Bovine serum albumin, antigenic

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

A PCP antigen was produced by coupling the hapten 4-[(l-piperi-dinyl )cyclohexyl]benzoic acid sulfate 1/4 M hydrate (figure 2) to bovine serum albumin using 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide HC1 in aqueous dimethylsulfoxide at pH 4.0 to 4.5. The antibodies produced against this antigen have been shown to be fairly specific for PCP versus PCP metabolites (Owens et al. 

Proteins may be covalently attached to the latex particle by a reaction of the chloromethyl group with a-amino groups of lysine residues. We studied this process (17) using bovine serum albumin as a model protein - the reaction is of considerable interest because latex-bound antigens or antibodies may be used for highly sensitive immunoassays. The temperature dependence of the rate of protein attachment to the latex particle was unusually small - this rate increased only by 27% when the temperature was raised from 25°C to 35°C. This suggests that non-covalent protein adsorption on the polymer is rate determining. On the other hand. the rate of chloride release increases in this temperature interval by a factor of 17 and while the protein is bound to the latex particle by only 2 bonds at 25°C, 22 bonds are formed at 35°C. 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

Smith, D.F., Zopf, D.A., and Ginsburg, V. (1978) Carbohydrate antigens Coupling of oligosacchanide phenethylamine-isothiocyanate derivatives to bovine serum albumin. In Methods in Enzymology, (V. Ginsburg, ed.), Vol. 50, pp. 169-171. Academic Press, New York. 

An alternative approach to the production of subunit vaccines entails their direct chemical synthesis. Peptides identical in sequence to short stretches of pathogen-derived polypeptide antigens can be easily and economically synthesized. The feasibility of this approach was first verified in the 1960s, when a hexapeptide purified from the enzymatic digest of tobacco mosaic virus was found to confer limited immunological protection against subsequent administration of the intact virus. (The hexapeptide hapten was initially coupled to bovine serum albumin, used as a carrier to ensure an immunological response.)... 

The use of biological materials as coatings for piezoelectric crystals was first demonstrated by Shons et al. [237], who immobilized bovine serum albumin (BSA) on a crystal precoated with a 30% solution of Nyebar C, a low-surface energy plastic. The rationale of using this solution as the coating material is that proteins adsorbed on low-energy surfaces retain their antigenic properties. Exposure of the BSA-coated crystal to a solution... 

Figure 7. Structure and location of the six regions of bovine serum albumin (BSA) and of human serum albumin (HSA) that we have shown to carry antigenic sites. It Is not Implied that the antigenic sites comprise the entire size of the regions shown, but rather that they fall within these regions. Reproduced with permission from Refs. 9, 10, and 11.

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