Prediction of maximum absorption activities with polyclonalantibodies immobilized on magnetic submicron particles torecognize positive mutants / 西安交通大学学报(医学版)

ABSTRACT

Objective To observe polyclonal antibodies immobilized on magnetic submicron particles (MSP) as affinity adsorbents and test the reliability of predicted maximum adsorption activity of an enzyme/mutant from a cell lysate (Vs) in recognizing positive mutants.Methods Escherichia coli alkaline phosphatase (ECAP) and Pseudomonas Aeruginosa arylsulfatase (PAAS) were purified by affinity chromatography to serve as immunogens for the preparation of their antisera, which after fractionation by 33％ ammonium sulfate and DEAE-cellulose chromatography yielded the respective polyclonal antibodies.After activation of COOH on MSP, polyclonal antibodies of each enzyme were immobilized to give MSP-polyAb.Activities of an adsorbed enzyme were measured with a chromogenic substrate of 4-nitrphenol by determining absorbance at 405nm after the termination of reaction by alkali.Based on the response curve of activities of the adsorbed enzyme to protein quantities of a lysate, Vs was predicted for comparison.Results The maximum adsorption quantity of ECAP or PAAS on the respective MSP-polyAb was about 2.0mg/g.Specific activity of ECAP after affinity purification was about 70-fold of that of PAAS.ECAP mutant R168K showing about 50％ activity improvement versus ECAP was recognized by comparison of Vs predicted with only 2.5μg of MSP-polyAb;with PAAS mutant G138S as the starting one, the use of 10.0μg of MSP-polyAb to predict Vs recognized the mutants bearing more than 20％ activity improvement.Conclusion With an optimized quantity of MSP-polyAb to predict Vs, weak positive mutants of an enzyme of low activity can be recognized when activities of the adsorbed enzyme/mutant are reliably measured.