基于基质辅助激光解吸电离飞行时间质谱技术无需抗体富集直接检测血清M蛋白方法的建立

Establishment of a direct detection method for serum M-proteins without antibody enrichment based on MALDI-TOF MS technology

目的基于基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)技术建立一种无需抗体富集直接检测血清M蛋白的方法,并评估其检测效能。方法方法学建立。收集在首都医科大学附属北京朝阳医院申请M蛋白鉴定检测的患者检验后剩余血清标本712份。用TCEP还原剂处理IgG、IgA纯品获得免疫球蛋白轻链,初步确定检测方法。收集20名健康体检人群检验后剩余血清标本,确定κ和λ轻链离子峰质荷比范围。设置8个平行管和8个批次进行批内和批间重复性评价;选取23例M蛋白阳性标本进行10倍、100倍和200倍稀释,采用所建MALDI-TOF MS方法检测M蛋白,评价灵敏度;采用IFE、SPE和MALDI-TOF MS 3种方法同时检测M蛋白,计算MALDI-TOF MS方法与IFE和SPE的符合率。结果批内和批间重复性均为100%;对23份M蛋白阳性标本的原倍、10倍、100倍和200倍稀释标本进行测定,MALDI-TOF MS对M蛋白的检测限为0.06~0.18 g/L;在712份标本中,以IFE为金标准,SPE与MALDI-TOF MS总的符合率分别为85.9%和92.3%,SPE与MALDI-TOF MS的阳性符合率分别为72.8%和99.7%;在M蛋白的不同型别中,MALDI-TOF-MS在IgA、IgD、IgM、游离轻链型和双克隆组中对M蛋白的检出率为100%,而SPE对IgM、IgA和游离轻链型标本的检出率分别只有66.7%、58%和19.5%;IgG组有1份阳性标本MALDI-TOF MS未检出。对23份M蛋白阳性标本进行原倍、10倍、100倍和200倍稀释后,10倍稀释时MALDI-TOF MS的检出率为100%,IFE为96%;100倍和200倍稀释时,IFE的检出率分别为MALDI-TOF MS的28%和23%。结论本研究成功建立了血清M蛋白的MALDI-TOF MS检测方法,该法检测通量高、速度快,具有良好的灵敏度、特异性和符合率。

Objective:To establish a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the direct detection of serum M protein without antibody enrichment, and to assess its detection performance.Methods:Method establishment. A total of 712 waste serum samples were collected from patients who applied for the M protein identification test in Beijing Chaoyang Hospital affiliated to Capital Medical University. The immunoglobulin light chain was obtained by reduction of IgG and IgA by TCEP, and the detection method was preliminarily determined. The waste serum samples from 20 healthy people were collected to determine the range of mass-to-charge ratios of κ and λ light chain ions. 8 parallel tubes and 8 batches were set up for intra-and inter-batch reproducibility evaluation. 10-fold, 100-fold and 200-fold diluted M protein from 23 positive samples were detected by established MALDI-TOF MS method, and its sensitivity was evaluated. 3 methods of IFE, SPE and MALDI-TOF MS were used to detect M protein simultaneously, and the coincidence rate between MALDI-TOF MS and IFE and SPE was calculated.Results:The repeatability within and between batches was 100%, respectively. The original, 10-, 100-and 200-fold dilutions of 23 M protein-positive samples were determined, and the detection limit of MALDI-TOF MS for M protein was 0.06-0.18 g/L. IFE as the gold standard, the overall coincidence rates of SPE and MALDI-TOF MS were 85.9% and 92.3%, respectively, and the positive coincidence rates of SPE and MALDI-TOF MS were 72.8% and 99.7%, respectively, of the 712 samples. Among the different types of M-proteins, MALDI-TOF-MS agreed 100% with IFE M-protein results for IgA, IgD, IgM, free light chain type and biclonal group, while the agreements of SPE for IgM, IgA and free light chain samples were only 66.7%, 58% and 19.5%, respectively. One positive sample in the IgG group was not detected by MALDI-TOF MS. 23 M-proteins positive samples were diluted by original, 10, 100 and 200 times to access the sensitivity of MALDI-TOF MS method. The coincidence rate of MALDI-TOF MS was 100% and IFE was 96% at 10-fold dilution. The coincidence rate of IFE was 28% and 23% of MALDI-TOF MS at 100-fold and 200-fold dilution, respectively.Conclusions:A MALDI-TOF MS method for the detection of serum M-proteins was successfully established. This method has the advantages of high detection throughput, fast speed, good sensitivity, specificity and coincidence rate.