Compositions and serological methods including bovine serum albumin polymers

#The present invention relates to novel serum albumin compositions, to their preparation and use, and to novel polymer components of such compositions.

#In blood serology, the knowledge of the presence of particular antibodies is very important. In blood transfusions, the knowledge of the presence of certain antibodies is essential. Presence of undetected antibodies may cause a transfusion reaction which may be fatal. Thus, when an antibody concentration is so low that they are absorbed by the cells instead of agglutinating it, it is often misinterpreted as compatibility when in fact it was a sensitizing injection. When a second transfusion is given a week to ten days later, a fatal reaction may occur. Even when the reaction is not fatal, it may reduce the normal life-span of transfused, incompatible cells necessitating subsequent transfusions. Such problems may be avoided by prior knowledge of all or most of the antibodies by the provision of a more sensitive antibody detection means.

#Serum or plasma albumins have been used in blood group serology for about 30 years as a means to aid in the detection of some antibodies in blood serum which escape detection when the blood serum to be tested is contacted with a saline or isotonic solution of red blood cells. Agglutination of red cells by antibodies have been classified as (1) those occurring in low protein or low dielectric constant media such as isotonic salt solutions and (2) those occurring in high protein or high dielectric constant media. The antibodies reacting under the former conditions have been identified as belonging to the IgM class of immune globulins, while those reacting under the latter conditions have been identified as belonging to the IgG class The theoretical explanation advanced for the ability of the protein to promote specific agglutination is that it raises the dielectric constant of the medium, thereby reducing the zeta potential of the cells and shortening the average intercellular distance to less than the distance necessary to be spanned by the IgG antibody molecules for agglutination. The protein employed in the detection medium is mammalian serum albumin, particularly bovine serum albumin, because of the availability of large quantities of beef blood. However, available bovine serum albumin solutions have been quite variable in their ability to raise the dielectric constant of water and therefore in their ability to promote the necessary agglutination in the detection of antibodies. The variability has not necessarily been avoided by employing higher concentrations of albumin. Moreover, available bovine serum albumin solutions are not of sufficient potentiating capability to avoid overlooking the existence of certain antibodies.

#The following examples illustrate the invention but are not to be construed as limiting.

#Fourteen milliliters of 0.1 N hydrochloric acid is added to 500 milliliters of a 2% aqueous bovine albumin solution (of albumin content equal to or greater than 98% and consisting of 90% monomer and 10% dimeric aggregate). The pH after the addition is 5.4 To the resulting solution is added with stirring at ambient temperature, 5 milliliters of a 0.588 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (ratio of carbodiimide to albumin, 20:1) and the stirring continued for about 60 minutes to obtain polymerized bovine serum albumin product in the reaction mixture. At this time, 5 milliliters of 0.588 M glycine is added with stirring to the reaction mixture at ambient temperature and the stirring contained for about 15 minutes to quench the polymerization. Fifty milliliters of 0.1 N HCl is added to adjust the pH to about 5.0. Thereafter, 382 milliliters of methanol (previously cooled to 0.degree. C.) is added with stirring over a period of about 30 minutes to the reaction mixture while the temperature is maintained between about -2.degree. C. and -5.degree. C., and the stirring continued for about 15 minutes after completion of the addition, whereupon the polymerized albumin product precipitates. The precipitate is recovered by centrifugation at 9000 r.p.m. for 30 minutes in a Model RC 2 Sorvall centrifuge and amounts to 20.4 grams of polymerized bovine serum albumin. The latter is reconstituted with a minimum volume of distilled water to obtain a 30.3% solution of polymerized bovine serum albumin.

#A 0.7 milliliter portion of the 30.3% polymerized bovine serum albumin solution is admixed with 0.3 milliliter of a 0.9% sodium chloride solution to obtain a 22% polymerized serological bovine albumin solution. Polyacrylamide disc electrophoresis analysis (7% gel at pH 7.2) of this solution shows the following concentrations of monomers and polymers in percent by weight: 71% monomer, 21.2% dimer, 4.8% trimer, 1.1% tetramer and 1.9% higher polymers.