RESUMO
Papain, bromelin and ficin can be standardized by the casein method for use in red blood cell antibody screening tests. The minimal and optimal enzyme activity for detecting blood group antibodies in donors, using the new Automated Pre-Transfusion Blood Testing System Olympus PK7200 by the two-stage method for all three enzymes, is one casein unit. One casein unit of proteinase activity changed the red blood cell surface charge to a low plateau value as measured by electrophoretic mobility and sialic acid content, and removed sterically inhibiting structures of surface protein as detected by SDS-PAGE.
Assuntos
Doadores de Sangue , Antígenos de Grupos Sanguíneos/imunologia , Bromelaínas/normas , Ficina/normas , Isoanticorpos/sangue , Papaína/normas , Autoanálise/métodos , Bromelaínas/metabolismo , Caseínas/metabolismo , Eletroquímica , Membrana Eritrocítica/química , Membrana Eritrocítica/imunologia , Membrana Eritrocítica/metabolismo , Ficina/metabolismo , Hemaglutinação , Humanos , Ácido N-Acetilneuramínico , Papaína/metabolismo , Ácidos Siálicos/sangueRESUMO
Because substrate specificities differ between proteolytic enzymes and because knowledge of the optimal enzyme activity levels is necessary in order to standardize procedures used in antibody screening, a study was made of the best common assay method for the routinely employed enzymes bromelain, papain and ficin. Casein degradation was found better suited to this purpose than azoalbumin. With standardization achieved, a useful two-phase bromelain inhibitor technique was devised using bromelain at 20 casein units of activity. This method improved upon the one-stage bromelain technique in terms of sensitivity, freedom from false positive reactions and it compared well with the two-phase papain inhibitor technique.
Assuntos
Albuminas/metabolismo , Tipagem e Reações Cruzadas Sanguíneas/métodos , Bromelaínas/metabolismo , Caseínas/metabolismo , Cisteína Endopeptidases/metabolismo , Ficina/metabolismo , Papaína/metabolismo , Bromelaínas/antagonistas & inibidores , Estudos de Avaliação como Assunto , Ficina/antagonistas & inibidores , Técnicas Imunoenzimáticas , Leucina/farmacologia , Papaína/antagonistas & inibidores , Especificidade por SubstratoRESUMO
Human sera contain bromelin inhibitors in irregular antibody screening techniques. In order to identify and purify these inhibitors, pooled human sera were precipitated with ammonium sulfate, followed by DEAE-cellulose and Sephadex G-150 column chromatography. Proteinase activity and inhibitory activity to bromelin were observed in two protein fractions, these were identified as alpha 2-macroglobulin and the S-2 thiol proteinase inhibitor.
Assuntos
Anticorpos/análise , Bromelaínas/antagonistas & inibidores , Inibidores Enzimáticos/sangue , Cisteína Endopeptidases , Endopeptidases , Testes de Hemaglutinação , Humanos , Métodos , Inibidores de Proteases , alfa-Macroglobulinas/análiseRESUMO
A simple standardization method for bromelin used in routine one-stage antibody screening is described. Bromelin proteinase activity was assayed using casein as the substrate, and converted to units. The use of proteinase activity units for standardization of bromelin resolves differences between commercial preparations.