Abstract：
OBJECTIVE: To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA.
METHODS: Healthy horses (n = 28) and horses with intestinal inflammation (n = 10).
METHODS: Feces were suspended in buffer to create fecal supernatant. Serum and fecal supernatant were analyzed using ELISA kits validated for the detection of MPO and CP in equine serum. Assay validation steps included intra- and interassay variability (coefficient of variation [CV]), dilution linearity, spike recovery, and sample type correlation. Variations in sample handling protocols (centrifugation speed, extraction buffer, and filtration) were evaluated.
RESULTS: 17 paired fecal and serum samples were used for initial analysis (10 healthy horses, 7 colitis). Previously reported sample handling protocols resulted in detectable MPO and CP but poor CV, linearity, and spike recovery. There was a linear correlation between serum and fecal samples for CP but not MPO. There was a significant difference between the concentration and CV of alternative sample handling protocols for CP and MPO, with improved CV for CP (2.1% to 18.6%) but not MPO (14.4% to 53.4%). Processing fresh feces with a fecal extraction buffer and filtration of supernatant resulted in the best CV (0.5% to 3.8%) and recovery (45% to 64%) for CP. Detection of MPO was inconsistent regardless of method.
CONCLUSIONS: There are few reliable diagnostic modalities for inflammation of the equine large colon. Findings support quantification of CP in equine feces using the described ELISA kit and protocol. With additional study to establish reference interval and clinical utility, the fecal inflammatory biomarker CP may allow for noninvasive quantification of intestinal inflammation in horses.
METHODS: Healthy horses (n = 28) and horses with intestinal inflammation (n = 10).
METHODS: Feces were suspended in buffer to create fecal supernatant. Serum and fecal supernatant were analyzed using ELISA kits validated for the detection of MPO and CP in equine serum. Assay validation steps included intra- and interassay variability (coefficient of variation [CV]), dilution linearity, spike recovery, and sample type correlation. Variations in sample handling protocols (centrifugation speed, extraction buffer, and filtration) were evaluated.
RESULTS: 17 paired fecal and serum samples were used for initial analysis (10 healthy horses, 7 colitis). Previously reported sample handling protocols resulted in detectable MPO and CP but poor CV, linearity, and spike recovery. There was a linear correlation between serum and fecal samples for CP but not MPO. There was a significant difference between the concentration and CV of alternative sample handling protocols for CP and MPO, with improved CV for CP (2.1% to 18.6%) but not MPO (14.4% to 53.4%). Processing fresh feces with a fecal extraction buffer and filtration of supernatant resulted in the best CV (0.5% to 3.8%) and recovery (45% to 64%) for CP. Detection of MPO was inconsistent regardless of method.
CONCLUSIONS: There are few reliable diagnostic modalities for inflammation of the equine large colon. Findings support quantification of CP in equine feces using the described ELISA kit and protocol. With additional study to establish reference interval and clinical utility, the fecal inflammatory biomarker CP may allow for noninvasive quantification of intestinal inflammation in horses.
摘要：
目的：优化和评价酶联免疫吸附试验检测马粪便中炎症生物标志物髓过氧化物酶（MPO）和钙卫蛋白（CP）的方法。
方法:健康马(n=28)和患有肠道炎症的马(n=10)。
方法:将粪便悬浮在缓冲液中以产生粪便上清液。使用ELISA试剂盒分析血清和粪便上清液，该试剂盒已验证用于检测马血清中的MPO和CP。测定验证步骤包括测定内和测定间变异性(变异系数[CV])，稀释线性度，尖峰恢复，和样本类型相关性。样品处理方案的变化(离心速度、提取缓冲液,和过滤）进行评估。
结果：17个配对的粪便和血清样本用于初始分析（10个健康马，7结肠炎）。先前报道的样品处理方案导致可检测的MPO和CP，但CV较差，线性度和尖峰恢复。血清和粪便样本之间的CP而不是MPO之间存在线性相关。CP和MPO的替代样品处理方案的浓度和CV之间存在显着差异，CP的CV改善（2.1％至18.6％），但MPO没有改善（14.4％至53.4％）。用粪便提取缓冲液处理新鲜粪便并过滤上清液导致CP的最佳CV(0.5%至3.8%)和回收率(45%至64%)。MPO的检测与方法无关。
结论：马大结肠炎症的可靠诊断方法很少。研究结果支持使用所述ELISA试剂盒和方案定量马粪便中的CP。通过额外的研究来建立参考区间和临床效用，粪便炎症生物标志物CP可用于马肠道炎症的非侵入性定量.
方法:健康马(n=28)和患有肠道炎症的马(n=10)。
方法:将粪便悬浮在缓冲液中以产生粪便上清液。使用ELISA试剂盒分析血清和粪便上清液，该试剂盒已验证用于检测马血清中的MPO和CP。测定验证步骤包括测定内和测定间变异性(变异系数[CV])，稀释线性度，尖峰恢复，和样本类型相关性。样品处理方案的变化(离心速度、提取缓冲液,和过滤）进行评估。
结果：17个配对的粪便和血清样本用于初始分析（10个健康马，7结肠炎）。先前报道的样品处理方案导致可检测的MPO和CP，但CV较差，线性度和尖峰恢复。血清和粪便样本之间的CP而不是MPO之间存在线性相关。CP和MPO的替代样品处理方案的浓度和CV之间存在显着差异，CP的CV改善（2.1％至18.6％），但MPO没有改善（14.4％至53.4％）。用粪便提取缓冲液处理新鲜粪便并过滤上清液导致CP的最佳CV(0.5%至3.8%)和回收率(45%至64%)。MPO的检测与方法无关。
结论：马大结肠炎症的可靠诊断方法很少。研究结果支持使用所述ELISA试剂盒和方案定量马粪便中的CP。通过额外的研究来建立参考区间和临床效用，粪便炎症生物标志物CP可用于马肠道炎症的非侵入性定量.
