Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology.
The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or =99% purity, a yield of 25.0 +/- 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60 degrees C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37 degrees C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.
Tanaka
K
K
Divisão de Pesquisa e Desenvolvimento Industrial, Fundação Pró-Sangue Hemocentro de São Paulo, Av. Enéas C. Aguiar 155, 05403-000 São Paulo SP, Brazil. tanaka.k@bol.com.br
Sawatani
E
E
Shigueoka
E M
EM
Dias
G A
GA
Nakao
H C
HC
Arashiro
F
F
D000372:Agglutination Tests / Q000379:methods; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D004202:Disinfectants / Q000494:pharmacology; D004564:Electrocoagulation; D004588:Electrophoresis, Cellulose Acetate; D005557:Formaldehyde / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D006388:Hemagglutinins; D011108:Polymers; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification*
D004202:Disinfectants; D006388:Hemagglutinins; D011108:Polymers; D005557:Formaldehyde; D012710:Serum Albumin, Bovine; D005976:Glutaral