RADIOIMMUNOASSAY (RIA) & ENZYME-LINKED

IMMUNOSORBENT ASSAY (ELISA)

MOHAMMED MOBEEN

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

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

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.

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.

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).

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.

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.

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.

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).

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

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.

Preparation of the Specific Antibody

Antigen

Injected intradermally into rabbits or guinea pigs

Antibody production

Antibodies recovered from the serum

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.

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.

Assay ProcedureAdd 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

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.

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”.

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

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.

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.

Types of ELISA1. 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.

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.

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

Indirect ELISAMicrotitre 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

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).

Advantages and Disadvantages of ELISA

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.

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.

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.

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/ApplicationSupportKnowledgebase/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.

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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