ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA.
Clin Chem. 2010 Sep;56(9):1496-9. doi: 10.1373/clinchem.2010.143156. Epub 2010 Jul 2.
Although the benefits of quantifying serum squamous cell carcinoma antigen (SCCa) have been reported, SCCa reagents were no longer available in the US by the late 1990s. Because SCCa quantification still has demonstrated clinical utility, we developed and validated a microtiter plate-based ELISA for measuring SCCa in serum.
We coated microtiter strips overnight with capture anti-SCCa monoclonal antibody, washed the wells, added blocking buffer, and lyophilized the strips. For detection, we used a biotinylated anti-SCCa detection antibody, streptavidin/horseradish peroxidase conjugate, and tetramethylbenzidine/H(2)O(2) substrate. A novel blocking reagent against human antimouse antibodies (HAMA) was evaluated. A reference interval was established with sera from healthy individuals and was confirmed in smokers.
The assay was linear to 40 microg/L SCCa (slope, 1.00; y intercept, 0.695; R(2), 0.996) with a detection limit of 0.3 microg/L. The intraassay imprecision results [mean (CV)] were 2.5 microg/L (3.4%), 18.0 microg/L (3.0%), and 30.7 microg/L (2.4%); interassay imprecision results were 2.0 microg/L (9.9%), 20.0 microg/L (7.6%), and 36.3 microg/L (3.5%). A correlation analysis against an established automated assay generated a slope of 0.976 and a y intercept of -0.193 microg/L (r(2) = 0.916). An upper reference limit of 2.1 microg/L SCCa was established at 95% confidence level, with no difference observed in smokers. No correlation between SCCa concentration and age was observed (r(2) = 0.0003). At a blocking reagent concentration of 5 mg/L, HAMA interference was eliminated in 3 samples known to produce falsely increased SCCa results.
This SCCa ELISA demonstrates acceptable performance characteristics for quantifying serum SCCa and is effective in eliminating HAMA interference.
虽然已经报道了量化血清鳞状细胞癌抗原（SCCa）的益处，但到 20 世纪 90 年代末，美国已经不再提供 SCCa 试剂。由于 SCCa 的定量分析仍然具有临床应用价值，我们开发并验证了一种基于微量滴定板的 ELISA 方法，用于测量血清中的 SCCa。
我们将捕获抗 SCCa 单克隆抗体包被在微量滴定板条上过夜，然后清洗孔，加入封闭缓冲液，并将条带冻干。用于检测，我们使用生物素化的抗 SCCa 检测抗体、链霉亲和素/辣根过氧化物酶缀合物和四甲基联苯胺/H2O2 底物。评估了一种针对人抗鼠抗体（HAMA）的新型阻断试剂。用来自健康个体的血清建立参考区间，并在吸烟者中进行了验证。
该测定法对 SCCa 的线性范围为 40μg/L（斜率为 1.00；y 截距为 0.695；R2 为 0.996），检测限为 0.3μg/L。批内精密度结果[平均值（CV）]分别为 2.5μg/L（3.4%）、18.0μg/L（3.0%）和 30.7μg/L（2.4%）；批间精密度结果分别为 2.0μg/L（9.9%）、20.0μg/L（7.6%）和 36.3μg/L（3.5%）。与已建立的自动化测定法的相关分析产生了 0.976 的斜率和-0.193μg/L 的 y 截距（r2=0.916）。在 95%置信水平下，建立了 SCCa 浓度为 2.1μg/L 的上参考限，在吸烟者中未观察到差异。未观察到 SCCa 浓度与年龄之间存在相关性（r2=0.0003）。在 5mg/L 的阻断试剂浓度下，消除了 3 份已知会产生假性 SCCa 升高结果的样本中的 HAMA 干扰。
该 SCCa ELISA 具有用于定量血清 SCCa 的可接受的性能特征，并且有效消除了 HAMA 干扰。
