Immunochemicaltechniques

IMMUNOCHEMICAL TECHNIQUES
IMMUNOCHEMICAL TECHNIQUES, Immune System, immunology, TECHNIQUES, Microbiology, Online pharmacy, pharmd, doctor of pharmacy, elisa,

1. IMMUNOCHEMICAL TECHNIQUES

2. I mmunochemical techniques - identification of a substance (especially a protein) by its action as an antigen.

3. An antibody , also known as an immunoglobulin , is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen . Each tip of the "Y" of an antibody contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision Antigen and Antibody

4. Enzyme-linked immunosorbent assay ( ELISA ), is one sub-type of solid-phase enzyme immunoassay ( EIA) . The term enzyme immunoassay includes all assay based on the measurement of enzyme labeled antigen or antibody. EIA are two basic types : homogeneous : EMIT ( enzyme multiplied immunoassay technique . It is used only for drug identification. Enzyme-linked immunosorbent assay ( ELISA ), Heterogeneous : ELISA it is used to detected the antigen or antibody . ELISA is named because the technique involves the use of an immunosorbent- an absorbing material specific for one of the components of the reaction, the antigen or antibody.

5. ELISA is usually done using 96 well microtitre plates . there are three type of ELISA Indirect assay : example used in detection of Rotavirus antigen in feces . Sandwich assay: example used in detection of HIV antibody in serum Competitive assay: example used in detection of HIV antibody in serum Generally , the antibody or antigen fix to well plate then ag or ab is added along with the enzyme and finally suitable substrate is added. The result is noted by the occurrence of colour . LETS SEE ONE BY ONE ELISA

6. Indirect assay

7. Indirect assay Example : rotavirus in foecus sample ( ag ) Goat antibody ( ab ) Enzyme: alkaline phosphatase Subs: paranitrophenyl phosphate

8. Sandwich assay

9. Sandwich assay Unknown ab : anti HIV antibody Ag:HIV antigen Know ab : goat antihuman immunoglobulin Enzyme: horse serum peroxidase Subs: O- phenylene diamine dihydrochloride

10. Competitive assay

11. Several variation of ELISA have been developed few examples are dipstick method ELISA Cylinder or cassette ELISA

12. Radioimmunoassay is based on the antigen-antibody reaction in which a trace amount of the radiolabeled antigen competes with endogenous antigen for limited binding sites of the specific antibody against this antigen. The accuracy of RIA is based on the reasonable assumption that the radiolabeled antigen and the native antigen have similar affinities for the specific antibody . Radioimmunoassay

13. Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure concentrations of antigens by use of antibodies for example , hormone levels in the blood Lets see procedure RIA methods are again becoming popular . It is generally more simple to perform than a bioassay

14. A specific antibody produced by hybridoma cell is called MONOCLONAL ANTIBODY. Hybrid cell resulting from the fusion of a myeloma cell with an antibody producing B-lymphocyte. Diagnostic tests Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot test and immuno dot blot tests detect the protein on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells... Monoclonal Antibody

15. Therapeutic treatment It is used in the treatment of Autoimmune disease like rheumatoid arthritis, chorns disease. It is also used in the treatment of Cancer. Monoclonal Antibody

16. Monoclonal Antibody

17. Monoclonal Antibody

18. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample . Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualize the location of the antibodies . This tech is used to detect the pathogens like c onococci ( std ), Corynebacterium (diphtheria), Morbillivirus (Measles). Fluorescence Immuno Assay

19. There are two types of FIA Direct assay : example it used in diagnosis of rabies virus ag in brain smear. Indirect assay : example it used in diagnosis of treponemal ab ( syphilis) Fluorescence Immuno Assay

20. Direct Fluorescence Immuno Assay

21. Fluorescence Immuno Assay

22. Indirect Fluorescence Immuno Assay

23. CHEMILUMINESCENCE IMMUNO ASSAY CLIA is a more economical and conventional method used as an alternative to ELISA. It plays a vital role in the examination of pituitary hormones mainly STH , TSH . As far as now ,CLIA has the potential that it can be used in various diagnostic and research uses than ELISA and FIA. Chemiluminescence (sometimes " chemoluminescence ") is the emission of light with limited emission of heat (luminescence), as the result of a chemical reaction.

24. PRINCIPLE INVOLVED IN CLIA This is similar to ELISA . In ELISA , we use a known anti body whereas here we use a MONOCLONAL ANTIBODY . Similarly we use colorimeter to read the colour intensity whereas here we use luminometer to read the light intensity.

25. REFERENCES MICROBIOLOGY by MICHAEL J.PELCZAR FIFTH EDITION TEXTBOOK OF MICROBIOLOGY AND IMMUNOLOGY by C.K.J PANIKER SEVENTH EDITION