Simultaneous determination of alpha-fetoprotein and free beta-human chorionic gonadotropin by element-tagged immunoassay with detection by inductively coupled plasma mass spectrometry

BACKGROUND: An inductively coupled plasma mass spectrometry (ICP-MS)-based immunoassay has been proposed independently by Baranov et al. (Anal Chem 2002;74:1629-36) and our group, but the applicability of this method for multianalyte analysis in clinical samples has not been fully illustrated. We developed a dual-label immunoassay method for the simultaneous determination of alpha-fetoprotein (AFP) and free beta-human chorionic gonadotropin (hCGbeta) in human serum. METHODS: Monoclonal antibodies immobilized on microtiter plates captured AFP and hCGbeta, which were detected by use of Eu(3+)-labeled anti-AFP and Sm(3+)-labeled anti-hCGbeta monoclonal antibodies. Eu(3+) and Sm(3+) were dissociated from the immunocomplex with HNO(3) solution (10 mL/L) and delivered by peristaltic pump to the ICP mass spectrometer. RESULTS: The measurable ranges of AFP and hCGbeta were 4.6-500 and 5.0-170 microg/L, respectively, with detection limits of 1.2 and 1.7 microg/L (3 SD above mean of zero calibrator), respectively. The intraassay imprecision (CV) for AFP was 8.3%, 4.0%, and 2.7% at 16.3, 86, and 354 microg/L, respectively, and the interassay CV was 10%, 5.7%, and 3.5%. For hCGbeta, the intraassay CV was 5.4%, 6.4%, and 3.1%, respectively, at 10.5, 45.2, and 105 microg/L, and the interassay CV was 7.2%, 8.0%, and 3.7%. Comparison with IRMAs for AFP and hCGbeta yielded correlation coefficients (r(2)) of 0.97 and 0.95. CONCLUSIONS: Two proteins can be measured simultaneously by immunoassays using two rare earth elemental tags (Eu(3+) and Sm(3+)) and ICP-MS detection. The multielement capability and the multiple potential elemental labels make ICP-MS attractive for multianalyte immunoassays. Implementation of ICP-MS-linked immunoassays may be relatively straightforward because the labeling and immunoreaction procedures have been well developed for clinical time-resolved immunofluorometric assays.     

Magnetic control of an electrochemical microfluidic device with an arrayed immunosensor for simultaneous multiple immunoassays

BACKGROUND: Methods based on magnetic bead probes have been developed for immunoassay, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. METHODS: We synthesized magnet core/shell NiFe(2)O(4)/SiO(2) nanoparticles and fabricated an electrochemical magnetic controlled microfluidic device for the detection of 4 tumor markers. The immunoassay system consisted of 5 working electrodes and an Ag/AgCl reference electrode integrated on a glass substrate. Each working electrode contained a different antibody immobilized on the NiFe(2)O(4)/SiO(2) nanoparticle surface and was capable of measuring a specific tumor marker using noncompetitive electrochemical immunoassay. RESULTS: Under optimal conditions, the multiplex immunoassay enabled the simultaneous detection of 4 tumor markers. The sensor detection limit was <0.5 microg/L (or <0.5 kunits/L) for most analytes. Intra- and interassay imprecisions (CVs) were <4.5% and 8.7% for analyte concentrations >5 mug/L (or >5 kunits/L), respectively. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross-talk (CV) between neighboring sites was <10%. CONCLUSION: This immunoassay system offers promise for label-free, rapid, simple, cost-effective analysis of biological samples. Importantly, the chip-based immunosensor could be suitable for use in the mass production of miniaturized lab-on-a-chip devices and open new opportunities for protein diagnostics and biosecurity.     

Simultaneous multianalyte ELISA performed on a microarray platform

BACKGROUND: A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Here we describe the construction and use of such a multiplex ELISA to measure prostate-specific antigen (PSA), alpha1-antichymotrypsin-bound PSA (PSA-ACT), and interleukin-6 (IL-6). METHODS: We silanized glass plates and printed the appropriate capture antibodies to allow for the construction of "sandwich" ELISA quantification assays. We examined specificity of the assay for appropriate antigen, assembled calibration curves, and obtained PSA concentrations for 14 human serum samples. We compared the serum PSA concentrations derived through the use of our array with values obtained independently using a standard ELISA method. RESULTS: R2 values generated by our microarray for the PSA and PSA-ACT calibration curves were 0.989 and 0.979, respectively. Analyte concentrations used for the construction of these curves were 0.31-20 microg of protein/L of diluent. IL-6 calibration curve concentrations were 4.9-300 ng of IL-6/L of diluent. The R2 value for the IL-6 calibration curve was 0.983. The 14 human serum samples screened by this micro-ELISA technique for PSA concentrations generated a regression equation (linear) with a slope of 0.83 +/- 0.10 and intercept of 0.74 +/- 0.70 (R2 = 0.88). CONCLUSIONS: Multiplexed ELISA arrays are a feasible option for analyte quantification in complex biologic samples.     

Multianalyte immunoassay based on spatially distinct fluorescent areas quantified by laser-excited solid-phase time-resolved fluorometry.

We describe a new multianalyte immunoassay principle and apply it to the simultaneous immunoassay of lutropin, follitropin, choriogonadotropin, and prolactin in serum. The method is based on the coating of distinct areas of polystyrene with analyte-specific antibodies. These antibodies react with the analyte and immobilize it in a specific area while another biotinylated antibody also reacts with the analyte to form a sandwich. After addition of streptavidin labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid, fluorescent areas are formed, the intensity of which is related to the amount of each analyte present in the sample. The fluorescent areas are quantified on the dry solid phase with laser-excited time-resolved fluorometric measurements. The assays developed are highly sensitive, precise, and accurate. We believe that this system shows potential for multianalyte immunoassay of diverse groups of compounds in disciplines such as endocrinology, infectious disease, hematology, and oncology.     

Enhancing a clenbuterol immunosensor based on poly(3,4-ethylenedioxythiophene)/multi-walled carbon nanotube performance using response surface methodology

Clenbuterol (CLB) is an illegal antibiotic for livestock, which is misused as a growth promoter drug. In this study, an immunosensor modified with poly(3,4-ethylenedioxythiophene) (PEDOT), multi-walled carbon nanotubes (MWCNT) and anti-clenbuterol antibody (Ab) was developed for the detection of CLB. A screen-printed carbon electrode (SPCE) was modified with PEDOT/MWCNT as a sensor platform before immobilizing Ab for specific CLB binding through a competitive-type immunoassay. Free CLB in the sample solution competed with clenbuterol-horseradish peroxide (CLB–HRP) to bind with Ab. A high current signal was obtained after optimization of the electrochemical immunoassay conditions (pH, incubation temperature, antigen (Ag) incubation time and % blocking) using the response surface methodology/central composite design (RSM/CCD). The developed immunosensor is highly reproducible and sensitive with good storage stability, which are necessary for practical application. In real sample application, this immunosensor produces comparable results with liquid chromatography-mass spectrometry; thus, it is useful for CLB screening and monitoring in real meat samples.

A clenbuterol immunosensor was developed with a poly(3,4-ethylenedioxythiophene)/multi-walled carbon nanotube-modified screen-printed carbon electrode and optimized using response surface methodology.     

Adoption of array technologies into the clinical laboratory

Array-based methods are making substantial contributions to the discovery of disease biomarkers and are fueling the growth of multianalyte testing for disease diagnosis and treatment. The distillation of high-density array results into sets of signature markers promises to improve disease staging, risk stratification and treatment decisions. To accommodate the growing requirement for multiplex testing, clinical laboratories are converting several single-analyte tests into array-based formats. However, adoption of array technologies provides several challenges to the laboratory, which must evaluate these new formats, train laboratory personnel, market the new services and obtain reimbursement for new analytes. Liquid-bead arrays are an attractive format for routine clinical diagnostics due to a combination of appropriate analyte density, simultaneous array decoding and detection, and flexibility for rapid customization. In this review, the suitability of several array platforms to diagnostic testing and applications of liquid-bead arrays for cystic fibrosis testing, multidisease carrier status assays and leukemia subtyping are discussed. As our understanding of the clinical utility of new or established biomarkers and recommendations for testing change, flexibility and adaptability of array platforms will be imperative. Future development of novel assay formats and improved quantitation will expand the number of diseases tested and lead to further integration into the diagnostic laboratory.     

Prostate-specific antigen detection by using a reusable amperometric immunosensor based on reversible binding and leasing of HRP-anti-PSA from phenylboronic acid modified electrode

BACKGROUND: In recent years, many automated immunoassay analyzers have been developed for accurate diagnosis of various disease states and to improve effective drug administration. Amperometric immunoassay has been increasingly applied to laboratory medicine due to its ease in automation, rapid speed and low detection limits. It is important to develop reusable immunologically-sensitive elements for prostate-specific antigen (PSA) detection. METHODS: The strategy for the immunosensor construction is based on the enzyme-conjugated prostate-specific antibody (HRP-anti-PSA) reversible binding with a self-assembled phenylboronic acid monolayer on gold. RESULTS: After incubating an HRP-anti-PSA modified electrode in a PSA solution, a decrease in the electrocatalytic response of the HRP-anti-PSA modified electrode to the reduction of H(2)O(2) is observed. The photometric activity assays show that this decrease of the electrocatalytic response arises from the formation of immunocomplexes of HRP-conjugated anti-PSA and its antigen, not from the loss of bound HRP-anti-PSA from the electrode surface. Analytical performances and optimal conditions of the described immunosensor are also investigated. Under the optimal conditions, the amperometric immunosensor shows a linear increase of the relative intensity in 2 PSA concentration range from 2 to 15 ng/ml and 15 to 120 ng/ml, respectively. CONCLUSION: This method could be used for rapid analysis of PSA and potentially other antigens.     

Evaluation of a particle-enhanced turbidimetric immunoassay for the measurement of immunoglobulin E in an ILab 900 analyzer.

BACKGROUND: The measurement of immunoglobulin E (IgE) in serum is widely used in the diagnosis of allergic reactions and parasitic infections. We describe here a fully automated assay for human IgE suitable for routine application in a general chemistry analyzer. METHODS: We used an ILab 900 analyzer. This instrument automates a particle-enhanced immunoturbidimetric assay with an analysis time of 9 min. RESULTS: The assay was linear in the range 4-1000 kIU/L (r = 0.9998). The intra- and interassay CVs at 57, 235, and 434 kIU/L were <3.5% and <7.4%, respectively. The detection limit was 4 kIU/L. Hemoglobin (相似文献   

Establishment of reference distributions and decision values for thyroid antibodies against thyroid peroxidase (TPOAb), thyroglobulin (TgAb) and the thyrotropin receptor (TRAb).

BACKGROUND: The National Academy of Clinical Biochemistry (NACB) stresses that the reference intervals for thyroid peroxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb) and thyroid stimulating hormone (TSH)-receptor antibodies (TRAb) should be based on young men who lack certain risk factors and have serum TSH between 0.5 and 2.0 mIU/L. However, some young men without any of the risk factors have autoantibodies, and cannot be identified by the present tools. A model for reference intervals and cut-off values should not be influenced by the prevalence of risk factors. METHODS: We developed a model of "composite logarithmic Gaussian distributions" and tested it in 1441 well-characterised subjects without clinically overt thyroid disease. RESULTS: TPOAb and TgAb could be measured in all individuals. The 97.5% upper limits 1) on a traditional non-parametric scale, 2) according to the NACB criteria, and 3) for our model were 284, 24 and 9.8 kIU/L for TPOAb, and 84, 22 and 19 kIU/L for TgAb, respectively. The decision value (defined as the concentration corresponding to 0.1% false positives) was 15 kIU/L for TPOAb and 31 kIU/L for TgAb. Concentrations above our reference intervals affected the corresponding distribution of TSH values. For TRAb the upper reference limits were 1) 0.75 and 2) 0.75 IU/L, while our model was not applicable to TRAb because only 2-3% of the results were above the functional assay sensitivity. CONCLUSIONS: In contrast to the NACB guidelines, our model for TPOAb and TgAb is more robust, as it is independent of the characteristics of the reference population.     

Silicon-based biosensors for rapid detection of protein or nucleic acid targets

BACKGROUND: We developed a silicon-based biosensor that generates visual, qualitative results or quantitative results for the detection of protein or nucleic acid targets in a multiplex format. METHODS: Capture probes were immobilized either passively or covalently on the optically coated surface of the biosensor. Intermolecular interactions of the immobilized capture probe with specific target molecules were transduced into a molecular thin film. Thin films were generated by enzyme-catalyzed deposition in the vicinity of the surface-bound target. The increased thickness on the surface changed the apparent color of the biosensor by altering the interference pattern of reflected light. RESULTS: Cytokine detection was achieved in a 40-min multiplex assay. Detection limits were 4 ng/L for interleukin (IL)-6, 31 ng/L for IL1-beta, and 437 ng/L for interferon-gamma. In multianalyte experiments, cytokines were specifically detected with signal-to-noise ratios ranging from 15 to 80. With a modified optical surface, specificity was also demonstrated in a nucleic acid array with unambiguous discrimination of single-base changes in a 15-min assay. For homozygous wild-type and homozygous mutant samples, signal-to-noise ratios of approximately 100 were observed. Heterozygous samples yielded approximately equivalent signals for wild-type and mutant capture probes. CONCLUSIONS: The thin-film biosensor allows rapid, sensitive, and specific detection of protein or nucleic acid targets in an array format with results read visually or quantified with a charge-coupled device camera. This biosensor is suited for multianalyte detection in clinical diagnostic assays.     

Screening autoantibody profiles in systemic rheumatic disease with a diagnostic protein microarray that uses a filtration-assisted nanodot array luminometric immunoassay (NALIA)

BACKGROUND: We developed a cost-efficient modular system for multiplex analysis of the multiple autoantibodies that characterize systemic rheumatoid diseases. METHODS: The nanodot array luminometric immunoassay (NALIA) system consists of conventional 96-well membrane-bottomed plates in which antigens or antibodies are adsorbed onto the underside of the membrane. Current arrays use a 5 x 5 format (25 dots/well), which allows 10 analytes to be measured in duplicate: double-stranded DNA (dsDNA), centromere protein B (CENP-B), PCNA, Sm, Sm ribonucleoprotein (Sm-RNP), U1-snRNP, Scl70, SSA/Ro, SSB/La, Jo-1, and controls. The test fluid, control sera, and subsequent reagents are drawn through the membrane. The captured analytes are quantified by monitoring chemiluminescence with a charge-coupled device (CCD) and analyzed with commercial array software. RESULTS: The assay can detect <20 x 10(3) IU/L of anti-dsDNA. The interwell CV was 10%-14%. There was an 83% concordance (kappa = 0.56) between the NALIA results obtained for anti-dsDNA assayed by beta-testing in a routine immunology diagnostic laboratory and the results obtained with a conventional ELISA reagent set. The concordance values for Ro, La, Sm, and RNP were 98% (kappa, 0.92), 93% (kappa, 0.41), 97% (kappa, 0.62), and 97% (kappa, 0.73), respectively. CONCLUSION: The NALIA approach promises to provide a highly economical platform for a wide range of applications that require assays of multiple analytes. The degree of concordance of our results with a conventional reagent set was no less than that occurring between different commercial products. A sample of serum from a finger stick provides a volume sufficient to perform the array assay.     

乙型肝炎病毒血清标志物GICA检测性能的比对分析

目的评估乙型肝炎病毒血清标志物（HBV—M）胶体金免疫层析法（GICA）的分析性能。方法收集雅培i1000免疫检测系统检测的HBV—M阳性标本653例、阴性标本103例和HBV—M标准品,以化学发光免疫测定法（CLIA）检测结果为标准,分别用酶联免疫吸附试验（ELISA）试剂和5种GICA试剂检测,统计分析检测结果。结果检测HBV—M敏感性：ELISA和GICA检测HBV—M的最低检测限分别为乙型肝炎表面抗原（HBsAg）0．5IU／mL和4～8IU／mL、乙型肝炎表面抗体（抗HBs）10mlU／mL和15～30mlU／mL、乙型肝炎e抗原（HBeAg）1NCU／mL和2～4NCU／mL、乙型肝炎e抗体（抗HBe）2NCU／mL和64～256NCU／mL、乙型肝炎核心抗体（抗HBc）2IU／mL．和32—256IU／mL。检测标本HBV—M的符合率：ELISA分别为87．4％、99．0％、87．7％、96．6％、100．0％,GICA分别为69．6％～71．7％、69．0％～72．0％、70．5％～73．9％、42．2％～49．1％、50．0％～60．0％。检测HBV—M特异性：除GICA柃测抗HBs的特异性略差外,其余各HBV—M的检测特异性均是可接受。,结论GICA检测HBV—M低浓度的标本,漏检率很高,只能用于GICA检测线性范围里HBsAg的筛查,不能作为临床诊断乙型肝炎和疗效考核的依据。     

Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers.

We developed a chemiluminescent enzyme immunoassay (CLEIA) to quantify such tumor markers as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA19-9, and CA125. We used a novel chemiluminescent substrate, a derivative of 1,2-dioxetane phosphate (AMPPD), to measure alkaline phosphatase as a labeling enzyme to Fab' fragments of antibody. Regardless of the solid phase, i.e., polystyrene beads (6 mm diameter) or ferrite-coated particles (0.3 microns diameter), the standard curves within the dynamic ranges of the conventional RIA or enzyme immunoassay (EIA) were linear in all cases except for those with AFP. Use of the ferrite particles further shortens the immunoreaction, so the assay can be performed in 30 min. In addition, the relationships between concentrations of the marker and chemiluminescent signals for CA19-9, CA125, and CEA were linear up to concentrations about 10-fold greater than the ordinary dynamic ranges. Intra- and interassay CVs (averages for individual analyte) were 2.2%-4.9% and 2.0%-5.8%, respectively. In an analysis of serum samples, results of the CLEIA correlated reasonably well with those of RIA or EIA. The lower limit of detection by CLEIA with ferrite particles was 390 arb. units/L for CA19-9, 990 arb. units/L for CA125, 0.06 micrograms/L for CEA, and 0.03 micrograms/L for AFP. Thus, the sensitivity increased to between two- and 10-fold that of RIA or colorimetric EIA, depending on the analytes.     

Analysis of nicotine, 3-hydroxycotinine, cotinine, and caffeine in urine of passive smokers by HPLC-tandem mass spectrometry

BACKGROUND: A method is described for the simultaneous analysis of nicotine and two of its major metabolites, cotinine and 3-hydroxycotinine, as well as for caffeine from urine samples. The method was developed to assess exposure of restaurant and hotel workers to environmental tobacco smoke. METHODS: The method includes sample pretreatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyzer. Sample pretreatment followed standard protocols, including addition of base before liquid-liquid partitioning against dichloromethane on a solid matrix, evaporation of the organic solvent using gaseous nitrogen, and transferring to HPLC vials using HPLC buffer. HPLC separation was run on-line with the electrospray ionization-tandem mass spectrometric detection. RESULTS: The detection limits of the procedure were in the 1 microg/L range, except for nicotine (10 microg/L of urine). Still lower detection limits can be achieved with larger sample volumes. Recoveries of the sample treatment varied from 99% (cotinine) to 78% (3-hydroxycotinine). CONCLUSIONS: The method described is straightforward and not labor-intensive and, therefore, permits a high throughput of samples with excellent prospects for automation. The applicability of the method was demonstrated in a small-scale study on restaurant employees.     

Quantitative, wide-range, 5-minute point-of-care immunoassay for total human chorionic gonadotropin in whole blood

BACKGROUND: Human chorionic gonadotropin (hCG) is among the most common analytes available for point-of-care immunotesting, with most assays currently based on simple manual assay devices. However, as the importance of good analytical performance of rapid assays is increasingly emphasized, more sophisticated immunoassay techniques are needed to meet the future challenges of rapid yet quantitative POC testing. METHODS: We developed a simple, dry-reagent, all-in-one immunoassay for the quantitative measurement of hCG in whole blood, plasma, or serum. The noncompetitive assay equally measures intact, nicked, and hyperglycosylated hCG as well as nonnicked and nicked hCG beta-subunit with a rapid and simple procedure consisting of a 5-min, one-step incubation and, subsequent to washing, the measurement of time-resolved fluorescence directly from a wet well surface. RESULTS: The assay had a detection limit (background + 3 SD) of 0.4 IU/L hCG. The within-run CV was <15% down to 2 IU/L, and the assay was linear to 6000 IU/L. The within- and between-run CVs in heparinized whole blood and plasma were 相似文献   

Drug analysis at the 1988 Olympic Winter Games in Calgary.

A comprehensive drug testing program was carried out during the 16 days of the 1988 Olympic Winter Games in Calgary, Canada. State-of-the-art technology was applied, involving high-resolution gas chromatography, high-performance liquid chromatography, gas chromatography/mass spectrometry, fluorescence polarization immunoassay, and radioimmunoassay. Samples from selected athletes were screened for five drug classes: stimulants, narcotic analgesics, anabolic steroids, beta-blockers, and diuretics. In addition, samples were also screened for local anesthetics, corticosteroids, beta-human choriogonadotropin, and cannabinoids. During the 16-day event, 428 urine samples were processed and 3090 screening procedures performed. We describe the methods for analysis at the Olympic Games and present the results.     

化学发光免疫定量检测狂犬病毒抗体方法的建立

目的建立化学发光免疫法检测人血清中狂犬病毒抗体。方法以纯化抗原包被固相,配以碱性磷酸酶标记抗原及金刚烷胺作为发光底物,采用双抗原夹心法检测血清中抗体。结果该方法的敏感性为0.2 IU/m l,线性范围为0~200 IU/m l,变异系数<10%,回收率在90%~110%之间。结论化学发光法定量测定人血清中狂犬病毒抗体是一种灵敏、准确、重复性好,操作简单的方法,适用于临床检测。     

两种HBV DNA检测试剂的比较

目的 分析两种试剂在HBV DNA定量检测中的相关性,评价其在不同病毒载量时的检测性能.方法 用人AB型血清将WHO第2代HBV DNA国际标准品(编号:97/750)配制成不同浓度的10份样本,10份样本的浓度分别为1×106、5×106、1×105、5×104、1×104、5×103、1×103、5×102、1×102、1×101 kIU/L.采用深圳匹基生物工程股份有限公司生产的HBV DNA荧光定量PCR检测试剂(PG试剂)和美国罗氏公司生产的定量PCR试剂(罗氏试剂)检测78份HBV感染者血清、30份健康献血者血清和10份不同浓度范围的WHO标准品中HBV DNA含量.对两种试剂检测HBV DNA结果相关性进行分析;对试剂在不同病毒载量时的检测性能进行评价;并对漏检情况进行分析.两种试剂每批检测均设有阴性质控、弱阳性质控和强阳性质控.结果 两种试剂对WHO HBV DNA标准物质稀释血清均能正确检出,罗氏试剂能检出最高稀释度样本浓度为2.00(kIU/L,lg),PG试剂能检出最高稀释度样本浓度为3.00(kIU/L,lg);两种试剂检测结果存在线性相关(R2=0.938 7,P<0.01),罗氏试剂检测上限与理论值相符,大于检测上限的标本经稀释重复测定,罗氏试剂检测结果[(8.35±0.20)kIU/L,lg]高于PG试剂检测结果[(7.73±0.42)klU/L,lg],差异有统计学意义(t=3.776,P<0.05).两种试剂检测108份血清标本中HBV DNA含量,罗氏试剂检测值[(5.88±1.64)kIU/L,lg]高于PG试剂检测值[(5.25±1.55)kIU/L,lg],差异有统计学意义(t=12.297,P<0.01);检测高HBV病毒载量[(>5.00～≤7.00)kIU/L,lg和(>7.00～≤9.00)kIU/L,lg]组,两种试剂的相关性较高(R2分别为0.779 7、0.603 7,P均<0.01);对低HBV病毒载量(>3.00～≤5.00 kIU/L,lg)组,两种试剂的相关性较低(R2=0.417 3,P<0.01);病毒载量为>3.00～≤4.00 kIU/L,lg组,PG试剂的漏检率为33.3%(5/15);病毒载量为>1.08～≤3.00 kIU/L,lg组,PG试剂未检出.结论 PG试剂与岁氏试剂检测结果虽存在一定差距,但相关性较好.两种试剂低病毒载量标本的相关性要低于高病毒载量标本,PG试剂检测HBV DNA的线性范围较窄.

Abstract：

Objective To evaluate clinical significance of two real-time fluorescence quantitative PCR kits for quantitative detection of HBV DNA and detection performance at different viral load levels.Methods A series of calibrators with different concentrations(1×106,5×105,1×105,5×104,1×104,5×103,1×103,5×102,1×102,1×101 kIU/L) were prepared with AB-type sera using the second generation WHO international standard (NIBSC code:97/750). HBV viral load in the sera of 78 patients,30 healthy blood donors and 10 calibrators were detected by real-time fluorescence quantitative PCR HBV DNA test kit from PIJI Bio-Technical Development Company Ltd (PG kit) and Cobas AmpliPrep/Cobas TaqMan HBV test kit. The correlation of the two methods was evaluated, and the performance of the two kits different viral load levels was evaluated. The false negative rate was analyzed. Negative control, low positive control and high positive control were included in every batch. Results Both two kits showed the correct results for the 10 specimens from the WHO international standards. The lowest detection limit of HBV DNA for Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit were 2.00 (kIU/L, lg) and 3.00 (kIU/L,lg) ,respectively. There was linear correlation between the results from Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit ( R2=0.938 7, P < 0.01 ), the upper limit of Roche kit had coincided with theoretical value. The samples with HBV DNA level above the upper limit of detection were diluted and retested to obtain the precise result. The result form Roche Cobas AmpliPrep/Cabas TaqMan HBV test [(8.35±0.20) kIU/L, lg] was higher than that from PG kit [(7.73±0.42 ) kIU/L, lg] (t=3. 776, P <0.05) . The detection of 108 serum samples showed that the level of HBV DNA detected by Roche Cobas AmpliPrep/Cobas TaqMan HBV test [(5.88±1.64) kIU/L, lg] was higher than that by PG kit [(5.25±1.55 kIU/L,lg] (t=12. 297 ,P <0.01 ). The correlation coefficients were high in samples with high HBV viral load[HBV DNA(>5.00 and≤7.00) kIU/L,Ig,R2=0. 779 7, P <0.01 ;HBV DNA( >7.00 ands≤9.00) kIU/L,lg,R2=0.603 7, P <0.01]. The correlation coefficient was low in samples with low HBV viral load[HBV DNA ( > 3.00 and≤5.00) kIU/L, lg, R2=0. 417 3, P <0.01 )]. When HBV DNA ( >3.00 and≤4.00) kIU/L,lg,the false negative rate was 33.3% (5/15). When HBV DNA ( > 1.08and≤3.00) kIU/L,lg,none of positive samples was detected with PG kit. Conclusions PG kit is not as good as Cobas AmpliPrep/Cobas TaqMan HBV test . The linear correlation between the results from the two kits is good. The correlation between the results detected with PG kit and Cobas AmpliPrep/Cobas TaqMan HBV test is higher in the high viral load groups than in the low viral load group. It is suggested that PG kit had a narrower linear range.     

Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in plasma and urine by use of a chiral cellulose-derivative column

This assay allows simultaneous determination of the enantiomers of both disopyramide and its active metabolite, mono-N-dealkyldisopyramide, in 1 mL of plasma or 0.1 mL of urine within approximately 35 min by HPLC with a chiral cellulose-derivative column and ultraviolet detection. Recoveries for the analytes and the internal standard (racemic verapamil) with an extraction from alkalinized plasma or urine into diethyl ether were greater than 90%. Intra- and interassay CVs for disopyramide enantiomers were less than 5.5% at 2.5 mg/L in plasma and less than 6.5% at 25 mg/L in urine; for mono-N-dealkyldisopyramide enantiomers they were less than 6.3% and less than 8.9%, respectively. Intra- and interassay relative errors for determining these analytes in plasma and urine at 2.5 and 25 mg/L, respectively, ranged from -5.9% to +2.5%. The calibration curves for the respective analytes were linear (r = 0.995 or greater, P less than 0.01) from 0.025 to 5.0 mg/L in plasma and from 0.5 to 10 mg/L in urine. The lower detection limits (signal-to-noise ratio of 3) for S(+)-disopyramide and the other analytes were 0.010 and 0.025 mg/L, respectively. We evaluated clinical applicability of this method by determining steady-state plasma concentrations and urinary excretions of the respective analytes in a pediatric patient being treated with racemic disopyramide.     

Enzyme immunoassay of the glycoprotein tropic hormones--choriogonadotropin, lutropin, thyrotropin--with solid-phase monoclonal antibody for the alpha-subunit and enzyme-coupled monoclonal antibody specific for the beta-subunit

Monoclonal antibody technology has made it possible to produce homogeneous populations of antibodies to discrete determinants on an antigen surface. We have produced monoclonal antibodies to the alpha-subunit and beta-subunits of the glycoprotein hormones choriogonadotropin, thyrotropin, and lutropin, and developed two-site simultaneous enzyme-linked immunospecific assays for these hormones. The anti-alpha-subunit monoclonal antibody was used as the solid-phase (coated tube) capture antibody for all three hormones; the anti-beta-subunit monoclonal antibodies were coupled to horseradish peroxidase (EC 1.11.1.7). Cross reactions between the closely related choriogonadotropin and lutropin were apparently greater in this method than in RIA, with use of the same antibodies. Ka of the antibodies did not appear to be as critical to sensitivity of the sandwich assay as it was for RIA. The lower limit of detection was 0.2 microgram/L after a 2-h incubation with serum sample at room temperature and a 30-min incubation with enzyme substrate at room temperature after washing away excess enzyme conjugate. Within-assay precision (CV) was very good, less than 6%.