Créminon C, Frobert Y, Habib A, Maclouf J, Pradelles P, Grassi J
CEA, Service de Pharmacologie et d'Immunologie, DRIPP, Centre d'Etudes SACLAY, Gif-sur-Yvette, France.
Biochim Biophys Acta. 1995 Feb 9;1254(3):333-40. doi: 10.1016/0005-2760(94)00196-6.
Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.
针对从公羊精囊纯化得到的组成型环氧化酶（COX-1）制备了多克隆抗血清和六种不同的单克隆抗体（mAb）。免疫印迹实验表明，多克隆抗血清和4种mAb能强烈识别血小板提取物中的人COX。使用不同组合的mAb建立了不同的双位点免疫分析方法来检测公羊COX-1。这些分析在包被有一个mAb的96孔微量滴定板中进行，另一个mAb（共价标记有乙酰胆碱酯酶（AChE））作为示踪剂。一种组合（固相CX-101 + CX-105-AChE）表现出最佳灵敏度，能显著检测低至23 pg/ml（0.3 fmol/ml的绵羊COX-1）的浓度。不幸的是，该分析与血小板提取物中的人COX-1交叉反应性较差。另一种组合（固相CX-111 + CX-110-AChE）对人COX-1具有良好的识别能力，但与公羊COX-1的交叉反应性较差。最后，选择纯化的抗COX-1 IgG包被和CX-110-AChE作为最佳折衷方案，因为观察到其既有良好的灵敏度（检测限为113 pg/ml的公羊COX-1），又在两种物种的COX-1之间具有显著的交叉反应性。同时，用对应于人COX-1氨基末端部分的12个氨基酸的肽段在兔中制备多克隆抗体。这些多克隆抗体经过亲和纯化，并用于开发以CX-110-AChE为示踪剂的另一种COX-1双位点免疫分析方法。这两种分析方法用于分析人血小板和培养的人脐静脉细胞（HUVEC）中的COX-1含量。从灵敏度和特异性方面比较了每种分析方法获得的结果。通过分析先前经分子筛色谱分离的血小板和HUVEC提取物来检查两种分析方法的有效性。
