Radioimmunoassay& enzyme linked immunosorbent

Июль 26, 2022

Главная
Медицина
Radioimmunoassay& enzyme linked immunosorbent

Содержание

2. Principle of Radioimmunoassay Principle: Uses an immune reaction [Antigen – Antibody reaction] to estimate a ligand
3. Advantages & Disadvantages of RIA Advantages Highly specific: Immune reactions are specific High sensitivity : Immune
4. Requirements for RIA Preparation & characterisation of the Antigen [Ligand to be analysed] Radiolabelling of the
5. Preparation & Radiolabelling of the Antigen Antigens prepared by.. Synthesis of the molecule Isolation from natural
6. Preparation of the Specific Antibody Antigen injected intradermally into rabbits or guinea pigs ? antibody production
7. Development of the Assay System A crucial step is separation of unbound antigens This achieved by
8. Assay Procedure Add known amounts of the test sample + labelled antigen into the microtitre wells
9. Enzyme Linked Immunosorbent Assay Principle: Uses an immune reaction like RIA Differs from RIA in detection
10. Advantages of ELISA Sensitive: nanogram levels or lower Reproducible Minimal reagents Qualitative & Quantitative Qualitative ?
11. Types of ELISA Noncompetitive binding assay or Sandwich method Antigen measuring system [Titrewells coated with antibodies
12. Noncompetitive or Sandwich Assay Antigen measuring system Titre wells coated with suitable antibody Add patient sample
13. Noncompetitive or Sandwich Assay Antibody measuring system Titre wells coated with suitable antigen Add patient sample
14. Competitive binding assay Titrewells coated with antibodies Known quantities of patient sample containing antigen + antigen
15. Enzyme labels Enzyme labels should have high specific reactivity Should be easily coupled to ligands &
17. Скачать презентацию

Слайд 2

Principle of Radioimmunoassay
Principle: Uses an immune reaction [Antigen – Antibody reaction]

to estimate a ligand
Ag + Ag* + Ab ? AgAb + Ag*Ab + Ag + Ab*
Unbound Ag* and Ag washed out
Radioactivity of bound residue measured
Ligand conc is inversely related to radioactivity
[Ag : ligand to be measured ; Ag* radiolabelled ligand]

Слайд 3

Advantages & Disadvantages of RIA
Advantages
Highly specific: Immune reactions are specific
High sensitivity

: Immune reactions are sensitive
Disadvantages
Radiation hazards: Uses radiolabelled 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.

Слайд 4

Requirements for RIA
Preparation & characterisation of the Antigen [Ligand to be

analysed]
Radiolabelling of the Antigen
Preparation of the Specific Antibody
Development of Assay System

Слайд 5

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 !

Слайд 6

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

Слайд 7

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

Слайд 8

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

Слайд 9

Enzyme Linked Immunosorbent Assay
Principle:
Uses an immune reaction like RIA
Differs from

RIA in detection method
Detection based on
Enzyme catalysed reaction OR
Fluorescent probe
NOT radioactivity [great advantage!]

Слайд 10

Advantages of ELISA
Sensitive: nanogram levels or lower
Reproducible
Minimal reagents
Qualitative & Quantitative
Qualitative

? Eg HIV testing
quantitative assays ? Eg Ther. Drug Monitoring
Greater scope : Wells can be coated with Antigens OR Antibodies
Suitable for automation ?high speed
NO radiation hazards

Слайд 11

Types of ELISA
Noncompetitive binding assay or Sandwich method
Antigen measuring system [Titrewells

coated with antibodies ; Enzyme labelled antibodies]
Antibody measuring system [Titrewells coated with antigens ; Enzyme labelled antiantibodies]
Competitive binding assay [Titrewells coated with antibodies ; Enzyme labelled antigens]

Слайд 12

Noncompetitive or Sandwich Assay
Antigen measuring system
Titre wells coated with suitable

antibody
Add patient sample containing the antigen
Incubate: till antigen antibody reaction is complete
Wash? remove unbound antigen
Add Antibody labelled with Enzyme
Incubate till antigen binds labelled antibody
Wash ? remove unbound labelled antibody
Add substrate ; incubate
Enzyme + Substrate ? Product ? measure colour
Colour proportional to antigen in patient sample

Слайд 13

Noncompetitive or Sandwich Assay
Antibody measuring system
Titre wells coated with suitable antigen
Add

patient sample containing the antibody
Incubate: till antigen antibody reaction is complete
Wash? remove unbound antibody
Add Antiantibody labelled with Enzyme
Incubate till labelled antiantibodies binds antigen-antibody complex
Wash ? remove unbound labelled antiantibody
Add substrate ; incubate
Enzyme + Substrate ? Product ? measure colour
Colour proportional to antibody in patient sample

Слайд 14

Competitive binding assay
Titrewells coated with antibodies
Known quantities of patient sample containing

antigen + antigen labelled with enzyme
Incubate: till antigen antibody reaction is complete
Wash? remove unbound antigens
Add substrate ; incubate
Enzyme + Substrate ? Product ? measure colour
Colour inversely related to antigen in patient sample

Слайд 15

Enzyme labels
Enzyme labels should have high specific reactivity
Should be easily coupled

to ligands & the labelled complex must be stable
The reactivity should be retained after linking of the enzyme to the antigen/antibody
The chosen enzymes should not be normally present in the patient samples
Examples of enzyme labels
Horse radish peroxidase, Alkaline phosphatase, Glucose oxidase

Radioimmunoassay&amp; enzyme linked immunosorbent

Содержание

Principle of RadioimmunoassayPrinciple: Uses an immune reaction [Antigen – Antibody reaction]

Advantages & Disadvantages of RIAAdvantagesHighly specific: Immune reactions are specificHigh sensitivity

Requirements for RIAPreparation & characterisation of the Antigen [Ligand to be

Preparation & Radiolabelling of the Antigen Antigens prepared by.. Synthesis of

Preparation of the Specific AntibodyAntigen injected intradermally into rabbits or guinea

Development of the Assay SystemA crucial step is separation of unbound

Assay ProcedureAdd known amounts of the test sample + labelled antigen

Enzyme Linked Immunosorbent AssayPrinciple: Uses an immune reaction like RIADiffers from

Advantages of ELISASensitive: nanogram levels or lowerReproducibleMinimal reagentsQualitative & Quantitative Qualitative

Types of ELISANoncompetitive binding assay or Sandwich methodAntigen measuring system [Titrewells

Noncompetitive or Sandwich Assay Antigen measuring systemTitre wells coated with suitable

Noncompetitive or Sandwich AssayAntibody measuring systemTitre wells coated with suitable antigenAdd

Competitive binding assayTitrewells coated with antibodiesKnown quantities of patient sample containing

Enzyme labelsEnzyme labels should have high specific reactivityShould be easily coupled

Похожие презентации