Antoine C, Lellouche J P, Maclouf J, Pradelles P
Service de Pharmacologie et d'Immunologie, DRIPP, Gif sur Yvette, France.
Biochim Biophys Acta. 1991 Oct 10;1075(2):162-8. doi: 10.1016/0304-4165(91)90247-e.
We have developed sensitive solid phase enzyme immunoassays (EIA) to analyze quantitatively leukotrienes (LTs) using acetylcholinesterase from Electrophorus electricus as a label for LTB4, LTC4 and LTE4. However, because of problems specific to LTs, we used different coupling procedures to prepare LTs conjugates necessary for the production of antibodies and for the preparation of enzymatic tracers. For the immunogens, all LTs were coupled to bovine serum albumin using glutaraldehyde (ethylene diamine was used to add an amino group to LTB4). Immunizations in rabbits were done following classical procedures. For the enzymatic tracers, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate was selected to conjugate the LTs via their amino groups to acetylcholinesterase. Titers of the different antisera ranged from 1:30,000 (LTE4), 1:40,000 (LTC4) to 1:50,000 (LTB4) and sensitivities (IC50) were 5.5 pg, 4.3 pg and 2.4 pg, respectively. Cross reactivities were also examined against other LTs. Sensitivities and specificities of the different systems were dependent on the conditions of incubation (temperature). Validation of the technique was done (i) after spiking known amounts of LTC4 in plasma and measuring the substance added after prior extraction and purification, (ii) by analyzing the supernatant of human neutrophils suspended in buffer or in plasma, (iii) by measuring LTE4 in urine. Due to the background provided by these complex matrixes, quantitation was performed after addition of [3H]LTs for recovery, protein precipitation, extraction by Sep-PakR and purification by HPLC. Measurement of LTs can be done in biological fluids with the same ease and advantages as other enzyme immunoassays that we have previously developed for eicosanoids analysis.
我们已经开发出灵敏的固相酶免疫分析法（EIA），以电鳐的乙酰胆碱酯酶作为白三烯B4（LTB4）、白三烯C4（LTC4）和白三烯E4（LTE4）的标记物，对这些白三烯进行定量分析。然而，由于白三烯存在一些特定问题，我们采用了不同的偶联方法来制备用于抗体生产和酶标记物制备所需的白三烯结合物。对于免疫原，所有白三烯均使用戊二醛与牛血清白蛋白偶联（使用乙二胺给LTB4添加一个氨基）。按照经典程序对兔子进行免疫。对于酶标记物，选择4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯，通过白三烯的氨基将其与乙酰胆碱酯酶偶联。不同抗血清的效价范围为1:30,000（LTE4）、1:40,000（LTC4）至1:50,000（LTB4），灵敏度（IC50）分别为5.5 pg、4.3 pg和2.4 pg。还检测了与其他白三烯的交叉反应性。不同系统的灵敏度和特异性取决于孵育条件（温度）。通过以下方式对该技术进行验证：（i）在血浆中加入已知量的LTC4，在预先提取和纯化后测量添加的物质；（ii）分析悬浮在缓冲液或血浆中的人中性粒细胞的上清液；（iii）测量尿液中的LTE4。由于这些复杂基质产生的背景干扰，在加入[3H]白三烯进行回收率测定、蛋白质沉淀、用Sep-PakR萃取和通过高效液相色谱法纯化后进行定量分析。与我们之前开发的用于类花生酸分析的其他酶免疫分析法一样，白三烯的测量可以在生物体液中轻松进行，且具有相同的优势。
