Human Serum Specimens Research Articles

Combination assay of lung cancer associated serum markers using surface-enhanced Raman spectroscopy.

The development of an ultrasensitive analysis technique for the combination assay of cancer associated markers is an effective method for the early detection of tumor. Herein, we report a highly sensitive and specific SERS-based sandwich immunoassay for the simultaneous detection of two protein markers associated with lung cancer, including carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE). Two bright SERS tags are prepared by surface modifications of flower-like gold nanoparticles with Raman molecules and target-specific antibodies, and SERS-active magnetic nanoparticles labelled with mixed antibodies are used as immune substrates for capturing the targets and further separating the sandwich structured immune complexes from the mixture. Immunoassays for joint detection of CEA and NSE using both buffer and human serum specimens are performed and the assay results indicate that the proposed SERS-based combination assay of the two markers shows good specificity and ultrahigh sensitivity. The limit of detections of CEA and NSE in human serum specimens are 1.48 pg mL-1 and 2.04 pg mL-1, respectively. The proposed SERS immunoassay is expected to be used for multiple-marker assays of any other tumors and to provide a reliable method for the early screening of cancers in clinic.

Nanogold-penetrated poly(amidoamine) dendrimer for enzyme-free electrochemical immunoassay of cardiac biomarker using cathodic stripping voltammetric method

Methods based on immunoassays have been developed for cardiac biomarkers, but most involve the low sensitivity and are unsuitable for early disease diagnosis. Herein we design an electrochemical immunoassay for sensitive detection of myoglobin (a cardiac biomarker for acute myocardial infarction) by using nanogold-penetrated poly(amidoamine) dendrimer (AuNP-PAMAM) for signal amplification without the need of natural enzymes. The assay was carried out on the monoclonal mouse anti-myoglobin (capture) antibody-anchored glassy carbon electrode using polyclonal rabbit anti-myoglobin (detection) antibody-labeled AuNP-PAMAM as the signal tag. In the presence of target myoglobin, the sandwiched immunocomplex could be formed between capture antibody and detection antibody. Accompanying AuNP-PAMAM, the carried gold nanoparticles could be directly determined via stripping voltammetric method under acidic conditions. Under optimal conditions, the detectable electrochemical signal increased with the increasing target myoglobin in the sample within a dynamic working range from 0.01 to 500 ng mL−1 with a detection limit of 3.8 pg mL−1. The electrochemical immunoassay also exhibited high specificity and good precision toward target myoglobin. Importantly, our strategy could be applied for quantitative monitoring of myoglobin in human serum specimens, giving well matched results with those obtained from commercialized enzyme-linked immunosorbent assay (ELISA) method.

Metabolomics Identifies Biomarker Pattern for Early Diagnosis of Hepatocellular Carcinoma: from Diethylnitrosamine Treated Rats to Patients.

Early diagnosis of hepatocellular carcinoma (HCC) remains challenging to date. Characteristic metabolic deregulations of HCC may enable novel biomarkers discovery for early diagnosis. A capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS)-based metabolomics approach was performed to discover and validate potential biomarkers for HCC from the diethylnitrosamine-induced rat hepatocarcinogenesis model to human subjects. Time series sera from the animal model were evaluated using multivariate and univariate analyses to reveal dynamic metabolic changes. Two independent human cohorts (populations I and II) containing 122 human serum specimens were enrolled for validations. A novel biomarker pattern of ratio creatine/betaine which reflects the balance of methylation was identified. This biomarker pattern achieved effective classification of pre-HCC and HCC stages in animal model. It was still effective in the diagnosis of HCC from high-risk patients with cirrhotic nodules, achieving AUC values of 0.865 and 0.905 for two validation cohorts, respectively. The diagnosis of small HCC from cirrhosis with an AUC of 0.928 highlighted the potential for early diagnosis. This ratio biomarker can also improve the diagnostic performance of α-fetoprotein (AFP). This study demonstrates the efficacy of present strategy for biomarker discovery, and the potential of metabolomics approach to provide novel insights for disease study.

Bioconjugation of peroxidase-like nanostructures with natural enzyme for in-situ amplified conductometric immunoassay of tissue polypeptide antigen in biological fluids

A new conductometric immunoassay based on biofunctionalized prussian blue-gold hybrid nanostructure (PBGN) was designed for sensitive determination of tissue polypeptide antigen (TPA). The immunosensor was prepared by immobilizing anti-TPA capture antibody on the interdigitated transducer, while the PBGN was used for bioconjugation of detection antibody and horseradish peroxidase (HRP). Upon target TPA introduction, the sandwiched immunocomplex was formed between capture antibody and detection antibody on the transducer. Accompanying the PBGN, the carried HRP and prussian blue catalyzed iodine ion into iodine relative to H2O2-KI system, thus resulting in the variations of local conductivity. Under optimal conditions, the immunosensor exhibited good conductometric responses toward target TPA, and allowed the detectable concentration as low as 0.28pgmL−1. The precision, specificity and reproducibility were acceptable. Importantly, our strategy was also utilized for analysis of 13 human serum specimens, giving well matched results with those obtained from human TPA ELISA kit.

Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy.

Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL(-1), with a low detection limit (LOD) of 0.32 pg mL(-1). Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format.

Discovery and verification of serum differential expression proteins for pulmonary tuberculosis

Pulmonary tuberculosis (PTB) is a chronic disease and has remained a severe threat to public health. Valuable biomarkers for improving the detection rate are crucial for controlling this disease. The purpose of this study was to discover potential biomarkers in sera from PTB patients compared with pneumonia patients and normal healthy controls. A total of 336 human serum specimens were enrolled in this study. Differentially expressed proteins were identified using iTRAQ method combining with MALDI-TOF-MS. Data was analyzed using relative bioinformatics methods. Potential biomarkers were further validated by IHC, ELISA and Western blot. As a result, 489 non-redundant proteins were identified in the sera, and 159 of which could be quantified by calculating their iTRAQ ratios. Compared to the controls, 26 differentially expressed proteins were recognized among PTB patients, including 16 overexpressed proteins and 10 downregulated proteins. Analysis of their functional interactions revealed that 12 proteins appeared in the center of the functional network. One of these key proteins, sex hormone binding globulin (SHBG), was found to be significantly elevated among PTB patients as compared with the controls examined by IHC, ELISA and Western blot. This result was consistent with the iTRAQ result. An independent blinded testing set to examine serum SHBG by ELISA achieved an accuracy of 78.74%, sensitivity of 75.6% and specificity of 91.5% in diagnosing PTB. In summary, iTRAQ in combination with MALDI-TOF-MS technology can efficiently screen differentially expressed proteins in sera from the PTB patients. SHBG is suggested to be a possible and novel serum biomarker for PTB.

HCR-stimulated formation of DNAzyme concatamers on gold nanoparticle for ultrasensitive impedimetric immunoassay

A novel signal-amplified impedimetric immunosensing strategy was successfully developed for ultrasensitive detection of low-abundance proteins (carcinoembryonic antigen, CEA, used as a model) based on hybridization chain reaction (HCR)-stimulated formation of DNAzyme concatamers on nanogold particle accompanying enzyme-triggered biocatalytic precipitation. The assay was carried out on capture antibody-modified electrode by using gold nanoparticle heavily functionalized with initiator strand and detection antibody as the signal-transduction tag with a sandwich-type immunosensing format. In the presence of target CEA, the formed immunocomplex underwent an unbiased strand-displacement reaction by the initiator strand on the gold nanoparticle between two auxiliary single-stranded DNA with the hemin aptamer. Upon hemin introduction, numerous DNAzyme molecules were formed on the concatamers and nanogold particle, which could catalyze 4-chloro-1-naphthol to produce an insoluble precipitation on the electrode, thereby resulting in the amplification of impedimetric signal. Under the optimal conditions, the immuno-HCR assay exhibited good impedimetric responses for the detection of target CEA in the working range from 1.0pgmL−1 to 20ngmL−1 with a detection limit of 0.42pgmL−1. In addition, the immuno-HCR assay was validated by measuring six human serum specimens for target CEA, receiving a highly matched correction between the obtained results by the immuno-HCR assay and the commercialized ELC-based immunoassay method.

Development and performance evaluation of a novel quantitative fluorescence immunochromatography for NT-proBNP

Objective To develop a rapid quantitative detecting assay for point-of-care testing (POCT) of N-terminal pro-brain natriuretic peptide（NT-proBNP）in serum by the fluorescence immunochromatographic technology. Methods Applying double-antibody sandwich assay to establish the quantitative NT-proBNP kit.The performance of quantitative NT-proBNP kit was evaluated by the sensitivity, specificity, accuracy, precision, stability and clinical effectiveness.It compared the research kit and conference kit by the parallel experience in the 1 056（605 males, 451 females）serum specimen collected from Guangdong Provincial People′s Hospital, Sun Yat-sen Memorial Hospital and Children′s Hospital of Zhengzhou between February 2013 to April 2014.Statistical significance of the results was assessed by correlation analysis, linear regression, receive operating characteristic (ROC) curve analysis, negative and positive consistent. Results The report range of the NT-proBNP kit was 18-35 000 ng/L.The coefficient of variation (CV) values for low, median and high concentration calibrators respectively were all less than 15%.Common interfering substances in human serum specimens such as bilirubin, triglyceride and cholesterol were found no significant affect on NT-proBNP antigen detection and the CV were no more than 15%.According to the results of detection for calibrators, the shelf time of the NT-proBNP diagnostic kit should be longer than 12 months.The NT-proBNP kit and reference kit had good correlation (Y=1.048 9X developed reference +121.54, R2=0.956 6, n=1 056) to detect the target protein through the parallel experiments and the deviation of the quantitative results of clinical serum samples showed no statistical significance (Z=0.88, P=0.379>0.05).The clinical assays of two different diagnostic kits showed good consistency based on the ROC curve evaluation which is compared by two cut-off values (300 and 450 ng/L).The areas under ROC curve were 0.981 and 0.978 respectively. Conclusions A novel NT-proBNP chromatographic quantitative immunofluorescence detection method was developed in this study.The performance evaluation data indicated that the kit is suitable for rapid detection of serum NT-proBNP.（Chin J Lab Med，2014,37：842-846） Key words: Peptide fragments; Natriuretic peptide, brain; Immunochromatography; Heart failure; Fluorescence

Nickel-functionalized reduced graphene oxide with polyaniline for non-enzymatic glucose sensing

We have developed a new class of organic–inorganic hybrid nanostructures based on the use of reduced graphene oxide (rGO), polyaniline, and a nickel metal nanostructure. It was applied to efficient non-enzymatic sensing of glucose based on its electrocatalytic oxidation. Scanning electron microscopy and energy-dispersive X-Ray were employed to characterize the material. It is shown that the doped polyaniline plays an important role in the formation of the hybrid nanostructures. Improved analytical performance is found when the hybrid nanostructures were placed on a glassy carbon electrode and used for non-enzymatic sensing of glucose at a typical working potential of +450 mV and a pH value of 13. Features include a fast response (~2 s), high sensitivity (6,050 μA mM−1 cm−2), a linear range from 0.1 μM to 1.0 mM, and a low detection limit (0.08 μM). The response to glucose follows a Michaelis-Menten kinetic behavior, and the KM value was determined to be 0.241 μM. Reproducibility and specificity are acceptable. Fructose and maltose do not interfere significantly. Importantly, the methodology was validated and evaluated for the analysis of 15 spiked human serum specimens, receiving in a good accordance with the results obtained by the non-enzymatic glucose sensing and the commercialized personal glucose meter.

Redox and catalysis ‘all-in-one’ infinite coordination polymer for electrochemical immunosensor of tumor markers

Prostate-specific antigen (PSA), as a glycoprotein enzyme encoded in humans by the KLK3 gene, is one of the most important biomarkers for the diagnosis and prognosis of prostate cancer. Herein, a new electrochemical immunosensor for sensitive determination of PSA was designed by using redox and catalysis ‘all-in-one’ infinite coordination polymer (PtNP@ICP) as signal tag on the polyamidoamine dendrimers modified electrode interface. To construct such ‘all-in-one’ PtNP@ICP nanostructures, the coordination polymerization was fully carried between metal ions and polydentate bridging ligands, and the PtNP was encapsulated into the ICP in the process of polymerization. The prepared PtNP@ICP nanocatalyst was characterized by transmission electron microscope (TEM), energy dispersive X-ray spectrometry (EDX), ultraviolet and visible (UV–vis) spectrophotometry and Fourier transform infrared spectroscope (FTIR). And the synthesized PtNP@ICP was utilized as signal tag for the label of PSA. With a sandwich-type immunoassay format, the conjugated signal tag on the transducer increased with the increasing PSA concentration in the sample thus enhancing the signal of the electrochemical immunosensor due to the catalytic reduction toward H2O2 of the enveloped PtNP. Under optimal conditions, the current was proportional to the logarithm of PSA concentration ranging from 0.001 to 60ng/mL. The detection limit (LOD) was 0.3pg/mL at 3sB. The immunosensor displayed an acceptable reproducibility, stability and selectivity. In addition, the methodology was evaluated with human serum specimens receiving good correlation with results from commercialized enzyme-linked immunosorbent assay (ELISA) method.

Metabolomics Study of Hepatocellular Carcinoma: Discovery and Validation of Serum Potential Biomarkers by Using Capillary Electrophoresis–Mass Spectrometry

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies. The lack of effective screening methods for early diagnosis has been a longstanding bottleneck to improve the survival rate. In the present study, a capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS)-based metabolomics method was employed to discover novel biomarkers for HCC. A total of 183 human serum specimens (77 sera in discovery set and 106 sera in external validation set) were enrolled in this study, and a "serum biomarker model" including tryptophan, glutamine, and 2-hydroxybutyric acid was finally established based on the comprehensive screening and validation workflow. This model was evaluated as an effective tool in that area under the receiver operating characteristic curve reached 0.969 in the discovery set and 0.99 in the validation set for diagnosing HCC from non-HCC (health and cirrhosis). Furthermore, this model enabled the discrimination of small HCC from precancer cirrhosis with an AUC of 0.976, highlighting the potential of early diagnosis. The biomarker model is effective for those a-fetoprotein (AFP) false-negative and false-postive subjects, indicating the complementary function to conventional tumor marker AFP. This study demonstrates the promising potential of CE-MS-based metabolomics approach in finding biomarkers for disease diagnosis and providing special insights into tumor metabolism.

Target-induced aptamer release strategy based on electrochemical detection of staphylococcal enterotoxin B using GNPs-ZrO2-Chits film

A novel electrochemical aptasensor was developed for ultrasensitive detection of staphylococcal enterotoxin B (SEB) by combining signal amplification and target-induced aptamer release strategy. A gold electrode was modified with a nanocomposite made of gold nanoparticles reduced in situ, zirconia nanoparticles, and chitosan. The SEB aptamer was hybridized by anchoring the capture probe on the modified gold electrode surface through AuS binding. In the presence of SEB, the capture probe-aptamer duplex was compelled to open, releasing the aptamer from the electrode. The resulting single-strand capture probe was hybridized with a biotinylated detection probe and labeled with streptavidin-horseradish peroxidase, producing an ultrasensitive enzyme-catalyzed electrochemical signal. Under optimal conditions, the amperometric responses were proportional to the SEB concentrations ranging from 2 to 512ngmL−1, with a detection limit of as low as 0.24ngmL−1 (S/N=3). The aptasensor exhibited good stability, outstanding reproducibility, and high selectivity. The as-prepared aptasensor was used to analyze SEB in human serum specimens, and validated through enzyme-linked immunosorbent assay method. Analytical results suggest that the developed assay is a promising alternative approach for detecting SEB in clinical diagnosis.

A disposable dry film photoresist-based microcapillary immunosensor chip for rapid detection of Epstein–Barr virus infection

Although there has been considerable progress in the development of engineering principles for miniaturized diagnostic devices, a substantial challenge remains to construct an integrated platform combining the advantages of low-cost production, easy handling and sensitive detection technique. We here report a new approach to disposable, polymeric bioanalytical microdevice: an immunosensor fabricated in Vacrel® 8100, a photoresist film that enables wafer-level production via simple lamination and photolithography in a non-cleanroom setting. The incorporated procedures of facile reagent delivery, fluid handling and amperometric detection using microfluidics allowed replicating the workflow of benchtop ELISA technique. The presence and accessibility of surface carboxyl groups enabled fast and versatile immobilization of biomolecules via amide crosslinking. We investigated the covalent bonding yield utilizing glucose oxidase and IR spectroscopy. To evaluate the platform, we show specific detection of an IgG-class antibody to Epstein–Barr virus (EBV) infection from a 5μl human serum specimen with only a 2min read-out time, which demonstrated 97.5% agreement with results obtained from a reference microplate format. Our approach offers the opportunity for rapid and low-cost microfluidic assays that enables easy-to-use point-of-care biomarker detection in clinically relevant samples.

Simultaneous Electrochemical Multiplexed Immunoassay of Biomarkers Based on Multifunctionalized Graphene Nanotags

AbstractA sensitive and feasible multiplexed immunoassay protocol for simultaneous electrochemical determination of carcinoembryonic antigen (CEA) and alpha‐fetoprotein (AFP) was developed using multifunctionalized graphene nanotags on a cyclodextrin‐modified immunosensor. The sensor was fabricated via the guest–host chemistry between the immobilized cyclodextrin and anti‐CEA/anti‐AFP antibodies. Two nanotags including cyclodextrin‐thionine‐graphene and cyclodextrin‐ferrocene‐graphene nanostructures were synthesized based on a wet chemistry method. The synthesized nanotags were employed for the labelling of horseradish peroxidase (HRP)–anti‐AFP and HRP–anti‐CEA conjugates, respectively. With a sandwich‐type assay format, the signal was monitored through the labelling of HRP toward catalytic reduction of H2O2 at various peak potentials in the presence of thionine and ferrocene, respectively. Under optimal conditions, the multiplexed immunoassay enabled the simultaneous determination of CEA and AFP with wide working ranges of 0.001–60 ng mL−1 for AFP and 0.003–40 ng mL−1 for CEA. The detection limits were 0.5 pg ml−1 for AFP and 0.8 pg ml−1 for CEA (at 3 sB). No obvious nonspecific adsorption and cross‐talk were observed during a series of analyses to detect target analytes. Intra‐ and inter‐assay coefficients of variation (CV) were below 9.5 %. Importantly, the methodology was also evaluated for the analysis of AFP and CEA in the clinical human serum specimens, receiving a good relationship between the developed immunoassay and a commercialized electrochemiluminescence enzyme‐linked immunoassay.

Preliminary study of MALDI-TOF mass spectrometry-based screening of patients with the NSCLC serum-specific peptides

背景与目的早期诊断是提高肺癌生存率的关键，传统的肺癌诊断技术仍存在一定局限性。鉴于近年来以质谱为核心技术的肿瘤蛋白组学在癌症诊断方面的初步研究，本研究探索性应用基质辅助激光解析电离飞行时间质谱（matrix assisted laser desorption ionization-time of flight-mass spectrometry, MALDI-TOF-MS）分析非小细胞肺癌（non-small cell lung cancer, NSCLC）患者和健康人群的血清差异多肽，以建立NSCLC的血清分类模型。方法将年龄和性别匹配的133例NSCLC患者和132例健康者血清标本按照3:1的比例随机分为两组：训练组由100例NSCLC患者和100例健康者血清标本组成，用以建立分类模型；测试组由33例NSCLC患者和32例健康者血清标本组成，用以验证模型。采用铜离子鳌合纳米磁珠提取血清多肽、MALDI-TOF-MS技术检测得到质谱图。ClinProToolsTM统计软件分析训练组NSCLC患者与健康者之间的多肽图谱，从中筛选出一组差异多肽并建立分类模型，最后用测试组对模型进行盲样验证。结果在训练组中观察到血清质荷比（m/z）在1, 000 Da-10, 000 Da范围内有131个差异多肽信号峰，在此范围内共得到14个有统计学意义的差异多肽峰（P < 0.000, 001; AUC≥0.9），其中NSCLC患者与健康者相比，表达上调的多肽有2个，表达下调的有12个，由统计软件筛选出3个多肽峰（7, 478.59 Da、2, 271.44 Da、4, 468.38 Da）建立分类模型，然后对测试组进行验证，其盲样验证敏感性100%，特异性96.9%，准确率98.5%。结论本组研究显示NSCLC患者与健康人群的血清多肽存在差异，应用MALDI-TOF-MS技术可建立NSCLC的血清多肽分类模型且小规模验证具有较好的敏感性和特异性，希望大规模验证模型，并与传统诊断方法对照或结合，进而尝试建立一种新的NSCLC早期诊断模式。

Sensitive electrochemical immunoassay of IgG1 based on poly(amido amine) dendrimer-encapsulated CdS quantum dots

Herein, we designed a new electrochemical immunoassay protocol for the detection of IgG1, as a model biomolecule, by using poly(amidoamine) dendrimer-encapsulated CdS quantum dots (DEQD) as molecular tags. Initially, monoclonal anti-human IgG1 specific antibody (mAb1) was anchored on a bovine serum albumin-modified glassy carbon electrode preferably selective to capture Fab of IgG1 from the sample. For the determination, monoclonal anti-human IgG1 (Fc specific) antibody (mAb2) was covalently bound onto the as-prepared DEQD based on a typical carbodiimide coupling. With a sandwich-type immunoassay format, the subsequent anodic stripping voltammetric detection of cadmium released under acidic condition from the coupled quantum dots (QD) was conducted in 2-mL pH 2.5 HCl containing 0.01 M KCl. Under optimal conditions, the dynamic concentration range of the developed immunoassay spanned from 0.1 pg mL−1 to 100 ng mL−1 IgG1 with a detection limit (LOD) of 0.06 pg mL−1 toward IgG1. The electrochemical immunoassay showed good reproducibility, selectivity and stability. The analysis of human serum specimens were in good accordance with the results obtained by a referenced enzyme-linked immunosorbent assay (ELISA) method.

Sensitive and simultaneous detection of different disease markers using multiplexed gold nanorods

A multiplexed bioanalytical assay is produced by incorporating two types of gold nanorods (GNRs). Besides retaining the desirable features of common GNRs LSPR sensors, this sensor is easy to fabricate and requires only a visible–NIR spectrometer for detection. This assay can simultaneously detect different acceptor–ligand pairs by choosing the proper GNRs possessing various LPWs in a wide detection wavelength range and can be developed into a high-throughput detection method. This bioanalytical assay allows easy detection of human serum specimens infected by S. japonicum and tuberculosis (TB) from human serum specimens (human serum/Tris–HCl buffer ratio=1:104) without the need for sample pretreatment. The technique is very sensitive compared to other standard methods such as indirect hemagglutination assays (IHA) that require a serum concentration ratio of larger than 1:20 and enzyme-linked immunosorbent assays (ELISA) requiring a ratio larger than 1:100. This methodology can be readily extended to other immunoassays to realize wider diagnostic applications.

Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8 μU/ml. Recoveries ranged between 33.2 % and 51.7 %. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301 μU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog®, Novolog®, Apidra®, Lantus®, and Levemir® in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics.

Evaluation of a method for the simultaneous detection of multiple tumor markers using a multiplex suspension bead array

ObjectiveTo achieve higher tumor detection efficiency, we evaluated a multiplex assay for TM analysis based on the Luminex-100 multiplex suspension bead array. DesignThe assay simultaneously determined the concentrations of nine TMs in 1114 human serum specimens (546 patients with tumors, 158 patients with non-tumor inflammatory diseases, and 410 normal controls). The nine TMs were AFP, CEA, CA125, CYFRA 21‐1, CA242, f-PSA, t-PSA, NSE and free β-hCG. The multiplex suspension bead assays were compared with conventional methods used in clinical laboratories. ResultsThe Luminex assay has the same levels of sensitivity, specificity and accuracy in the prediction of positive tumor specimens as conventional methods. ConclusionMultiplex suspension bead arrays have promising applications in clinical laboratories.

The new Roche Vitamin D Total assay: fit for its purpose?

Measurement of serum 25-hydroxyvitamin D [25(OH)D] is used to assess vitamin D status. We evaluated the analytical performance of a new automated assay, Elecsys Vitamin D Total (Roche Diagnostics, Mannheim, Germany), based on competitive protein binding. The Elecsys assay was tested for imprecision, linearity and functional sensitivity at three test-sites and compared to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, a high-performance liquid chromatography (HPLC) method and the Liaison 25(OH) Vitamin D Total immunoassay (Diasorin). Imprecision testing with human serum specimens showed within-run CVs of ≤6% and between-run CVs of ≤8%. The assay was linear from 33 up to at least 111 nmol/L and showed equivalent 25(OH)D levels for matched serum and heparinized plasma samples. The assay correlated reasonable to well with LC-MS/MS (r=0.93; y=1.07x-5.04 nmol/L), HPLC (r=0.91, y=0.90x+3.03 nmol/L) and the Liaison assay (r=0.86, y=1.19x+2.80 nmol/L). Some of the samples showed large between-method differences. The new Elecsys assay fulfilled present analytical performance requirements and showed close agreement to other well-established methods for 25(OH)D analysis, making it fit for routine assessment of vitamin D status.