RESUMO
Objective To establish an assay for detecting α2-macroglobulin activity in Cohn fraction Ⅳ.Methodsα2-M reacted with trypsin to form α2-M-trypsin complex.After the chromogenic substrate Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride ( BAPNA) was added, absorption at 410 nm was detected with the microplate reader .α2-M activity in Cohn fractionⅣwas quantitatively detected according to the established standard curve of plasma α2-M activity. Result Several critical parameters in this assay were optimized .A standard curve of plasma α2-M activity was established . According to this standard curve ,α2-M activity in Cohn fraction Ⅳsample was detected to be 1.578 PU/ml.Conclusion Using normal human plasma as the reference material , theα2-M activity in Cohn fractionⅣcan be detected through chro-mogenic substrate assay.This study provides a simple method to detect α2-M activity during the purification process of α2-M from Cohn fraction Ⅳ.
RESUMO
Objective To study the determination of biological activity of ?_1-AT with chromogenic substrate and its influential factors using fresh pooled normal human plasma as reference standard.Methods Measuring absorption value of reactions between residual trypsin and BAPNA at 405nm,after ?1-AT inhibition by excess trypsin.Fresh pooled normal human plasma was used as reference standard to calculate the biological activity of?1-antitrypsin.Results Good linear correlation was obtained when the plasma was diluted to 1/50 to 1/100.PEG4000,sucrose,S/D(Tween80/TNBP) and sodium caprylate did not influence the biological activity of ?1-AT,but?1-AT activity was increased by about 20% when the concentration of sodium citrate was above 0.125mol/L.Conclusion The experiment proved that?1-AT biological activity was determined using fresh pooled human plasma as reference standard,the method is stable and reliable.Except sodium citrate,all of the materials used in the assay did not influence the determination of?1-AT activity.