血清抗氨基甲酰化蛋白自身抗体定量检测微阵列化学发光免疫分析法的建立及应用

Establishment and application of quantitative detection of serum anti-carbamylated protein autoantibodies by microarray chemiluminescence immunoassay

目的基于微阵列蛋白芯片技术自建检测抗氨基甲酰化蛋白(Car P)抗体的化学发光免疫分析法,对该方法的检测性能进行评价,并探讨该指标在类风湿性关节炎(RA)患者的初步临床应用价值。方法自建抗Car P抗体定量检测方法,评价该方法的精密度、最低检测限、线性范围、特异性等。以120例健康对照人群血清抗Car P抗体水平的第95分位为临界值,分析RA组、非RA组抗Car P抗体水平及阳性率;分析RA组抗Car P抗体与疾病活动性指标间的关联性。结果该方法检测高值和低值样本的精密度均<15%;线性范围可达(3. 31～1448. 18) AU/m L,抗Car P抗体与抗环瓜氨酸肽(CCP)抗体几乎不存在交叉反应。与健康对照组相比,RA组、抗CCP抗体阳性RA组和关节痛组患者抗Car P抗体水平显著升高,未分化结缔组织病组也升高。与健康对照组抗Car P抗体5%阳性率相比,RA患者的阳性率达28. 21%,抗CCP抗体阳性RA组阳性率达32. 2%;关节痛组阳性率为38. 89%,均显著升高;其余疾病组间则无统计学差异。RA患者抗Car P抗体水平与类风湿因子(RF)水平和血沉(ESR)均呈弱相关,与CRP和Ig G水平均呈中等相关。结论建立的定量检测抗Car P抗体的蛋白芯片化学发光法具有良好的检测精密度和较宽的线性范围,敏感性和特异性较好。抗Car P抗体检测对RA诊断和疾病的活动性评价有价值。

Objective To develope a chemiluminescence immunoassay based on microarray protein chip technology for detecting the anti-carbamylated protein( Car P) antibody. We aimed to evaluate the detection performance of this methodand to explore its preliminary clinical application value of this index in patients with rheumatoid arthritis( RA). Methods Aquantitative detection method for anti-CarP antibody was established to evaluate the precision,minimum detection limit,linearrange and specificity of the method. The 95 th position of anti-CarP antibody level in serum of 120 healthy controls was definedas the cutoff value. The anti-CarP antibody level and positive rate in RA group and non-RA group were analyzed. And thecorrelation between anti-CarP antibody and disease activity index in RA group was analyzed. Results The precision of thismethod for detecting high-level sample and low-level sample was less than 15%;The linear range could reach( 3.31 ~1448.18) AU/m L,and there was almost no cross-reaction between anti-Car P antibody and anti-CCP antibody. Comparedwith healthy control group,the level of anti-CarP antibody in RA group,anti-CCP antibody positive RA group and joint paingroup was significantly higher,and that in undifferentiated connective tissue disease group was also higher. Compared withthe 5% positive rate of anti-CarP antibody in healthy control group,the positive rate of RA patients was 28. 21%,anti-CCPantibody positive RA patients was 32. 2%,joint pain group was 38. 89%,which were significantly higher. There was nostatistical difference between other disease groups. The level of anti-CarP antibody was weakly correlated with level ofrheumatoid factor( RF) and erythrocyte sedimentation rate( ESR) in RA patients,but it was moderately correlated with CRPand Ig G level. Conclusion The protein chip chemiluminescence method for quantitative detection of anti-CarP antibody hasgood detection precision and wide linear range,and has good sensitivity and specificity. Anti-CarP antibody detection isvaluable for RA diagnosis and disease activity evaluation.