has been applied in different fields, including environmental monitoring, clinical diagnosis, food safety and pharmaceutical analysis, as a promising approach for selective, sensitive, rapid and simple analysis.
- 29. Counting immunoassay(CIA) During incubation, the beads bind to a variety of antigens and jointly form a large mass, but some beads are not bound. The whole solution passes through a cell counter, with only unbound beads counted. The amount of unbound beads is inversely proportional to that of antigens.
- 30. GENERAL PROCEDUREOF RIA A known quantity of an antigen is made radio active frequently by labeling it with gamma radioactive isotopes of iodine attached to tyrosine. This radio labeled antigen is then mixed with a known amount of antibody for that antigen and as a result the two chemically bind to one another.
- 34. CRP CRPis an acute phase reactant and a general marker of systemic inflammation. Well-known inflammatory markers includes, C-reactive protein Complete blood count Albumin Cytokines Erythrocyte sedimentation rate.
- 35. Additional phenotype-definingmarkers included CD4 CD8+ CCR7 CD28 CD62L CD45RA T- cells.
- 36. The CD4+T cell gene signature in progressors was enriched for cytotoxicity-related genes GZMH, GZMB, GZMA, NKG7, FCRL6, SLAMF7, CX3CR1, and PRF1, among others. The most commonly mutated gene in people with cancer is p53 or TP53. More than 50% of cancers involve a missing or damaged p53 gene. Most p53 gene mutations are acquired. Germline p53 mutations are rare, but patients who carry them are at a higher risk of developing many different types of cancer.
- 37. Radioimmunotherapy (RIT)represents a selective internal radiation therapy, that is, the use of radionuclides conjugated to tumor-directed monoclonal antibodies (including those fragments) or peptides. In a clinical field, two successful examples of this treatment protocol are currently extended by 90Y-ibritumomab tiuxetan (Zevalin) and 131I-tositumomab (Bexxar), both of which are anti-CD20 monoclonal antibodies coupled to cytotoxic radioisotopes and are approved for the treatment of non-Hodgkin lymphoma patients. In order to reduce the unnecessary exposure and to enhance the therapeutic efficacy, various biological, chemical, and treatment procedural improvements have been investigated in RIT. This review outlines the fundamentals of RIT and current knowledge of the preclinical/clinical trials for cancer treatment.
- 38. Radioimmunoassays areused for detecting the concentration of a specific antigen. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies. The advantages of RIA are its relative simplicity and the high sensitivity provided by the use of radioactive compounds.
- 39. RIA isused in the assay of drug like amphetamine,barbiturates,digitoxin,morphine etc. In analysis of vitamine like riboflavin,folic acid. Serum gastrin radioimmunoassay (RIA) is a sensitive and specific method suitable for measurement of circulating concentrations of this peptide hormone, which is a major regulator of gastric acid secretion. The RIA technique may be useful for early (disease diagnosis) of viral infections and for confirmation of response to immunization without the need for a blood sample, as well as for the study of the secretory immune response in very young and older subjects.
- 40. Radioimmunoassay (RIA)is a technique in which researchers use radioactive isotopes as traceable tags to quantify specific biochemical substances from blood samples. Immunoassays have been widely used in many important areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetic and bioequivalence studies in drug discovery and pharmaceutical industries.