Godara Savina, Pundir C S
Biochemistry Research Laboratory, Department of Biochemistry & Genetics, MD University Rohtak, India.
Indian J Med Res. 2008 Apr;127(4):370-6.
BACKGROUND & OBJECTIVE: Although the measurement of oxalate in urine and serum by Amaranthus leaf oxalate oxidase immobilized on free arylamine glass beads is highly sensitive and specific, the handling of glass beads is tedious and cumbersome. The present study was undertaken to overcome this problem.
Partially purified Amaranthus spinosus leaf oxalate oxidase was immobilized through diazotization onto arylamine glass beads affixed on the surface of a plastic strip by a non reactive fixative and employed for oxalate determination in urine and serum samples collected from healthy individuals and urinary stone formers.
The immobilized enzyme retained 56 per cent of its initial activity with a conjugation yield of 40 mg/g support. The strip bound enzyme showed maximum activity at pH 3.5 when incubated at 40 degrees C for 15 min. The minimum detection limit of the method was 0.01 mM/l in the urine and 2.5 microM/l in the serum. The analytical recovery of added oxalate was 97.7+/-1.2 per cent in urine and 92.0+/-2.4 per cent in serum. Within and between assay coefficient of variation (CV) were 4.6 and 5.2 per cent in urine and 7.4 and 5.8 per cent in serum respectively. A good correlation for oxalate in urine (r1= 0.99) and in serum (r2= 0.92) was obtained between Sigma kit method and the present method. The strip could be reused 150 times over a period of 2 months, when stored at 4 degrees C in reaction buffer.
INTERPRETATION & CONCLUSION: Immobilization of Amaranthus leaf oxalate oxidase on to affixed glass beads provided enormous ease in its reuse for determination of oxalate in urinary and serum samples.
尽管固定在游离芳胺玻璃珠上的苋菜叶草酸氧化酶用于尿液和血清中草酸盐的检测具有高灵敏度和特异性，但玻璃珠的处理繁琐且麻烦。本研究旨在克服这一问题。
通过重氮化将部分纯化的刺苋叶草酸氧化酶固定在由非反应性固定剂固定在塑料条表面的芳胺玻璃珠上，并用于检测从健康个体和尿路结石患者采集的尿液和血清样本中的草酸盐。
固定化酶保留了其初始活性的56%，结合产率为40mg/g载体。当在40℃孵育15分钟时，条带结合的酶在pH 3.5时显示最大活性。该方法的最低检测限在尿液中为0.01mM/l，在血清中为2.5μM/l。添加草酸盐的分析回收率在尿液中为97.7±1.2%，在血清中为92.0±2.4%。尿液中批内和批间变异系数（CV）分别为4.6%和5.2%，血清中分别为7.4%和5.8%。Sigma试剂盒法与本方法在尿液（r1 = 0.99）和血清（r2 = 0.92）中的草酸盐检测上具有良好的相关性。当在反应缓冲液中于4℃储存时，该条带可在2个月内重复使用150次。
将苋菜叶草酸氧化酶固定在固定的玻璃珠上极大地方便了其重复用于尿液和血清样本中草酸盐的检测。
