背景:再生岛衍生蛋白(REG)在脓毒症患者中上调,胰腺炎,和胃肠道疾病。REG家族的一个成员,即REG3E,最近在狗的胰腺组织和血浆中发现,在胰腺炎和脓毒症中高表达。
目的:我们的目的是开发和验证一种检测犬血液中REG3E浓度的ELISA方法。
方法:使用重组犬REG3E蛋白和在豚鼠和兔中产生的多克隆抗犬REG3E抗体,建立了间接夹心ELISA。使用从犬血浆中纯化的蛋白质的蛋白质印迹和质谱分析评估抗体特异性。检测验证包括稀释线性评估,并行性,尖峰恢复,重复性和再现性,稳定性,干扰,血清和肝素化血浆的比较。
结果:抗体与REG3E特异性结合,没有与其他蛋白质交叉反应的证据。ELISA的检测限为15ng/mL,定量下限为30ng/mL。该测定法显示出良好至优异的线性,稀释和混合平行,和恢复,平均观察到的预期比率为104%,107%,102%,92%,分别,也没有钩效应的证据.在三个不同水平下,重复性的变异系数≤8.5%,重复性的变异系数≤14.3%。血浆中REG3E的测量结果不受不同储存条件的显著影响,冻融循环,溶血,血脂血症,或者黄疸.肝素化血浆和血清样品中的REG3E浓度之间没有显着差异。
结论:犬REG3EELISA具有可接受的精密度,准确度,线性度和测量犬血浆和血清中REG3E的可重复性。
BACKGROUND: Regenerating island-derived proteins (REG) are upregulated in people with sepsis, pancreatitis, and gastrointestinal diseases. One member of the REG family, namely REG3E, was recently identified in pancreatic tissue and plasma of dogs, with high expression in pancreatitis and sepsis.
OBJECTIVE: We aimed to develop and validate an ELISA to measure REG3E concentrations in canine blood.
METHODS: An indirect sandwich ELISA was developed using recombinant canine REG3E protein and polyclonal anti-canine REG3E antibodies raised in guinea pigs and rabbits. Antibody specificity was assessed using western blot and mass spectrometric analysis of protein purified from canine plasma. Assay validation included evaluation of dilutional linearity, parallelism, spiking recovery, repeatability and reproducibility, stability, interferences, and comparison of serum and heparinized plasma.
RESULTS: Antibodies bound specifically to REG3E with no evidence of cross-reactivity with other proteins. The limit of detection of the ELISA was 15 ng/mL, and the lower limit of quantification was 30 ng/mL. The assay demonstrated good to excellent linearity, dilutional and mixing parallelism, and recovery, with mean observed-to-expected ratios of 104%, 107%, 102%, and 92%, respectively, and no evidence of a hook effect. Coefficients of variation were ≤8.5% for repeatability and ≤14.3% for reproducibility at three different levels. Measurements of REG3E in plasma were not significantly influenced by different storage conditions, freeze-thawing cycles, hemolysis, lipemia, or icterus. There was no significant difference between REG3E concentrations in heparinized plasma and serum samples.
CONCLUSIONS: The canine REG3E ELISA has acceptable precision, accuracy, linearity, and reproducibility for the measurement of REG3E in canine plasma and serum.