RESUMEN
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.
Asunto(s)
Donantes de Sangre , Antígenos de Grupos Sanguíneos/inmunología , Bromelaínas/normas , Ficaína/normas , Isoanticuerpos/sangre , Papaína/normas , Autoanálisis/métodos , Bromelaínas/metabolismo , Caseínas/metabolismo , Electroquímica , Membrana Eritrocítica/química , Membrana Eritrocítica/inmunología , Membrana Eritrocítica/metabolismo , Ficaína/metabolismo , Hemaglutinación , Humanos , Ácido N-Acetilneuramínico , Papaína/metabolismo , Ácidos Siálicos/sangreRESUMEN
Proteolytic enzyme preparations and techniques used routinely in blood group serology for the detection of atypical patient antibodies prior to transfusion vary widely and are often poorly standardised. Recent advances have been made in the use of biochemical methods to standardise and stabilise the potency of the enzyme preparations used. A joint working party of the International Council for Standardization in Haematology (ICSH) and the International Society of Blood Transfusion (ISBT) has investigated possibilities for the provision of standards for the protease preparations and techniques. The specification for these standards was that the performance of enzyme reference preparation in the reference technique should be of equivalent sensitivity to the ICSH/ISBT LISS spin indirect antiglobulin test using a titration series of a reference weak anti-D, and be free from false-positive reactions. The working party circulated materials for evaluation in inter-laboratory trials, followed by a laboratory workshop meeting to achieve agreement on the specification for reference materials and methods. Reference freeze-dried papain at 0.6 azoalbumin units and weak anti-D preparations (91/562) have been prepared and validated to meet these specifications. The performance of a test enzyme preparation in the technique for which it is recommended for use should be at least equal to that of the reference papain preparation, by the reference two-stage technique in terms of sensitivity, using a titration series of the reference anti-D, and freedom from false-positive reactions, using six fresh inert sera. The reference papain and weak anti-D can also be used to calibrate the level of proteolytic activity required in other procedures in blood group serology, such as new technology methods for antibody detection, and automated and microplate cell grouping procedures. These preparations and an agreed method for their use are now available from listed centres as ICSH/ISBT and Food and Drug Administration reference materials.
Asunto(s)
Tipificación y Pruebas Cruzadas Sanguíneas/normas , Bromelaínas/normas , Prueba de Coombs/normas , Papaína/normas , Globulina Inmune rho(D)/sangre , Reacciones Falso Positivas , Liofilización , Humanos , Agencias Internacionales , Estudios Multicéntricos como Asunto , Estándares de Referencia , Sensibilidad y Especificidad , Estados Unidos , United States Food and Drug AdministrationRESUMEN
In-vitro activity of 14 commercial pancreatin preparations, commonly used in the Federal Republic of Germany, were tested. All had been declared by their manufacturers to contain more than 6000 FIP (Fédération International Pharmaceutique) units of lipase and to be acid resistant. The declared lipase and amylase amounts were found to be present in 11 of the 14 preparations. Three of the 14 preparations, said to be acid resistant were found not to be so in buffer with falling pH values between 4.0 and 2.5, so that there occurred an, at times marked, loss of enzyme activity. Most noticeable was the poor solubility of most preparations at pH 6.6. Only three of the 14 liberated their total enzyme content within 60 minutes, as they should for theoretical reasons, based on the relatively short duodeno-cecal transit time.
Asunto(s)
Extractos Pancreáticos/normas , Amilasas/análisis , Amilasas/normas , Bromelaínas/análisis , Bromelaínas/normas , Ácido Deshidrocólico/análisis , Ácido Deshidrocólico/normas , Dimetilpolisiloxanos/análisis , Dimetilpolisiloxanos/normas , Combinación de Medicamentos/análisis , Combinación de Medicamentos/normas , Concentración de Iones de Hidrógeno , Lipasa/análisis , Lipasa/normas , Extractos Pancreáticos/análisis , Pancreatina/análisis , Pancreatina/normas , Solubilidad , Tripsina/análisis , Tripsina/normasRESUMEN
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.