The following presentation contains helpful information regarding Radioimmunoassay (RIA) and Enzyme-Linked Immunosorbent Assay (ELISA), including their history, introduction, advantages, procedures and applications.

1. RADIOIMMUNOASSAY
(RIA) & ENZYME-LINKED
IMMUNOSORBENT ASSAY
(ELISA)
MOHAMMED MOBEEN

2. Contents
 Radioimmunoassay (RIA):
1. Principle of Radioimmunoassay
2. General Procedure of Radioimmunoassay
3. Schematic for Radioimmunoassay
4. Merits and Demerits of RIA
5. Instrumentation for RIA
6. Requirements of RIA
7. Preparation & Radiolabelling of the Antigens
8. Preparation of the Specific Antibody
9. Development of the Assay System
10.Assay Procedure
11.Applications of RIA

3. Contents
 Enzyme-Linked Immunosorbent Assay (ELISA):
1. Principle
2. Ideal Characteristics of Enzyme Labels
3. Advantages of ELISA
4. Types of ELISA
5. Direct ELISA or Double Antibody Sandwich Assay
6. Indirect ELISA
7. Common ELISA formats (Direct vs. Sandwich Assays)
8. Advantages and Disadvantages of ELISA
9. Applications of ELISA
 Conclusion
 References

4. Radioimmunoassay (RIA)
Introduction:
• Radioimmunoassay was introduced by Berson and Yalow in 1959 as an in vitro
assay for insulin in human plasma.
• It is a serological method based on immunological antigen-antibody reactions.
• It makes use of radiolabeled substrate to measure trace amounts of haptens
(drugs, hormones, steroids, antigens etc.) in the biological samples.
• It is preferred when the trace quantities of the substrate is difficult to measure
by other analytical techniques.

5. Principle of Radioimmunoassay
• Principle: The basic principle of radioimmunoassay is competitive binding,
where a radiolabelled antigens ("tracer") compete with a unlabelled antigens
for a fixed, limited number of binding sites on the specific antibody or
receptor binding sites.
Fig. 1. Principle of a competitive binding radioimmunoassay. Radiolabeled antigen ("tracer") added to an antibody specific to the
antigen leads to formation of an antigen-antibody complex. Unlabeled antigen from a sample or standard solution can also bind
antibody, leading to unlabeled antigen-antibody complex. In the radioimmunoassay, the amount of radiolabeled antigen (tracer) is
held constant. Increasing amounts of unlabeled antigen in the sample will compete with tracer for binding to the antibody, leading to
more unlabeled antigen-antibody complex.

6. General Procedure of Radioimmunoassay
Fig. 2. a) Sample containing a high amount of antigen. The unlabeled antigen competes for binding to the antibody in the tube or well.
b) Sample containing no or low amounts of antigen. Antibody is bound by the radiolabeled antigen (tracer).

7. Schematic for Radioimmunoassay
Fig. 2. Schematic for a radioimmunoassay. Radioactive antigen ("tracer") is added to the antibody, followed by addition of unlabeled
antigen (from sample or from standard). The antigen-antibody complexes formed are precipitated using a precipitating reagent (in
the example shown, a secondary antibody) to separate bound and free tracer.

8. Merits and Demerits of RIA
• Merits:
• Useful for measuring trace amounts of antibodies, drugs and their
metabolites, hormones, nucleic acids, prostaglandins, proteins, viral
antigens, vitamins etc. in biological samples.
• Requires very small amounts of sample i.e. only 5-50 picomoles/ml.
• Simple, rapid, precise, accurate, specific and convenient method.
• Reliable and reproducible.
• Highly specific: Immune reactions are specific.
• High sensitivity: Immune reactions are sensitive.

9. Merits and Demerits of RIA
• Demerits:
• Requires special counting equipment.
• Radiation hazards: Uses radiolabelled reagents.
• Requires skilled personnel and is an expensive process.
• Laboratories require special license to handle radioactive materials.
• Requires special arrangements for:
• requisition and storage of radioactive materials.
• radioactive waste disposal.

10. Instrumentation for RIA
• Centrifuge: For separating the bound radiolabelled antigens from the
unbound radioalabelled antigens.
• Radioactive counters:
1. Scintillation counters: Radioactive isotopes that emit β-radiatons are
used (Eg: 3 H, 14 C).
2. Gamma counters: Radioactive isotopes that emit γ-radiations are used
(Eg: 124 I, 125 I, 131 I).

11. Requirements for RIA
1. Preparation and characterisation of the antigen (ligand to be analysed)
2. Radiolabelling of the antigen
3. Preparation of specific antibody
4. Development of assay system

12. Preparation and Radiolabelling of the Antigens
• Antigens prepared by:
• Synthesis of the molecule
• Isolation from natural sources
• Radiolabelling (Tagging procedure):
• 3 H, 14 C or 125 I are used as radioactive tags.
• Antigens are tagged to 3 H, 14 C or 125 I.
• Tagging should NOT affect the antigenic specificity & antigenic activity.

13. Preparation of the Specific Antibody
Antigen
Injected intradermally
into rabbits or guinea pigs
Antibody production
Antibodies recovered
from the serum

14. Preparation of the Specific Antibody
• Some ligands are not antigenic:
• Ligands like hormones, steroids and drugs are known as “haptens”.
• Eg: Gastrin, Morphine.
• Haptens which are conjugated to albumin are antigenic in nature.

15. Development of the Assay System
• Separation of unbound antigens from the mixture is a very crucial step.
• This achieved by binding the antibodies to the microtitre well’s surface (solid
phase RIA).
• Antigens bound to the fixed antibodies remain stuck to the inner surface.
• Decanting and washing the well removes unbound antigens.
• Other techniques of separation – centrifugation.

16. Assay Procedure
Add known amounts of test sample + radiolabeled antigens to the microtitre wells
Incubate
Allow the reaction to complete
Decant and wash the contents of the well
All unbound antigens removed
Radioactivity remaining in the microtitre wells is measured by a counter (GM
counter, scintillation counter etc)
Intensity of radioactivity is inversely correlated with the concentration of antigens
present in the test sample
Sensitive to very low concentrations of antigens

17. Applications of RIA
• Used to assay drugs like amphetamine, barbiturates, clonazepam,
chlordiazepoxide, digoxin, digitoxin, flurazepam, hydromorphone,
hydrocodone, LSD, morphine, pentazocine.
• Analysis of vitamins like riboflavin and folic acid.
• Analysis of hormones like aldosterone, testosterone, dihydrotestosterone,
estrone, insulin, glucagon, growth hormones, thyroxin, triiodothyronine and
tropic hormones like ACTH, FSH, LH etc.
• Blood bank screening for hepatitis-B surface antigen.
• In the early detection of cancer and tuberculosis.
• In testing the functioning of thyroid gland using 137 I.
• In the diagnosis and treatment of peptic ulcers, thyroid disorders etc.
• In determining RBC volume and whole blood volume.

18. Enzyme Linked Immunosorbent Assay (ELISA)
Introduction:
• ELISA was developed in 1970 to overcome the limitations of RIA.
• It is an in vitro non-isotopic immunoassay that makes use of enzyme-linked
anti-globulins and substrates for the detection and quantification of antigens
or antibodies in biological fluids.
• In place of radioactive isotopes, ELISA uses an enzyme as a label which gives
the assay its name as “enzyme-linked”.

19. Principle of ELISA
• ELISA is based on specific interaction between antigen and their
corresponding antibodies.
Immunoreactant (either antigen or antibody) is coated to a solid phase support in microtitre wells
Respective antigen or antibody is added to it
Enzyme linked antibody is added to the above formed antigen-antibody complex
Colourless substrate is added to the well
Enzyme catalyses the colorless substrate to a colored product
Detection either visually or spectrophotometrically
Optical density of the resulting color is proportional to the concentration of analyte in the sample

20. Ideal characteristics of enzyme labels
• Enzyme labels should have high specific reactivity.
• These should be easily coupled to ligands and the labelled complex must be
stable.
• The reactivity should be retained after linking of the enzyme to the
antigen/antibody.
• The chosen enzymes shouldn’t be normally present in the patient’s samples.
• Examples of enzyme labels: Horse radish peroxidase, Alkaline phosphatase,
Glucose oxidase.

21. Advantages of ELISA
• Very sensitive, even towards nanogram levels or lower.
• Reproducible.
• Minimal reagents are required.
• Can be used for both qualitative & quantitative purposes:
• Qualitative assays: Eg: HIV testing
• Quantitative assays: Eg: Therapeutic Drug Monitoring
• Has a much wider scope; the wells can be coated with either antigens or
antibodies.
• Suitable for automation; is a high speed process.
• No radiation hazards.

22. Types of ELISA
1. Direct ELISA or Double Antibody Sandwich Assay:
• Used to detect the presence of antigens and is preferred when the
antibody labelled enzyme can directly react with the antigen.
• Ex: Detection of Helicobacter pylori in human stool.
2. Indirect ELISA:
• Used to detect the presence of antibodies.
• Ex: Diagnosis of HIV infection.

23. Types of ELISA
• Other variants of the standard ELISA also exist:
 ELISPOT (enzyme-linked immunospot assay) refers to ELISA-like capture
and measurement of proteins secreted by cells that are plated in PVDF-
membrane-backed microplate wells. It is a "sandwich" assay in which the
proteins are captured locally as they are secreted by the plated cells, and
detection is with a precipitating substrate. ELISPOT is like a Western blot in
that the result is spots on a membrane surface.
 In-cell ELISA is performed with cells that are plated and cultured overnight
in standard microplates. After the cultured cells are fixed, permeabilized
and blocked, target proteins are detected with antibodies. This is an
indirect assay, not a sandwich assay. The secondary antibodies are either
fluorescent (for direct measurement by a fluorescent plate reader or
microscope) or enzyme-conjugated (for detection with a soluble substrate
using a plate reader).
• ELISA is nearly always performed using 96-well or 384-well polystyrene plates
and samples in solution (i.e., biological fluids, culture media or cell lysates).
This is the platform discussed in the remainder of this article.

24. Direct ELISA or Double Antibody Sandwich Assay
Microtitre wells are coated with suitable purified antibodies
Biological sample containing the antigens is added to the wells
Incubation is done till the antigen-antibody reaction is complete
Washing is done to remove any unbound antigens
Addition of enzyme-labelled antibody
Incubation is done till an antibody-antigen-labelled antibody complex i.e. a layered
sandwich is formed
Washing to remove any unbound labelled antibody
Addition of a colourless substrate and subsequent incubation
Enzyme + Substrate  Product  Colour is measured visually or spectrophotometrically
Colour is proportionally related to antigens in the biological sample

25. Indirect ELISA
Microtitre wells are coated with suitable inactivated antigens
Biological sample containing the antibodies is added to the wells
Incubation is done till the antigen-antibody reaction is complete
Washing is done to remove any unbound antibodies
Addition of enzyme-labelled anti-antibody or anti-human immunoserum globulin (anti-HISG)
Incubation is done till an antigen-antibody-labelled anti-antibody complex is formed
Washing is done to remove any unbound labelled anti-antibody
Addition of a colourless substrate and subsequent incubation
Enzyme + Substrate  Product  Colour (optical density) is measured spectrophotometrically
Colour is proportionally related to antibodies in the biological sample

26. Common ELISA formats (Direct vs. Sandwich Assays)
Fig. 4. Common ELISA formats. In the assay, the antigen of interest is immobilized by direct adsorption to the assay plate or by first
attaching a capture antibody to the plate surface. Detection of the antigen can then be performed using an enzyme-conjugated
primary antibody (direct detection) or a matched set of unlabeled primary and conjugated secondary antibodies (indirect detection).

27. Advantages and Disadvantages of ELISA

28. Applications of ELISA
• It is a major diagnostic tool in screening donated blood for the presence of
HIV-1 and HIV-2 viruses, hepatitis-C antibodies, hepatitis-B antibodies and
hepatitis-B antigen.
• Human chorionic gonadotropin (HCG) levels in urine can be measured using
ELISA to detect pregnancy within few days of conception.
• Used to detect serum antibodies to Mycobacterium tuberculosis
(Tuberculosis), Mycobacterium leprae (Leprosy), Brucella (Brucellosis),
Salmonella (Enteric diseases), Treponema pallidum (Syphilis), Vibrio cholera
(Cholera) and Yersinia (Plague).
• To detect serum antibodies produced against a variety of parasites causing
amoebiasis, malaria, Chagas disease and toxoplasmosis.
• To determine the time for ovulation by measuring LH levels.
• To assess thyroid activity by measuring thyroid stimulating hormone (TSH),
triiodothyronine (T3) and thyroxin (T4) levels.

29. Applications of ELISA
• To detect HIV, syphilis, chlamydial infections, hepatitis-B and C, toxoplasmosis,
UTI etc.
• To detect snake poisoning.
• To detect fungal diseases like candidiasis and aspergillosis.
• To assay oncoproteins like α-2-hepatoglobulin.
• To detect potential allergens in food and house dust.
• To measure rheumatoid factors and other auto-antibodies in autoimmune
diseases such as lupus erythematosus (LE), thyroiditis etc.
• To detect illicit drugs like cocaine, opiates and Δ9-tetrahydrocannabinol.

30. Conclusion
• Both RIA and ELISA are very accurate and effective analytical tools.
• ELISA happens to be an advancement of RIA, taking over the disadvantages
that RIA possessed.
• Even with their respective disadvantages, both the methods are still being
used and at the same time being further developed to minimize the errors and
maximize the productivity.
• Thus, it could be said that both these immunological assay methods are useful
in their own ways and have played an important role in the advancement of
pharmaceutical analytical techniques.

31. References
• ELISA; https://en.wikipedia.org/wiki/ELISA
• Radioimmunoassay; https://en.wikipedia.org/wiki/Radioimmunoassay
• An Overview of ELISA; Thermofisher Scientific;
https://www.thermofisher.com/ie/en/home/life-science/protein-
biology/protein-biology-learning-center/protein-biology-resource-
library/pierce-protein-methods/overview-elisa.html
• Radioimmuno-Assays; Perkin-Elmer;
http://www.perkinelmer.com/Resources/TechnicalResources/ApplicationSuppo
rtKnowledgebase/radiometric/radioimmunoassay.xhtml
• Yalow, ItS. and Berson, S.A.; Immunoassay of endogenous plasma insulin in
man; J. Clin. Invest., 1960; 39:1157-75.
• Ekins ItP.; The estimation of thyroxine in human plasma by an electrophoretic
technique; Clin. Chim. Acta.; 1960; 5:453-65.

32. References
• Odell, W.D., Daughadsy, W.H. (eds).; Principles of competitive protein binding
assays; Philadelphia i.B.; Lippincott Co.; 1971.
• Murphy, B.E.P., Pattee, Ci. and Gold, A.; Clinical evaluation of a new method
for the determination of serum thyroxine; J. Clin. Endocrinol. Metab.; 1966;
26:247-56.
• Hunter, W.M. and Greenwood, P.C.; Preparation of 1-131 labeled growth
hormone of high specificactivity; Nature (London); 1962; 194:495-96.
• Greenwood, F.C., Hunter, W.M. and Glover, iS.; The preparation of 1-131
labeled human growth hormone of high specific radioactivity; Biochem. 3.;
1963; 89:114-23.
• Rosa, U.; Protein radioiodination by an electrolyte technique; Strahlentherapie
(Sonderb)., 1965; 60:258-61.

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