In this slideshare i have discussed about the digoxin, its property,different analytical methods of digoxin and the general procedure of immunoassay of digoxin. Also additionally i have added 10 mcq related to the topic with multiple options and correct option as well.

1. IMMUNOASSAY OF DIGOXIN
JAYHIND L BHARTI
M PHARM- 1ST YEAR
PHARMACOLOGY DEPARTMENT
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2. What is immunoassay?
Consist of 2 words:
1)IMMUNO/IMMUNE 2)ASSAY
Immuno refers to an immune response Assay refers to a test
that causes the body to generate antibodies
IMMUNOASSAY
Thus immunoassay is a test that utilizes immunocomplexing when antibodies and
antigens are brought together.
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3. INTRODUCTION OF IMMUNOASSAY
 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 immunoassay is a test that uses antibody and antigen complexes as a mean
of generating a measurable result.
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4. What is Digoxin
 Digoxin, also known as digitalis, is a purified cardiac glycoside, extracted from
the plant Digitalis lanata.
 Digoxin is widely used in the treatment of various heart conditions :
 cardiac arrythmias
 atrial fibrillation & atrial flutter
 cardiac failure that can not be controlled by other medication.
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5. Why we perform digoxin immunoassay?
 Digoxin has a narrow range of safe and effective concentrations in serum.
 Although the therapeutic and toxic concentrations overlap,
measurement of digoxin levels helps to maintain effective concentrations and to
diagnose and prevent overdosage.
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6. Analytical methods used in immunoassay of digoxin
Methods used to monitor serum digoxin concentrations include :
a) Radioimmunoassay (RIA)
b) Enzyme Immunoassay ( EIA)
c) Cloned Enzyme Donor Immunoassay (CEDIA)
d) Fluorescence Polarisation Immunoassay (FPIA)
e) Enzyme Multiplied Immunoassay (EMIT)
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7. a)Radioimmunoassay (RIA)
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8. b) Enzyme Immunoassay ( EIA)
 The general principle of this technique is based on the binding of conjugated
enzyme molecule with specific antibodies to detect and quantify the presence
of either antigens or antibodies in the test sample.
 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
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9. General procedure of immunoassay
1. 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
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10. Cont..
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.
9. Add 0.350ml (350μl) of wash buffer (see Reagent Preparation Section), decant (tap and
blot) or aspirate.
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 SHAKETHE PLATE AFTER SUBSTRATE ADDITION
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11. Cont..
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.
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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12. REFERENCES
 https://www.slideshare.net/AfreenHashmi2/immunoassay-of-digoxin
 https://tech.snmjournals.org/content/jnmt/1/2/18.full.pdf
 https://www.slideshare.net/Rashmi116/immunoassay-of-digoxin-244207357
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13. MCQ
1)What is the use of digoxin?
a. Treat heart failure
b. Treat anxiety
c. Treat copd
d. Treat infection
2) An antigen antibody complex is known
a. Antibody-complex
b. complex drug
c. Immuno-complex
d. Antigen antibody complex
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14. 3) Digitalis is obtained from which plant?
a. Digitalis purpurea
b. Foxglove
c. Digitalis lanata
d. Senna leaves
4)Which is not the analytical method used in immunoassay of digoxin
a. Radioimmunoassay (RIA)
b. Enzyme Immunoassay ( EIA)
c. Cloned Enzyme Donor Immunoassay (CEDIA)
d. Chemo immunotheraphy (CIT)
5) In enzyme immunoassay the label on the tracer is an
a. bacteria
b. enzyme
c. toxin
d. radio active particle
6) RIA Stands for
a. regulatory impact assessment
b. radiology imaging associates
c. radio immunoassay
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15. 7)Why reagents are added in same order during the procedure of digoxin?
a. to minimize the reaction time difference between wells
b. to maximize the reaction time difference between wells
c. to neutralize the reaction time difference between wells
d. to diminise the reaction time difference between wells
8) Digoxin has a narrow range of safe and effective concentrations in serum?
a. true
b. false
9) At what wavelength the absorbance of digoxin should be read?
a. 250
b. 350
c. 450
d. 550
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