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1. Immunoassay of Digoxin
Presented by-
Afreen Hashmi
M.Pharm.(Pharmacology)
1st sem
Enroll no. A8454918005
Guided by-
Mr. Vivek Srivastava
Assistant Professor-II
Department of Pharmacology
Amity Institute of Pharmacy
AMITY UNIVERSITY UTTAR PRADESH
AMITY INSTITUTE OF PHARMACY
LUCKNOW
2018-20
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2. Content
 Immunity
 Classification of immunity
 Immune & Assay
 Introduction
 Principle
 Procedure
 Classification of immunoassay
 Advantages of immunoassay
 Disadvantages of immunoassay
 Uses of immunoassay
 Digoxin
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3. Immunity
 Immunity is the ability of an organism to resist a particular infection or toxin
by the action of specific antibodies or sensitized WBC.
 Resistance shown by the host to micro-organism. It also shows resistance to
toxic molecules or foreign cells.
 The immune system produces antibodies or cells that can deactivates
pathogens.
 Fungi, protozoa, bacteria & viruses all are potential pathogens.
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4. Classification of immunity
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5. Immune & Assay
Immuno Assay
Immune response that
causes the body to
generate antibodies
test
Immunoassay is a test that utilizes
immunocomplexing when antibodies
and antigens are brought together
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6. Introduction
 Immunoassay are analytical methods based on the specific immuno-
reaction between antibody(Ab) and antigen(Ag) for the determination of
amount of either reactant in the solution. An antigen antibody complex is
known as an immuno-complex.
 An immune assay is a test that uses antibody and antigen complexes as a
mean of generating a measurable result.
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7. Principle
 Immuno assay methods are based on a competitive binding between a fixed
amount of labelled form of an analyte and available amount of unlabeled
sample analyte for a limited amount of binding sites on a highly specific
anti-analyte antibody.
Reagents required immunoassay development
 Antibodies
 Antigen
 Signal-generating labels
 Separation matrice
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8. Antibodies
Indirect Attack of Antibodies:
The most effective way for antibodies to destroy antigens is by activating complement
proteins to help attack the antigens.
These proteins can do a variety of things. They can rupture the invading cells, promote
clumping of the cells, or weaken the foreign antigens. These proteins react in the way
that is most efficient for dealing with the antigen that is recognized.
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Antibodies are also known as Immunoglubulin,
Y-shaped molecules of glycoproteins
manufactured by the body that help fight against
foreign substances called antigen.
Antibodies attack antigens in two ways: indirectly
and directly.

9. Direct Attack of Antibodies
Antibodies also attack antigens by directly binding to or attacking the membrane
of an antigen.
This physical reaction, called an antigen-antibody reaction, causes the cells to
clump together.
This agglutination makes it easier for other white blood cells to destroy the
invading antigen. This is not as effective as the indirect route.
Antigen
Antigen are any substances that stimulates immune to produce antibodies
Antigens can be Bacteria, viruses or fungi that causes infection or disease.
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10. Labels
 All immunoassays require the use of labeled material in order to
measure the amount of antigen or antibody present.
 The labelled component of immunoassay is sometimes called
Tracer
Examples of a label
 A radioactive compound,
 An enzyme that causes a change of color in a solution.
 or a substance that produces light.
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11. Procedure
When these immuno analytical reagents are mixed and incubated
analyte is bound to the antibody forming an immune complex.
This complex is separated from the unbound reagent fraction by physical or
chemical separation technique.
Analysis is achieved by measuring the label activity (e.g. radiation,fluorescence
or enzyme) in either bound or free fraction.
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12. Classifications Of Immuno
Assay
 Competitive Immunoassay
 Homogeneous
 Heterogeneous
 Non –Competitive Immunoassay
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13. Competitive immunoassay
 These Are Reagent (Ag) Excess Immunoassay
 In This Assay A Fixed Amount Antibody And Fixed Amount Labelled Antigen
And Unlabelled Antigen Variable Amount Was Taken.
 Here Labelled And Unlabelled Antigen Both Competes With Each Other For
Binding To A Fixed Amount Antibody.
Homogeneous competitive immunoassay
 In This , Unlabelled Analyte In A Sample
 Competes With Labelled Analyte To Bind An Antibody
 The Amount Of Labelled Unbound Analyte Then Measured.
 The Amount Of Labelled Unbound Analyte Is Proportional To The Amount Of
Analyte In The Sample.
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14. Heterogeneous competitive immunoassay
 In this assay, unlabelled analyte in a sample competes with labelled analyte
to bind an antibody.
 The unbound analyte is separated or washed away, and the remaining
labelled, bound analyte is measured.
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15. Non-competitive immunoassay
 These are antibody excess immunoassay.
 In this , fixed amount of antigen and a variable amount of unlabelled antibody
and fixed amount of labelled antibody was taken.
 The unknown analyte in the sample binds with labelled antibodies.
 The unbound, labelled antibodies are washed away and the bound labelled
antibodies are measured.
 The intensity of the signal is directly proportional to the amount of unknown
analyte.
 The amount of labelled antibody on the site is then measured.
 It will be directly proportional to the concentration of the analyte because
labelled antibody will not bind if analyte is not present in the unknown sample.
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17. Advantages of immunoassay
 High sensitivity-Low detection limit
 High specificity –Detect specific compound
 Safe and simple
 Fast Tests (between 5 minutes and 1hour)
 Cost effective
 Tests can yield quantitative or qualitative data
Disadvantages of immunoassay
 May not be sensitive to be certain compounds
 Some chemists are reluctant to use immunoassay due to its
biological basis and their unfamiliarity with it
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18. Uses of immunoassay
 These measures the presence or concentration of macromolecule or a small molecule
in a solution through the use of an antibody or an antigen. For ex. in analyte fluids
urine and serum.
 These are used in sports anti-doping laboratories to test athletes, blood samples for
prohibited recombinant human growth hormone.
 These are used in analysis of metabolites or biomarkers which indicate disease
diagnosis.
 Used in measurements of very low concentrations of low molecular weight drugs.
 Used in therapeutic drug monitoring.
 In clinical pharmacokinetic.
 Used in bioequivalence studies in drug discovery and pharmaceutical industries.
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19. Digoxin
 Digoxin is the primary cardiac glycoside extracted from the foxglove plant,
Digitalis lanata .
 It is used in the
-treatment of CHF because of its inotropic effects on the myocardium.
- treatment of atrial fibrillation because of its chronotropic effects.
-treatment of atrial flutter because of its positive inotropic effects
-treatment paroxysmal atrial tachycardia because of its positive inotropic
effects
 Pharmacological Class :- Cardiac Glycoside
 Therapeutic Class :- Inotrope, antiarrhythmic
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20. Mechanism of Action
The positive inotropic effects is caused by the binding to Na+ K+
ATPase activated adenosine phosphate
inhibition of Na pump
decreased transport of Na+ out of myocardial cells ( increased
intracellular conc)
calcium entry and decrease calcium elimination
enhanced myocardial contractility.
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21. Side effect
 Dizziness
 Mental disturbances
 Diarrhea
 Headache
 Nausea
 Vomiting
 Anorexia
 Cardiac dysrhythmia
 Arrhythmia in children (consider a toxicity)
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22. How Digoxin Corrects Heart Failure
 Due to FOC CO RBF (relieve oliguria)
 Due to better tissue perfusion relieve cyanosis
 Due to blockade of AV node, conducting tissues slow ventricular rate
relieve tachycardia [stronger slower heartbeat( increased efficiency)
without increased demand]
 Due to CO better emptying of the ventricles increased venous
rectum better damage from the tissues with relieve of congestion in the
lungs and liver and reduction of oedem relief of dyspnea
 Better results are obtained in pateints with atrial fibrillation that with normal
rhythm
 Narrow therapeutic index (safety margin) side effect common
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23. Why Digoxin Is Used In Atrial Fibrillation,
Flutter ?
#In Atrial Flutter
 Due to its cardioselective parasympathomimetic effects on atrioventricular
conduction
 Its controls an excessively high ventricular rate
 Improvement is due to direct effect of digitalis as well a the effect of vagus on the
SA node and conducting tissue
#In Atrial Fibrillation
 Reduction of ventricular rate is the best measure of glycoside effect.
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24. Analytical Methods
 Enzyme Immunoassay ( EIA)
 Cloned Enzyme Donor Immunoassay (CEDIA)
 Enzyme Multiplied Immunoassay (EMIT)
 Fluorescence Polarisation Immunoassay (FPIA)
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25. Enzyme Immunoassay ( EIA)
 In this method, the label on the tracer is an enzyme.
 The catalytic properties of enzymes allows the detection and quantitation of small
quantities of the drug.
Cloned Enzyme Donor Immunoassay (CEDIA)
 The method uses an antibody to detect the drug to be measured.
 A label is used to measure the binding reaction.
 It uses genetically engineered fragment of the enzyme β-galactosidase as the label.
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26. Enzyme Multiplied Immunoassay (EMIT)
 The method uses activity as an enzyme glucose 6-phosphate
dehydrogenases chemically coupled to a drug molecule.
 Activity of the enzyme is inhibited when the drug is bound to a specific antibody.
 The extent of enzyme activity reflects the proportion of enzyme labelled drug
which is not bound to antibody, which in turn reflects the no. of binding sites
occupied by unlabelled drug and hence the drug conc in the sample.
Fluorescence Polarisation Immunoassay
(FPIA)
 The method uses specific antibodies to isolate the desired analyte.
 Small fluorescent molecule, excited with polarised vertical light, rotates rapidly
and emits polarised light in comparison to molecules.
 The intensity of polarised light is a measure of conc of the analyte.
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27. Procedure for Immunoassay of Digoxin
 Format the microplates’ wells for each serum reference calibrator, control and patient
specimen to be assayed in duplicate. Replace any unused microwell strips back into
the aluminum bag, seal and store at 2-8°C.
 2. Pipette 0.025ml (25μl) of the appropriate serum reference calibrator, control or
specimen into the assigned well.
 3. Add 0.050 ml (50μl) of Digoxin Enzyme Reagent to all the wells
 4. Swirl the microplate gently for 20-30 seconds to mix.
 5. Add 0.050 ml (50μl) Digoxin Biotin Reagent to all wells
 6. Swirl the microplate gently for 20-30 seconds to mix.
 7. Cover and incubate for 30 minutes at room temperature.
 8. Discard the contents of the microplate by decantation or aspiration. If decanting, blot
the plate dry with absorbent paper.
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28.  9. Add 0.350ml (350μl) of wash buffer (see Reagent Preparation Section), decant (tap and
blot) or aspirate. Repeat two (2) additional times for a total of three (3) washes. An
automatic or manual plate washer can be used. Follow the manufacturer’s instruction
for proper usage. If a squeeze bottle is employed, fill each well by depressing the
container (avoiding air bubbles) to dispense the wash. Decant the wash and repeat two
(2) additional times.
 10. Add 0.100 ml (100μl) of working substrate solution to all wells (see Reagent
Preparation Section). Always add reagents in the same order to minimize reaction time
differences between wells.
 DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION
 11. Incubate at room temperature for fifteen (15) minutes.
 12. Add 0.050ml (50μl) of stop solution to each well and gently mix for 15-20 seconds.
Always add reagents in the same order to minimize reaction time differences between
wells.
 13. Read the absorbance in each well at 450nm (using a reference wavelength of 620-
630nm. The results should be read within thirty (30) minutes of adding the stop
solution.
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29. References
 Tripathi, KD. 2008. Essential of Medical Pharmacology. 6th Editin. India: Jaypee
Brothers Medical Publishers (P) Ltd.
 A.F.Davidson, H.L. Walton, D. J. Pinto, R.E.Immunoassay,New York ,1988, pp.60.
 https://www.youtube.com/watch?v=zy_fK1P2odM
 https://www.ncbi.nlm.nih.gov/pubmed/14727944
 https://www.slideshare.net/hhnoel/drug-therapy-of-congestive-heart-failure
 https://www.slideplayer.com
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