Development and validation of an ELISA to measure regenerating island-derived protein 3E in canine blood

IF 1.2 4区农林科学 Q3 VETERINARY SCIENCES Veterinary clinical pathology Pub Date : 2024-06-02 DOI:10.1111/vcp.13352

Laureen M. Peters, Theresia Reding Graf, Luca Giori, Meike Mevissen, Rolf Graf, Judith Howard

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Abstract

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.

Objectives

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.

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开发并验证测量犬血液中再生岛衍生蛋白 3E 的 ELISA。

背景：再生岛衍生蛋白（REG）在败血症、胰腺炎和胃肠道疾病患者体内上调。最近在狗的胰腺组织和血浆中发现了 REG 家族的一个成员，即 REG3E，它在胰腺炎和败血症中表达较高：我们旨在开发并验证一种酶联免疫吸附试验（ELISA），以测定犬血液中的 REG3E 浓度：方法：使用重组犬 REG3E 蛋白和在豚鼠和兔子体内培养的多克隆抗犬 REG3E 抗体，开发了一种间接夹心 ELISA。通过对从犬血浆中纯化的蛋白质进行 Western 印迹和质谱分析来评估抗体的特异性。检测验证包括稀释线性、平行度、加标回收率、重复性和再现性、稳定性、干扰以及血清和肝素化血浆比较的评估：结果：抗体与 REG3E 有特异性结合，没有证据表明与其他蛋白质有交叉反应。ELISA 的检测限为 15 纳克/毫升，定量下限为 30 纳克/毫升。该检测方法的线性度、稀释和混合平行度以及回收率都非常好，平均观察到的结果与预期的结果之比分别为 104%、107%、102% 和 92%，没有证据表明存在钩状效应。三个不同水平的重复性变异系数≤8.5%，重现性变异系数≤14.3%。血浆中REG3E的测定结果不受不同储存条件、冻融循环、溶血、脂血或黄疸的显著影响。肝素化血浆和血清样本中的REG3E浓度没有明显差异：犬 REG3E 酶联免疫吸附测定法在检测犬血浆和血清中的 REG3E 时具有可接受的精密度、准确度、线性和重现性。

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来源期刊

Veterinary clinical pathology 农林科学-兽医学

CiteScore

1.70

自引率

16.70%

发文量

133

审稿时长

18-36 weeks

期刊介绍： Veterinary Clinical Pathology is the official journal of the American Society for Veterinary Clinical Pathology (ASVCP) and the European Society of Veterinary Clinical Pathology (ESVCP). The journal''s mission is to provide an international forum for communication and discussion of scientific investigations and new developments that advance the art and science of laboratory diagnosis in animals. Veterinary Clinical Pathology welcomes original experimental research and clinical contributions involving domestic, laboratory, avian, and wildlife species in the areas of hematology, hemostasis, immunopathology, clinical chemistry, cytopathology, surgical pathology, toxicology, endocrinology, laboratory and analytical techniques, instrumentation, quality assurance, and clinical pathology education.