Date post: | 12-Apr-2017 |
Category: |
Education |
Upload: | praveen2793 |
View: | 31 times |
Download: | 0 times |
ESTIMATION OF HORMONES BY RIA (Radioimmunoassay)
Submitted to Submitted by Dr. M. Narayana Swamy V.Praveen Professor & Head MVHK 1648 Dept. of Veterinary Physiology Veterinary College of Bangalore Hebbal
Introduction • Radioimmunoassay (RIA) is a very sensitive in vitro
assay technique used to measure concentrations of antigens (for example, hormone levels in the blood) by use of antibodies
• For this method one need a pure preparation of known antigen / antibody, or both, in order to standardize the assay.
• To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine.
Principle• The principle of RIA is based on the theory that in the absence
of an unlabeled antigen or hormones (H), the labeled radioactive hormones (H*) has maximal opportunity to react with limited number of antibody binding site(Ab).
• Antibody have bivalent binding site so one antibody molecule can bind two hormone molecules.
• If some of the limited antibody combining sites are allowed to react with the unlabeled hormones, fewer sites will be left to react with the labeled hormones, which results in a decrease in antibody – bound radioactivity.
Contd…• This data may be graphed on an arithmic scale and a standard
curve is made. • The amount of hormone in unknown sample is determined by
comparisions with the standard curve.
Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ab*
[Ag : ligand to be measured ; Ag* radiolabelled ligand] Uses an Antigen – Antibody reaction to estimate a ligand
Requirements 1. Preparation & characterisation of the Antigen [Ligand to be
analysed] 2. Radiolabelling of the Antigen3. Preparation of the Specific Antibody4. Development of Assay System
Preparation & Radiolabelling of the Antigen
Antigens prepared by.. ◦Synthesis of the molecule ◦ Isolation from natural sources
Radiolabelling [Tagging procedure]◦ 3 H 14 C 125 I are used as radioactive tags◦Antigens are tagged to 3 H 14 C 125 ◦Tagging should NOT affect Antigenic specificity &
Antigenic activity !
Preparation of the Specific Antibody
• Antigen injected intradermally into rabbits or guinea pigs antibody production
• Antibodies recovered from the serum• Some ligands are not Antigenic – Hormones, Steroids, Drugs HAPTENS– Eg: Gastrin, Morphine, – Haptens conjugated to albumin antigenic
Development of the Assay System• A crucial step is separation of unbound antigens• This achieved by binding the antibodies to the microtitre well
surface [Solid phase RIA]• Antigens bound to the fixed antibodies remain stuck to the
inner surface• Decanting & washing the well removes unbound antigens• Other techniques of separation: Centrifugation
Assay Procedure• Add known amounts of the test sample + labelled antigen
into the microtitre wells• Incubate allow the reaction to reach completion• Decant & wash contents of the well removes all unbound
antigens• Radioactivity remaining in the Microtitre wells measured by a
Counter [GM counter , Scintillation counter etc]• Intensity of radioactivity is inversely correlated with the conc
of antigens in the test sample• Sensitive to very low conc. of antigens
Advantages
• Radioimmunoassay is widely-used because of its great sensitivity.
• Using antibodies of high affinity, it is possible to detect a few picograms (10−12 g) of hormone in the tube.
• The greater the specificity of the antiserum, the greater the specificity of the assay
Limitations
• The main drawbacks to radioimmunoassay are the expense and hazards of preparing and handling the radioactive antigen.
• Both 125I or 131I emit gamma radiation that requires special counting equipment
• The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4).
RIAAdvantages◦ Flexibility◦ Sensitivity◦ Size
Disadvantages◦ Toxicity◦ Shelf life◦ Disposal costs
SCHEMATIC REPRESENTATION OF RIA
STANDARD CURVE FOR RIA
QUALITY CONTROL PARAMETERS IN RIA
• In the process of hormone estimation the quality control in terms of sensitivity, specificity and precision are required to be carried out for each hormone.
• Sensitivity – it is generally regarded as hallmark of good assay. Its defined as minimum amount of hormone (lowest detectable limit) distinguishable from zero concentration.
• Precision – Its also known as reproducibility, is a measure of variation observed between repeated determination of the same sample in different assay.
• Specificity – Specificity of an assay is defined as the degree to which an assay responds to substance other than for which the assay is designed.
INSTRUMENTATION
Thank you