Abstracto

Protein quantification using several short chimeric standards without purification

Hung Chai

The post-weaning multi-systemic wasting syndrome is brought on by porcine circovirus. Despite the availability of commercial vaccines, the development of vaccines that are more cost-efficient and effective is anticipated. Serological distinction between naturally infected and vaccineexposed animals is made possible by the use of chimeric antigens. In this study, chimeric fusion proteins derived from the multimerizing subunit of the mouse polyomavirus capsid protein VP1 and a circovirus capsid antigen Cap were purified and characterised using asymmetric flow field-flow fractionation (AF4) in conjunction with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Different elution profiles, such as decreasing cross-flow and constant cross-flow, were examined (linearly and exponentially). The comparison of the hydrodynamic radius measured by online DLS allowed for the evaluation of the ideal sample retention, separation effectiveness, and resolution. According to the findings, using dual elution profiles (exponential and constant cross-flow rates) speeds up separation time, avoids unfavourable sample-membrane interaction, and produces superior resolution. Additionally, the data reveal no sample deterioration during the AF4 separation and no self-associations of the individual pentameric particles into bigger clusters. Transmission electron microscopy morphological evaluations and the Rg/Rh ratios for various fractions are well correlated. The individual fractions were also submitted to offline analysis, such as batch DLS, TEM, and SDSPAGE, followed by Western blot, in addition to the online examination.