Whole-bacterial cell enzyme-linked immunosorbent assay for cell-bound Moraxella bovis pili

Type‐IV pili spectroscopic markers: Applications in the quantification of piliation levels inMoraxella boviscells by a FT‐IR ANN‐based model

“…Pili were recovered from piliated cells by mechanical shearing and purified as previously described [17]. The purity of the purified pili was confirmed by 15% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining.…”

“…A sequential competitive bact-ELISA was used to quantify the cellular piliation level of M. bovis suspensions as described previously [17].…”

“…In a previous work we demonstrated that this could be due to the piliation loss that occurs as a consequence of the shear forces generated in cell environments when they are grown in mechanically stirred bioreactors [15]. Therefore, in M. bovis vaccine production, it is crucial to monitor cell-bound pili levels during bacterial growth to verify whether the bacterial cells produced maintain their immunogenic properties [11,15,17]. Consequently, we tested if piliation levels of samples withdrawn from stirred bioreactors could be estimated by the FT-IR ANN-based system developed.…”

“…Rapid and reliable detection and quantification of the piliated phenotype in M. bovis is crucial to guarantee the efficacy of vaccine formulations and also important for any study of pathogenicity and epidemiology of IBK [11]. Current techniques for identifying piliated or nonpiliated cells include electron microscopy, slide agglutination (SA), tandem-crossed immunoelectrophoresis (TCIE) and immunological tests such as immunodiffusion and enzyme-linked immunosorbent assay (ELISA) [16][17][18]. These techniques are the only tools available up to now to evaluate cell-bound pili.…”

“…These techniques are the only tools available up to now to evaluate cell-bound pili. Although these approaches are in general accurate, experiments are expensive and time consuming; therefore, they are normally not appropriate to be applied in industrial or field conditions [17][18][19]. Importantly, as M. bovis pili antigens are currently arranged into seven serogroups (A to G), and antibodies against each serotype exhibit no cross reaction among them, seven different ELISA methods should be developed to detect each bacterial pilus serotype.…”