神经生长因子的化学发光标记与检测

以辣根过氧化物酶（ＨＲＰ）和吖啶酯（ＡＥ）为化学发光标记试剂分别标记神经生长因子（ＮＧＦ）单克隆抗体，经分离纯化制成标记抗体（ＨＲＰ－Ａｂ，ＡＥ－Ａｂ），采用化学发光免疫分析法（ＣＬＩＡ）对ＮＧＦ进行检测，其检出限为０．５ｎｇ／ｍＬ，线性范围为２～１２８ｎｇ／ｍＬ．１０例样本分别用ＣＬＩＡ和ＲＩＡ进行检测，其结果无显著性差异．     

Study of blood collection and sample preparation for analysis of vitamin D and its metabolites by liquid chromatography–tandem mass spectrometry

The analysis of vitamin D status, with special emphasis on 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, is gaining interest in clinical studies due to the classical and non-classical effects attributed to this prohormone. In this research, the influence of the two steps preceding determination (viz. sample collection and preparation) on the quantitative analysis of vitamin D and its more important metabolites has been studied. Two preparation approaches, deproteination and solid-phase extraction (SPE), have been evaluated in terms of sensitivity to delimit their application, thus establishing that detection of 1,25-dihydroxyvitamin D cannot be addressed by protein precipitation. Concerning sample collection, serum and plasma reported high accuracy (above 83.3%) for vitamin D and metabolites, while precision, expressed as relative standard deviation, was below 12.9% for all analytes in both samples. Statistical analysis revealed that serum and plasma provided similar physiological levels for vitamin D₃, 24,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃, while significantly different levels were obtained for 1,25-dihydroxyvitamin D₃, always higher in plasma than in serum. Sample collection and treatment have proved to be significant in the analysis of vitamin D and its relevant metabolites.     

Determination of estradiol in human serum using magnetic particles-based chemiluminescence immunoassay

A magnetic particles (MPs)-based chemiluminescence immunoassay (CLIA) with high sensitivity, specificity, and reproducibility was proposed for the evaluation of estradiol (E₂) in human sera. The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and the horseradish peroxidase (HRP)-luminol-H₂O₂ chemiluminescent system with high sensitivity was chosen as the detection system. The method showed specific recognition to E₂, without cross-reaction for the major steroids, including estrone (E₁), estriol (E₃), dihydrotestosterone (DHT), androstenedione, and testosterone (T), which was commonly found in human serum. The addition of sodium trichloracetate (Na-TCA) in the enzyme buffer as a blocking agent contributed to the realization of direct analysis of E₂ in human serum without extraction. Besides, the effects of several physicochemical parameters, including the dilution ratios of E₂-6-HRP conjugate and anti-E₂ polyclonal antibody, immunoreaction time, chemiluminescent (CL) substrate volume, volume of MPs, and CL reaction time, were studied and optimized. The proposed method had a detection limit of 2.51 pg mL⁻¹ with a larger working range of 15-1000 pg mL⁻¹. The inter-assay and intra-assay coefficient of variation (CV) were both less than 15%. The average recoveries of three different spiked concentration samples were 93.3, 106 and 101%, respectively. The method has been successfully applied to the determination of E₂ in 105 human sera and showed a good correlation compared with the commercial radioimmunoassay (RIA) kit with a correlative coefficient of 0.9892. This method has exhibited great potential in the fabrication of diagnostic kit and could be used in the clinical analysis of E₂ in human serum.     

孔雀石绿分子印迹96孔检测板的制备及应用研究

为更加快速灵敏简单的检测水产品中的孔雀石绿残留,通过原位聚合法,以10%的乙腈甲苯溶液为溶剂,MAA为功能单体、EGDMA为交联剂(模板分子与之比例为1:4:20),制备分子了印迹聚合物。通过包被MIP到96孔板上研发了以化学发光法检测的MG-MIP-96孔检测板,其标准曲线为y=-29197.49x + 1945998.45,R2 = 0.9416,线性范围时30-66.64ng/ml。检测限为3.355ng/ml,批内变异系数范围为7.38%-18.07%,批内回收率范围为103.41%-144.45%。批间变异系数范围为12.45%-17.52%,批间回收率范围为100.07%-143.20%。以孔雀石绿结构类似物副品红(PA)和结晶紫(CV)进行特异性检测的交叉率分别为27.97%、30.97%。说明此新型检测方法具有很大的研究价值和实际应用价值。     

Regulatory compliance vs. NEXUS quality for laboratory tests

In the U.S., all clinical laboratory testing is regulated by the Clinical Laboratory Improvement Amendments (CLIA) (). The CLIA link test quality and adherence to a body of testing regulations intended to ensure accurate, reliable, and timely patient test results. The goal of the CLIA legislation was to ensure a minimum, fundamental level of quality. In the context of “NEXUS,” quality must “go beyond getting the ‘right’ answer on the ‘right’ patient that can be interpreted against ‘right’ reference values. CLIA regulations with specific minimum, performance requirements, or safeguards, are designed to prevent testing errors. The US Institute of Medicine found that testing processes fail as a result of human error, lack of documentation, and lack of test management. In the latest (2004) interpretations of CLIA regulations, the minimum quality control requirement continues to be analyzing at least two external, liquid quality control materials per test per day. In 1995, we proposed that the responsibility for achieving quality test results shifts from the sole purview of the laboratory director to an “alliance” of laboratory professionals, manufacturers, and regulators. The EQC (equivalent quality control) concept as proposed is a positive step in achieving this alliance. With the obvious lack of scientific and statistical robustness, EQC falls far short of ensuring quality. Achieving the “NEXUS Vision” for quality laboratory testing will not come solely from laboratory professionals. The NEXUS is about how to ensure the full-quality assessment of the testing process – pre-analytical, analytical, and post-analytical.Presented at the 10th Conference Quality in the Spotlight, March 2005, Antwerp, Belgium.     

Is CLIA 2003 the quality alliance envisioned between manufacturers,laboratory professionals,and regulatory agencies?

The Clinical Laboratory Improvement Amendments of 1988 (CLIA88) had a groundbreaking effect on laboratory professionals, instrument and reagent manufacturers, and regulators in the United States, and by association, worldwide. CLIA88 defined new levels of responsibility for all three of these stakeholders in modern laboratory testing. As a result, we envisioned a CLIA-driven approach to regulations, which in effect, created a three-member quality alliance. In 2003, the long awaited CLIA updates to the quality control and quality assurance requirements were published. The revisions in CLIA 2003 will herald an era in which manufactures take responsibility for meeting new standards of quality (improved accuracy and precision or reduced total allowable error) in the clinical laboratory and, especially, at point of care where testing is performed by non-laboratorians. The European (Bureau International des Poids ed Mesures) efforts at traceability, the National Committee of Clinical Laboratory Standards efforts at estimating total allowable error, and the new CLIA 2003 quality systems approach for quality requirements return to the fundamental concepts of accuracy to assess the efficacy of clinical laboratory testing.Presented at the 8th Conference on Quality in the Spotlight, 17–18 March 2003, Antwerp, Belgium     

A secondary antibody format chemiluminescence immunoassay for the determination of estradiol in human serum

A competitive immunoassay for estradiol (E2) based on secondary antibody format was established. The donkey anti-rabbit IgG was used as the secondary antibody to coat micro-plates, and the horseradish peroxidase (HRP)-luminol-H₂O₂ chemiluminescent system with high sensitivity was chosen as the detection system. The addition of sodium trichloroacetate (CCl₃COONa) in the enzyme buffer as a replaceable packing material can realize directly analysis of E2 in human serum without extraction, which improved reproducibility and resolution of the assay. Additionally, the method showed specific recognition of estrogen, without cross-reaction for the major steroids (estrone (E1), estriol (E3), dihydrotestosterone (DHT), androstenedione, testosterone (T)) commonly found in human serum. The chemiluminescence immunoassay with secondary antibody can be applied to detect E2 with good precision at concentrations as low as 1.48 pg mL⁻¹. The proposed method has been successfully applied to the determination of E2 in 97 human sera and showed a good correlation compared with the commercially radioimmunoassay (RIA) kit with a correlative coefficient of 0.9881. This method has exhibited great potential in the fabrication of diagnostic kit and can be used in the clinical analysis of E2 in human serum.     

Chemiluminescence immunoassay for chloramphenicol

In recent years, various chemiluminescent clinical immunoassay kits have been widely applied to the detection of hormones. However, a kit for chloramphenicol (CAP) is often absent from most commercial product lists, even though it is important to control the levels of CAP residues in foodstuffs too. Therefore, we describe a simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of CAP in foodstuffs. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled antigen is horseradish peroxidase (HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/ml), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8% and <20%, respectively, and the analytical recovery of the method was 87–100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The detection limit for the method was 0.1–10 ng/ml, and it displayed good linearity.