Immunoassay ELISA_ppt.pptx

Introduction
Principle
Types
Applications

1. ADITYA BANGALORE INSTITUTE OF PHARMACY DEPARTMENT OF PHARMACOLOGY Modern Pharmaceutical Analysis TOPIC ON: Immunoassays, RIA and ELISA Submitted by : RajeshYadav (M Pharm)

2. contents Introduction Principle Types Applications Reference

3. Introduction Analytical method or Biochemical test An antibody: antigen complex is also known as an immuno-complex Highly specific “lock and key” system: antibody –antigen reaction

4. “ Immuno ”& “assay” “ Immuno ” refers to an immune response that causes the body to generate antibodies, and “Assay” refers to a test. Thus, an immunoassay is a test that utilizes immunocomplexing when antibodies and antigens are brought together

5. Immunoassays are different from other types of laboratory tests, such as colorimetric tests , because they use antibody:antigen complexes to generate a signal that can be measured.

6. Immunoassay: Antibodies, Antigens and Analytes An antibody is a protein that is produced by the body in response to an “invading” (foreign) substance. An Antigen is the substance that the body is trying to fight off (eliminate or reduce) by mounting an immune response. An analyte is anything measured by a laboratory test. In immunoassay testing, the analyte may be either an antibody , or an antigen .

7. Immunoassays Immunoassays utilize one or more select antibodies to detect analytes of interest. The analytes being measured may be those that are naturally present in the body (such as a thyroid hormone), those that the body produces but are not typically present (such as a cancer antigen), or Those that do not naturally occur in the body (such as an abused drug).

8. Antibodies Antibodies possess high a) specificity and b) affinity for a specific antigen . It is the specific binding of an antibody to an antigen that allows the detection of analytes by a variety of immunoassay methods.

9. Structure of Antibodies Antibodies ( Ab ) are a type of protein called immunoglobulins . The most common one is immunoglobulin G ( IgG ). IgG is a protein composed of two main structural and functional regions

10. Principle Technique which incorporates the binding reaction of a target substance (antigen) with an antibody Antibodies bind to different natural and synthetic antigens in the body such as carbohydrates, lipids, proteins and nucleic acids.

11. TYPES:- • Competative immunoassays. • Non- competative immunoassays. • Homogenous immunoassays. • Heterogenous immunoassays

12. Homogeneous vs Heterogeneous Immunoassay Methods Immunoassay methods that require separation of bound Ab -Ag*complex are referred to as heterogeneous immunoassays. Those that do not require separation are referred to as homogeneous immunoassays

13. Radioimmunoassay Oldest type of immunoassay Sensitive method ( ng to pg concentration) Less specific Qualitative as well as Quantitative analysis Radioisotope

14. Radioimmunoassay can detect substance like : • Hormones • Vitamins • Serum Protein • Drugs • Infective Agent

15. Principle 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)

16. Advantages of Radioimmunoassay • Specific • Sensitive • Convenient • Reliable • Reproducible

17. Disadvantages of Radioimmunoassay • Prolonged reaction time (in days) • Radioisotopes are costly. • Possible health hazards due to handling of radioisotopes. • Limited assay range. • Lack of direct linear relationship between analyte concentration and signal response. • Lengthy counting time.

18. Application Of Radioimmunoassay i )Detection of Narcotic Drugs ii)Radioimmunoassay of Hydromorphone & Hydrocodone in Human Plasma iii) Radioimmunoassay of Flunisolide in human plasma iv) Detection of Digoxin v) Thyroid Testing

19. ELISA Linked to an enzyme A test that uses antibodies and color change to identify a substance Involves at least one antibody with specificity for a particular antigen

20. Principle The basic principle of an ELISA is to use an enzyme to detect the Ag- Ab binding . The enzyme converts a colorless substrate ( chromogen ) to a colored product, indicating the presence of Ag- Ab binding

21. Types: i )Direct ELISA

22. ADVANTAGES OF DIRECT DETECTION Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated DISADVANTAGES OF DIRECT DETECTION Immunoreactivity of the primary antibody may be reduced as a result of labeling. Labeling of every primary antibody is time-consuming and expensive . Little signal amplification .

23. ii) Indirect ELISA

24. ADVANTAGES OF INDIRECT DETECTION Wide variety of labeled secondary antibodies are available commercially. Immunoreactivity of the primary antibody is not affected by labeling. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification DISADVANTAGES OF INDIRECT DETECTION Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal. An extra incubation step is required in the procedure

25. iii) Sandwich ELISA

26. Advantages Assay is quantitative , amount of viral antigen can be detected Assay has high sensitivity and specificity More samples can be tested at the same time Disadvantages Need ELISA reader for result interpretation; not possible under field conditions The method is time consuming and labourious .

27. iv) Competitive ELISA

28. ADVANTAGES Suitable for complex (crude or impure) samples, since the antigen does not require purification prior to measurement. DISADVANTAGES Each antigen may require a different method to couple it to the enzyme

29. COMPARISON BETWEEN VARIOUS TYPES OF ELISA

30. Applications HIV test Detection of Mycobacterium antibodies in tuberculosis Detection of rotavirus in feces Detection of hepatitis B markers in serum Detection of enterotoxin of E. coli in feces

31. Reference Pharmaceutical Drug Analysis by Ashutosh Kar ; 2005 P .485 – 504 Yalow R, Berson S - Immunoassay of endogenous plasma insulin in man;1960 P .39 – 115 The journal of nuclear medicine; 1979 P .748- 752 Ibrahim A. Darvish . Review on Immunoassay methods and their application in pharmaceutical analysis –International journal of biomedical science; 2006 P .217 - 220