Berkova N, Karawajew L, Korobko V, Behrsing O, Micheel B, Shamborant O, Stukatcheva E, Shingarova L
Laboratoire d'endocrinologie de la reproduction, Hôpital Saint-François d'Assise, Québec, Canada.
Biotechnol Appl Biochem. 1996 Apr;23(2):163-71.
The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved in several pathological processes, and human recombinant TNF-alpha (hrTNF-alpha) is available for testing in preclinical and clinical research. The purpose of this work was the creation of tetradomas producing bispecific anti-hTNF-alpha/anti-HRP (horseradish peroxidase) antibodies and the development of a rapid and sensitive solid-phase enzyme immunoassay. Monoclonal antibodies obtained against hrTNF-alpha could recognize both natural and recombinant hTNF-alpha. The four chosen hybridomas produced IgG1 with an affinity constant of the order of 10(-9) M. Three of them recognized different epitopes. The clone selected for fusion with the hybridoma producing anti-HRP antibodies secreted antibodies against portion 30-50 of the hTNF-alpha N-terminal amino acid residues as found by Western-blot analysis with mutant and chimaeric proteins. The tetradoma producing bispecific anti-hTNF-alpha/anti-HRP antibodies was identified using a fluorescence-activated cell sorter. Bispecific antibodies were isolated by hydroxyapatite chromatography. A sandwich ELISA was developed: one of the monoclonal anti-TNF-alpha antibodies was absorbed to the solid phase as the catcher and was detected by a bispecific anti-hTNF-alpha/anti-HRP antibody. The detection limit of the assay was 1 ng/ml. With such ELISA, the level of hTNF-alpha could be conveniently estimated in different samples containing either natural or recombinant hTNF-alpha in an experimental environment or in clinical trials.
细胞因子肿瘤坏死因子-α（TNF-α）参与多种病理过程，人重组TNF-α（hrTNF-α）可用于临床前和临床研究测试。本研究的目的是制备产生双特异性抗hTNF-α/抗辣根过氧化物酶（HRP）抗体的四元瘤，并开发一种快速灵敏的固相酶免疫测定法。获得的抗hrTNF-α单克隆抗体可识别天然和重组hTNF-α。所选的四个杂交瘤产生IgG1，其亲和常数约为10^(-9) M。其中三个识别不同的表位。通过与产生抗HRP抗体的杂交瘤融合而选择的克隆分泌针对hTNF-α N端氨基酸残基30-50部分的抗体，这是通过对突变体和嵌合蛋白进行蛋白质印迹分析发现的。使用荧光激活细胞分选仪鉴定产生双特异性抗hTNF-α/抗HRP抗体的四元瘤。通过羟基磷灰石色谱法分离双特异性抗体。开发了一种夹心ELISA：将一种抗TNF-α单克隆抗体作为捕获剂吸附到固相上，并用双特异性抗hTNF-α/抗HRP抗体进行检测。该测定法的检测限为1 ng/ml。使用这种ELISA，可以方便地在实验环境或临床试验中含有天然或重组hTNF-α的不同样品中估计hTNF-α的水平。
