Piñeiro Matilde, Lampreave Fermín, Alava María A
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Vet Immunol Immunopathol. 2009 Feb 15;127(3-4):228-34. doi: 10.1016/j.vetimm.2008.10.318. Epub 2008 Nov 1.
Measurement of acute phase proteins (APPs) levels in blood is increasingly being used for monitoring health and welfare in farm animals. In this work a sandwich-type ELISA for the quantification of pig Major Acute phase Protein (Pig-MAP), one of the main APP in pigs, has been developed and validated. Two Pig-MAP specific monoclonal antibodies were developed in mouse. One of the monoclonal antibodies was fixed to microtiter plates and the other was coupled to horseradish peroxidase and used as detection antibody. To calibrate the assay dilutions of a standard pig serum of known Pig-MAP concentration were added to the plate in each assay. The assay showed good accuracy, kept linearity under dilution and recovery was proportional. The detection limit was 0.1 microg/mL. Precision was adequate with coefficients of variation lower than 8% for both inter and intra-assays. A good linear correlation between Pig-MAP concentration values obtained by ELISA and by radial immunodiffusion, used as reference method, was found (r = 0.978; beta = 1.02). Pig-MAP concentration was analysed in serum samples obtained from two pig herds of different health status (10 animals per age and herd, of 10, 12, 14, 18 weeks of age). Mean values obtained in the farm of low health status were higher than the obtained in the farm of high health status (p<0.001). In the farm of high health status, mean Pig-MAP concentration remained constant at the different ages analysed (mean values of 0.83+/-0.18 mg/mL) whereas in the farm of low health status differences between age groups were found. In this farm (low health status) mean values for the total of pigs analysed were of 1.68+/-0.74 mg/mL.
血液中急性期蛋白（APPs）水平的检测越来越多地用于监测农场动物的健康和福利状况。在本研究中，已开发并验证了一种夹心型酶联免疫吸附测定法（ELISA），用于定量猪主要急性期蛋白（Pig-MAP），它是猪体内主要的APP之一。在小鼠体内制备了两种Pig-MAP特异性单克隆抗体。其中一种单克隆抗体固定在微量滴定板上，另一种与辣根过氧化物酶偶联用作检测抗体。每次测定时，将已知Pig-MAP浓度的标准猪血清稀释液加入板中以校准测定。该测定法显示出良好的准确性，在稀释条件下保持线性，回收率呈比例关系。检测限为0.1μg/mL。精密度良好，批内和批间变异系数均低于8%。发现ELISA法与用作参考方法的放射免疫扩散法所获得的Pig-MAP浓度值之间具有良好的线性相关性（r = 0.978；β = 1.02）。对来自两个健康状况不同的猪群（每个年龄和猪群各10只动物，年龄分别为10、12、14、18周）的血清样本进行了Pig-MAP浓度分析。健康状况较差的猪场中获得的平均值高于健康状况良好的猪场（p<0.001）。在健康状况良好的猪场中，所分析的不同年龄阶段的Pig-MAP平均浓度保持恒定（平均值为0.83±0.18mg/mL），而在健康状况较差的猪场中，不同年龄组之间存在差异。在该猪场（健康状况较差），所分析的猪的总平均值为1.68±0.74mg/mL。
