Development of a Method to Obtain Monospecific Antibodies Directed Against a 31 kD Protein of Pseudomonas syringae pv. syringae

Abstract

We describe a simple method of purifying Pseudomonas syringae pv. syringae (P. s. pv. s.) specific polyclonal antibodies which are directed against a cell surface protein sized 31 kD. The actual purification step of the polyclonal antibodies occurs with denatured proteins after an SDS-PAGE gel electrophoresis and western blotting. Polyclonal antibodies were obtained which recognized a 31 kD protein. On a western blot no cross reaction of the purified polyclonal ‘monospecific 31 kD antibodies’ with other proteins from the same strain was observed. However, a positive “monospecific 31 kD antibody” reaction was only visible when either protein extracts from several selected pathogenic P. s. pv. s. strains from different hosts, or two isolates of Pseudomonas syringae pv. pisi (P. s. pv. p.) were used. An indication that this 31 kD protein is specific to P. s. pv. s. is that no cross reaction was found with protein extracts from the tested pathogenic and non-pathogenic pseudomonads. However, a differentiation of the tested P. s. pv. s. and P. s. pv. p. isolates was possible when cells were grown above 30 °C. Then protein extracts from P. s. pv. s. revealed the lack of the 31 kD protein but not P. s. pv. p. protein extracts. Polyclonal antibodies raised in rabbits against the 31 kD protein band from the western blot proved to be specific enough to agglutinate whole P. s. pv. s. strain R32 cells or for detecting a single 31 kD protein band on a western blot when whole cell protein extracts of P. s. pv. s. strain R32 were used.