Fluoro immunoassay ppt

This document contains the detailed explanation of Fluoro Immunoassay.

1. SUBMITTED BY- SK AZIZ UDDIN. SUBMITTED TO- DR. RIKESHWAR PRASAD DEWANGAN. COURSE- M.PHARM. SUBJECT- PHARMACEUTICAL ANALYSIS. BATCH-2020-2022. COLLEGE NAME- JAMIA HAMDARD. FLUORO IMMUNOASSAY

2. Introduction Principle of fluro immunoassay Types of immuno fluorescence Advantages & Disadvantages Applications Limitation CONTENTS

3. IMMUNITY DEFINITION- Immunity is the capability of multi cellular organisms to resist harmful microorganisms. Immunity involves both specific and non specific components.

4. Introduction Immunoassay- : Immunoassay is a test that uses antibodies and antigen complexes as a means of generating a measurable result. An antibody : antigen complex is also known as an immune complex. An immunoassay is a test that utilizes immune complex when antibodies and antigens are brought together. Immunoassays are used to quantify molecules of biological interest based on the specificity and selectivity of antibody reagents generated.

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6. Competitive immunoassay In a competitive format, unable analyte (usually the antigen) in the test sample is measured by its ability to compete with the labelled antigen in the immunoassay. It is less label measured in the means more of the unlabelled (test antigen) antigen is present.

7. NON COMPETITIVE IMMUNOASSAY In non competitive assay , the measurement of the labelled analyte (usually the antibody) is directly proportional to the amount of antigen present in the sample. Non competitive assay formats can use either one step or two step methods. In two step assays format, there are wash steps in which the sandwich binding complex is isolate and wash to remove excess unbound labelled antigen.

8. DIFFERENCE COMPETITIVE VERSUS NON COMPETITIVE IMMUNOASSAY

9. HOMOGENEOUS IMMUNOASSAY Homogenous methods have been generally applied to the measurement of small analytes such as abused and therapeutics drugs. Since homogenous methods do not require the separation of the bound Ab - Ag* from the free Ag*,there are generally much easier and faster to perform.

10. HETEROGENOUS IMMUNOASSAY In the heterogeneous immunoassays the labelled, unbound analyte is separated or washed away, and the remaining labelled , bound analyte is measured.

11. TECHNIQUES OF IMMUNOASSAY

12. IMMUNO FLUORESCENCE Immuno fluorescence is a technique allowing the visualization of a specific antigen by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate(FITC). The specific antibodies are labelled with a compound (FITC) that makes them glow an apple-green colour when observed microscopically under ultraviolet light.

13. FLUORESCENCE Fluorescence is the property of certain molecules to absorb light at one wave length and emit light at longer wave length when it is illuminated by light of a different wavelength. The fluorescence can be visualized using fluorescence microscopy. The IF technique allows for a visualization of the presence as well as the distribution of target molecules in a sample.

14. PRINCIPLE

15. CONTINUE;- An immunofluorescence assay is based on following principles steps;- Specific antibodies bind to the protein of interest Fluorescent dyes are coupled to these immune complexes in order to visualize the protein of interest using microscopy Immunofluorescence is a powerful approach for getting insight into cellular structures and processes using microscopy. specific proteins can be assessed for their expression and location, making immunofluorescence indispensable for scientist to solve many cell biological questions.

16. Immunofluorescence staining Direct immunofluorescence ;- staining in which primary antibody is labelled with fluorescence dye. Indirect immunofluorescence -; Staining in which a secondary antibody labelled with fluorochrome is used to recognize a primary antibody.

17. Direct immunofluorescence Direct immunofluorescence uses a single antibody that is chemically linked to a fluorophore . The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be detected via microscopy

18. Advantages of direct immunofluorescence This technique has several advantages over indirect immunofluorescence because of the direct conjugation of the antibody to the fluorophore . This residues the number of steps in the staining procedure making the process faster and can reduce background signal by avoiding some issues with antibody cross-reactivity .

19. Indirect immunofluorescence Indirect immunofluorescence uses two antibodies; the unlabelled primary antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore , recognises the primary antibody and binds to it.

20. Advantages of indirect immunofluorescence Gives an amplification effect- more label per molecule of target protein. Requires only one labelled antibody to identify many proteins- same labelled secondary antibody can be used to bind to many different proteins. A different primary antibody is used for each target protein. variable part of primary antibody binds to specific part of target protein. The secondary antibody binds to the constant part of the primary antibody. Therefore a sample of the same batch of secondary antibody can bind to many different primary antibodies.

21. Application Immunofluorescence can be used on tissue sections, cultured cell lines, or individual cells, and may be used to analyse the distribution of proteins and small biological and non-biological molecules. Immunofluorescence can be used in combination with other , non-antibody methods of fluorescent staining, for example, use of DAPI,( 4’,6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to A-T rich regions in DNA, to label DNA. It also play a key role in the diagnosis of autoimmune disorder. This technique has a number of different biological applications including evaluation of cells in suspension,cultured cells,tissue , beads.

22. Limitation Quality and concentration of the antibody. Proper handling of the specimen. Choice of secondary antibodies. Fluorophores undergoes photo bleaching as they are exposed to light.

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