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Immunological Assay.pptx
This document discusses immunoassays and two common types - radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). RIA involves labeling an antigen or antibody with a radioactive material to measure it in a mixture. It is very sensitive but involves radiation hazards. ELISA uses an enzyme-linked antibody or antigen to detect the presence of a substance. It is a plate-based assay that is sensitive, reproducible, and does not use radiation. Both methods are used for applications like disease detection, drug monitoring, and analyzing hormones and metabolites.
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Immunological Assay.pptx
- 1. Immunological Assay Dr. Ashwani K.Dhingra Professor Guru Gobind Singh College of Pharmacy, Yamuna Nagar
- 3. Immunoassay • Immunoassay meansa method to measure any particular substance in a mixture using its specific-binding antibody. • One of the merits of immunoassay is that we can measure a substance that is present in a mixture of various contaminants. • Immunoassays have become very popular in view of their high sensitivity, safety, economy and simple instrument requirements. Types : 1. RIA (Radio Immune Assay) 2. ELISA (Enzyme Linked Immunosorbent Assay)
- 4. RIA (Radio Immune Assay) The technique wasintroduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay. The sensitivity range is 0.0006–0.006 μg antibody/ml. It is a very sensitive in-vitro assay technique in which antibody or antigen is labelled with a radioactive material (like ¹²⁵I, ³H)
- 5. Principle It involves threeprinciples: • An immune reaction i.e., antigen, antibody binding. • A competitive binding or competitive displacement reaction. (It gives specificity) • Measurement of radio emission. (It gives sensitivity)
- 7. Procedure Known quantityof an antigen is made radioactive, frequently by labelling it with γ-radioactive isotopes of iodine. This radio labelled antigen is then mixed with a known amount of antibody for that antigen. A sample of serum from a patient containing an unknown quantity of same antigen is added. This causes the unlabelled (cold) antigen from serum to compete with radio labelled antigen (hot) for antibody binding site. As the concentration of cold antigen is increased, more of it binds to antibody, displacing the radio labelled variant and reducing the ratio of antibody bound radio labelled antigen to free radio labelled antigen.
- 8. The bound antigensare then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a γ counter. The concentration of the test antigen can be calculated from the ratio of the bound and total antigen labels, using a standard dose response curve. From these data, a standard binding curve can be drawn. After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly.
- 9. Advantages •Highly specific: Immunereactions are specific •High sensitivity : Immune reactions are sensitive Disadvantages •Radiation hazards: Uses radiolabeled reagents •Requires specially trained persons •Labs require special license to handle radioactive material •Requires special arrangements for requisition, storage of radioactive material, radioactive waste disposal. APPLICATIONS •Detection of narcotic drugs •Blood bank screening for the hepatitis (a highly contagious condition) virus
- 10. Measurement ofgrowth hormone levels, immunoglobulin's, tumour markers Tracking of the leukaemia virus. Diagnosis and treatment of peptic ulcers Endocrinology – Insulin, HCG, Vasopressin – Detects Endocrine Disorders – Physiology of Endocrine Function Pharmacology – Morphine – Detect Drug Abuse or Drug Poisoning Study Drug Kinetics. Clinical Immunology – Antibodies for Inhalant Allergens – Allergy Diagnosis Oncology – Carcino-embryonic Antigen – Early Cancer Detection and Diagnosis APPLICATION S
- 11. ELISA (Enzyme Linked Immunosorbent Assay) ELISA is abiochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In this method the antigen or antibody is conjugated to an enzyme. It is a plate based assays designed for detecting and quantifying substances such as peptides, proteins, antibodies, antigens and hormones. It involves detection of ‘analyte’ in a liquid sample using liquid reagent (wet lab) or dry strips (dry lab). The test can be done in polystyrene tubes (macro-ELISA) or polyvinyl micro titre plates (micro-ELISA).
- 12. Basic Principle Based on BasicImmunology Response. • Lock and Key Concept Antigen (key): substance when introduced into the body produces antibodies. Antibody (lock): protein in the body that is used by immune system to identify and neutralize foreign targets (referred to as antigens). Key fits into the lock. • Enzyme conjugate substrates Enzyme that converts colourless substrates to a colored product –Bound to the antibody that is part of the antibody-antigen complex. Bound to a secondary antibody that binds with the antibody- antigen complex.
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- 14. Types of ELISA Direct ELISA IndirectELISA Sandwich ELISA Competitive ELISA
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- 20. • Sensitive: nanogramlevels or lower • Reproducible • Minimal reagents • Qualitative & Quantitative Qualitative – HIV testing Quantitative assays – Therapeutic drug monitoring (TDM) Greater scope: Wells can be coated with Antigens OR Antibodies • NO radiation hazards • Fast - 90 samples tested in 2-3 hr. • Sensitivity (up to 10 pg/mL). ADVANTAGE S
- 21. Detection of HIVantibodies in serum. Detection of mycobacterial antibodies in tuberculosis. Detection of rotavirus in faeces. Detection of hepatitis B markers in serum. Detection of enterotoxin of E coli in faeces. Detection of potential food allergens. Analysis of hormones, vitamins, metabolites, diagnostic markers • like ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids, Therapeutic drug monitoring: like Barbiturates, morphine, digoxin. APPLICATIONS