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Use of Horseradish‐Peroxidase Labelled Antibodies in ELISA for Plant Virus Detection
Author(s) -
Polák J.,
Krcarístek J.
Publication year - 1988
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/j.1439-0434.1988.tb01009.x
Subject(s) - horseradish peroxidase , chromatography , peroxidase , chemistry , bovine serum albumin , antiserum , tris , buffer solution , nuclear chemistry , biochemistry , antibody , biology , enzyme , immunology
Detection of plant viruses by ELISA using horseradish peroxidase for antibody labelling (ELISA‐peroxidase) has been standardized by evaluating variants of the procedure, regarding composition and concentration of buffers and additives. Immunoglobulins (IgG) are isolated from antisera by precipitation with ammonium‐sulphate and by purification with DEAE‐52 (Whatman) cellulose. IgG are conjugated with horseradish peroxidase by a modified oxidation‐periodate method. In ELISA‐peroxidase 0.05 M carbonate‐bicarbonate coating buffer pH 9.6 has been substituted by 0.01 M carbonate buffer pH 9.2. Extraction buffer is used with 0.5% bovine serum albumin (BSA), without polyvinylpyrrolidone (PVP). Samples are diluted in, phosphate buffered saline (PBS) pH 7.2 with 0.05% Tween 20 and 0.5% BSA. IgG are conjugated with horseradish peroxidase, diluted in 0.1 M Tris‐HCl, pH 7.4 with 0.05% Tween 20 and 1% BSA. The substrate is incubated in the darkness for 20 min at room temperature. ELISA‐peroxidase proved to be equivalent in sensitivity and specificity with ELISA using alkaline phosphatase for antibody labelling. Its advantage is a lower cost of chemicals used in the test.