De Groote D, Fauchet F, Jadoul M, Dehart I, Raher S, Gevaert Y, Lopez M, Gathy R, Franssen J D, Radoux D
Department of Endocrinology, University of Liège, Sart Tilman, Belgium.
J Immunol Methods. 1994 Jan 3;167(1-2):253-61. doi: 10.1016/0022-1759(94)90094-9.
A new monoclonal antibody-based ELISA for leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) measurements is described. The sensitivity (56 pg/ml after 4 h incubation, 14 pg/ml after 24 h incubation), precision (intra-assays < 5%), reproducibility (interassay < 10%), and accuracy (recoveries, ranging between 98 and 119%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances guarantee accurate cytokine measurement in biological fluids such as serum, plasma, synovial fluid, follicular fluid, urine and culture supernatants. Using the assay, LIF/HILDA was measurable in supernatants after in vitro whole blood stimulation with phytohemagglutinin (PHA), OKT3, and phorbol myristate acetate (PMA) but not with lipopolysaccharide (LPS) or Ca ionophore. LIF/HILDA production was not measurable until after 24 h of culture, when cytokine levels were seen to increase linearly in the supernatant to reach values of up to 40 ng/ml after 96 h of culture. Finally, a good correlation was found (r = 0.96; p < 0.0001; y = 23.1x + 233) between the LIF/HILDA values obtained using the ELISA and DA-1a bioassay.
本文描述了一种基于单克隆抗体的新型酶联免疫吸附测定法（ELISA），用于测量白血病抑制因子/DA细胞人白细胞介素（LIF/HILDA）。该测定法的灵敏度（孵育4小时后为56 pg/ml，孵育24小时后为14 pg/ml）、精密度（批内变异系数<5%）、重现性（批间变异系数<10%）和准确性（在多种液体中的回收率在98%至119%之间），加上其在稀释试验中的出色表现，以及在可能存在交叉反应物质时无干扰，保证了在血清、血浆、滑液、卵泡液、尿液和培养上清液等生物液体中准确测量细胞因子。使用该测定法，在用植物血凝素（PHA）、OKT3和佛波醇肉豆蔻酸酯乙酸酯（PMA）体外刺激全血后，上清液中可检测到LIF/HILDA，但用脂多糖（LPS）或钙离子载体刺激时则检测不到。直到培养24小时后才能检测到LIF/HILDA的产生，此时细胞因子水平在上清液中呈线性增加，培养96小时后达到高达40 ng/ml的值。最后，发现使用ELISA获得的LIF/HILDA值与DA-1a生物测定法之间具有良好的相关性（r = 0.96；p < 0.0001；y = 23.1x + 233）。
