Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrations
Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrations
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DOI：
10.1007/s00726-012-1244-8
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发表时间：
2012-07-01
期刊：
影响因子：
3.5
通讯作者：
Hauske, Sibylle J.
中科院分区：
文献类型：
--
作者：
Adelmann, Katja;Frey, Dirk;Hauske, Sibylle J.
目前，血清肌肽酶（CN-1）的测量主要通过评估酶活性来进行。该方法耗时，不太适合大系列样品，并且可能与CN-1蛋白浓度的测量不一致。为了克服这些限制，我们开发了使用不同抗CN-1抗体的夹心ELISA测定法，即，ATLAS（多克隆IgG）和RYSK 173（单克隆IgG 1）。使用基于ATLAS的测定，在血清以及EDTA和肝素血浆中检测到相似量的CN-1。与基于ATLAS的测定相比，基于RYSKS 173的测定以显著较低的浓度检测所有个体血清中的CN-1（范围：0.1-1.8 vs. 1-50 μ g/ml，基于RYSK vs. ATLAS，P < 0.01）。使用基于RYSK的测定法在EDTA血浆中的CN-1检测增加，尽管与ATLAS相比浓度显著较低。在肝素血浆中，使用基于RYSK的测定法也检测不到CN-1。在基于RYSK的测定中，向血清中添加DTT使CN-1的检测增加到几乎达到基于ATLAS的测定中发现的水平。两种ELISA测定都是高度可重复的（对于基于RYSK和ATLAS的测定，分别为R：0.99，P <0.01和R：0.93，P < 0.01）。基于ATLAS的测定结果显示与CN-1活性正相关（R：0.62，P < 0.01），而基于RYSK的测定结果并非如此。然而，CN-1活性和在基于RYSK的测定中检测到的CN-1比例之间存在负相关，即，用基于RYSK的测定法检测的CN-1/用基于ATLAS的测定法检测的CN-1 × 100%（Spearman-Rang相关系数：-0.6，P < 0.01），表明基于RYSK的测定法最可能检测具有低CN-1活性的CN-1构象。RYSK 173和ATLAS抗体在蛋白质印迹中反应相似，与PNGase处理无关。RYSK 173在血清中的结合不是由于差异N-糖基化，如突变CN-1 cDNA构建体所证明的。总之，我们的研究表明酶活性和CN-1蛋白浓度在ELISA中有很好的相关性，并表明血清中存在不同的CN-1构象。这些不同构象的相关性仍然是难以捉摸的，需要在进一步的研究中解决。
Serum carnosinase (CN-1) measurements are at present mainly performed by assessing enzyme activity. This method is time-consuming, not well suited for large series of samples and can be discordant to measurements of CN-1 protein concentrations. To overcome these limitations, we developed sandwich ELISA assays using different anti-CN-1 antibodies, i.e., ATLAS (polyclonal IgG) and RYSK173 (monoclonal IgG1). With the ATLAS-based assay, similar amounts of CN-1 were detected in serum and both EDTA and heparin plasma. The RYSKS173-based assay detected CN-1 in serum in all individuals at significantly lower concentrations compared to the ATLAS-based assay (range: 0.1-1.8 vs. 1-50 mu g/ml, RYSK- vs. ATLAS-based, P < 0.01). CN-1 detection with the RYSK-based assay was increased in EDTA plasma, albeit at significantly lower concentrations compared to ATLAS. In heparin plasma, CN-1 was also poorly detected with the RYSK-based assay. Addition of DTT to serum increased the detection of CN-1 in the RYSK-based assay almost to the levels found in the ATLAS-based assay. Both ELISA assays were highly reproducible (R: 0.99, P < 0.01 and R: 0.93, P < 0.01, for the RYSK- and ATLAS-based assays, respectively). Results of the ATLAS-based assay showed a positive correlation with CN-1 activity (R: 0.62, P < 0.01), while this was not the case for the RYSK-based assay. However, there was a negative correlation between CN-1 activity and the proportion of CN-1 detected in the RYSK-based assay, i.e., CN-1 detected with the RYSK-based assay/CN-1 detected with the ATLAS-based assay x 100% (Spearman-Rang correlation coefficient: -0.6, P < 0.01), suggesting that the RYSK-based assay most likely detects a CN-1 conformation with low CN-1 activity. RYSK173 and ATLAS antibodies reacted similarly in Western blot, irrespective of PNGase treatment. Binding of RYSK173 in serum was not due to differential N-glycosylation as demonstrated by mutant CN-1 cDNA constructs. In conclusion, our study demonstrates a good correlation between enzyme activity and CN-1 protein concentration in ELISA and suggests the presence of different CN-1 conformations in serum. The relevance of these different conformations is still elusive and needs to be addressed in further studies.