Macanovic M, Lachmann P J
Molecular Immunopathology Unit, MRC Centre, Cambridge, UK.
Clin Exp Immunol. 1997 May;108(2):220-6. doi: 10.1046/j.1365-2249.1997.3571249.x.
A new radial enzyme diffusion (RED) method for the measurement of DNase activity in serum and urine is described. The sensitivity of the assay is in the range of 15.6-500 ng/ml. The assay is based on the hydrolysis of double-stranded (ds) DNA (or nucleosomes) in agarose. The specificity of the reaction for DNase I was established by showing that either EDTA in the reaction buffer or G-actin abolished DNase activity. Being a functional assay, RED has advantages over radioimmunoassay (RIA) or ELISA, since antigenic assays may also measure complexes of DNase with actin. This method was used to measure DNase activity in the sera and urine of lupus-prone mice (NZB/NZW F1 hybrids, aged 4-6 weeks). Serum DNase activity in these mice was significantly lower (mean 9 ng/ml) than in control, normal mice of the same age and sex (mean 37 ng/ml). Concentration of DNase in the urine of 4-6-week-old female NZB/NZW F1 hybrids (24 ng/ml) was significantly lower then in control mice (521 ng/ml). The RED method was used to measure the concentration of actin as the DNase inhibitor in serum. G-actin in the presence of ATP binds DNase and inhibits its nucleolytic activity. Since ATP is necessary for the actin inhibition of DNase I, this shows that there is actin as well as DNase I in the serum. Actin is not only ATP-dependent, but also heat-labile. Heating the sera for 10 min at 50 degrees C increases DNase activity. This is an alternative method for measuring the concentration of actin in the serum. An almost identical estimate of actin concentration in sera of normal mice was found from the difference of DNase activity in the presence or absence of ATP (mean actin concentration = 21 ng/ml) or from the difference of DNase activity in heated and non-heated serum (mean actin concentration 18 ng/ml). We were not able to demonstrate DNase inhibitors in the urine of either control or NZB/W F1 hybrid mice.
本文描述了一种用于测量血清和尿液中DNase活性的新型径向酶扩散（RED）方法。该检测方法的灵敏度范围为15.6 - 500 ng/ml。该检测基于琼脂糖中双链（ds）DNA（或核小体）的水解。通过证明反应缓冲液中的EDTA或G - 肌动蛋白可消除DNase活性，确定了该反应对DNase I的特异性。作为一种功能检测方法，RED比放射免疫分析（RIA）或酶联免疫吸附测定（ELISA）具有优势，因为抗原检测也可能测量DNase与肌动蛋白的复合物。该方法用于测量易患狼疮小鼠（4 - 6周龄的NZB/NZW F1杂种小鼠）血清和尿液中的DNase活性。这些小鼠的血清DNase活性（平均9 ng/ml）显著低于相同年龄和性别的对照正常小鼠（平均37 ng/ml）。4 - 6周龄雌性NZB/NZW F1杂种小鼠尿液中DNase的浓度（24 ng/ml）显著低于对照小鼠（521 ng/ml）。RED方法用于测量血清中作为DNase抑制剂的肌动蛋白浓度。存在ATP时，G - 肌动蛋白结合DNase并抑制其核酸水解活性。由于ATP是肌动蛋白抑制DNase I所必需的，这表明血清中存在肌动蛋白以及DNase I。肌动蛋白不仅依赖ATP，而且对热不稳定。在50℃下将血清加热10分钟会增加DNase活性。这是一种测量血清中肌动蛋白浓度的替代方法。通过存在或不存在ATP时DNase活性的差异（平均肌动蛋白浓度 = 21 ng/ml）或加热和未加热血清中DNase活性的差异（平均肌动蛋白浓度18 ng/ml），发现正常小鼠血清中肌动蛋白浓度的估计值几乎相同。我们无法在对照或NZB/W F1杂种小鼠的尿液中证明存在DNase抑制剂。
