Department of microbiology microbial food technology group a diploma in quality assurance in microbiology diploma



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Complement fixation test


The complement fixation test is an immunological medical test that can be used to detect the presence of either specific antibody or specific antigen in a patient's serum. It was widely used to diagnose infections, particularly with microbes that are not easily detected by culture methods, and in rheumatic diseases. However, in clinical diagnostics labs it has been largely superseded by other serological methods such as ELISA and by DNA-based methods of pathogen detection, particularly PCR.

Process


The CF test uses sheep red blood cells (sRBC), pre-bound by anti-sRBC antibody, and serum (usually from guinea pig) as a source ofcomplement, which is a system of serum proteins that react with antigen-antibody complexes. If this reaction occurs on a cell surface, it will result in the formation of trans-membrane pores and therefore destruction of the cell. Accordingly, if the antibody-sensitized sRBC are brought into contact with active complement, they will undergo disintegration (hemolysis).

Complement will also react with antigen-antibody complexes in solution. The complement is thereby expended and can no longer trigger hemolysis; inhibition of complement hemolysis therefore indicates the presence of antigen-antibody complexes. A patient's serum containing a certain antibody (specific for, say, rubella virus) will yield antigen-antibody complexes after addition of the corresponding antigen (inactivated rubella virus in our example). Complement added to the mixture will be consumed, and sensitized sRBC added subsequently will not undergo hemolysis. Therefore, absence of hemolysis constitutes a positive CF test (patient's serum contains the antibody of interest).


Testing for antigen


While detection of antibodies is the more common test format, it is equally possible to test for the presence of antigen. In this case, the patient's serum is supplemented with specific antibody to induce formation of complexes; addition of complement and indicator sRBC is performed as before.

Quantitative testing


The test can be made quantitative by setting up a series of dilutions of patient serum and determining the highest dilution factor that will still yield a positive CF test. This dilution factor corresponds to the titer

Western blot


Western blot analysis of proteins separated by SDS-PAGE.[1]

The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

There are now many reagent companies that specialize in providing antibodies (both monoclonaland polyclonal antibodies) against tens of thousands of different proteins.[4] Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biologybiochemistryimmunogenetics and other molecular biology disciplines.

Other related techniques include using antibodies to detect proteins in tissues and cells byimmunostaining and enzyme-linked immunosorbent assay (ELISA).

The method originated from the laboratory of George Stark at Stanford. The name Western blotwas given to the technique by W. Neal Burnette[5] and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termednorthern blotting and the detection of post-translational modification of protein is termed eastern blotting.


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