BP Singh

168 Shamsher Singh and R.K. Klletarpalhost tissue with enhanced sensitivity.described below.These techniques area) Enzyme linked immunosorbent assay (ELISA) ; ELISA isrelatively simple, rapid, very sensitive and requires small amountof antiserum. Of the various forms of ELISA tests, the doubleantibody sandwich form (DA-ELISA) is most commonly used. Inthis case virus specific C\ntibodies are adsorbed to a solid surface(microtiter plates) and to this is added the sample suspected tocontain the viral antigen which is followed by the addition ofenzyme labelled specific antibody. The labelled antibodies bind tothe antigen which is already round to the coating antibodies.Finally the substrate is hydrolysed and the development of colourin the end product is measured as absorbance value in a multiscanspectrophotometer. The colour change in the substrate isproportional to the amount of enzyme present which in turn isproportional to antigen concentration.The repetitive nature of most of the operations involved inELISA make this technique well suited to automation and permitssimultaneous testing of a large number of samples.b) Dot-immunobinding assay (DIBA) : It is a variant of ELISAtest wherein, instead of using microtitre plates as solidsupport,nitrocellulose membranes are used. A few microlitres ofextract of infected samples are blotted on this membrane which isthen submerged in primary antibody (crude antiserum). Theprecipitated specific antibody is then detected with enzymelabelled second antibody or protein A.DIBA has an edge over conventional ELISA as it does notrequire any special equipment, it is relatively inexpensive, itrequires only a crude specific antiserum to each of the viruses anda single universal enzyme conjugate. Above all the blottedmembranes can be mailed to long dis~ances for further processingin a centralized laboratory.c) Immunosorbent electron microscopy (lSEM) : It is a verysensitive technique coupling serology with electron microscopy.In this case the electron microscope grids are pretreated withspecific antiserum which facilitates the adherence of virus particleson the grid by several folds. The virus particles are then