This document discusses immunoassay and its use in measuring digoxin levels. It begins with an introduction to immunoassay, describing how it uses antibody-antigen complexes to generate measurable results. It then covers the principles, procedures, classifications (competitive vs non-competitive), advantages, disadvantages and uses of immunoassay. The document also discusses digoxin, its mechanism of action, side effects, and provides the procedure for its immunoassay measurement.

1. Dr. APJ ABDUL KALAM TECHNICAL UNIVERSITY
(Formerly Uttar Pradesh Technical University) LUCKNOW
Noida Institute of Engineering and Technology
(Pharmacy Institute)
SUBMITTED TO:- SUBMITTED BY:-
DR. SAUMYA DAS SONI KUMARI
ASSOCIATE PROFESSOR M.PHARM (PHARMACOLOGY)
NIET (PHARMACY INSTITUTE) NIET (PHARMACY INSTITUTE)
IMMUNOASSAY OF DIGOXIN

2. INDEX
 Introduction
 Principle
 Procedure
 Classification of Immunoassay
 Advantage, Disadvantage and Uses
 Digoxin and its mechanism of action
 Immunoassay of Digoxin
 Reference

3. 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.
Reference:-

4. 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 in immunoassay
development
 Antibodies
 Antigen
 Signal-generating labels
 Separation matrice

5. 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.

6. CLASSIFICATION OF IMMUNOASSAY
 Competitive Immunoassay
Homogeneous
Heterogeneous
 Non -Competitive Immunoassay

7. 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.

8. Heterogenous 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.

9. 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 analyte directly proportional to the
amount of unknown
 The amount 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.

10. Advantages of immunoassay
• High sensitivity-Low detection limit
• High specificity -Detect specific compound
• Safe and simple
• Fast Tests (between 5 minutes and l hour)
• 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.

11. 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.

12. 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
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
Reference:- https://en.wikipedia.org/wiki/Digoxin

13. 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.)
Increase calcium entry and decrease calcium elimination
enhanced myocardial contractility.

14. SIDE EFFECTS
 Dizziness
 Mental disturbances
 Diarrhea
 Headache
 Nausea
 Vomiting
 Anorexia
 Cardiac dysrhythmia
 Arrhythmia in children (consider a toxicity)

15. Procedure for Immunoassay of Digoxin
 Format the microplate 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 of the appropriate serum reference calibrator,
control or specimen into the assigned well.
 3. Add 0.050 ml of Digoxin Enzyme Reagent to all the wells
 4. Swirl the microplate gently for 20-30 seconds to mix.
 5. Add 0.050 ml 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 plate dry with absorbent paper.

16.  9. Add 0.350ml of wash buffer, decant (tap and blot) or aspirate.
Repeat two additional times for a total of three 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 additional times.
 10.Add 0.100 ml 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 minutes.
 12. Add 0.050ml 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
minutes of adding the stop solution.

17. REFERNCE
 Tripathi, KD. 2008. Essential of Medical Pharmacology.
6'* Editin. India: Jaypee Brothers Medical Publishers (P)
Ltd.
 A Davidson, ML. Walton, D. J. Pinto,
R.E.Immunoassay,New York ,1988, pp.60.
 https://www.ncbi.nlm.nih.gov/pubmed/14727944
 https://www.slideshare.net/hhnoel/drug-therapy-of-
congestive-heart-failure
 https://www.slideplayer.com