Soler Lourdes, García Natalia, Andrés Marta, Armengol Ramón, Lampreave Fermín, Alava María A, Piñeiro Matilde
Acuvet Biotech, C/ Bari 25 dpdo, 50197, Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Pedro Cerbuna 12, 50009, Zaragoza, Spain.
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Pedro Cerbuna 12, 50009, Zaragoza, Spain.
Vet Immunol Immunopathol. 2019 Nov;217:109922. doi: 10.1016/j.vetimm.2019.109922. Epub 2019 Aug 8.
Inter alpha trypsin inhibitor heavy chain 4 (ITIH4) is a serum protein belonging to the Inter alpha trypsin inhibitor (ITI) family, which was previously characterized by our group as a new APP in cattle. This protein was firstly described in pigs where is known to be a major acute phase protein, also denominated Pig-MAP. Increases of ITIH4 of up to 12 times the pre-infection values were previously reported in the serum of heifers with experimentally induced summer mastitis. ITIH4 was detected in the milk of cows with mastitis by western blot, but the method previously used to quantify this protein, radial immunodiffusion, was not sensitive enough to quantify it in milk samples. In this study we developed an ELISA method which allows the quantification of bovine ITIH4 in serum and milk samples. Previously developed antibodies were used to perform the assay, including anti bovine ITIH4 polyclonal antibodies and a monoclonal antibody against pig ITIH4 that also recognizes the bovine homologous protein. The ELISA developed showed an adequate precision, with inter and intra- assay coefficients of variation lower than 10% for serum and milk samples. The assay keeps linearity under dilution for both serum and milk samples. A good agreement was observed between the values measured by ELISA and radial immunodiffusion in serum samples.
α-胰蛋白酶抑制剂重链4（ITIH4）是一种血清蛋白，属于α-胰蛋白酶抑制剂（ITI）家族，我们团队之前将其鉴定为牛体内一种新的急性时相蛋白（APP）。该蛋白最初在猪身上被描述，已知是一种主要的急性期蛋白，也被称为猪急性期蛋白（Pig-MAP）。先前报道，在实验性诱导夏季乳腺炎的小母牛血清中，ITIH4增加至感染前值的12倍。通过蛋白质印迹法在患乳腺炎奶牛的乳汁中检测到了ITIH4，但之前用于定量该蛋白的方法——放射免疫扩散法，对乳汁样本进行定量时灵敏度不够。在本研究中，我们开发了一种酶联免疫吸附测定（ELISA）方法，可对血清和乳汁样本中的牛ITIH4进行定量。使用先前开发的抗体进行该检测，包括抗牛ITIH4多克隆抗体和一种抗猪ITIH4单克隆抗体，该单克隆抗体也能识别牛同源蛋白。所开发的ELISA方法具有足够的精密度，血清和乳汁样本检测的批内和批间变异系数均低于10%。该检测方法在血清和乳汁样本稀释后仍保持线性。ELISA法和放射免疫扩散法测定血清样本的值之间观察到良好一致性。
