detailed note on types with diagrams ,advantages and disadvantages ,applications

1. IMMUNOLOGICAL
ASSAYS
DEEKSHA.R.PAI
FIRST M.PHARM(QUALITY
ASSURANCE)
SRINIVAS COLLEGE OF
PHARMACY
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2. IMMUNOASSAY
• Immunoassay means a method to measure any particular
substance in a mixture using its specific-binding antibody.
• One of the merits of immunoassay is that we can measure a
substance that is present in a mixture of various contaminants.
• Immunoassays have become very popular in view of their high
sensitivity, safety, economy and simple instrument
requirements.
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3. ELISA
• ELISA is a biochemical technique used mainly in
immunology to detect the presence of an antibody or an
antigen in a sample.
• In this method the antigen or antibody is conjugated to an
enzyme.
• It is a plate based assays designed for detecting and
quantifying substances such as peptides, proteins, antibodies,
antigens and hormones.
• It involves detection of ‘analyte’ in a liquid sample using
liquid reagent (wet lab) or dry strips (dry lab).
• The test can be done in polystyrene tubes (macro-ELISA) or
polyvinyl microtitre plates (micro-ELISA).
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4. ELISA
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5. BASIC PRINCIPLES
Based on Basic Immunology Response
•Lock and Key Concept
–Antigen (key): substance when introduced into the body
produces antibodies
–Antibody (lock): protein in the body that is used by immune
system to identify and neutralize foreign targets (referred to as
antigens)
–Key fits into the lock
•Enzyme conjugate substrates
–Enzyme that converts colourless substrates to a colored product
–Bound to the antibody that is part of the antibody-antigen
complex OR
–Bound to a secondary antibody that binds with the antibody-
antigen complex
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6. ADVANTAGES OF ELISA TEST
• Sensitive: nanogram levels or lower
• Reproducible
• Minimal reagents
• Qualitative & Quantitative
Qualitative – Eg: HIV testing
Quantitative assays - Eg: TDM
• Greater scope : Wells can be coated with Antigens OR
Antibodies
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7. • NO radiation hazards
• Fast—90 samples tested in 2-3 hr
• Sensitivity (up to 10 pg/mL)
• Specificity (sample with high concentration contaminants)
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8. • Four common ELISA tests –based on the binding structure
between the Antibody and Antigen.
1.Direct-ELISA
2.Indirect-ELISA
3.Competitive-ELISA
4. Non-competitive-ELISA(Sandwich-ELISA)
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9. Direct ELISA
• It uses the directly labelling the antibody itself.
• Microwell plates are coated with a sample containing the
target antigen, and the binding of labelled antibody is
quantitated by a colourimetric, chemiluminescent, of
fluorescent end-point.
1. Apply a sample of known antigen to a surface, often in the
well of a microtitre plate. The antigen is fixed to the surface
to render it immobile.
2. The plate wells or other surface are then coated with blocking
buffer.
3. Detecting antibody(labelled by an enzyme), usually diluted in
blocking buffer, is applied to the plate for binding to the
antigen coated on the plate.
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11. • The plate is washed, so that unbound antibody is removed.
After this wash, only the antibody-antigen complexes remain
attached to the well.
• Apply a substrate which is converted by the enzyme to elicit a
chromogenic or fluorescent signal.
• View/quantify the result using a spectrophotometer or other
optical device.
• Positive and negative controls should always be included in
the test
• At every step, incubation and washing is done to wash off
unbound reagents.
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12. Advantages of Direct ELISA:
• Quick methodology since only one antibody is used.
• Cross-reactivity of secondary antibody is eliminated.
Disadvantage of Direct ELISA:
• Immuno-reactivity of the primary antibody may be reduced as
a result of labelling.
• Labelling of energy primary antibody label from one
experiment to another.
• Little signal amplification.
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13. Indirect ELISA
• For antibody detection, the wells of microtitre plates are
coated with antigen.
• Sera to be tested are added in these coated wells.
• If antibody is present,antigen-antibody reaction takes place.
• To detect this reaction, a goat antihuman immunoglobulin
antibody conjugated with an enzyme is added.
• A substrate is added and enzyme acts on substrate to produce a
colour .
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15. Advantages of Indirect ELISA :
• High sensitivity: More than one labelled antibody is bound
per antigen molecule;
• Flexible: Different primary detection antibodies can be used
with a single labelled secondary antibody;
• Cost-saving: Fewer labelled antibodies are required.
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17. • A Sandwich ELISA
1. Plate is coated with a capture antibody;
2. sample is added, and any antigen present binds to capture
antibody;
3. detecting antibody is added, and binds to antigen;
4. enzyme-linked secondary antibody is added, and binds to
detecting antibody;
5. substrate is added, and is converted by enzyme to
detectable form.
6. The positive results produces color which can be read by
ELISA reader. 17

18. Sandwich ELISA advantages:
• High specificity, since two antibodies are used the
antigen/analyte is specifically captured and detected.
• Suitable for complex samples, since the antigen does not
require purification prior to measurement.
• Flexibility and sensitivity, since both direct and indirect
detection methods can be used.
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19. COMPETITIVE ELISA TEST
 Here competition occurs between two antibodies for the same
antigen.
 It has been used for detection of HIV antibodies.
 The microtiter plate wells are coated with HIV antigen.
 Sera to be tested is added to these wells and incubated at
37degree C and washed
 Antigen-Antibody reaction occurs
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21.  To detect this reaction ,enzyme labelled specific HIV
antibodies are added
 These antibodies remain free because there is no antigen left
to react
 Substrate is added but there is no enzyme to act .Hence
positive results show no color.
 If serum to be tested is negative for antibodies, antigen is
there to combine with enzyme conjugated antibodies and
reacts with substrate to produce colour.
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22. Competitive ELISA advantages:
• High specificity, since two antibodies are used the
antigen/analyte is specifically captured and detected
• Suitable for complex samples, since the antigen does not
require purification prior to measurement
• Flexibility and sensitivity, since both direct and indirect
detection methods can be used
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23. APPLICATIONS
• Detection of HIV antibodies in serum.
• Detection of mycobacterial antibodies in tuberculosis.
• Detection of rotavirus in faeces.
• Detection of hepatitis B markers in serum.
• Detection of enterotoxin of E coli in faeces.
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24. • Detection of potential food allergens.
• Analysis of hormones, vitamins, metabolites, diagnostic
markers
• Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone,
vitamin B12, prostaglandins, glucocorticoids,
• Therapeutic drug monitoring:
• Eg :Barbiturates, morphine, digoxin.
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25. RADIOIMMUNOASSAY
Introduction:
• The technique was introduced in 1960 by Berson and Yalow
as an assay for the concentration of insulin in plasma.
• It represented the first time that hormone levels in the blood
could be detected by an in vitro assay.
• The sensitivity range is 0.0006–0.006 μg antibody/ml
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26. RIA
RADIOIMMUNOASSAY(RIA) is a very sensitive invitro assay
technique in which antibody or antigen is labelled with a
radioactive material(125I).
• It is used to measure concentrations of antigens using
specificity of antigen-antibody binding and quantization using
radioactivity.
• It is also called as binder-ligand assay where binder is the
component to which radioactive material is labelled and
ligand(antigen or antibody) is the component which is to be
detected.
• It is more specific and sensitive method and can detect antigen
upto picogram quantities.
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27. PRINCIPLE OF RIA
It involves three principles:
• An immune reaction i.e. antigen, antibody binding.
• A competitive binding or competitive displacement reaction.
(It gives specificity)
• Measurement of radio emission. (It gives sensitivity)
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29. PROCEDURE
 A known quantity of an antigen is made radioactive, frequently
by labelling it with γ-radioactive isotopes of iodine attached to
tyrosine.
 This radio labelled antigen is then mixed with a known amount
of antibody for that antigen.
 A sample of serum from a patient containing an unknown
quantity of same antigen is added.
 This causes the unlabelled(cold) antigen from serum to
compete with radio labelled antigen(hot) for antibody binding
site. 29

30.  As the concentration of cold antigen is increased , more of it
binds to antibody, displacing the radio labelled variant and
reducing the ratio of antibody bound radio labelled antigen to
free radio labelled antigen.
 The bound antigens are then separated from the unbound ones,
and the radioactivity of the free antigen remaining in the
supernatant is measured using a γ counter.
 The concentration of the test antigen can be calculated from
the ratio of the bound and total antigen labels , using a
standard dose response curve.
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31.  From these data, a standard binding curve, like the one shown
in red, can be drawn.
 The samples to be assayed (the unknowns) are run in parallel.
 After determining the ratio of bound to free antigen in each
unknown, the antigen concentrations can be read directly from
the standard curve.
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32. Advantages & disadvantages
Advantages
• Highly specific: Immune reactions are specific
• High sensitivity : Immune reactions are sensitive
Disadvantages
• Radiation hazards: Uses radiolabelled reagents
• Requires specially trained persons
• Labs require special license to handle radioactive material
• Requires special arrangements for requisition, storage of
radioactive material ,radioactive waste disposal.
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33. APPLICATIONS
• Detection of narcotic drugs
• Blood bank screening for the hepatitis (a highly contagious
condition) virus.
• Measurement of growth hormone levels, immunoglobulin's,
tumour markers
• Tracking of the leukaemia virus
• Diagnosis and treatment of peptic ulcers and
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34. • Endocrinology
– Insulin, HCG, Vasopressin
– Detects Endocrine Disorders
– Physiology of Endocrine Function
• Pharmacology
– Morphine
– Detect Drug Abuse or Drug Poisoning
• Study Drug Kinetics.
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35. • Clinical Immunology
– Antibodies for Inhalant Allergens
– Allergy Diagnosis
• Oncology
– Carcino-embryonic Antigen
– Early Cancer Detection and Diagnosis
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36. References
 Gupte S. The short textbook of medical microbiology. 8th ed.,
150-52.
 Michael J P, Pelczar E C S. Microbiology. 5th ed., 734-36
 Chakraborthy P. Textbook of microbiology. 2nd ed., NCBA
Ltd, 190-95
 Baveja C P. Textbook of Microbiology. 2nd ed., 116-20
 Kokare C R. Pharmaceutical Microbiology. 2nd ed., Nirali
Prakashan, 23.14.
 Ashutoshkar. Pharmaceutical Drug Analysis. 2nd ed., 485-90.
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