2. Immunoassay
Immunoassays are bioanalytical methods in which the quantitation of the
analyte depends on the reaction of an antigen (analyte) and an antibody.
TYPES OF IMMUNOASSAYS
Enzyme immunoassay
Fluoro immunoassay
Luminescence immunoassay
Optical immunoassay
Radio immunoassay
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3. Enzyme immunoassay
enzyme immunoassay includes all assays based on the measurement of enzyme
labelled antigen or antibody.
Homogeneous EIA: no need to separate the bound and free fractions so that the
test can be completed in one step. Eg. enzyme multiplied immunoassay technique
(EMIT)
Heterogeneous EIA: It requires the separation of the free and bound fractions
either by centrifugation or by absorption on solid surfaces and washing. Eg.
Enzyme Linked Immunosorbent Assay (ELISA).
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4. Enzyme Linked Immunosorbent
Assay (ELISA)
This technique involves the use of an immunosorbent, an absorbing
material specific for one of the components of the reaction, the antigen
or antibody.
Procedure:
wells of a microtiter plate are coated with antibody
After thorough washing, the particulate is added incubated overnight at 4⁰C
or for 2 hours at 37⁰C
wells are washed and guinea pig antiserum added labeled with alkaline
phosphatase, added and incubated at 37⁰C for one hour.
After washing, a suitable substrate (para nitrophenyl phosphate) is added at
room temperature positive produce yellow color.
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6. USES OF ELISA
HIV detection
Infectious diseases like hepatitis, cytomegalovirus Ig M, Ig G, dengue Ig G,
influenza etc.
Rotavirus detection in fecal specimens and enterotoxin of E. coli in feces.
Mycobacterial antibody detection in tuberculosis
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7. Enzyme multiplied immunoassay
technique
In this type of assay, a sample of interest with the analyte (drug) is added
to a fixed quantity of enzyme bound drug, and the anti-drug antibody.
After addition of substrate, absorbance measurements are taken at time
intervals to determine the speed of the enzyme reaction.
PROCEDURE
Mix sample containing drug with antibody
Add fixed quantity of enzyme bound drug
Add substrate and cofactor
Measure absorbance 15-45 seconds after substrate addition
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8. 8
USES OF EMIT
Determination of serum digoxin, cocaine etc.
Used for drugs, hormones and metabolite determination
Used for whole blood, serum or urine.
Assay for anti-epileptic drugs in serum
9. ADVANTAGES OF EIA
Rapid and relatively cost effective
Specific and sensitive assays of wide applicability
No radiation hazards.
Separation step may not be required
Manipulations are simple
DISADVANTAGES OF EIA
They are species specific
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10. FLUORO IMMUNOASSAY
Fluorescence is the property of absorbing light rays of one particular
wavelength and emitting rays with a different wavelength
This technique is mainly used to identify specific antibodies or antigen.
Antibody identification is usually performed on blood (serum).
In this technique visualization of a specific protein or antigen in cells or
tissue sections by binding a specific antibody
fluorescent dye such as fluorescein isothiocyanate is used
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12. Indirect immunofluorescence
The test serum is incubated with antigen immobilized on a 96-well plate
or microscope slide. Secondary antibody labeled with fluorescence are
added
After washing any bound secondary antibodies can be detected by
shining UV light on the slide
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14. LUMINISCENCE IMMUNOASSAY
Luminescence is a generic form that covers a range of processes which
produces light.
Chemiluminescence is light emission that arises during the course of a
chemical reaction. Bioluminescence is a special type of
chemiluminescence found in biological systems in which a catalyst
protein increases the efficiency of chemiluminescent reaction.
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15. CHEMILUMINESCENT IMMUNOASSAY
It involves a luminescent substance as a label. The growing success of this technique in
pharmaceutical analysis due to its high performance, low detection limits, and good
precision.
It is the emission of light caused by a chemical reaction typically an oxidation reaction
PROCEDURE
Monoclonal antibody coated well, test specimen added
HRP (horseradish peroxide) labeled antibody conjugate
Incubate for 1 hour at 37⁰C
Remove unbound enzyme labeled antibody add chemiluminescence reagent
Read relative light unit with luminometer.
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17. APPLICATION
Determination of bioanalyte in clinical specimen
Chemiluminescence detection in liquid chromatography
Chemiluminescence detection in capillary electrophoresis
Chemiluminescence in DNA analysis
ADVANTAGES
Chemiluminescent assay has an excellent sensitivity comparable to EIA & RIA
very little reagent is used, they are also quite inexpensive to perform
The relatively high speed of detection also means a faster turnaround time.
DISADVANTAGES
False result may be obtained if there is lack of precision in injection of the
H2O2 or if some biological materials such as urine or plasma cause
quenching of light emission
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18. OPTICAL IMMUNOASSAY(OIA)
Optical immunoassay is based on the interaction of antigen-antibody complexes
on inert surfaces. Specific binding of antibody increases the thickness of the
reactants on the surface and changes the color of light reflected from the surface
Procedure:
The test sample was mixed with bioanalytical antibody solution in the ratio of 1:1(v/v)
and incubated for 2 min
The mixture 40ug was placed on the center of the chip and incubated for 10 min
the chip was then rinsed, dried and HRP- avidin conjugate was dispensed onto each chip
and incubated for 5 min
Substrate solution was dropped onto center of the chip after rinsing and incubated for 5
min. After final washing and drying cycle, the results were observed and recorded
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20. APPLICATION
For snake toxin and venom detection.
For snake species identification
For detection of Streptococcal Pharyngitis
ADVANTAGES
It is an easily interpretable method
More sensitive than routine culture method
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21. RADIO IMMUNOASSAY
The principle of RIA is based on the antigen- antibody reaction in which
radiolabeled antigen (exogenous) competes with endogenous antigen for
the limited sites of the specific antibody against the same antigen.
The radiolabeled antigen should have similar biological activity and
immunogenicity like that of native antigen
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22. Procedure
The assay is carried out in well plates where the inner surface of well is coated
with antibodies.
Radioactive antigen is added to all these wells which forms a complex with
antibodies.
The calibrator (unlabeled antigen of known concentration) is added to multiple
wells.
Antigen of unknown concentration(sample) is also added to one of the wells.
Competition between labeled and unlabeled antigen takes place due to limited
binding sites present in antibodies.
After washing unreacted antigen and antibody measured for radioactivity using
gamma or scintillation counter
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24. APPLICATION
Blood bank screening for the hepatitis virus
Early cancer detection
Measurement of growth hormone level
Diagnosis and treatment of peptic ulcer
RIA of thyroid hormones
RIA of fertility related hormones
Tumour marker
RIA of drugs
Drugs such as phenytoin, theophylline, cyclosporine, morphine, gentamycin, anti-
depressants etc. are estimated by RIA.
Non clinical application
Measurement of aflatoxins in food items. Aflatoxins are the secondary metabolites of the
fungi, aspergillus. Aflatoxins are potentially carcinogenic and hence monitoring levels of
aflatoxins contamination is mandatory.
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25. ADVANTAGES
High specificity and hence there are no interference from other substances.
High sensitivity-up to few pictograms of antigen can be determined when
antibodies with high affinity are used.
High precision
Applicable to wide variety of compounds in various fields-pharmacology,
endocrinology, oncology, epidemiology, clinical immunology etc.
DISADVANTAGES
Special expertise and safety precautions are required as radioactive materials
are used and disposed
Expensive compared to other methods, as radioactive materials are used
Development of specific antibodies to antigen
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26. CONCLUSION
Immunoassays are bioanalytical methods have been widely used in many
important areas of pharmaceutical analysis such as diagnosis of diseases,
therapeutic drug monitoring, clinical pharmacokinetic and bioequivalence
studies in drug discovery and pharmaceutical industries.
The importance and widespread of immunoassay methods in
pharmaceutical analysis are attributed to their inherent specificity, high-
throughput, and high sensitivity for the analysis of wide range of analytes
in biological samples.
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27. REFERENCE
Jenni Punt, Sharon A. Stranford, Patricia P. Jones, Judith A. Owen;
Immunology, 8th edition; W.H Freeman and company; New York.
KENNETH MURPHY & CASEY WEAVER; Janeway’s Immunobiology 9th edition;
Blackwell Scientific Publication; London,
RONALD J. HARBECK, JERI TEAGUE, JOURNAL OF CLINICAL MICROBIOLOGY,
Novel, Rapid Optical Immunoassay Technique for Detection of Group A
Streptococci from Pharyngeal Specimens: Comparison with Standard Culture
Methods, Apr. 1993, p. 839-844.
Marja E. Koivunen, Richard L. Krogsrud; Principles of Immunochemical
Technique Used in Clinical Laboratories; August 2006 _ Volume 37. Number
8.
Ibrahim A. Darwish; International journal of Biomedical science, Immunoassay
Methods and their Applications in Pharmaceutical Analysis: Basic
Methodology and Recent Advances, vol. 2; no. 3; September 2006
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