Capillary electrophoretic immunoassay for alpha-fetoprotein with chemiluminescence detection

A capillary electrophoretic immunoassay with chemiluminescence detection (CEIA-CL) using a non-competitive format for analyzing tumor marker alpha-fetoprotein (AFP) has been developed. In this method, antigen (Ag) AFP reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ab*-Ag complex produced in the solution are separated by CE in a separation capillary. Then they catalyze the reaction of enzyme substrate luminol and H(2)O(2) in a reaction capillary following the separation capillary. Parameters affecting the CE separation and CL detection were investigated. Under the optimal conditions, the free Ab* and the Ab*-Ag complex were well separated within 4 min, the linear range and the detection limit (S/N=3) for AFP were 5-500 ng/ml and 0.85 ng/ml (1.2 x 10(-11)M), respectively. The proposed method has been applied satisfactorily in the analysis of human sera samples.     

Non‐competitive immunoassay for luteinizing hormone in human serum using capillary electrophoresis with chemiluminescence detection

A non‐competitive immunoassay based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of luteinizing hormone (LH) in human serum. The work involved the development of separation and CL conditions, allowing for routine analysis of serum samples. In this study, horseradish peroxidase (HRP)‐labelled monoclonal anti‐LH can catalyse the luminol–hydrogen peroxide reaction. The determined LH can react with excessive amount of HRP‐labelled anti‐LH. Within 14 min, free enzyme conjugate and immune complex could be separated in alkaline borate buffer by means of a high voltage (15 kV). To improve sensitivity, a series of measures were adopted, including the choice of para‐iodophenol as a CL enhancer, unique design in detect window. Under the optimal conditions, the calibration curve for LH was established in the concentration range 1–200 mIU/mL and the detection limit was 0.08 mIU/mL. Compared with ELISA, this method decreased the detection limit by about 12 times, and it has been successfully employed in the determination of LH in human serum. Copyright © 2007 John Wiley & Sons, Ltd.     

Capillary electrophoretic enzyme immunoassay with electrochemical detection using a noncompetitive format

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) using a noncompetitive format has been developed. In this method, antigen (Ag) reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ag-Ab* complex produced in the solution are separated by capillary zone electrophoresis in a separation capillary. Then they catalyze enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB(Red)) and H(2)O(2) in a reaction capillary following the separation capillary. The reaction product, TMB(Ox), can be determined using amperometric detection on a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, a significant amount of TMB(Ox) can be produced for detection. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. A tumor marker (CA15-3) was used as a model, in order to test the method. The concentration LOD of CA15-3 is 0.024 U/ml, which corresponds to a mass detection limit of 1.3x10(-7) U.     

Expression of hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-HBs antibody

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying a hexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5 % of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100 % with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.     

Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen

An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin magnetic nanoparticles and used for targeting the HBsAg. By the addition of horseradish peroxidase conjugated with secondary antibody (HRP–HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed into 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred into an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to the HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear toward HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at a signal/noise ratio of 3.     

慢性乙型肝炎病毒感染自然史中HBsAg和HBsAb共存血清学模式分析

目的分析慢性乙肝病毒感染者HBsAg和HBsAb共存模式中血清学指标、HBV-DNA和肝酶等指标与自然病程的关系,探讨其临床意义。方法回顾性分析2016年重庆医科大学附属第一医院HBsAg和HBsAb双阳性患者的血清学指标、HBV-DNA和ALT、GGT检测结果,并对其感染的自然病程进行分析。结果 2016年该院HBsAg和HBsAb双阳性患者共520例,占全部HBV感染者的2.80%,占总送检标本数的0.42%。可分期的184例双阳性患者中,免疫耐受期47例(25.54%),免疫清除期17例(9.24%),低复制期108例(58.70%),再活动期12例(6.52%),HBsAg、HBsAg/HBsAb比值、HBV-DNA、ALT和GGT水平差异均有统计学意义(P<0.05),低复制期患者HBsAg/HBsAb比值均低于其他患者(P<0.05)。不同分期患者HBsAb、年龄和性别比较差异无统计学意义(P>0.05),且HBsAb水平均较低。284例资料完整HBsAg和HBsAb共存病例中HBV-DNA阳性136例,占47.89%。HBsAg浓度与HBV-DNA载量成正相关(r=0.295,P<0.05),HBsAb浓度与HBV-DNA载量之间没有显著相关性(r=0.04,P>0.05)。结论 HBsAg和HBsAb共存患者并不少见,与性别无关,可发生在各个年龄阶段,以低复制期患者为最多。HBsAg和HBsAb共存患者中HBsAb多以低浓度形式存在,且浓度与自然病程无关。HBsAb的出现并非代表患者体内病毒复制停止,在诊断及治疗HBsAg和HBsAb共存模式的乙肝病毒感染者时仍需结合HBV-DNA载量来判断感染状态。     

Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans.

Free radical scavenging effects of the cellular protein extracts from two strains of Deinococcus radiodurans and Escherichia coli against O2-, H2O2 and *OH were investigated by chemiluminescence (CL) methods. The cellular protein extracts of D. radiodurans R1 and KD8301 showed higher scavenging effects on O2- than that of E. coli. D. radiodurans R1 and KD8301 also strongly scavenged H2O2 with an EC50 (50% effective concentration) of 0.12 and 0.2 mg/mL, respectively, compared to that of E. coli (EC50 = 3.56 mg/mL). The two strains of D. radiodurans were effective in scavenging *OH generated by the Fenton reaction, with EC50 of 0.059 and 0.1 mg/mL, respectively, compared to that of E. coli (EC50 > 1 mg/mL). Results from the chemiluminescence assay of *OH-induced DNA damage and the plasmid pUC18 DNA double-strand break (DSB) model in vitro showed that D. radiodurans had remarkably inhibitory effect on the *OH-induced oxidative damage of DNA. The scavenging effects of D. radiodurans on reactive oxygen species (ROS) played an important role in the response to oxidation stress and preventing against DNA oxidative damage, and may be attributed to intracellular scavenging proteins, including superoxide dismutase (SOD) and catalase.     

Determination of alkaloids in Sinomenium acutum by field‐amplified sample stacking in capillary electrophoresis with chemiluminescene detection

A simple and rapid capillary electrophoresis (CE) with an acidic potassium permanganate chemiluminescence (CL) detection method was developed to determine three alkaloids (curine, sinomenine and magnoflorine) simultaneously. A laboratory‐built CE–CL detection interface was used. The field‐amplified sample stacking technique was applied to the online concentration of alkaloids. Experimental conditions for CE separation and CL detection were investigated in detail to acquire optimum conditions. Under optimal conditions, the three alkaloids were baseline separated within 6 min, and the detection limits (S/N = 3) ranged from 0.03 µg/mL to 0.49 µg/mL. This method was successfully applied to determine the above three alkaloids in Sinomenium acutum, and the result of the determination of sinomenine was in good agreement with those given by high‐performance liquid chromatography and CE methods. In addition, a possible CL reaction mechanism of sinomenine–KMnO₄–H₂SO₄ was proposed. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of ultra‐high sensitivity bioluminescent enzyme immunoassay for hepatitis B virus surface antigen using firefly luciferase

Hepatitis B virus (HBV) infection continues to be a global public health concern. Efficient diagnosis of HBV surface antigen (HBsAg) is useful for identification of infection, treatment and prevention of transfusion‐transmitted viral infections. Seronegative window reduction afforded by a highly sensitive measurement methodology is necessary as a small quantity of virus with infection risk exists for the period characterized by undetectable HBsAg following HBV infection. In this study, a bioluminescent enzyme immunoassay (BLEIA) for HBsAg was developed employing firefly luciferase as a labeling enzyme and a two‐step sandwich immunoassay method. The cut‐off value (10 mIU/mL) was 50‐fold more sensitive relative to conventional chemiluminescent enzyme immunoassay based on luminol luminescence involving peroxidase as the labeling enzyme and the identical antibodies. Preliminary clinical data for this BLEIA revealed that the HBV seroconversion panel derived sequentially from HBV‐infected human blood was detected 11 days following window closure from the first bleed, whereas detection occurred 14–25 days following window closure with the three conventional commercial kits. Copyright © 2009 John Wiley & Sons, Ltd.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

Development of a compact capillary electrophoresis–chemiluminescence system with ultra‐fast peroxyoxalate reaction to monitor the hydrolysis of rhodamine 6G

A simple and effective capillary electrophoresis–chemiluminescence (CE–CL) detection system was developed based on an ultra‐fast bis(2,4,6‐trichlorophenyl)oxalate (TCPO) chemiluminescence (CL) reaction (0.6 s duration) that avoided overlapping peaks and peak tailing. Through a series of static injection experiments, this unusually rapid CL reaction was ascribed to the catalytic effect of imidazole in the tetrahydrofuran solvent, which has been rarely utilized in such investigations. A possible mechanism is given to explain the results. Under the optimized conditions, rhodamine 6 G (R6G) and its hydrolysis product (R6G‐COOH) could be efficiently separated through electrophoresis in 7 min, with sensitive CL detection in the proposed CE–CL system. In this way, the alkaline hydrolysis of R6G was monitored, followed by estimation of relative rate constants and activation energy. This finding and application should be helpful in further study for the TCPO CL reaction, and revealed an attractive opportunity for simplifying the CE–CL system, such as in a microchip device. Copyright © 2011 John Wiley & Sons, Ltd.     

Development of a dual‐label time‐resolved fluoroimmunoassay for the detection of α‐fetoprotein and hepatitis B virus surface antigen

Time‐resolved fluorometry of lanthanide chelates is one of the most useful non‐isotopic detection techniques and has been used in numerous applications in biomedical science. We developed a time‐resolved fluoroimmunoassay (TRFIA) to quantify α‐fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg) in human serum. Based on a two‐site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co‐coated in 96 microtitration wells and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu³⁺‐ and Sm³⁺‐McAbs were added and fluorescence signals of Sm³⁺ and Eu³⁺ tracers were collected. Detection limits of AFP and HBsAg were 0.09 mIU/L and 0.01 µg/L, respectively. Measurement ranges of AFP‐TRFIA and HBsAg‐TRFIA were 1–1000 mIU/L and 0.2‐150 µg/L, respectively. Intra‐ and inter‐assay coefficients of variation of AFP‐TRFIA were 3.3‐4.1% and 5.7‐7.2% and for HBsAg‐TRFIA were 2.9‐3.9% and 4.9‐6.8%, respectively. Linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. For the endurance test, Eu‐labeled McAbs were stable for at least one year at ?20°C and the results of the TRFIA with the same reagents were also reproducible after one year. The availability of a highly sensitive, reliable and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg, thereby providing diagnostic value in various clinical conditions and could be applied for clinical use. Copyright © 2012 John Wiley & Sons, Ltd.     

Separation of bovine serum albumin and its monoclonal antibody from their immunocomplexes by sodium dodecyl sulfate–capillary gel electrophoresis and its application in capillary electrophoresis-based immunoassay

A non-competitive immunoassay was performed by sodium dodecyl sulfate–capillary gel electrophoresis with UV detection using bovine serum albumin (BSA) and monoclonal anti-BSA. BSA, anti-BSA and their immunocomplexes were well resolved under non-denaturing conditions. A linear calibration curve was obtained and can be used for the quantification of anti-BSA. The limit of detection of anti-BSA was 0.1 μM under the present conditions. Compared with capillary zone electrophoresis, we believed that this method has the potential to be used as a more general format for performing capillary electrophoresis-based immunoassay of medium- and large-sized analytes.     

Post-chemiluminescence behaviour of Ni2+, Mg2+, Cd2+ and Zn2+ in the potassium ferricyanide-luminol reaction.

A new chemiluminescence (CL) reaction was observed when Ni2+, Mg2+, Cd2+ or Zn2+ was injected into the reaction mixture after the finish of the CL reaction of alkaline luminol and potassium ferricyanide. This reaction is described as a post-chemiluminescence (PCL) reaction. The possible mechanism for the PCL was proposed based on studies of the CL kinetic characteristic and the CL spectra. The experimental conditions of the CL reactions were optimized and the feasibility of using the reaction to analyse these metal ions was evaluated. The PCL reaction method operates in the ranges: 1 x 10(-7)-8 x 10(-6) g/L Ni2+; 3 x 10(-6)-2 x 10(-4) g/L Mg2+; 8 x 10(-7)-1 x 10(-4) g/L Cd2+; and 2 x 10(-4)-2 x 10(-3) g/L Zn2+, with detection limits of 4 x 10(-8) g/mL, 1 x 10(-6) g/mL, 3 x 10(-7) g/mL, 8 x 10(-5) g/mL, respectively.     

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.     

Iron Deficiency,a Risk Factor for Thyroid Autoimmunity During Second Trimester of Pregnancy in China

ObjectivePrevious studies have reported an association between iron deficiency (ID) and increased thyroid peroxidase antibody (TPO-Ab) during early pregnancy. The objective of this study was to explore the relationship between ID and thyroid dysfunction, as well as thyroid autoantibodies, during the second trimester of pregnancy.MethodsA total of 1,592 pregnant women (13 to 28 weeks gestation) were enrolled in this cross-sectional study. According to serum ferritin (SF) concentrations, they were divided into ID (SF <20 μg/L) or non-ID (SF ≥20 μg/L) groups. Logistic regression analysis was used to evaluate the association between ID and subclinical hypothyroidism (thyroid-stimulating hormone [TSH] >4.0 mIU/L and free thyroxine [FT4] within the reference range) and thyroid autoimmunity.ResultsThe prevalence of ID was 23.43% (373/1,592). Compared with the non-ID group, the ID group had lower FT4 levels (13.94 pmol/L [8.91 to 29.82 pmol/L] versus 14.63 pmol/L [8.22 to 47.24 pmol/L]; P<.001]) and higher TSH levels (1.85 mIU/L [0.01 to 7.84 mIU/L] versus 1.69 mIU/L [0.01 to 10.2 mIU/L]; P<.05). Logistic regression analysis confirmed ID as a risk factor for increased thyroglobulin antibody (TG-Ab) (odds ratio 1.974; 95% confidence interval 1.065, 3.657; P<.05), but not for subclinical hypothyroidism or increased TPO-Ab.ConclusionID is associated with increased TG-Ab during the second trimester of pregnancy.     

鱼山镇1岁以上人群不同年龄组乙肝表面抗原携带率与乙肝疫苗接种关系的调查

研究鱼山镇乙肝疫苗接种率不同人群的乙肝病毒表面抗原(HBsAg)携带率的差异。将该镇所有户籍登记在册人员按出生日期分成3组,再按随机抽样法对每个年龄组抽取一定数量的人组成样本,调查乙肝疫苗接种史;对每位参加者采集静脉血5ml,无菌分离血清,使用固相放射免疫试剂和酶联免疫试剂检测乙肝病毒表面抗原和抗乙肝病毒表面抗原抗体(HBsAb,抗-HBs)。小年龄组、中年龄组、大年龄组乙肝疫苗接种率依次为80.00%、50.46%、25.36%,差异具有统计学意义(χ2=262.24,P<0.005);小年龄组、中年龄组、大年龄组HBsAg携带率依次为2.07%、11.93%、17.87%,差异具有统计学意义(χ2=77.48,P<0.005);小年龄组、中年龄组、大年龄组HBsAb依次为43.38%、24.77%、10.95%,差异具有统计学意义(χ2=107.28,P<0.005)。乙肝疫苗接种率在小年龄组、中年龄组、大年龄组依次降低,HBsAg携带率依次增高,HBsAb阳性率依次降低。因此,接种乙肝疫苗对人群有较好的保护作用,人群乙肝疫苗接种率越高,HBsAb阳性率越高,HBsAg携带率越低。     

白细胞介素17 在妊娠合并乙肝中的表达

目的：观察白细胞介素17（Interleukin-17,IL-17）在乙肝孕妇和正常孕妇胎盘中表达差异来反映胎盘内的细胞免疫应答的情 况,为外源性细胞因子在增强机体免疫、清除病毒及阻断和治疗HBV宫内感染提供新的科学依据。方法：选择择期剖宫产的足月 妊娠妇女46 例,其中血清HBsAg（+）30 例,血清HBsAg（-）的对照组16 例,采用免疫组化方法检测胎盘组织IL-17 的表达。结 果：IL-17 主要表达在胎盘绒毛的合体滋养层细胞胞浆、毛细血管内皮细胞胞浆以及基质中。血清HBsAb（+）孕妇与HBsAb（-）孕 妇比较,无显著差异（P＞0.05）,HBsAg（+）乙肝孕妇与对照组的表达比较差异有统计学意义（P＜0.05）,HBeAg（+）孕妇与HBeAg （-）孕妇的表达比较差异有统计学意义（P＜0.05）。结论：IL-17 在乙肝孕妇胎盘组织中的表达高于正常孕妇,IL-17 的表达不但加 强了机体的防御功能,且能促进炎性反应。     

Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels

A simple and sensitive method for in situ amplified electrochemical immunoassay of human serum IgG has been developed by using double-codified nanosilica particles as labels based on horseradish peroxidase-doped nanosilica particles (HRP-SiO(2)) with the conjugation of anti-IgG antibodies (anti-IgG-SiO(2)-HRP). With the sandwich-type immunoassay format, the linear range of the developed immunosensor by using anti-IgG-SiO(2)-HRP as tracer and hydrogen peroxide (H(2)O(2)) as enzyme substrate is 0.01-15 nmol/L IgG with a detection limit of 5.0 pmol/L, while the assay sensitivity by directly using HRP-labeled anti-IgG as secondary antibodies is 1.0-10 nmol/L with a detection limit of 0.1 nmol/L IgG. The reproducibility, stability and specificity of the proposed immunoassay method were acceptable. The IgG concentrations of the clinical serum specimens assayed by the developed immunosensor show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) method.     

一种联合检测乙型肝炎病毒前S1抗原与核心抗原方法的建立及其与病毒核酸检测结果的一致性

本研究以与血清中HBV DNA含量高度相关的两种HBV抗原(前S1抗原与核心抗原)为靶标,建立了联合检测这两种HBV核酸相关抗原(NRAg)的双抗体夹心法ELISA试剂.对系列稀释血清的检测表明,该试剂的平均分析灵敏度为103.2基因组拷贝/mL(95%可信限102.2-4.2基因组拷贝/mL),显著高于前S1抗原或核心抗原的单独检测.对994份HBsAg阴性血清的检测结果表明NRAg ELISA的特异性为99.7%(95%可信限:99.1%～99.9%).对271份临床慢性肝炎血清进行检测,结果NRAg ELISA与HBV DNA结果的总符合率达96.3%(95%可信限:93.3%～98.2%),NRAg ELISA的读值/临界值比(S/CO)与HBV基因组拷贝数呈正相关.利用NRAg试剂,发现了1例HBsAg"a"抗原表位突变的变异株.这些结果显示HBV NRAg ELISA与HBV DNA具有高度相关性,并能够检测出HBsAg抗原变异株,有望成为HBsAg变异株筛选的有力工具,并为广大基层医疗单位提供一种便捷的替代HBV DNA定性检测的手段.