5th Medical Clinic, Clinical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167, Mannheim, Germany.
Amino Acids. 2012 Jul;43(1):143-51. doi: 10.1007/s00726-012-1244-8. Epub 2012 Feb 17.
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 μg/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 × 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.
目前，血清肌肽酶（CN-1）的测量主要通过评估酶活性来进行。这种方法耗时耗力，不适合大量样本，并且与 CN-1 蛋白浓度的测量结果不一致。为了克服这些限制，我们使用不同的抗 CN-1 抗体（即 ATLAS（多克隆 IgG）和 RYSK173（单克隆 IgG1））开发了夹心 ELISA 测定法。使用基于 ATLAS 的测定法，在血清和 EDTA 和肝素血浆中均检测到了相似量的 CN-1。基于 RYSKS173 的测定法在所有个体的血清中检测到的 CN-1 浓度明显低于基于 ATLAS 的测定法（范围：0.1-1.8 与 1-50μg/ml，RYSK-与 ATLAS-，P<0.01）。与基于 ATLAS 的测定法相比，EDTA 血浆中 RYSK 基测定法检测到的 CN-1 增加，尽管浓度明显较低。在肝素血浆中，基于 RYSK 的测定法也难以检测到 CN-1。向血清中添加 DTT 可使基于 RYSK 的测定法中 CN-1 的检测增加到几乎与基于 ATLAS 的测定法相同的水平。两种 ELISA 测定法均具有高度的可重复性（R：0.99，P<0.01 和 R：0.93，P<0.01，分别用于 RYSK-和 ATLAS-基于测定法）。基于 ATLAS 的测定法的结果与 CN-1 活性呈正相关（R：0.62，P<0.01），而基于 RYSK 的测定法则不是。但是，CN-1 活性与基于 RYSK 的测定法中检测到的 CN-1 比例之间存在负相关，即基于 RYSK 的测定法中检测到的 CN-1/基于 ATLAS 的测定法中检测到的 CN-1×100%（Spearman-Rang 相关系数：-0.6，P<0.01），表明基于 RYSK 的测定法最有可能检测到具有低 CN-1 活性的 CN-1 构象。Western blot 显示 RYSK173 和 ATLAS 抗体的反应相似，与 PNGase 处理无关。用突变型 CN-1 cDNA 构建体证明，RYSK173 在血清中的结合不是由于差异 N-糖基化所致。总之，我们的研究表明 ELISA 中酶活性与 CN-1 蛋白浓度之间具有良好的相关性，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不清楚，需要在进一步的研究中解决。
