Anti-BSA

For each antigen, three groups of ten mice were immunized subcutaneously on day 1 with 0.2 mL of the entrapped formulation, 0.2 mL of the crosslinked formulation or 0.2 mL of a CFA- antigen emulsion. A secondary immunization was administered on day 14 with 0.2 mL of the same Adjuvax formulations or 0.2 mL of an IFA-antigen emulsion. On day 27, serum was collected and analyzed for anti-P55 antibody or anti-BSA antibody by ELISA. 

Figure 5. Comparison of Freund s Adjuvant to Adjuvax formulations in stimulating antibody response to BSA. Relative antibody titers between adjuvant groups at day 27 were determined by measuring the absorbance at 450 nm of a 1 500 dilution of anti-BSA immune sera by ELISA.

FIGURE 14.6 Typical stripping voltammograms for (a) nanocrystal-labeled antibodies and (b-f) magnetic bead-Ab-Ag-Ab-nanocrystal complexes, (b) Response for a solution containing dissolved ZnS anti-/32-microglobulin, PbS-anti-BSA, and CdS-anti-IgG conjugates (reproduced from [29] with permission). 

Monoclonal antibody-binding affinity was determined by microchip-based capillary electrophoresis with LIF detection (33). The mixing was carried out off-chip, and the on-chip separations were performed in less than 60 s (Fig. 9). A Scatchard plot analysis resulted in an affinity constant for the monoclonal anti-BSA antibody to fluorescently labeled BSA (BSA ) of... 

X 107 M 11 for a 1 1 stoichiometric ratio. Two affinity complexes were separated. One complex was identified by the Scatchard method as having a 1 1 stoichiometric ratio (complex 1). The other complex (complex 2) is proposed to have a stoichiometry with an excess of anti-BSA to BSA, ... 

Fig. 9 Series of electropherograms obtained on-chip from mixtures of 361 nM anti-BSA antibody with different concentrations of labeled BSA (BSA ). The ratio of total concentration of BSA to total concentration of antibody (Ag/Ab) is given at the top of each electropherogram. Two main complex peaks are identified. Essentially no BSA is seen in A, while BSA dominates in F. (Reprinted with permission from Ref. 33. Copyright 1998 Wiley-VCH.)...

The secondary bonds, which may be formed much more slowly than the primary bonds, actually contribute more to the overall affinity. For example, the primary (Coulombic) bond between bovine serum albumin (BSA) and anti-BSA IgG is 3.3kcalM 1 whereas the secondary bond (van der Waals) is 28kJ, for a total AH = 42 kJ. Because the formation of the secondary bond is much slower, it is easier to prevent formation of the strong complex rather than to try to dissociate it. This is one reason why the competitive immunoassays yield results that correlate with the equilibrium-binding constants, but any such direct-binding assays have to rely on the measurement of the initial rate of binding. 

BSA Selective binding to anti-BSA antibody in electropolymerised polypyrrole film on SPCE surface Impedance... 

Homogeneous immunoassay is conducted entirely in solutions. In this format, separation of the labeled antibody (Ab) and the antibody-antigen (Ab-Ag) complex is required, and this is usually achieved using CE separations. For instance, separation of Cy5-BSA from unreacted Cy5 and the complex (formed from Cy5-BSA and anti-BSA) was achieved in a flow-through sampling chip [567]. 

In addition, affinity CE separation was carried out to resolve monoclonal anti-BSA and BSA in dilute mouse ascites fluid. Then the affinity constant of this antigen-antibody interaction was measured [1004]. 

Fig. (10). Agar diffusion of different proteins and a lipid conjugated with lactose against anti-lactose antibodies (well L) and against anti-BSA antibodies (Well BS) wells 1-6 contained Lac-poly, Lac-BSA, Lac-sphingosine, Lac-ORA, Lac-HGG and BSA Chemical modification of the antigen by periodate oxidation or borohydride reduction can effect an agar diffusion against anti-gum arabic antibodies (Se), GA=gum arabic Bl=blank.

Fig. (41). Agar diffusion pattern B = BSA, A = immune serum, P = purified anti-fucose Abs, C = purified anti-BSA Abs, F = fiicose-BSA.

Fig. (43). Isoelectrofocusing and agar diffusion of anti-Fuc and anti-BSA antibodies.

Marzocchi-Machado CM, Polizello AC, Azzolini AE, Lucisano-Valim YM. The influence of antibody functional affinity on the effector functions involved in the clearance of circulating immune complexes anti-BSA IgG/BSA. Immunol Invest 1999 28(2-3) 89—101. 

Fig. 10.—Reactivity of anti-galactose antibodies (well Ai) and anti-BSA antibodies (well A2) with BSA (1), periodate-oxidized /3-Gal-BSA (2), and /3-Gal-BSA (3). Inhibition of anti-galactose antibodies (Ab) by galactose (D) and glucose (E), and reference (C). Wells 4-7 contain decreasing amounts of /3-Gal-BSA. (Reprinted from Journal of Immunological Methods, Volume 75, J. H. Pazur and S. A. Kelly, pp. 107-116, copyright 1984 with kind permission of Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)...

Diffusion in agar performed by the conventional method shows a strong reaction between the antibody and Man-BSA, but not with BSA (Fig. 12, plates A). However, both compounds give precipitin complex with anti-BSA serum. Oxidation of the antigens with periodate no longer gives a precipitin reaction with the anti-mannose antibodies, but has no effect on the anti-BSA antibodies. Hapten-inhibition results are also shown in Fig. 12, plates C and D. These tests are conducted with the purified antibody... 

Fig. 12.—Reactivity of anti-a-mannose antibodies and anti-BSA antibodies with the native and periodate-oxidized mannose-glycoconjugate and BSA (plates A and B). M = Man-BSA, xM = periodate-oxidized Man-BSA, B = BSA, xB = periodate-oxidized BSA, A = antimannose antibodies, A2 = anti-BSA antibodies. Plates C and D show hapten inhibition of purified anti-mannose antibodies (A ) with mannose (L). The amount of antigen in the outer wells ranges from 20 /u.g in well 1 to 1 /u.g in well 6. [Reprinted with permission from J. H. Pazur, B. Liu, and T. Witham. J. Protein Chem., 13 (1994) 59-66.]...

Fig. 13.—A Agar diffusion of immune sera (Se), 1-thio-D-mannose antibodies (A ), and anti-BSA antibodies (A2) against Man-S-BSA (/) and BSA (2). B Agar-diffusion plate of anti-Man-S-antibodies and antibodies oxidized by peroxypropanoic acid for 0, 4, and 8 h. C, D, E, and F Hapten inhibition by agar diffusion, A = purified anti-Man-S antibodies I = antibodies + p-nitrophenyl 1 -thio-a-D-mannopyranoside 12 = antibodies + D-mannose I3 = antibodies + ethyl 1-thio-a-o-mannoside 1 to 6, outer wells contain decreasing concentration of Man-S-BSA. (Reprinted with permission from Journal of Protein Chemistry, Volume 9, J. H. Pazur, B. Liu, Nan Q Li, and Y. C. Lee, pp. 143-150, copyright 1990 Journal of Protein Chemistry.)...

The anti-fucose antibodies were eluted by 0.5 M L-fucose solution from the first column and the anti-BSA antibodies by elution with 0.5 M ammonium thiocyanate from the second. Agar diffusions are shown in the plates in Fig. 14. 

On gel electrophoresis, the purified antibody preparations yield single bands when the gel is stained for proteins. However, on gel isoelectrofocus-ing, differences in the results maybe noted, as shown in Fig. 15. Several protein isomers were present in each antibody preparation, with 7 isomers being detected in the anti-BSA antibodies (B) and 11 isomers in the anti-fucose antibodies (F). The coupled electrofocusing-agar diffusion method showed that each isomer of the anti-fucose set possessed the same antibody activity with the antigen, a-L-fucosyl-BSA. 

Fig. 16.—Inhibition of anti-Fuc and anti-BSA antibodies by fucose. A, = anti-fucose antibodies, I = anti-fucose antibodies + fucose, A2 = BSA antibodies, L = BSA antibodies + fucose.

For BSA assay, to 1.5 ml of buffer-BSA solution, typically 40 fi of a 1 100 dilution of rabbit anti-BSA (2.4 mg/ml) and 40 /aI of a 1 10 dilution of urease-BSA conjugate were added. For cAMP assay, 30 ju.1 of a 1 10 dilution of the (NH4)2S04 fraction of cAMP antiserum (0.5 mg/ml) and 30/al of a 1 10 dilution of enzyme-cyclic nucleotide conjugate were added to 1.0 ml of buffer or buffer-nucleotide standard. 

Fig. 4. Calibration curve obtained for bovine serum albumin (BSA), by monitoring amount of urease-BSA conjugate bound to anti-BSA antibody. Data were obtained with tubes containing 1.5 ml of BSA standard, 40 fil of 1 100 anti-BSA serum, 40 /al of 1 10 urease-BSA conjugate, and 300 1 of insolubilized goat anti-rabbit y-globulin suspension, all prepared in 10 mAf Tris-HCl-EDTA, pH 7.5.

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