ria

1. Radioimmunoassay

2. Introduction
What is immunoassay?
• Radio immunoassay (RIA) is a radio-analytical technique with remarkable sensitivity and a high
degree of specificity that is widely used for the estimation of a variety of molecules present in
complex matrices.
• This technique is used over a wide spectra of substances such as hormones, steroids, vitamins, drugs,
tumor markers and viral antigens.

3. History
This isotopic measuring method was developed in 1959 by two Americans, biophysicist
ROSALYN YALOW and physician SOLOMON A. BERSON.
RIA combines the specificity of an antigen-antibody reaction with a sensitivity of radioactive
measurements.
This technique used for detection of micro quantities of protein, viral antigens, antibodies,
structural proteins, vitamins and drug and their metabolites.
It can also be used for detection of pictogram quantities (10-12 g) of biological constituents
present in biological fluid.
RIA is used in place of bioassay in various branches of science like biochemistry,
microbiology, hematology and clinical pharmacology.

4. REQUIREMENTS FOR DEVELOPMENT OF RIA
1. Pure antigen: for standards (µg)
tracer production (tens of µg) Ab production (hundreds
of µg)
2. Tracer: self-made or commercial
3. Specific, high-affinity antibody: self-made or commercial
4. Amethod to separate bound and free antigen
5. Optional: a system to extract the antigen from the sample.

5. Principle
• RIA works on basic principle of biochemistry that competitive binding between
antigens for same antibody binding site.
• The competition of an analyte with its radio isotopically labeled counterpart for a
limited amount of antibody, the specific reagent, is the underlying principle of this
technique. Increasing the analyte concentration inhibits the binding of the labeled
analyte to the antibody.
Ag +Ag* +Ab AgAb +Ag*Ab +Ag
UnboundAg* andAg washed out
Radioactivity of bound residue measured
Ligand conc. is inversely related to radioactivity
Ag: ligand to be measured
Ag*: radio labelled ligand

7. Principle
• The concentration of the unknown analyte is thus obtained by comparing its
inhibitory effect on the binding of the labeled analyte to that of a known standard.
• After determining the ratio of bound to free antigen in each unknown, the antigen
concentrations can be read directly from the standard curve (as shown).

8. DEVELOPMENT OFTHEASSAY
SYSTEM
1. Acrucial step is separation of unbound antigens.
2. This is achieved by binding the antibodies to the microtitre well surface [Solid
phase RIA]
3. Antigens bound to the fixed antibodies remain stuck to the inner surface.
4. Decanting & washing the well removes unbound antigens.
5. Other techniques of separation: Centrifugation.

9. ASSAYPROCEDURE
Add known amounts of the test sample + labelled antigen into the micro-titre wells
Incubate
allow the reaction to reach completion
Decant & wash contents of the well
removes all unbound antigens

11. METHODS IN RIA:
1. Radio labelling of theAntigen or radio labelled production.
2. Preparation & characterisation of theAntigen [Ligand to be analysed] .
3. Preparation of the SpecificAntibody.
4. Development ofAssay System or separation techniques.

12. 1.PREPARA
TIONAND RADIO LABELING OFANTIGEN
• Antigen preparation:
 Synthesis of the molecule
 Isolation from natural sources
• Radiolabelling [Tagging procedure] :
 Two most commonly used radio labels in RIA 3H and 125I although 75Se, 32P, 14C
and 131I have also been used but these have some limitations that are
32P and 57Co = limited by stereo chemical aspects because drug naturally contain
phosphorous and cobalt.
14C = low specific activity field
131I = Short decay half life and radio degradation character

13. • On the other hand 3 H and 125 I have advantages as follows:
3 H = Direct incorporate into molecular structure, half lives(12 years)
125 I= High activity and ease of counting (half lives 60 days)
• Technique of labelling of drug or antigen with 3H and 125I:
Specific tritium labels are generally obtained by reducing an appropriate precursor
in presence of 3H.
Labelling of drug with 125I include chloramines-T, monochloride exchange and
enzymatic iodination methods. The most suitable iodination procedure is
depending on the stability of the drug and the specific activity that is sufficient to
meet the sensitivity requirements of assay.

14. 2. PREPARATION PURIFICATION OF DRUG-PROTEIN
CONJUGATE (LIGAND-ANTIGEN) TO BEANALYZED
• It is possible to find the specific antibody for drug(antigen) as a result of sensitivity
reaction
• Several drugs which have induced antibodies production due their inherent
antigenicity like – penicillin, strychnine, tetracycline, sulphonamide and
procainamide. However their low specificity and limited availability makes their use
rather improbable.
• In most cases the drugs (analysed antigen) are bind with suitable carrier protein to
make conjugated antigen immunogenic.
• There are several reactive groups on protein carrier which can used for the purpose of
conjugation of standard drug(antigen). These groups include the terminal amino and
carboxyl groups, ε-amino group of lysine, the carboxyl group of aspartic and glutamic
acid, the phenolic group of tyrosine.

15. • The most commonly used methods for conjugation are as follows:
• Carbodimide and glutaraldehyde reaction
• Carbony-diimidazole reaction
• Scotten-baumann reaction
• Mannich reaction
• Diazotization reaction
• The method chosen for conjugated will depend on the functional groups available
for coupling the drug to protein and no. of drugs molecules which are to be
coupled to protein.

16. 3. PREPARATION & CHARACTERIZATION OF THE
SPECIFICAND HIGHAFFINITYANTIBODIES
• Preparation:
 Once the pure antigen prepared then it is emulsified into equal volumes of
saline and Freud’s adjuvant (contain alum ppts, natural detergents, mineral oils,
killed mycobacterium) to get final concentration of 10 to 50mg/ml.
 One ml of the emulsion is injected intradermally, subcutaneously, and/or
intramuscularly at weekly or monthly intervals into multiple sites of a suitable
animal species such as rat, guinea pigs or rabbits. A suitable animal species is
usually dictated by the size of the animal facilities.
 Animals are tested after 3-4 weeks and then blood is collected and separated.
The resulting blood containing antibody called antiserum. It is directly used in
assay and should be stored at 4°c.

17. • Characterization:
 Characterization of antiserum done by
fractionation, immunoadsorption or
immunosaturation technique.

18. 4.DEVELOPMENT OF SUITABLE SEPARATION TECHNIQUES
TO SEPARATE FREE FROM BOUND STANDARD DRUG
Several methods that employ physiochemical and immunological separation have been devised as
follows –
• Physical methods: Filtration, Chromatography, Electrophoresis, charcoal- dextran adsorption, and
ion exchange resin.
• Disadvantages: It is tend to be time dependent and harsh so they may remove bound drug from
antibody during separation.
• Chemical method: Organic solvents such as ethanol, dioxane and polyethylene glycol (PEG), and
salts such as sodium, zinc, ammonium sulfate.
• Disadvantages: Chemical precipitation may precipitate free as well as bound drug during
separation depending on physicochemical nature of drug.
• Second antibody method: it is most physiologic procedure to precipitate bound antigen. This
method employs, an antibody against gamma globulin of the animal species used to produce
antidrug antibody to the plasma serum to precipitate drug-antibody complex.
• Disadvantages: This technique have required prolong incubation time.

19. DIAGRAMMATIC REPRESENTATION OF RIA

20. OTHER RELATED IMMUNOASSAY
• Related methods include the use of antibodies to specifically bind antigenic
substances.
• They do not include competitive protein binding assays (CPBA) or receptor site
binding assay (RSBA).
• The most commonly related method is Immunoradiometric (IRM) – the technique
which employs an excess of labelled antibody to quantify the drug in the system.
The principle of the system displayed in figure as below.
SP
S +Ab* SAb* +Ab* SAb* + SPAb*
• S is unknown or standard antigen, Ab* is labelled antibody, and SP is the antigen
coupled to a solid support.
• SAb* fraction will remain in the supernatant, where as the SPAb* will be
coupled to the solid support.

21. Advantages:
• The antibody can be more readily labelled with high specific activity using 125I.
• No need of any specific separation technique as in RIA.
• Instead of using radio tracers several other methods rely on alternate labeling
procedures. They include hemaglutination-inhibition, fluorescence, and free
radical or spin label and last Enzyme Immuno-Assays.

22. APPLICATION OF RIA
Analysis of hormones, vitamins, metabolites, diagnostic markers: ACTH, FSH,
triiodothyronine (T3) and thyroxine (T4), Glucagons, Insulin, Testosterone, vitamin B12,
prostaglandins, glucocorticoids.
Therapeutic drug monitoring: Barbiturates, morphine, digoxin.
Diagnostic procedures for detecting infection: HIV, HepatitisA, B etc.
Tumour markers: RIA of tumour markers such as alpha-fetoprotein (AFP),
carcinoembrionic antigen (CEA), b-HCG for choroid -carcinoma, prostate specific
antigen (PSA) for prostate cancer, are available for detection and management of cancer.
 Non clinical application: Such as veterinary science, food processing industry, drug
industry, forensic science and environmental monitoring.