DOT BLOT ASSAY PRINCIPLE FREE

Lawson has mentioned that appropriate screening procedures have been conducted in order to certify any plant free of certain pathogen using ELISA, DBIA, TBIA, PCR, DNA probes. There are various immuno-diagnostic and molecular-diagnostic techniques presently available in field of virology and these are divided into two: Protein based techniques which include precipitation/agglutination tests, enzymes linked immunosorbent assay (ELISA), dot immunoblotting assay (DBIA) and tissue blot immunobinding assay (TBIA). The first was the advent of antibody based detection, in particular monoclonal antibodies and enzyme-linked immunosorbent assay. Technologies for the molecular detection of plant pathogens have already undergone two major breakthroughs well over the past three decades. However, progress in molecular biology, biochemistry and immunology has led to the development of many new, accurate, rapid and less labour-intensive methods of virus detection. A single diagnostic test or assay may provide adequate information on the identity of a virus but a combination of methods is generally needed which are specific, sensitive and inexpensive. Traditional diagnosis of plant viruses requires bioassay, an indicator plant, determination of host range, symptomatology, virus particle morphology (size and shape), and vector relations. The serological methods of diagnosis, detection and identification of viruses in plants play a vital role. Comparisons between different serological methods were demonstrated by many investigators Dot immunobinding was eight times more sensitive for detection of PVX and four times more sensitive for detection of PVS and PVY than DAS-ELISA. In an effort to reduce the cost of virus assays, different types of regular paper were evaluated as possible replacements for the commonly used nitrocellulose membrane (NCM) as the solid phase in the tissue-blot immunoassay (TBIA) were used. The possibility of using both sides of the nitrocellulose membrane (NCM) by tissue blot immuno assay (TBIA) for the detection plant viruses. Tissue blots were made by pressing with a firm and gentile force, the freshly cut tissue surface on nitrocellulose membranes. The technique of tissue blotting on nitrocellulose membrane was described for detection of plant viruses in infected plants. The above procedure termed dot blot immunobinding assay (DBIA). This dot assay was modified to detect protein by spotting the antigen on a nitrocellulose membrane and incubating the membrane in test antibody followed by incubation in peroxidase-conjugated second antibody to the first antibody, and by development in 4-chloro-1-naphthol. Blotting technique has become widely used for specific identification of nucleic acid and proteins. Single commercially available second antibody conjugate is utilized, thus eliminating the problems of preparing and storing many different conjugated antisera. The crude antiserum could be used, although it should be cross absorbed before to prevent spurious host reaction. It needs only to be macerated and added to the plate. The advantages of indirect ELISA are sample. This avoids the necessity of making specific enzyme conjugates for each antigen to be tested and eliminates the extreme specificity, thus allowing for quantitative evaluation of strain relationships. The efficiency of the ELISA technique was for practical purpose independent of the ratio of antibodies to antigen. It proved to be a very valuable detection tools for the plant viruses. The use of enzyme linked immunosorbent assay (ELISA) for the detection of plant viruses is well documented.

Dot blot assay principle