Frederiksen Pernille D, Thiel Steffen, Jensen Lisbeth, Hansen Annette G, Matthiesen Finn, Jensenius Jens Chr
Department of Medical Microbiology and Immunology, University of Aarhus, Wilhelm Meyers Allé, 8000 Aarhus, DK, Denmark.
J Immunol Methods. 2006 Aug 31;315(1-2):49-60. doi: 10.1016/j.jim.2006.07.001. Epub 2006 Jul 31.
Mannan-binding lectin (MBL) is attracting considerable interest due to its role in the immune defense. The high frequency of congenital MBL deficiency makes it feasible to evaluate clinical relevance through epidemiological investigations on fairly limited numbers of patients. MBL deficiency is determined by three mutant allotypes termed B, C and D in the coding region as well as mutations in the promoter region. It has been suggested that individuals, with deficiency-associated allotypes, may present significant amounts of low molecular weight MBL. We have compared the quantification of MBL by four commercially available assays with results obtained by our own in-house assays. Most assays are selectively sensitive for the wild type MBL (allotype A), but special combinations of antibodies also detect mutant forms of MBL. Thus a sandwich-type time-resolved immunoflourometric assay (TRIFMA), with a mouse monoclonal antibody (93C) as the catching and detecting antibody, shows B/B and D/D homozygous individuals to present signals corresponding to up to 500 ng MBL per ml (with plasma from an A/A individual as standard) as compared to less than 50 ng/ml and 200 ng/ml, respectively, when measured in other assays. In GPC at isotonic conditions the MBL in B/B and D/D individuals showed a Mr of 450 kDa. This MBL cannot bind to mannan. We further present a new method for quantifying the amount of MBL polypeptide chain. By applying plasma samples on SDS-PAGE at reducing conditions followed by Western blotting and quantification by chemiluminescense, this approach presents single polypeptide chains to the antibody independent of allotype differences in the collagen-like region. Titrations of recombinant MBL served as standard. In sera from homozygous mutants (O/O) the MBL concentrations estimated on Western blot were in the range of 100 to 500 ng/ml and correlated with that measured in the 93C-based TRIFMA.
甘露聚糖结合凝集素（MBL）因其在免疫防御中的作用而备受关注。先天性MBL缺乏症的高发生率使得通过对数量相对有限的患者进行流行病学调查来评估其临床相关性成为可能。MBL缺乏由编码区的三种突变同种异型（称为B、C和D）以及启动子区的突变决定。有人提出，具有与缺乏相关同种异型的个体可能会出现大量低分子量的MBL。我们将四种市售检测方法对MBL的定量结果与我们自己的内部检测结果进行了比较。大多数检测方法对野生型MBL（同种异型A）具有选择性敏感性，但抗体的特殊组合也能检测到MBL的突变形式。因此，一种以小鼠单克隆抗体（93C）作为捕获和检测抗体的夹心型时间分辨免疫荧光测定法（TRIFMA）显示，与其他检测方法测得的分别低于50 ng/ml和200 ng/ml相比，B/B和D/D纯合个体呈现出相当于每毫升高达500 ng MBL的信号（以A/A个体的血浆为标准）。在等渗条件下的凝胶渗透色谱法（GPC）中，B/B和D/D个体中的MBL显示分子量为450 kDa。这种MBL不能与甘露聚糖结合。我们还提出了一种定量MBL多肽链数量的新方法。通过在还原条件下将血浆样品应用于SDS-PAGE，然后进行Western印迹并用化学发光法定量，这种方法将单条多肽链呈现给抗体，而与胶原样区域的同种异型差异无关。重组MBL的滴定用作标准。在纯合突变体（O/O）的血清中，Western印迹法估计的MBL浓度在100至500 ng/ml范围内，并且与基于93C的TRIFMA中测得的浓度相关。
