Preparation and application of a novel monoclonal antibody specific for the heat shock protein 60 of Lawsonia intracellularis

Porcine proliferative enteropathy (PPE), an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs widespread. An accurate method for detecting L. intracellularis is particularly important for preventing and controlling PPE. Heat shock protein 60 (Hsp60) is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms. Thus, the purpose of the current investigation was to produce a novel L. intracellularis Hsp60 monoclonal antibody (mAb) useful for immunodiagnostics. Three hybridomas secreted anti-Hsp60 termed 3E5, 4E2, and 9G6 were generated, and the titers of ascitic fluids of 3E5, 4E2, 9G6 were 1:1024000, 1:2048000 and 1:2048000, respectively. The western blotting analysis demonstrated that rHsp60 was recognized by mAbs 3E5, 4E2 and 9G6. Subsequently, analyses of specificity showed all the mAbs were highly specific to L. intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs, such as Escherichia coli, Salmonella Choleraesuis, Salmonella Typhimurium, and Brachyspira hyodysenteriae. Furthermore, the mAbs were useful for detecting L. intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs. Our research will provide a foundation for the development of immunological diagnostic tests.