Biotin interference in susceptible CanAg Ca242 immunoassays and elimination method

Biotin as dietary supplement or therapy may lead to analytical interference in biotin-streptavidin immunoassay.When excess biotin is present in the specimen, the biotin molecules will saturate the streptavidin-binding sites, thus preventing the antibody-analyte sandwich from binding with the streptavidin-coated solid phase to generate assay signal after the washing phase. In order to investigate the biotin interference in CanAg Ca242, we compare the difference of experimental samples(spiked biotin solution) and control samples(spiked PBS solution). Pure biotin was dissolved in 0.01 M NaOH and stored at -20°C for reservation. Biotin stock diluted with PBS into working solutions with nine concentration gradients that were then spiked into three different baseline Ca242 samples to achieve the indicated final concentration of biotin (1000,500,250,125,62.5,31.25,15.63,7.81) ng/mL. Our data(Fig .1) shows three different concentration across the analysis of measurement ranges of Ca242 had significant interference from biotin, but the magnitude of the interference was variable at different concentration of biotin. The higher the biotin concentration, the more significant the reduction of Ca242 .The data from table 1 shows that the relative deviation between the baseline level and the biotin-spiked samples, the relative deviation greater than 10% was considered a significant difference according to the indoor quality target. The Ca242-Low level, cutoff level, and high level appeared significant difference respectively at biotin concentration of 15.63 ng/ml, 31.25 ng/ml and 62.5 ng/ml. At the biotin concentration of 250 ng/ml, the relative deviation was more than 99 %（Table 1）. Now that we have confirmed that biotin interferes with Ca242 detection, the next step is to find a feasible solution. Based on the strong affinity to streptavidin, we used streptavidin microparticles to “pre-bound” biotin. The samples were absorbed with magnetic microparticles coated with streptavidin. This reagent included in the Cobas® assays kits supplied by Roche and the streptavidin concentration is 0.72 mg/mL. After pretreatment, the results had recovered firmly to the baseline levels(Table 2).Meanwhile, we performed parallel experiments on the Mindray CL2000i. The data shows that biotin interference does not appear in the Mindray CL2000i intact Ca242 assay, which does not use the streptavidin/biotin method (Fig. 2).作为膳食补充剂或治疗用药的生物素（Biotin），可能会对生物素-链霉亲和素（streptavidin）免疫测定产生分析干扰。当样本中存在过量生物素时，生物素分子会占据链霉亲和素的结合位点，从而在洗涤步骤后，阻止抗体-分析物夹心复合物与包被链霉亲和素的固相载体结合，进而无法产生测定信号。 为探究生物素对糖链抗原242（CanAg Ca242）检测的干扰情况，本研究对比了实验样本（添加生物素溶液）与对照样本（添加PBS）的检测差异。将纯生物素溶解于0.01 M氢氧化钠溶液中，于-20℃冷藏保存备用。将生物素储备液用PBS稀释为9个浓度梯度的工作液，随后将其分别加入3份不同基线水平的Ca242样本中，使样本最终生物素浓度分别为1000、500、250、125、62.5、31.25、15.63、7.81 ng/mL。 本研究的数据（图1）显示，Ca242检测的3个不同浓度量程范围内均受到生物素的显著干扰，但干扰程度随生物素浓度不同存在差异：生物素浓度越高，Ca242检测值的降低越显著。表1的数据表明，根据室内质量控制目标，基线水平与加标生物素样本间的相对偏差大于10%即可认定为存在显著差异。低水平、临界值水平及高水平Ca242样本分别在生物素浓度为15.63 ng/mL、31.25 ng/mL和62.5 ng/mL时出现显著干扰。当生物素浓度达到250 ng/mL时，相对偏差超过99%（表1）。 在确认生物素会干扰Ca242检测后，本研究下一步旨在探寻可行的干预方案。基于生物素与链霉亲和素的高亲和力特性，我们使用链霉亲和素微球“预结合”生物素：将样本与包被链霉亲和素的磁性微球孵育吸附。该试剂来自罗氏（Roche）提供的Cobas®检测试剂盒，其链霉亲和素浓度为0.72 mg/mL。经此预处理后，检测结果可完全恢复至基线水平（表2）。同时，本研究在迈瑞CL2000i（Mindray CL2000i）化学发光免疫分析仪上开展了平行实验，数据显示，未采用生物素-链霉亲和素检测方法的迈瑞CL2000i完整Ca242检测体系中，未出现生物素干扰现象（图2）。