immunoassay of digoxin.pptx (analytical methods of immunoassay)

Immunoassays are bioanalytical methods in which the quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody.
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.

1. Immunoassay of   Digoxin  Presented By- Nikita Bankoti M.Pharm (Pharmacology) KUDOPS

2. INTRODUCTION Immunoassays are bioanalytical methods in which the quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody.   An immunoassay is a test that uses antibody and antigen complexes as a mean of generating a measurable result.

3. 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.

4. Reagents required for immunoassay development Antibodies Antigen Signal-generating labels Separation matrice

5. Digoxin Digoxin is also known as digitalis, it is the primary cardiac glycoside extracted from the foxglove plant, Digitalis lanata . Digoxin is widely used in the - Treatment of CHF because of its inotropic effects on the myocardium. Treatment of atrial fibrillation because of its chronotropic effects( affects heart rate). Treatment of atrial flutter and paroxysmal atrial tachycardia because of its positive inotropic effects (   increase the force of muscle contraction of the heart ).

6. Mechanism of action of digoxin

7. Analytical methods used in immunoassay Methods used to monitor serum digoxin concentration include: Enzyme Immunoassay (EIA) Radioimmunoassay (RIA) Cloned enzyme donor Immunoassay (CEDI) Enzyme multiplied Immunoassay (EMI) Fluorescence Polarisation Immunoassay (FPIA)

8. 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 antigen 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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10. Radioimmunoassay (RIA) The labelled antigen is mixed with the antibody at a concentration that saturates the antigen-binding sites of the antibody. As the concentration of the unlabeled antigen increases more labelled antigen will be replaced from the binding site. The sample is washed to remove unbound radioactive drug. The decrease in the amount of radio labelled antigen bound specific antibody in the presence of the test samples is measured by using a gamma counter to determine the amount of antigen present in the test sample. In std condition, amount of labelled antigen bound to the antibody decreases as the amount of unlabelled antigen increases in sample.

11. 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 beta-galactosidase as the label. The enzyme has two units, an enzyme acceptor and an enzyme donor. These units alone are inactive, but in solution, they become activated and reassemble. As a single unit, they can react with substrate. Drug bound to the enzyme donor competes with the drug or metabolite in the sample for the antibody binding site. If the drug bound to the enzyme donor binds to the antibody, it is prevented from reassembling with the enzyme acceptor and activating the enzyme. If the drug or metabolite is present in the sample, the unbound enzyme donor reassembles with the enzyme acceptor and reacts with the substrate to produce a change in absorbance.

12. Fluorescence Polarisation Immunoassay (FPIA) The principle of the assay is that a fluorescent dye (attached to an antigen) can be excited by plane-polarized light at the appropriate wavelength. The patient sample is incubated with a known quantity of the fluorescent-labelled drug and an antibody specific for the drug. The labelled and unlabelled drug compete for the binding sites of the antibody. Polarized light is emitted in certain angles depending on whether the fluorescent-labelled drug is bound to antibodies or not. Since this is a competitive assay, the greater the amount of drug in the sample, the lower the amount of fluorescence. The intensity of polarized light is a measure of concentration of the analyte.

13. Enzyme Multiplied Immunoassay (EMIA) Commonly used analytical method for immunoassay of digoxin. Methodology: Enzyme multiplied immunoassay is manufactured using recombinant DNA technology, used for the analysis of digoxin and its active metabolites in serum or plasma. The assay is based on competition between drug in the sample and drug labelled with recombinant glucose-6-phosphatedehydrogenase (rG6PDH) for antibody binding sites. Enzyme activity decreases upon binding to the antibody, so the drug concentration in the sample can be measured in terms of enzyme activity.

14. General procedure of immunoassay Specimen collection and preparation: Each assay requires serum or plasma. Sample volume is instrument dependent. Use fresh samples, if samples are to be tested within 8 hours of collection, they may be stored at room temperature (20-25 C). For transporting, maintain the sample temperature at 2-8 C. Samples can be stored refrigerated at 2-8 C for up to 7 days or stored frozen -20C for up to 6 months. Repeated freeze-thaw cycles should be avoided. If the samples contain particulate matter, fibrous material then vigorously mix sample in a vortex for at least 30 seconds or centrifuge sample at >2000rpm for 15 min.

15. Reagents used are- Sample (serum containing digoxin) Digoxin tracer reagent (digoxin-HRP in a buffer dye) Rabbit serum antibody (digoxin-ab) Washing buffer Signal agent (luminol in buffer)

16. Procedure for immunoassay of Digoxin 96- well microplate Pipette 0.025ml (25 μ l ) of the appropriate serum reference calibrator, control or specimen into the assigned wells. Add 0.050ml (50 μ l ) of digoxin enzyme reagent(HRP) to all the wells. Swirl the microplate gently for 20-30 seconds to mix. Add 0.050ml (50 μ l ) of digoxin antibody (rabbit serum) to all wells. Swirl the microplate gently for 20-30 seconds to mix. Cover and incubate for 30 min at room temperature.

17. Add 0.350ml (350 μ l ) of washing buffer and wash for 3 times.(an automatic or manual plate washer can be used) Add 0.100ml (100 μ l) of working signal reagents to all wells. (always add reagents in the same order to minimize reaction time difference between wells) Do not shake the plate after reagent addition. Incubate at room temperature for fifteen (15) mintutes. Read the relative light unit with chemiluminescence microplate reader. The results should be read within 30 minutes of adding the signal reagent.

18. “Thank you”