高效凝胶渗透色谱法检测马初乳中的免疫球蛋白G  

Determination of immunoglobulin G in mare colostrum by high-performance gel permeation chromatography

作　　者：吕岳文[1] 王红娟[1] 杨洁[1] 

机构地区：[1]新疆大学生命科学与技术学院,新疆乌鲁木齐830046

出　　处：《色谱》2011年第3期265-268,共4页Chinese Journal of Chromatography

基　　金：国家科技支撑计划重大项目(2006BAD04A11-06);新疆维吾尔自治区科技重大专项项目(200731134)

摘　　要：建立了高效凝胶渗透色谱(HPGPC)测定马初乳中免疫球蛋白G(IgG)含量的方法。采用TOSOH TSK-G4000PWXL色谱柱(300 mm×7.8 mm,5μm)分离,以0.05 mol/L磷酸盐缓冲液(pH 6.9)为流动相,流速0.8mL/min,检测波长280 nm,温度25℃。结果表明:免疫球蛋白G的线性范围为0.2～3.0 g/L(r2=0.999 5),平均回收率为97.47%,相对标准偏差(RSD)为1.22%,检出限(信噪比为10)为0.08 mg/L,方法的稳定性、精密度和重现性(以峰面积的RSD计)分别为2.86%、1.62%、1.82%。在优先满足小马哺育的前提下,采集新疆昭苏马场中两个不同品种马匹的马乳,于低温保存,在4℃和12 000 r/min下30 min内离心两次,制得乳清,测得第一次泌乳时,IgG含量在2 h时高达35.0～50.0 g/L,而在72 h后,马乳中IgG含量迅速下降为2.0～4.0 g/L。该方法前处理过程简单、快速,方法简便、准确、重现性好、精密度高,适合作为马初乳中IgG的检测方法。A direct high-performance gel permeation chromatographic （HPGPC） method for the determination of immunoglobulin G in mare colostrum was established. HPGPC separation was performed on a TOSOH TSK-G4000PWxL column （300 mm × 7.8 mm, 5 μm） with 0.05 mol/L phosphate buffer solution （pH 5.9） as the mobile phase at a flow rate of 0. 8 mL/min, and the column temperature was maintained at 25 ℃. The injection volume was 20 μL. At the detection wavelength of 280 nm, the linear range was from 0.2 to 3.0 g/L （r2 = 0. 999 5 ） with a detection limit of 0.08 mg/L （S/N = 10）. The recovery was 97.47% with a relative standard deviation （RSD） of 1. 22%. The RSDs of the peak area of stability, accuracy and reproducibility for the established method were 2.86%, 1. 52% and 1. 82%, respectively. Mare milk was collected from Zhaosu （China）, a complete collection was stored in an ice box, then sent to a laboratory and stored in a low temperature refrigerator. The whey milk was prepared by centrifugation two times at 12 000 r/min and 4 ℃ for 30 min. The whey protein was obtained from the middle layer. A 2 mL volume of the whey milk was mixed with 23 mL of mobile phase. The average contents of IgG were from 35.0 g/L to 50.0 g/L at the first lactation （2 h） , and the average contents of IgG were from 2.0 g/L to 4.0 g/L after 72 h. The relatively simple analytical method was proved to be accurate and precise in its application to mare colostrum.

关 键 词：高效凝胶渗透色谱法 免疫球蛋白G 马初乳 

分 类 号：O658[理学—分析化学]