The document discusses two types of immunoassays - ELISA and RIA. ELISA (enzyme-linked immunosorbent assay) is commonly used to detect antibodies, antigens, and proteins by immobilizing them on a microplate and detecting them using enzyme-linked antibodies. RIA (radioimmunoassay) uses radiolabeled antigens or antibodies to detect substances and can detect concentrations as low as nanograms or picograms per milliliter. Both techniques rely on the specificity of the antigen-antibody reaction and are used in clinical diagnostics, research, and quality control testing.

1. KUVEMPU UNIVERSITY
Sahyadri Science College, Shimoga
PG Department of Biotechnology
Topic : Immunoassays - ELISA and RIA
PRESENTED BY UNDER THE GUIDANCE
Ms. Bi Bi Ayesha Dr. Jamuna K S
I MSc, II Sem Lecturer
Sahyadri Science College Sahyadri Science College
Shimoga Shimoga

2. Contents
 Introduction
 ELISA
 Introduction
 History
 Principle of ELISA
 Procedure of ELISA
 Types of ELISA
 Advantages of ELISA
 Disadvantages of ELISA
 Applications of ELISA
 RIA
 Introduction
 History
 Principle of RIA
 Procedure of RIA
 Advantages of RIA
 Disadvantages of RIA
 Applications of RIA
 Conclusion
 References

3. Introduction
 Immunoassays are bioanalytical methods that use the specificity of an antigen-
antibody reaction to detect and quantify target molecules in biological samples.
 These methods are frequently used in clinical diagnostics, drug discovery, drug
monitoring, and food testing.
 The first immunoassay developed was described by Yalow and Berson.
 They used radiolabelled insulin to assess the concentration of insulin in human
plasma, and thus developed the first radioimmunoassay (RIA).
 Antigens were immobilized on a microplate well, incubated with antiserum, and
then the concentration of antibody in the antiserum was quantified using an
enzyme-linked anti-immunoglobulin antibody. This method is the enzyme-linked
immunosorbent assay (ELISA).

4. Enzyme-linked immunosorbent assay
(ELISA)

5. Enzyme-linked immunosorbent assay (ELISA)
 ELISA is an immunological assay commonly used to measure
antibodies, antigens, proteins and glycoproteins in biological
samples.
 Originally described by Peter Permann and Eva Engvall (1971),
the method enables analysis of protein samples immobilized in
microplate wells using specific antibodies.
 ELISAs are typically performed in 96-well or 384-well
polystyrene plates, which passively bind antibodies and
proteins.
 ELISA has been used as a diagnostic tool in medicine, plant
pathology, and biotechnology, as well as a quality control check
in various industries.

6. HISTORY

7. Principle of ELISA
 The most crucial element of an ELISA is a highly specific
antibody-antigen interaction.
 Antigen of interest isabsorbed on to surfacesorbent
 Antigenisrecognized onlybythespecific antibodyimmune
 Thisantibody isrecognized by second antibody (immuno)which has
enzyme attached (enzyme linked)
 Hencethename,Enzyme-linked immunosorbent assay
 Substrate reacts with enzyme to produce product usually
depicted by coloured.
 The development of colour using a chromogenic substrate
corresponds to the presence of the antigen.

8.  In an ELISA, an enzyme is linked to an antibody that has been
specifically raised against an antigen of interest.
 Biological samples containing the target antigen are allowed
to bind the enzyme-labeled antibody and the reaction is
visualized using an enzyme-specific substrate which is added
to the reaction mixture.
 The enzyme-specific substrate binds to the enzyme attached
to the antibody, generating a colored product which can be
detected using chromogenic or chemiluminescent imaging
techniques.
 ELISA has a high specificity and sensitivity, thus it is frequently
used for high-throughput screening of antibodies or drugs.
Principle of ELISA

9.  The most commonly used enzyme labels are horseradish peroxidase (HRP)
and alkaline phosphatase (AP).
 Other enzymes have been used as well; these include β-
galactosidase, acetylcholinesterase, and catalase.
 A large selection of substrates is available commercially for performing ELISA
with an HRP or AP conjugate.
 The choice of substrate depends upon the required assay sensitivity and the
instrumentation available for signal-detection (spectrophotometer,
fluorometer, or luminometer).

10. Procedure of ELISA
 An antibody is attached to a polystyrene plate
which is a solid surface and is attracted or has an
affinity towards bacteria, other antibodies and
hormones.
 A microtiter coated with antigen is filled with this
antigen-antibody mixture after which free
antibodies are removed by washing.
 A second antibody specific to primary antibody is
added which is usually conjugated with an
enzyme.
 Free enzyme-linked secondary antibodies are
removed by washing the plate.
 Finally, the substrate is added. The substrate is
converted by the enzyme to form a coloured
product, which can be measured by
spectrophotometry.

11. Types of ELISA
ELISA tests can be classified into three types depending upon the different
methods used for binding between antigen and antibodies, namely:
 Direct ELISA
 Indirect ELISA
 Sandwich ELISA
 Competitive ELISA

12. Direct ELISA
 In a direct ELISA, the antigen is bound to the bottom of the microplate well, and then
it is bound by an antibody that is specific to the antigen and also conjugated to an
enzyme or other molecule that enables detection.

13. Indirect ELISA
 In an indirect ELISA, the antigen is bound to the bottom of the microplate
well, then an antibody specific to the antigen is added.
 A secondary antibody, conjugated to an enzyme or other detection molecule,
is then bound to the first antibody.

14. Sandwich ELISA
 For the sandwich ELISA, two antibodies specific to two different epitopes on the
target antigen are used.
 The capture antibody is bound to the bottom of the microplate well and binds one
epitope of the antigen.
 The detection antibody binds to the antigen at a different epitope and is conjugated
to an enzyme that enables detection. (If the detection antibody is unconjugated,
then a secondary enzyme-conjugated detection antibody is required).

15. Competitive ELISA
 In a competitive ELISA, a reference antigen is bound to the bottom of microplate wells.
 Sample plus antibody are added to the wells, and if there is antigen present in the
sample, it competes with reference antigen for binding to the antibody. Unbound
material is washed away.
 The more antigen was in the sample, the less antibody ends up bound to the bottom
of the wells by the reference antigen, and the lower the signal.

16. Indirect ELISA
Sandwich ELISA
Competitive ELISA
Procedure

17. ELISPOT
(enzyme-linked immunospot assay)
 It 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.

18. In-cell ELISA
 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).

19. Variants of ELISA
 Although many variants of ELISA have been developed and used in different
situations, they all depend on the same basic elements:
 Coating/capture – direct or indirect immobilization of antigens to the
surface of polystyrene microplate wells.
 Plate blocking – addition of irrelevant protein or other molecule to
cover all unsaturated surface-binding sites of the microplate wells.
 Probing/detection – incubation with antigen-specific antibodies that
affinity-bind to the antigens.
 Signal measurement – detection of the signal generated via the direct
or secondary tag on the specific antibody.

20. ELISA plates
 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).
 When developing a new ELISA for a specific antigen, the first step is to
optimize the plate-coating conditions for the antigen or capture antibody.
 Plate coating is achieved through passive adsorption of the protein to the
plastic of the assay microplate. This process occurs though hydrophobic
interactions between the plastic and non-polar protein residues.

21. Different types of pre-coated microplates for
ELISA
ELISA plate Coating Applications
Modified polymer surfaces Various modifications to the plate
surface to increase hydrophobicity or
hydrophilicity
Enhance passive binding of biomolecules
based on their physiochemical characteristics
Antibody-binding plates Protein A, G, L, or A/G Binds to the FC region (VL for protein L) of
capture antibodies to properly orient while
leaving antigen binding capability
Biotin-binding plates Streptavidin or neutravidin Binds small biotinylated peptides and other
small molecules that are difficult to bind by
passive adsorption
Fusion-tag binding plates Glutathione (GST tag binding) or nickel
or copper coated (His tag binding)
Study of genetically engineered fusion
proteins or protein-protein interactions

22. Detection strategies for ELISA
Chromogenic
(colorimetric)
Fluorescence Chemiluminescence
Sensitivity
Equipment required Standard absorbance plate
reader
Fluorometer Luminometer plate reader
Enzyme HRP or AP Fluorescent tag or HRP
(with chemifluorescent
substrates)
HRP or AP
Advantages Direct visualization, high
reproducibility between
plates
High reproducibility
between plates, wide
dynamic range
Most sensitive detection
strategy, wide dynamic
range
Considerations Requires black microplates Requires opaque or black
microplates

23. Advantages of ELISA
 Results fetched from ELISA gives an accurate diagnosis of a particular disease
since two antibodies are used.
 Can be carried out for complex samples as the antigen is not required to get
purified to detect.
 It is highly responsive since direct and indirect analysis methods can be
carried out.
 It is a rapid test, yields results quickly.
 Possible detection for ELISA ranges from the quantitative, semi-quantitative,
standard curve, qualitative, calibration curve models etc.
 Easier to perform and uncomplicated process as compared to other assays
which require the presence of radioactive materials.

24. Disadvantages of ELISA
 Labour – intensive and expensive to prepare antibody
 Sophisticated techniques and expensive culture media are required.
 High possibility of false positive/negative.
 Insufficient blocking of immobilized antigen results in false results.
 Antibody instability.
 Refrigerated transport and storage are required as an antibody is a
protein.

25. Applications
 The presence of antibodies and antigens in a sample can be determined.
 It is used in the food industry to detect any food allergens present.
 To determine the concentration of serum antibody in a virus test.
 Several diseases are diagnosed using ELISA - e.g. Ebola, Pernicious anaemia, AIDS,
Rotavirus, Lyme disease, Syphilis, Toxoplasmosis, Zika virus, Carcinoma of the epithelial
cells, SARS-CoV-2 etc.
 During a disease outbreak, to evaluate the spread of the disease, e.g. during recent
COVID-19 outbreak, rapid testing kits are being used to determine presence of antibodies
in the blood sample.
 HCG protein which indicates pregnancy is detected by ELISA. A combination of blood or
urine sample and purified HCG linked to an enzyme is added to the system. If HCG is
absent in the test sample, then only the linked enzyme binds to the solid surface.
 The more the substance of interest is present, the more reaction takes place and less of
linked enzyme binds to the solid surface. These reactions are indicated usually with a
change in the colour of the solution.

26. Radioimmunoassay (RIA)

27. Radioimmunoassay (RIA)
 A radioimmunoassay (RIA) is an immunoassay that
uses radiolabeled molecules to quantify hormones, drugs, and viral antigens.
 A RIA is a very sensitive in vitro assay technique used to measure
concentrations of substances, usually measuring antigen concentrations by
use of antibodies.
 The radioimmunoassay technique, as the name implies, achieves sensitivity
through the use of radionuclides and specificity that is uniquely associated
with immunochemical reaction.
 In contrast, an immunoradiometric assay (IRMA) is an immunoassay that uses
radiolabeled molecules but in an immediate rather than stepwise way.
 A radioallergosorbent test (RAST) is an example of radioimmunoassay. It is
used to detect the causative allergen for an allergy.

28. History
 Developed in 1959 by Rosalyn Yalow and
Solomon Berson for measurement of
insulin in plasma.
 It represented the first time that hormone
levels in the blood could be detected by an
in vitro assay.
 In 1977 Yalow received the Nobel Prize for
her and Berson’s development of RIA

29. Radioimmunoassay can detect
substance like :
• Hormones
• Vitamins
• Serum Protein
• Drugs
• Infective Agent
Sensitivity of
Radioimmunoassay :
It can detect substance from a
range of Nano gram(ng) to Pico
gram(pg) concentration.
Qualitative as well as Quantitative analysis.

30. Principle of Radioimmunoassay (RIA)
 Radioimmunoassay uses radioisotope-labeled purified known antigen which
competes with unlabeled (unknown) antigen for binding sites on a known
amount of antibody.
 The Ag-Ab complexes that form between the antigen and antibody can then be
precipitated using the second antibody and the amount of radioactivity of the
bound complex is measured by means of radioisotope analyzers and
autoradiography.

31. Radioimmunoassay (RIA) method
 Radioimmunoassay is an old assay technique but it is still a
widely used assay and continues to offer distinct advantages
in terms of simplicity and sensitivity.
 RIA is a competitive binding assay. The target antigen is
labelled radioactivity and bound to its specific antibodies.
 Radioactivity: 125-I labels are usually applied although other
isotopes such as C14 and H3 have also been used.
 The antibody & labelled antigen are always present as
limiting factors and the concentrations of unlabelled antigens
(sample) under examination is increased continually

32. Advantages of RIA
 Radio immune assay is very sensitive technique used to measure
concentration of antigen without the need to use a bioassay.it can measure
one trillionth (10-12) of a gram of material per millilitre of blood.
 It is structurally specific as antigen: antibody reaction are highly specific.
 It is indirect method of analysis
 It is a saturation analysis as active reagent added in smaller quantity than that
of analyte.
 High possible to detect pictogram of antigen

33. Disadvantages of RIA
 Prolonged reaction time (in days) as a consequence highly diluted reagent is used.
 Radioisotopes are costly.
 Possible health hazards due to handling of radioisotopes.
 All the reagents must be added precisely.
 Limited assay range.
 Lack of direct linear relationship between analyte concentration and signal response.
 Difficulty of automation.
 Lengthy counting time.

34. Applications of RIA
 Endocrinology
 Insulin, HCG, Vasopressin
 Detects endocrine disorders
 Physiology of endocrine function
 Pharmacology
 Morphine
 Detect drug abuse or drug poisoning
 Study drug kinetics
 Epidemiology
 Hepatitis B
 Clinical Immunology
 Antibodies for inhalant allergens
 Allergy diagnosis
 Oncology
 Carcinoembryonic antigen
 Early cancer detection and diagnosis

35. Conclusion

36. References
 Kindt TJ, Goldsby RA, Osborne BA, Kuby J. Kuby immunology. Macmillan; 2007.
 Patrono C, Peskar BA, editors. Radioimmunoassay in basic and clinical
pharmacology. Springer; 1987 Jan 1.
 Rana SV. Biotechniques Theory & Practice. Rastogi Publications; 2008.
 Shan J, Toye P. Journal of Immunoassay and Immunochemistry. 2009.

37. Thank You