BSA and cBSA

BSA possesses a total of 59 lysine e-amine groups (with only 30—35 of these typically available for derivatization), 1 free cysteine sulfhydryl (with 17 disulfides buried within its three-dimensional structure), 19 tyrosine phenolate residues, and 17 histidine imidazolides. The presence of numerous carboxylate groups gives BSA its net negative charge (pi 5.1). 

BSA Containing Positively-Charged Amines and Negatively-Charged Carboxylates 

After the boost, the group of mice receiving the AV—cBSA conjugate generated over twice the antibody response as the group receiving the peptide conjugated to native BSA. 

A corollary to the use of cBSA as a carrier protein is that its increased immune response often abrogates the use of complete Freund s adjuvant, now a source of concern because of its potential side effects in animals. A relatively innocuous mixture with alum is usually all that is required as adjuvant to result in good antibody production. 

As mentioned previously for KLH, DMSO may be used to solubilize hapten molecules that are rather insoluble in aqueous environments. Conjugation reactions may be done in solvent/aqueous phase mixtures to maintain some solubility of the hapten 

BSA containing positively charged amines and negatively charged carboxylates 

Cationized BSA containing blocked carboxylates with additional positively charged amines 

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Figure 7. Particle size distributions in aqueous suspensions of A-300/BSA(Sigma) at Cs,o2 = 0.45 wt.% and Cbsa = 0.07 wt.% and pH (a) 3.84 and (b) 8.53 with respect to the light scattering intensity, particle volume and number.

The most common carrier proteins in use today are keyhole limpet hemocyanin (KLH MW 4.5 X 105 to 1.3 X 107), BSA (MW 67,000), aminoethylated (or cationized) BSA (cBSA), thyroglobulin (MW 660,000), ovalbumin (OVA MW 43,000), and various toxoid proteins, including tetanus toxoid and diphtheria toxoid. Other proteins occasionally used include myoglobin, rabbit serum albumin, immunoglobulin molecules (particularly IgG) from bovine or mouse sera, tuberculin purified protein derivative, and synthetic polypeptides such as poly-L-lysine and poly-L-glutamic acid. 

Figure 272 The effectiveness of cBSA as an immunogen can be seen by the comparison of specific antibody response in mice to arginine vasopressin coupled to both native and cationized BSA. The quantity injected was standardized according to the amount of arginine vasopressin present. The cationized carrier results in higher concentrations of antibody produced against the peptide than the immunogen made with native BSA.

A-300 and PVP/A-300 prepared with the solid residual of the centrifuged dispersion dried at room temperature, then heated at 350K for 3 h and pressed (28 x 2 mm, 10-16 mg) or PVP (0.5-0.6 mg) stirred with KBr ( 60 mg) were used to record the IR spectra by means of a Specord M80 (Karl Zeiss, Jena) spectrophotometer. BSA/A-300 samples (22 x 5 mm, 12mg) were prepared utilizing dried (at room temperature for 24 h), stirred and pressed solid residual of the centrifuged (6000 rpm for 30 min) BSA/silica dispersions (Csio = 5 wt. % and various Cbsa) exposed for 1 h. 

FIGURE 6.28 Dependence of interfacial free energy on concentrations of (a) BSA in the aqueous solution and (b) glucose at Cbsa=2 wt% (curve 1) or fructose at Cbsa= 1-5 wt% (curve 2) in the ternary systems BSA/ monosugar/water. 

FIGURE 6.32 Adsorption (curve 1) and desorption (curve 2) isotherms of saccharose on surface of (a) nanosilica and (b) BSA/nanosilica at Cbsa=515 mg/g. 

With increasing charge density, more efficient cellular uptake of the polycationic BSA derivatives has been found and the highest polycationic albumin cBSA-147 revealed the most efficient uptake into A549 cells [6]. Interestingly, even at high cBSA-147 concentrations only low cytotoxicity has been found. Cell uptake proceeds... 

Various amounts of BSA (Cbsa = 0-128 pmol dm" ) were added to the NaHA solution of which concentration was 0.1 w/v% (Cha = 1-2 pmol dm" ) and that of the carboxylic groups was 2.5 mmol dm". Phase separation did not occur even at the maximum Cbsa- The electrical mobilities of the mixed solutions were measured under... 

Figure 4 shows the results of U of the complexes as a function of the concentration of BSA Cbsa - In the region of Cbsa < 60 pmoldm , the values were almost constant. In the region of Cbsa > 60 pmol dm they increased with increasing Cbsa and attained the constant value at Cbsa = 122pmoldm" at which a saturated complex is considered to be formed. 

The relative viscosity rj i of the mixed solutions composed of NaHA and BSA was measured at 25 °C under J = 0.001 mol dm The results are shown in Fig. 5 as a function of Cbsa added to the NaHA solution (1.2 pmoldm ) which was the same solution as that for the electrical mobility. The values of /rei were found to decrease with increasing Cbsa- As mentioned above, with increasing Cbsa the values of bsa/ ha of the complexes and the molar masses increased. The molar masses were obtained by approximating all added BSA molecules bind to NaHA molecules. As shown in Fig. 5, the molar masses of the complexes increased linearly with increasing Cbsa from 8.5 x 10 to 9.25 x 10 gmol In spite of the increase of the molar mass of the saturated soluble complex as much as almost ten times of NaHA, the viscosity was found to decrease. This is considered to result from the change of the shape of the complexes, i.e., from a rod to a random-coil type. 

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