Hurdle technology

1. Radioimmunoassay
Presentation by group 5

2. Group members
● Nanduja.K.S
● Nivedya.K.R
● Nuhma Shibin.M
● Pearl Pinto
● Pournamy.S
● Reshma Sathish
● Rinsha.T
● Sandhra Victoriya Jose
● Sareena.S
● Saviyo Joseph

3. Immunoassay
An immunoassay is a test that uses antigen and antibody complexes
Antibody:antigen complex is also known as immune complex
“Immuno” refers to an immune response that causes the body to generate
antibodies
“Assay”refers to test
Immunoassay is a test that utilises immuno complexing when antibodies
and antigens are brought together

4. ANTIBODIES, ANTIGEN AND ANALYTES
An antibody is a protein that is produced by the bodyin response to
invading substances
Antibodies are produced as a part of bodies immune response to protect
itself
An antigen is the substance that a body is trying to fight off by mounting an
immune response
An immunogen is substance that elicits immune response eg drug protein
conjugate

5. Antibody, antigen and analytes
An analyte is anything measured by the laboratory test
In immunoassay test an analyte can be either an antigen or an antibody
Immunoassay use ine or more selected antibodies to detect analytes of
interest

6. Radio immuno assay
Radioimmunoassay is an elegant technique in analytical chemistry
If the substance to be analysed is in very low quantities in the orders of
micrograms, nanograms conventional methods like gravimetric and
calorimetric methods fail
RIA find extensive application in the Assay of many substances which are
present in trace amount in blood

7. PRINCIPLE
• Based on a competitive binding reaction between
-Fixed amount of a labeled analyte (Ag*)
-Variable amount of unlabeled sample analyte (Ag)
• For a limited amount of binding sites on a highly specific
antibody

8. Essential Components Of RIA

9. Development Of A Standard Curve

12. Radioimmunoassay Procedure

15. Separation Techniques
Physical Methods
● Gel filtration chromatography
● Electroporesis
● Charcol dextran adsorption
● Adsorption on ion exchange resin
Chemical methods
● Organic solvents such as
ethanol,PEG
● Salt such as ammonium,sulphate

16. Competetive radioimmuno assay
Competition between labelled and unlabelled antigen being detected for a
limited number of binding sites on antibody.
Depending on whether the solid phase is coated with either the antibody or
the antigen (analyte) its of two type
● Antigen-capture immunoassay
● Antibody-capture immunoassay

19. Non competetive immunoradiometri assay
● Analyte to be measured is sandwiched between two antibodies
● First antibody is coated onto solid support
● Second antibody is radiolabelled for detection
● The analyte bound by both antibody to form a sandwich complex
ADVANTAGE
- Faster reaction rate
- High sensitivity

21. TYPES OF RADIOIMMUNOASSAYS
1) Single Antibody RIA (Charcoal-dextran Separation)
● Used for measurement of steroid hormones.
● Inexpensive but less sensitive than other methods.

23. 2) Double Antibody RIA
● Precipitation of bound complexes with a second antibody.
● Used for both steroid and protein assays.
● Has good sensitivity.
● But requires an additional incubation period that can prolong assay time.

25. 3) Solid-phase RIA
● Used for protein and steroid hormones.
● Polystyrene test tubes, microtiter plates are used for this purpose.
● Provides simple way to separate bound and free reactant.

27. Advantages of RIA
● It is an extremely sensitive assay as it can measure antigen up to picogram
quantities.
● It is a highly specific test as the antibody-antigen reaction is highly specific.
● A large number can be processed.
● It is an indirect method of analysis.

28. Disadvantages of RIA
● There are possible health hazards due to handling of radiative materials.
● Radioactive iodine used is not a cheap reagent.
● All reagents should be added precisely
● Requires specially trained persons.
● Labs require special license to handle radioactive material.
● Short half-life time of the isotope.
● Expensive instrumentation for the counting of radioactivity.

29. Applications of RIA
● Measuring toxins in contaminated food.
● Narcotics detection or drug poisoning.
● Early cancer detection and diagnosis.
● Analysis of hormones, vitamins,metabolites etc...
● Detecting infections,peptic ulsers etc..
● Measuring “rheumatoid factors” and other auto antibodies in autoimmune diseases.
● Used for researches in brain chemicals called neurotransmitters

30. Conclusion
● RIA is an effective, precise and accurate method of quantifying
concentrations of an antigen. The method is advantageous for its speed,
specificity, high sensitivity, relative ease of performance, and the possibility of
performing a great number of parallel determinations using automation and
computer evaluation.
Immunoassay techniques in their most simple forms provide excellent
screening tools to detect adulteration and contaminations.