BSA is a stable globular protein with a molecular weight of approximately 66 kDa. BSA consists of a polypeptide chain of approximately 583 amino acid residues and does not contain any carbohydrates. Due to its compatibility with biological reactions, it is widely used in various biochemical processes.
BSA as a carrier protein to cross-link haptens and other weak antigens can make them more immunogenic in antibody production. It is very stable and highly soluble. In addition, BSA is large and complex enough for sufficient immunity.
Fig.1 The multivalent attachment of a thiodigalactoside derivative using BSA as the scaffold. (Zhang, et al., 2018)
CD BioGlyco provides development services for polysaccharides and BSA conjugates. Taking the development of Streptococcus pneumoniae Capsular Polysaccharide and BSA conjugates as an example, our development process is as follows.
Polysaccharides are dissolved in sodium borate buffer and activated by adding a solution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP) dissolved in an acetone/water mixture. The pH is immediately adjusted and maintained by continuous dropwise addition of NaOH solution. After the mixture is stirred for several minutes, a certain volume of HCl is added and allowed to stand after stirring. The mixture is passed rapidly through a column of Sephadex G-25 equilibrated in sodium bicarbonate buffer.
BSA is dissolved in buffer and added to the activated polysaccharide solution. The mixture is stirred gently for several hours. Adding buffer to block any remaining activation sites on unreacted polysaccharides and incubate. The final solution is lyophilized and stored until used in the purification procedure.
Conjugation progress is analyzed by high-performance size exclusion chromatography (HPSEC).
The strategy of hydrophobic interaction chromatography (HIC) (columns packed with Sephadex LH-20) is used to purify polysaccharide-protein conjugates. The lyophilized reaction mixture is dissolved in distilled water and applied at a certain flow rate to a column packed with Sephadex LH20 pre-equilibrated with distilled water. Elution is performed isocratically with distilled water.
The carbohydrate content in the conjugate as well as the pure polysaccharide is determined by the phenol-sulfuric acid method, and the protein concentration is determined by the bicinchoninic acid protein assay. The purity of the conjugates is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gels are stained for proteins and carbohydrates.
Female mice are immunized subcutaneously with appropriate amounts of native capsular polysaccharide or capsular polysaccharide-BSA conjugate in sterile saline. Mice inject with saline and BSA respectively are also used as controls. The second immunization is performed after the first immunization.
Enzyme-linked immunosorbent assay (ELISA): Coat flat-bottomed 96-well microtiter plates with capsular polysaccharide saline solution overnight. After washing the wells thoroughly with PBS, block the plate with PBS containing ovalbumin at room temperature. Wells are then washed again as above, and dilutions of individual sera from animals immunized with capsular polysaccharide, capsular polysaccharide-BSA, BSA, and PBS are added and incubated. Mouse antibodies are detected with rabbit anti-mouse IgG conjugated to horseradish peroxidase after washing the wells again.
Fig.2 The preparation process of S. pneumoniae capsular polysaccharide-BSA conjugate. (CD BioGlyco)
CD BioGlyco has developed a highly specialized Glyco™ Vaccine Development Platform and provides high-quality Carbohydrate-based Vaccine Development Service for our clients. We aim to provide customers with unparalleled conjugation services using BSA as the carrier. If you are interested in our services, please contact us for more details without any hesitation.
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