METHODOLOGICAL APOPROACH EXEMPLIFIED BY LACTOCOCCI: OPTIMIZATION OF BIOTECHNOLOGICAL PROCESSES BY MEANS OF ELISA VIA LOCALIZATION OF THE TARGET BIOPOLYMER AND SELECTION OF DURATION OF CULTURE GROWTH AND MEDIA COMPOSITION PROVIDING MAXIMUM YEALD

Abstract

Enzyme-linked immunoassay (ELISA) is suitable as a method for detection and quantification of bacterial biopolymers at condition that they have antigenic properties. Using of ELISA allows optimizing biotechnological processes via selection of the strain producing the biolimer of interest, localization of the biopolymer and selection of duration of culture growth and media composition providing maximum yeald. We elaborated the methodology for studing the time dependence of specific concentration (Csp) (calculated per cell) of the biopolymer of interest in cell fractions and culture media. The methodology includes four points: (i) selecting of bacteria applicable as objects of study; (ii) obtaining, preliminary preparation and quantification of cell fractions and specimens of culture media; (iii) design of ELISA suitable for studing Csp of intracellular antigens (contained in cell-wall free fraction), cell wall antigens and secretory antigens; (iv) methods for converting of ELISA data into Csp (in conventional units) and explanation of the results. We present the methodology using 4 strains of lactococci and rabbit polyclonal antibodies (PAb) against genus-specific antigens of LаÑtococcus lactis subsp. cremoris BIM B-493D (PAbanti-Ll 493D) as an example. We show that Csp of lactococcal antigens in cell fractions and culture media depends on strain, media composition and duration of culture growth. Distribution of antigens between cell fractions and media and time-dependance of Csp is unpredictable and for each strain/media should be determined individually. The methodology is suitable for any bacterium and any well-defined antigen under the condition that either PAb or mouse monoclonal antibodies specific for this antigen will be used. The methodology will find an application in biotechnology and research studies.