Hunt T C, Summers J A, Campos M G, Rimmer S J, Sturton G, Palfai S, Church M K
Immunopharmacology Group, Southampton General Hospital, U.K.
Clin Exp Allergy. 1993 May;23(5):425-34. doi: 10.1111/j.1365-2222.1993.tb00349.x.
Eosinophil major basic protein (MBP), purified from guinea pig eosinophil granules was used to raise five monoclonal antibodies (MoAbs). Their reactivity with MBP was confirmed by immunoblotting and indirect ELISA. Two of the MoAbs were used to develop a sensitive and specific antigen capture (sandwich) ELISA for guinea pig eosinophil MBP which gives an accurate and reproducible standard curve over the range of 10-10,000 ng/ml. The specificity of the ELISA for MBP was confirmed and its suitability for testing biological samples ascertained by measurement of MBP in bronchoalveolar lavage fluid (BALF) and plasma from guinea pigs sensitized and challenged with ovalbumin. The ELISA was also capable of detecting MBP in culture supernatants from purified eosinophil preparations challenged with calcium ionophore in vitro. One of the monoclonals could be used to strongly and specifically stain guinea pig eosinophils in immunocytochemistry, whilst all five could be used to visualize eosinophils in suspension in BALF or peritoneal lavage fluid by flow cytometry. There was no staining of other guinea pig leucocyte types, nor crossreactivity with human eosinophils by immunocytochemistry or flow cytometry.
从豚鼠嗜酸性粒细胞颗粒中纯化得到的嗜酸性粒细胞主要碱性蛋白（MBP），被用于制备五种单克隆抗体（MoAb）。通过免疫印迹和间接酶联免疫吸附测定（ELISA）证实了它们与MBP的反应性。其中两种单克隆抗体被用于开发一种针对豚鼠嗜酸性粒细胞MBP的灵敏且特异的抗原捕获（夹心）ELISA，该方法在10 - 10,000 ng/ml范围内能给出准确且可重复的标准曲线。通过在卵清蛋白致敏和激发的豚鼠的支气管肺泡灌洗液（BALF）和血浆中测量MBP，证实了该ELISA对MBP的特异性及其用于检测生物样品的适用性。该ELISA还能够检测体外经钙离子载体激发的纯化嗜酸性粒细胞制剂培养上清液中的MBP。其中一种单克隆抗体可用于在免疫细胞化学中强烈且特异地染色豚鼠嗜酸性粒细胞，而所有五种单克隆抗体均可通过流式细胞术用于观察BALF或腹腔灌洗液中悬浮的嗜酸性粒细胞。对其他豚鼠白细胞类型无染色，通过免疫细胞化学或流式细胞术也未与人嗜酸性粒细胞发生交叉反应。
