Abstract
To obtain specific monoclonal antibodies (MAbs) against Curtobacterium flaccumfaciens (Hedges 1922) Collins and Jones 1984, protein complexes of C. flaccumfaciens pv. flaccumfaciens NCPPB 390 strain, extracted by method (P-1) of De Boer and Wieczorek, and method (P-2) of Yakrus and Schaad were used as immunogen in mouse. Seven hybridoma lines (1P1, 2P1, 3P1, 4P1, 5P1, 6P1, and 7P1) were established with the P-1 antigen and four ones (1P2, 2P2, 3P2, and 4P2) with the P-2. Three MAbs from P-2-hybridoma lines (2P2, 3P2, and 4P2) showed high specificity against whole cells of C. flaccumfaciens in an indirect ELISA (ABC-AP-ELISA). The isotype of three MAbs was IgG3. All the MAbs from P-1-hybridoma line and 1 MAb from P-2-ones, which were classified as IgM, reacted with many strains of 4 genera of Gram positive bacteria used in this study on ABC-AP-ELISA. The epitopes included in the P-1 and P-2 were analyzed with purified MAb 1P1 and MAb 3P2. When digested with glucoamylase, antigenicity against these MAbs was retained. Further digestion with proteinase K or tripsin destroyed the antigenicity. On western immunoblot of P-1 and P-2, MAb 1P1 recognized two bands of molecular weight 25k and 59k, and MAb 3P2 recognized two bands of molecular weight less than 14.4k and 59k. Those epitopes are probably proteins because of their susceptibility to proteinase K and tripsin. The antibodies recognized epitopes present on a same protein or different proteins having a very similar molecular weight at 59k. They also recognized smaller one clearly different between two antibodies. Purified MAb 3P2 can be used for highly specific detection of C. flaccumfaciens, and all the MAb may be used for the analysis of epitopes in whole cells of some Gram positive bacteria related to C. flaccumfaciens.
