Neutralization Test for Virus

NEUTRALIZATION TEST FOR VIRUS Neutralization of a virus is defined as the loss of infectivity through reaction of the virus with particularized antibody. Virus and serum are mixed under appropriate setting and then inoculated into cell culture, eggs or animals. The presence of unneutralized virus may be detected by reactions such as CPE, haemadsorption/haemagglutination, plaque formation, unhealthiness in animals. The loss of infectivity is bought about by interference by the skip Ab with any one of the steps leading to the release of the viral genome into the legion cells.There are two types of counteraction- Reversible neutralisation reaction The neutralisation process can be reversed by diluting the Ab-Ag mixture inwardly a short time of the formation of the Ag-Ab complexes (30 mins). It is thought that bilateral neutralisation is due to the interference with attachment of virions to the cellular receptors eg. the attachment of the HA protein of flu viruses to sia lic acid. The process requires the saturation of the surface of the virus with Abs. permanent counteraction with time, Ag-Ab complexes usually give way more stable (several hours) and the process cannot be reversed by dilution. incomplete the virions nor the Abs are permanently changed in stable neutralization, for the unvaried components can be recovered. The neutralized virus can be reactivated by proteolytic cleavage. Stable neutralization has a various mechanism to that of reversible neutralization. It had been shown that neutralized virus can attach and that already attached virions can be neutralized.The phone number of Ab elements required for stable neutralization is considerably smaller than that of reversible neutralization, Kinetic evidence shows that even a single Ab molecule can neutralize a virion. Such neutralization is generally produced by Ab molecules that establish contact with 2 antigenic sites on dissimilar monomers of a virion, greatly increasing the s tability of the complexes. An example of stable neutralization is the neutralization of polioviruses, whereby, the attachment of the antibody to the viral capsid stabilizes the capsid and inhibits the uncoating and release of viral nucleic acid.Viral evolution must tend to select for mutations that change the antigenic determinants tough in neutralization. In contrast, other antigenic sites would tend to remain unchanged because mutations affecting them would not be selected for and could even be detrimental. A virus would thus evolve from an master key type to a variety of types, different in neutralization (and sometimes in HI) tests, but retaining some of the original mosaic of antigenic determinants recognizable by CFTs.Because of its high immunological specificity, the neutralization test is often the standard against which the specificity of the other serological techniques is evaluated. Before the neutralization test is carried out, the known components that are to be appl y must be standardized. To identify a virus isolate, a known pretitred antiserum is used. Conversely, to measure the antibody solution of an individual to a virus, a known pretitred virus is used. To titrate a known virus, serial tenfold dilutions of the isolate is prepared and inoculated into a pliable host system such as cell culture or animal.The virus ending titre is the reciprocal of the highest dilution of virus that infects 50% of the host system eg. 50% of cell cultures machinate CPE, or 50% of animals develop disease. This endpoint dilution contains one 50% tissue culture infecting dose (TCID50) or one 50% lethal dose (LD50) of virus per unit volume. The denseness of virus generally used in the neutralization test is light speed TCID50 or 100 LD50 per unit volume. The antiserum is titrated in the neutralization test against its homologic virus.Serial twofold dilutions of serum is prepared and mixed with an equal volume containing 100TCID50 of virus. The virus and ser um mixtures are incubated for 1 hour at 37oC. The time and temperature for brooding varies with different viruses. The mixtures are then inoculated into a susceptible host system. The endpoint titration contains one antibody unit and is the reciprocal of the highest dilution of the antiserum protecting against the virus. Generally 20 antibody units of antiserum is used in the neutralization tests.