Immunoassay and its appication

imunoassay

1. IMMUNOASSAYS TYPES AND APPLICATIONS Presented by, j.Prakash M.Pharm 1 st sem Dept. of Pharmaceutical analysis 1

2. Immunoassay Immunoassays are bioanalytical methods in which the quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. TYPES OF IMMUNOASSAYS Enzyme immunoassay Fluoro immunoassay Luminescence immunoassay Optical immunoassay Radio immunoassay 2

3. Enzyme immunoassay enzyme immunoassay includes all assays based on the measurement of enzyme labelled antigen or antibody. Homogeneous EIA : no need to separate the bound and free fractions so that the test can be completed in one step. Eg. enzyme multiplied immunoassay technique (EMIT) Heterogeneous EIA: It requires the separation of the free and bound fractions either by centrifugation or by absorption on solid surfaces and washing. Eg. Enzyme Linked Immunosorbent Assay (ELISA). 3

4. Enzyme Linked Immunosorbent Assay (ELISA ) This technique involves the use of an immunosorbent, an absorbing material specific for one of the components of the reaction, the antigen or antibody. Procedure: wells of a microtiter plate are coated with antibody After thorough washing, the particulate is added incubated overnight at 4⁰C or for 2 hours at 37⁰C wells are washed and guinea pig antiserum added labeled with alkaline phosphatase, added and incubated at 37⁰C for one hour. After washing, a suitable substrate (para nitrophenyl phosphate) is added at room temperature positive produce yellow color. 4

5. TYPES OF ELISA 5

6. USES OF ELISA HIV detection Infectious diseases like hepatitis, cytomegalovirus Ig M, Ig G, dengue Ig G, influenza etc. Rotavirus detection in fecal specimens and enterotoxin of E. coli in feces. Mycobacterial antibody detection in tuberculosis 6

7. Enzyme multiplied immunoassay technique In this type of assay, a sample of interest with the analyte (drug) is added to a fixed quantity of enzyme bound drug, and the anti-drug antibody. After addition of substrate, absorbance measurements are taken at time intervals to determine the speed of the enzyme reaction. PROCEDURE Mix sample containing drug with antibody Add fixed quantity of enzyme bound drug Add substrate and cofactor Measure absorbance 15-45 seconds after substrate addition 7

8. 8 USES OF EMIT Determination of serum digoxin, cocaine etc. Used for drugs, hormones and metabolite determination Used for whole blood, serum or urine. Assay for anti-epileptic drugs in serum

9. ADVANTAGES OF EIA Rapid and relatively cost effective Specific and sensitive assays of wide applicability No radiation hazards. Separation step may not be required Manipulations are simple   DISADVANTAGES OF EIA They are species specific 9

10. FLUORO IMMUNOASSAY Fluorescence is the property of absorbing light rays of one particular wavelength and emitting rays with a different wavelength This technique is mainly used to identify specific antibodies or antigen. Antibody identification is usually performed on blood (serum). In this technique visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody fluorescent dye such as fluorescein isothiocyanate is used 10

11. Direct immunofluorescence Staining cells with antibodies directly linked to flourochrome is known as direct immunofluorescence. 11

12. Indirect immunofluorescence The test serum is incubated with antigen immobilized on a 96-well plate or microscope slide. Secondary antibody labeled with fluorescence are added After washing any bound secondary antibodies can be detected by shining UV light on the slide 12

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14. LUMINISCENCE IMMUNOASSAY Luminescence is a generic form that covers a range of processes which produces light. Chemiluminescence is light emission that arises during the course of a chemical reaction. Bioluminescence is a special type of chemiluminescence found in biological systems in which a catalyst protein increases the efficiency of chemiluminescent reaction. 14

15. CHEMILUMINESCENT IMMUNOASSAY It involves a luminescent substance as a label. The growing suc­cess of this technique in pharmaceutical analysis due to its high performance, low detection limits, and good preci­sion. It is the emission of light caused by a chemical reaction typically an oxidation reaction PROCEDURE Monoclonal antibody coated well, test specimen added HRP (horseradish peroxide) labeled antibody conjugate Incubate for 1 hour at 37⁰C Remove unbound enzyme labeled antibody add chemiluminescence reagent Read relative light unit with luminometer. 15

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17. APPLICATION Determination of bioanalyte in clinical specimen Chemiluminescence detection in liquid chromatography Chemiluminescence detection in capillary electrophoresis Chemiluminescence in DNA analysis ADVANTAGES Chemiluminescent assay has an excellent sensitivity comparable to EIA & RIA very little reagent is used, they are also quite inexpensive to perform The relatively high speed of detection also means a faster turnaround time. DISADVANTAGES False result may be obtained if there is lack of precision in injection of the H 2 O 2 or if some biological materials such as urine or plasma cause quenching of light emission 17

18. OPTICAL IMMUNOASSAY(OIA) Optical immunoassay is based on the interaction of antigen-antibody complexes on inert surfaces. Specific binding of antibody increases the thickness of the reactants on the surface and changes the color of light reflected from the surface Procedure: The test sample was mixed with bioanalytical antibody solution in the ratio of 1:1(v/v) and incubated for 2 min The mixture 40ug was placed on the center of the chip and incubated for 10 min the chip was then rinsed, dried and HRP- avidin conjugate was dispensed onto each chip and incubated for 5 min Substrate solution was dropped onto center of the chip after rinsing and incubated for 5 min. After final washing and drying cycle, the results were observed and recorded 18

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20. APPLICATION For snake toxin and venom detection. For snake species identification For detection of Streptococcal Pharyngitis ADVANTAGES   It is an easily interpretable method More sensitive than routine culture method 20

21. RADIO IMMUNOASSAY The principle of RIA is based on the antigen- antibody reaction in which radiolabeled antigen (exogenous) competes with endogenous antigen for the limited sites of the specific antibody against the same antigen. The radiolabeled antigen should have similar biological activity and immunogenicity like that of native antigen 21

22. Procedure The assay is carried out in well plates where the inner surface of well is coated with antibodies. Radioactive antigen is added to all these wells which forms a complex with antibodies. The calibrator (unlabeled antigen of known concentration) is added to multiple wells. Antigen of unknown concentration(sample) is also added to one of the wells. Competition between labeled and unlabeled antigen takes place due to limited binding sites present in antibodies. After washing unreacted antigen and antibody measured for radioactivity using gamma or scintillation counter 22

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24. APPLICATION Blood bank screening for the hepatitis virus Early cancer detection Measurement of growth hormone level Diagnosis and treatment of peptic ulcer RIA of thyroid hormones RIA of fertility related hormones Tumour marker   RIA of drugs Drugs such as phenytoin, theophylline, cyclosporine, morphine, gentamycin, anti-depressants etc. are estimated by RIA. Non clinical application Measurement of aflatoxins in food items. Aflatoxins are the secondary metabolites of the fungi, aspergillus. Aflatoxins are potentially carcinogenic and hence monitoring levels of aflatoxins contamination is mandatory. 24

25. ADVANTAGES High specificity and hence there are no interference from other substances. High sensitivity-up to few pictograms of antigen can be determined when antibodies with high affinity are used. High precision Applicable to wide variety of compounds in various fields-pharmacology, endocrinology, oncology, epidemiology, clinical immunology etc.  DISADVANTAGES Special expertise and safety precautions are required as radioactive materials are used and disposed Expensive compared to other methods, as radioactive materials are used Development of specific antibodies to antigen 25

26. CONCLUSION Immunoassays are bioanalytical methods have been widely used in many important areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmaco­kinetic and bioequivalence studies in drug discovery and pharmaceutical industries. The importance and widespread of immunoassay methods in pharmaceutical analysis are attributed to their inherent specific­ity, high-throughput, and high sensitivity for the analysis of wide range of analytes in biological samples. 26

27. REFERENCE Jenni Punt, Sharon A. Stranford , Patricia P. Jones, Judith A. Owen; Immunology, 8 th edition; W.H Freeman and company; New York. KENNETH MURPHY & CASEY WEAVER; Janeway’s Immunobiology 9 th edition; Blackwell Scientific Publication; London , RONALD J. HARBECK, JERI TEAGUE, JOURNAL OF CLINICAL MICROBIOLOGY, Novel, Rapid Optical Immunoassay Technique for Detection of Group A Streptococci from Pharyngeal Specimens: Comparison with Standard Culture Methods, Apr. 1993, p. 839-844. Marja E. Koivunen , Richard L. Krogsrud ; Principles of Immunochemical Technique Used in Clinical Laboratories; August 2006 _ Volume 37. Number 8. Ibrahim A. Darwish; International journal of Biomedical science, Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances, vol. 2; no. 3; September 2006 27

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