Development of a rapid and sensitivity magnetic chemiluminescence immunoassay for DNA methyltransferase 1 in human serum

DNA methyltransferase 1(DNMT1)is a useful biomarker for lung cancer in early clinical diagnosis.A rapid magnetic chemiluminescence immunoassay(MCLIA)for DNMT1 in human serum has been developed.Horseradish peroxidase(HRP)-second-Ab was used to labeled polyclonal antibodies of anti-DNMT1.DNMT1 in sample integrates with specific immunomagnetic beads and can constitute a supersandwiched immunoreaction.In magnetic field,nonspecific materials can be separated.After luminescent substrate luminol-H2O2-BIP was added,the relative light unit(RLU)of HRP was detected and was discovered to be directly proportional to the content of DNMT1 in sample.The correlative variables involved in the MCLIA value were optimized and the methodological evaluation was carried out.After optimization,in the range of0.5–128 ng/mL,the linear regression equation was y=0.5014 x+1.769(x was logCDNMT1,y was relative luminescence units(RLU)/RLU0),and the limit of detection was 0.01 ng/mL.The RSD of intra-and interassays were 15.8%–16.9%and 14.3%–18.1%,respectively.The recovery was from 70.0%to 106.2%.Furthermore,paralleled with purchasable enzyme-linked immunosorbent assay(ELISA)kits,MCLEIA had lower detection limit,wider linear range and shorter detection time.Therefore,the MCLEIA established in this study could be used for the sensitive detection of DNMT1 in serum sample.

Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).

Micro-plate magnetic chemiluminescence immunoassay and its applications in carcinoembryonic antigen analysis

A micro-plate magnetic chemiluminescence immunoassay was developed for rapid and high throughput detection of carcinoembryonic antigens (CEA) in human sera. This method was based on a sandwich immunoreaction of fluorescein isothiocyanate (FITC)-labeled anti-CEA antibodies, CEA antigens, and horseradish peroxidase (HRP)-conjugated anti-CEA antibodies in mi- cro-plate. The immunomagnetic particles coated with anti-FITC antibodies were used as the solid phase for the immunoassay. The separation procedure was carried out by a magnetic plate adaptor and the luminol-hydrogen peroxide (H2O2)-HRP system was employed for the chemiluminescence detection. The proposed method combined the advantages of the micro-plate reactor and magnetic particle separation technology with the linear range of 5-250 ng mL·1. The detection limit of CEA was 0.61 ng mL·1. The coefficient of the variation was less than 7% and 13% for intra-assay and inter-assay precision, respectively. Compared with the commercial micro-plate chemiluminescent kit, the proposed method showed a good correlation.

Washing-free chemiluminescence immunoassay for rapid detection of cardiac troponin Ⅰ in whole blood samples

Chemiluminescence immunoassay(CLⅠA) has always been a great challenge in detecting cardiac troponin Ⅰ(c Tn Ⅰ) in whole blood samples without centrifugation because of the interference of red blood cells and low sensitivity. Ⅰn this study, the antigens and erythrocytes in the blood were captured by the antibodies immobilized on the magnetic particles, recognized by another biotinconjugated c Tn Ⅰ antibody and detected by streptavidin/acridine aster-conjugated polychloromethylstyrene microspheres(PCMS). After magnetic separation, the supernatant was transferred and measured. No significant difference was noted between the c Tn Ⅰ concentrations of the serum samples,plasma samples and whole blood. The prepared PCMS provided more functional areas to conjugate streptavidin and acridinium ester, so the immunoassay has highly sensitive, the limits of blank at0.012 ng/mL, and functional sensitivity at 0.019 ng/mL with a CV of 20%, and 0.058 ng/mL with a CV of 10%. Total precision of any sample type ranged from 2.62%~5.67%. The assay was linear over the studied range of 0.01-50.00 ng/mL, and no hook effect was found when c Tn Ⅰ concentrations reached 1900 ng/mL. No significant interference was noted with the potential endogenous interfering substances. Compared with the commercial kit(Abbott assay kit), the correlation coefficient was 0.9859. A washing-free CLⅠA was established for the rapid detection of c Tn Ⅰ in human whole blood, using erythrocyte capture antibodies-conjugated magnetic nanoparticles for eliminating the influence of erythrocytes and PCMS for signal amplification, which showed great potential in clinical application.

Rapid detection of Cyfra 21-1 by optical-biosensing based on chemiluminescence immunoassay using bio-functionlized magnetic nanocomposites

This letter reports a chemiluminescene immunoassay method combined with immunomagnetic separation to rapidly detect Cyfra 21-1, in which bio-functionlized magnetic nanocomposites were used as mobile substrate for capturing and isolating the cyfra 21-1 proteins. After the captured Cyfra 21-1 further reacted with horseradish peroxidase-conjugated anti-Cyfra 21-1 antibody to form a sandwich immunocomplex, the chemiluminescence would be produced as a result of addition of the chemiluminescent substrate. A home-made optical biosensor was designed to detect the chemiluminescence instead of other large instruments. There is a good linear response between the chemiluminescence intensity and the concentration of Cyfra 21-1 in the range from 0.2 to 50 ng/mL. The whole detection process including incubation, washing and detection could be performed within 45 min. The proposed method offers a simple, noninvasive and reliable tool for detecting non-small cell lung cancer and has potential application for clinical testing.

A New Immunoassay Method by Capillary Electrophoresis with Enhanced Chemiluminescence Detection

This paper described a new immunoassay method by capillary electrophoresis with enhanced chemiluminescence (CL) detection system based on luminol-hydrogen peroxide reaction catalyzed by horseradish peroxides (HRP). Using para-iodophenol as a CL enhancer, the detection limit of about 1×10-12 mol/L for HRP was achieved, which corresponded to 1.32×10-5 U/mL. In optimal conditions, the free HRP-labeled CA125 antibody (Ab*) and the bound enzyme-labeled complex (Ab*-Ag) were well separated by capillary electrophoresis within 4 min. The assay was successfully used to determine the contents of CA125 in human sera, which were associated with ovarian cancer, and the recoveries of the standard addition experiments were 96 to 109 %.

MAGLUMI X8全自动化学发光免疫分析仪配套的甲状腺功能试剂盒的性能验证

目的对MAGLUMI X8全自动化学发光免疫分析仪配套的甲状腺功能五项检测试剂盒进行性能验证。方法收集2023年2—3月从江县人民医院体检样本10例,根据中国国家卫生行业标准(WS/T 492-2016),对该检测系统促甲状腺素、总三碘甲状腺原氨酸、总甲状腺素、游离三碘甲状腺原氨酸、游离甲状腺素5个项目进行检测性能验证。结果被验证系统精密度、准确度、线性范围及参考范围性能指标均满足临床检测要求。结论被验证系统精密度、准确度、线性范围及参考范围性能指标均符合要求,可用于临床上甲状腺功能的诊断及监测。

Recent advances in electrogenerated chemiluminescence biosensing methods for pharmaceuticals

Electrogenerated chemiluminescence(electrochemiluminescence, ECL) generates species at electrode surfaces, which undergoes electron-transfer reactions and forms excited states to emit light. It has become a very powerful analytical technique and has been widely used in such as clinical testing, biowarfare agent detection, and pharmaceutical analysis. This review focuses on the current trends of molecular recognition-based biosensing methods for pharmaceutical analysis since 2010. It introduces a background of ECL and presents the recent ECL developments in ECL immunoassay(ECLIA), immunosensors, enzyme-based biosensors, aptamer-based biosensors, and molecularly imprinted polymers(MIP)-based sensors. At last, the future perspective for these analytical methods is briefly discussed.

Diagnosis of Toxoplasma gondii infection in pregnant women using automated chemiluminescence and quantitative real time PCR

Objective: To identify serodiagnosis and quantification of Toxoplasma gondii(T. gondii) infection among pregnant women in Salmas, northwest of Iran. Methods: In this crosssectional study, 276 blood samples were collected from pregnant women referred to the health care centers in Salmas city. The demographic variables were also recorded. Titers of antiToxoplasma IgM and IgG antibodies(Ab) were determined using the chemiluminescence immunoassay. Quantitative real-time PCR targeting the T. gondii repeated element gene was also performed on the blood sample. Results: Out of all, 19.92%(55/276) and 2.17%(6/276) patients were seropositive for anti-Toxoplasma IgG and IgM Ab, respectively. Moreover, the presence of T. gondii DNA was observed in 12.31%(34/276) blood samples. A significant relationship was observed between the IgG Ab seropositivity and contact with the cat as a risk factor(P=0.022). Conclusions: The seroprevalence rate of T. gondii infection in pregnant women is relatively low. Consequently, the seronegative pregnant women are at risk, and a considerable rate of positive blood samples for the presence of parasite's DNA should not be ignored. Besides, quantitative real-time PCR could be considered as an accurate method for diagnosis of acute toxoplasmosis especially when the precise results are of the most importance in pregnancy. Limiting contact with cats is also suggested for pregnant women.

Determination of theophylline concentration in serum by chemiluminescent immunoassay

Objective: This study aimed to establish chemiluminescent immunoassay (CLIA) for quantitative determination of theophylline levels in human serum. Methods: To measure the concentration of theophylline (n=122) and evaluate the assay.Results: The linear range of the CLIA method was 0.51～40 mg/L (Y=1.02X+0.44, r=0.995). The intra and inter CV (coefficient variance) of CLIA were 3.20% and 3.57%, respectively. The average recovery rate was 102.3%. This method was free from interference by brilirubin (＜200 μmol/L), hemoglobin (＜10 g/L), and triglycerides (＜15 mmol/L). Conclusion: This method is simple, convenient and precise for clinical pharmacokinetics study oftheophylline.

Rapid and Sensitive Chemiluminescent Enzyme Immunoassay for the Determination of Neomycin Residues in Milk

Immunoassays greatly contribute to veterinary drug residue analysis. However, there are few reports on detecting neomycin residues by immunoassay. Here, a rapid and sensitive chemiluminescent enzyme immunoassay （CLIEA） was successfully developed for neomycin residue analysis. CLIEA demonstrated good cross-reactivity for neomycin, and the IC50 value was 2.4 ng/mL in buffer.

Determination of Progesterone Receptor by Chemiluminescent Enzyme Immunoassay

A new method of chemiluminescent enzyme immunoassay (CLEIA) was developed and the standard curve and regression equation for determination of progesterone receptor (PR) made. The luminosity of tissue samples was tested and PR level was calculated by the regression equation. Correlation analysis revealed that there was a linear relationship between different concentrations of the standard PR samples and the corresponding values of luminosity: Y=3748+463.77X, γ=0 9958. The values of the luminosity in 38 cases of tumor tissues were determined with the highest being 267.32 fmol/mg, the lowest 3.69 fmol/mg and the mean 78.53 fmol/mg. The new method of CLEIA was a stable, creditable,specific and sensitive assay for determination of PR.

HBV、HCV、HIV血筛多中心研究免疫学灰区的核酸检测分析与临床特征研究

目的分析化学发光灰区标本的临床特征及核酸检测对化学发光灰区标本结果判断的指导性意义。方法收集2021年7月—12月全国不同地区的5家综合医院入院患者术前/输血前血源性传播疾病样本检测结果,对化学发光灰区检测结果的标本进行核酸检测结果及临床特征分析。结果5723例样本中总计检出HBV免疫灰区样本28例(占比0.49%),HCV灰区样本20例(占比0.35%)。经核酸检测验证,28例HBV灰区样本中,15例HBV样本核酸检测为阳性(占比53.5%),其HBcAb也均为阳性;13例HBV样本核酸检测为阴性(占比46.5%),其中HBcAb阳性4例。HBV与HCV免疫检测灰区在临床各个科室均有发现,出现HBV灰区样本最多的前三科室为骨科、妇科、泌尿科,灰区样本核酸验证假阳性最多的临床科室为妇科与骨科。HCV灰区样本最多的前三科室为泌尿、肾内、外科,且均为假阳性。HBV灰区样本患者临床诊断结果有35.7%(10/28)为肿瘤类疾病,HCV灰区样本患者临床诊断结果有40%(8/20)为肿瘤类疾病。结论化学发光法容易造成假阳性结果,应注意复检验证,且设置灰区并非必要。灰区样本可见于多个临床科室,具有一定的临床分布特征。核酸检测可以提高检测灵敏度并且更大限度保证结果的准确性,能够验证免疫检测灰区。

甲状腺患者生化免疫检验中化学发光免疫测定技术应用的价值分析

目的探讨化学发光免疫测定技术在甲状腺肿瘤疾病诊断中应用的价值。方法选取2021年2月—2023年5月连云港市妇幼保健院收治的158例疑似甲状腺肿瘤患者为研究对象,分别应用免疫放射分析法和化学发光免疫测定法进行检测。以病理诊断结果为金标准,比较两种检验方式对甲状腺癌的诊断结果及诊断效能(特异度、灵敏度和准确度)。结果在甲状腺肿瘤诊断中,化学发光免疫测定法的结果更接近金标准,其灵敏度(93.02%)高于免疫放射分析法(78.29%),差异有统计学意义(χ^(2)=11.390,P<0.05)。化学发光免疫测定法的特异度和准确度为86.21%、91.77%,高于免疫放射分析法,差异有统计学意义(χ^(2)=9.471、19.533,P均<0.05)。结论化学发光免疫测定法在甲状腺患者生化免疫检测中的应用更接近金标准,该检测方式的灵敏度、特异度和准确度较高,在疾病诊断中应用的价值较高。

化学发光免疫分析法测定患者血浆伏立康唑浓度的验证与评价

目的:验证并评价化学发光免疫分析法(CLIA)用于伏立康唑血药浓度的测定。方法:(1)验证CLIA法,进行检测限、重复性、线性、准确度及精密度5个方面的验证;(2)评价CLIA法,收集2023年1—2月该院进行伏立康唑血药浓度监测的113例患者的血浆样本,分别用超高效液相色谱-串联质谱法(UPLC-MS/MS)和CLIA法进行检测,采用配对t检验、组内相关系数(ICC)、Passing-Bablok回归和Bland-Altman分析比较两种方法的相关性及一致性。结果:经验证,CLIA法用于测定伏立康唑血药浓度符合分析技术要求,方法稳定可靠。CLIA法检测的血浆伏立康唑平均浓度为(3.843±2.056)μg/mL,高于UPLC-MS/MS法测得的(3.399±1.959)μg/mL,差异有统计学意义(P<0.01)。两种方法测定结果的ICC为0.932(P<0.001);Passing-Bablok回归方程为Y=0.0834+1.099 X(R=0.956,n=113),方程斜率为1.099(95%CI=1.030~1.161),截距为0.0834(95%CI=-0.062~0.251),两种方法测定值之间存在良好的线性相关关系。Bland-Altman分析显示,两种方法的平均偏差为0.442μg/mL(95%CI=0.331~0.558μg/mL),平均偏差百分比为14.49%(95%CI=11.47%~17.51%),一致性尚可接受。结论:CLIA法稳定可靠、操作简便、检测效率高,更适用于临床样本检测。CLIA法与UPLC-MS/MS法测定的结果相关性良好,一致性尚可,CLIA法可作为UPLC-MS/MS法的潜在替代方法用于临床检测伏立康唑血药浓度。

高灵敏化学发光法特异性检测人血清促甲状腺激素方法的建立

目的 通过双位点夹心化学发光免疫分析原理,建立一种高灵敏度测定人血清促甲状腺激素(thyroid stimulating hormone, TSH)的化学发光免疫分析方法。方法 将样本与预包被有抗促甲状腺激素(TSH)抗体的超顺磁珠,以及抗TSH-碱性磷酸结合物添加到反应管中共孵育,待测样本中的TSH通过夹心法与磁珠和抗TSH-碱性磷酸结合物与形成双位点复合物。与磁珠结合的复合物吸附到磁场上,用缓冲液洗去未结合的物质。加入发光底物3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐[3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1,2-dioxetane, AMPPD],测定相对光子强度(RLU),通过标准曲线计算待测样本中TSH的浓度。结果 本研究建立的TSH检测方法,最低检测限为0.003μIU/mL,批内CV小于1%,总CV小于3%,正确度样本实测值与靶值之间相对偏差均在±12.5%范围内,线性范围为0.005~100μIU/mL且相关系数(r)高于0.9900。开封稳定性试验的相对偏差<10%。结论 该方法检测血清TSH浓度具有灵敏度高、准确性好、线性范围广、稳定性强的优点,各项指标均达到临床检测要求,可以推广使用。

化学发光免疫分析法及在饲料检测中的应用

化学发光免疫分析法(CLIA)是将灵敏度较高的化学发光反应与特异性较强的免疫反应融合而产生的一种免疫分析方法,具有操作简便、自动化程度高、检测灵敏度高、检测时间短、检测范围宽等优势,被广泛应用于医学、环境检测、食品安全、药物分析检测等领域。伴随着CLIA的不断发展,其在饲料质量安全领域也表现出较好的发展前景。本文主要综述了CLIA的原理、分类、新技术以及其在饲料检测中的应用进展,以期为CLIA在饲料质量安全检测领域的更深入研究提供参考。

间接免疫荧光法、线性免疫印迹法、化学发光法单项和联合检测抗核抗体的临床价值

目的探讨间接免疫荧光法(IFA)、线性免疫印迹法(LIA)、化学发光法(CLIA)单独和联合检测抗核抗体(ANA)的临床价值。方法选取2022年8月—2023年7月在上海交通大学医学院附属第九人民医院进行ANA检测的患者4722例,其中自身免疫性疾病(AID)患者935例(AID组)、非AID患者3787例(非AID组)。采用IFA检测ANA,采用LIA、CLIA检测特异性ANA谱。采用Kappa一致性检验评估不同方法之间的一致性。采用受试者工作特征(ROC)曲线评价不同方法诊断AID的效能。结果AID组IFA、LIA、CLIA阳性检出率分别为67.5%、52.1%、44.8%,均高于非AID组(36.9%、20.1%、17.4%)(P<0.05)。IFA与LIA的一致性一般(Kappa=0.609),IFA与CLIA的一致性差(Kappa=0.276)。在不同类型疾病患者中,AID患者的一致性最高(IFA与LIA、CLIA的kappa值分别为0.628、0.444),其他疾病类型患者的一致程度最低(IFA与LIA、CLIA的kappa值分别为0.120、0.194)。IFA、LIA、CLIA单项检测诊断AID的曲线下面积(AUC)分别为0.707、0.662、0.655。IFA和LIA并联、IFA和CLIA并联检测诊断AID的AUC分别为0.711、0.699,IFA和LIA串联、IFA和CLIA串联检测诊断AID的AUC分别为0.677、0.647。结论IFA与LIA、CLIA的一致性不高,且检测结果不一致的情况更可能出现在非AID患者中。使用单项检测方法进行AID筛查时,应优先采用IFA检测ANA;若采用ANA和特异性ANA谱联合检测,以并联方式为宜。

女性雄性激素质谱多指标检测在多囊卵巢综合征诊断中的应用

目的探讨液相色谱串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)法在多囊卵巢综合征(polycystic ovary syndrome,PCOS)患者中高雄激素血症(hyperandrogenemia,HA)检出率,探讨不同种类雄激素在PCOS中的诊断价值。方法纳入2023年1月至2023年12月于首都医科大学附属北京妇产医院妇科内分泌门诊就诊合并雄激素过多临床表现的PCOS患者100例为试验组,同期就诊的健康备孕人群100例为对照组。LC-MS/MS法检测总睾酮(total testosterone,TT)、游离睾酮(free testosterone,FT)、生物活性睾酮(bioavailable testosterone,BIO-T)、雄烯二酮(androstenedione,A2)、17羟孕酮、双氢睾酮、脱氢表雄酮、硫酸脱氢表雄酮、性激素结合球蛋白指标,绘制受试者工作特征曲线,计算曲线下面积(area under curve,AUC),评价各雄激素水平对PCOS的诊断效能,化学发光免疫法(chemiluminescence immunoassay,CLIA)和LC-MS/MS 2种方法比较TT检测水平的诊断价值以及对HA的检出率。结果与对照组相比,试验组TT、黄体生成素(luteinizing hormone,LH)、LH/卵泡刺激素(follicle stimulating hormone,FSH)、抗苗勒管激素、空腹胰岛素、稳态模型的胰岛素抵抗指数均明显升高(P<0.05),FT的AUC诊断灵敏度最高为86.4%(P<0.05,95%CI:0.815~0.912),其次为TT的AUC为84.4%(P<0.05,95%CI:0.812~0.909)、BIO-T的AUC为82.2%(P<0.05,95%CI:0.792~0.896)、A2的AUC为84.2%(P<0.05,95%CI:0.790~0.895)。与CILA法相比,利用LC-MS/MS法检测TT诊断PCOS的灵敏度更高(AUC 0.891 vs 0.841),且对于合并雄激素过多临床征象的PCOS患者HA检出率明显高于CLIA法(P<0.05)。结论LC-MS/MS法对多种雄激素指标检测灵敏度高、准确性好,FT对于高雄激素临床表现的PCOS诊断价值最高,且该法可提高PCOS的检出率,实现HA的早期筛查,更适合临床推广应用。

免疫层析技术及化学发光免疫分析在梅毒检测中的应用价值比较

目的探究免疫层析技术及化学发光免疫分析在梅毒检测中的应用价值。方法选取2020年9月至2021年9月南昌市第一医院接收的100例住院及门诊患者作为研究对象,所有患者均采用化学发光免疫分析、免疫层析技术检测血清标本,以梅毒螺旋体明胶凝集试验(TPPA)检验结果为金标准,比较不同检测方法的阳性检出率、准确度、特异度、灵敏度、阴性预测值与阳性预测值。结果TPPA检测结果检出阳性13例,化学发光免疫分析法检出阳性12例,免疫层析技术检出阳性13例。免疫层析技术检测准确度高于化学发光免疫分析,差异有统计学意义(P<0.05);两种检测方法特异度、灵敏度、阴性预测值、阳性预测值比较差异无统计学意义。两种检测方法阳性检出率结果均显示,年龄<40岁、40~65岁患者阳性检出率低于年龄>65岁患者,差异有统计学意义(P<0.05)。结论免疫层析技术检测梅毒具有更高的检测效能,值得临床推广应用。