Rebecca C Bishop, Sarah M Graham, Sara L Connolly, Pamela A Wilkins, Annette M McCoy
{"title":"系统评估支持使用酶联免疫吸附法对马粪便中的钙粘蛋白进行定量,这是对马肠道炎症进行无创定量的第一步。","authors":"Rebecca C Bishop, Sarah M Graham, Sara L Connolly, Pamela A Wilkins, Annette M McCoy","doi":"10.2460/ajvr.24.04.0110","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA.</p><p><strong>Animals: </strong>Healthy horses (n = 28) and horses with intestinal inflammation (n = 10).</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Clinical relevance: </strong>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.</p>","PeriodicalId":7754,"journal":{"name":"American journal of veterinary research","volume":null,"pages":null},"PeriodicalIF":1.3000,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses.\",\"authors\":\"Rebecca C Bishop, Sarah M Graham, Sara L Connolly, Pamela A Wilkins, Annette M McCoy\",\"doi\":\"10.2460/ajvr.24.04.0110\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Objective: </strong>To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA.</p><p><strong>Animals: </strong>Healthy horses (n = 28) and horses with intestinal inflammation (n = 10).</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Clinical relevance: </strong>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.</p>\",\"PeriodicalId\":7754,\"journal\":{\"name\":\"American journal of veterinary research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.3000,\"publicationDate\":\"2024-07-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"American journal of veterinary research\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.2460/ajvr.24.04.0110\",\"RegionNum\":3,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/9/1 0:00:00\",\"PubModel\":\"Print\",\"JCR\":\"Q2\",\"JCRName\":\"VETERINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"American journal of veterinary research","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.2460/ajvr.24.04.0110","RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/9/1 0:00:00","PubModel":"Print","JCR":"Q2","JCRName":"VETERINARY SCIENCES","Score":null,"Total":0}
Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses.
Objective: To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA.
Animals: 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.
Clinical relevance: 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.
期刊介绍:
The American Journal of Veterinary Research supports the collaborative exchange of information between researchers and clinicians by publishing novel research findings that bridge the gulf between basic research and clinical practice or that help to translate laboratory research and preclinical studies to the development of clinical trials and clinical practice. The journal welcomes submission of high-quality original studies and review articles in a wide range of scientific fields, including anatomy, anesthesiology, animal welfare, behavior, epidemiology, genetics, heredity, infectious disease, molecular biology, oncology, pharmacology, pathogenic mechanisms, physiology, surgery, theriogenology, toxicology, and vaccinology. Species of interest include production animals, companion animals, equids, exotic animals, birds, reptiles, and wild and marine animals. Reports of laboratory animal studies and studies involving the use of animals as experimental models of human diseases are considered only when the study results are of demonstrable benefit to the species used in the research or to another species of veterinary interest. Other fields of interest or animals species are not necessarily excluded from consideration, but such reports must focus on novel research findings. Submitted papers must make an original and substantial contribution to the veterinary medicine knowledge base; preliminary studies are not appropriate.