A radial-partition fluoroimmunoassay for the detection of Hepatitis B surface antigen with the streptavidin/biotin complex

A sandwich technique based on the very affinity between avidin and biotin is used. Antibodies are covalently bound to glass-fibre on which the antigen is captured. The discs are then treated in sequence with monoclonal antibodies of high selectivity, species-specific anti-IgG (biotin-linked), streptavidin and β-galactosidase, (biotin-linked). The discs are then incubated with the substrate, 4-methylumbelliferyl-β-d-galactopyranoside and the fluorescence is measured in an automatic immunoassay system.     

Highly sensitive potentiometric immunosensor for hepatitis B surface antigen diagnosis

Immunosenors are of great interest because oftheir potential utility as specific, simple, label-free anddirect detection techniques and reductions in size, costand time of analysis compared with conventional im-munoassay techniques. The immunoassays with …     

Size exclusion chromatography of hepatitis B surface antigen particles

Summary To find the factors responsible for the broadening of the recombinant-hepatitis B surface-antigen peak in size-exclusion chromatography, the purified material was fractionated on preparative scale followed by multiple analysis of the separated fractions. The results from chromatographic analysis suggested the presence of large particle aggregates, probably tubular structures which, however, were not detected by electron microscopy. The antigen particles ranged from 16 to 32 nm in all the fractions, except two last fractions consisting of 16–24 nm particles. The relation ELISA/Lowry increased with increasing the fraction number, being a maximum in the fraction corresponding to the maximum of the chromatographic peak. Probably, the particles which are variable in size differ from each other with respect to the efficiency of protein assembly. Fractions collected in different regions of the peak were adsorbed on alum and injected in mice. The high antibody levels were produced without significant differences in immunogenicity between samples. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Ultra-sensitive immunosensor for detection of hepatitis B surface antigen using multi-functionalized gold nanoparticles

The signal amplification for analytical purposes has considerable potential in detecting trace levels of analytes for clinical, security or environmental applications. In the present report a strategy based on a sandwich type immunoassay system was designed for the detection of hepatitis B surface antigen which exploits the specific affinity interaction between streptavidin and biotin recognition systems. The method involves the specific coupling of multi-functionalized gold nanoparticles (bearing biotin and luminol molecules) to the streptavidin modified by secondary antibody. The chemiluminescent signal is produced by the gold nanoparticles in the presence of HAuCl₄ as catalyst and hydrogen peroxide as oxidant. The immunosensor was able to detect hepatitis B surface antigen in the linear concentration range from 1.7 to 1920 pg mL⁻¹ and the detection limit of 0.358 pg mL⁻¹, at signal/noise = 3.     

Oxygen electrode-based enzyme immunoassay for the amperometric determination of hepatitis B surface antigen

Specific antibodies labelled with glucose oxidase are immobilized onto a gelatin membrane, which is fixed over an oxygen electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen by the enzyme catalyzed reaction. The response increases linearly with increasing antigen concentration over the range 0.1–100 μg 1^?1. A microcomputer is used for data acquisition and processing.     

Preparation and characterization of biodegradable microspheres containing hepatitis B surface antigen

Poly-DL-lactide-poly(ethylene glycol) (PELA) microspheres containing Hepatitis B surface antigen (HBsAg) were elaborated by a solvent extraction method based on the formation of a double water/oil/water (w/o/w) emulsion. Microspheres were characterized in terms of morphology, size and size distribution, encapsulation efficiency, and the efficiency of microsphere formation (EMF). Transmission electron microscopy (TEM) and polyacrylamide gel electrophoresis (PAGE) were used to investigate the structural integrality of HBsAg encapsulated in PELA microspheres. The release profile was investigated by the measurement of antigen present in the release medium at various intervals. The PELA-10 microspheres displayed the highest antigen encapsulation efficiency (about 80%), and antigen molecules could be stabilized in the PELA-10 microspheres during the preparation process. It suggested that the PELA microspheres had a great potential as a new polymer adjuvant for HBsAg. The release of Hepatitis B surface antigen from poly-DL-lactide-poly(ethylene glycol) microspheres.     

基于双层纳米金修饰的高灵敏电位型乙肝表面抗原免疫传感器研究

基于电沉积和层层自组装技术，提出了一种新的生物分子固定化方法，研制成一种高灵敏电位型乙肝表面抗原免疫传感器。利用L-半胱胺酸（LCys）的双官能团结合双层纳米金，从而通过比表面积大，生物相容性好的纳米金胶吸附大量抗体，同时用聚乙烯醇缩丁醛（PVB）薄膜的笼效应把乙肝表面抗体（HBsAb）和纳米金固定在玻碳电极上，从而制得了高灵敏度、高稳定性的电位型免疫传感器。采用循环伏安法（CV）对电极的层层自组装过程进行了考察，并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是8．5～256．0ng／mL，线性相关系数为0．9978，灵敏度为89．0，检出限为3．1ng／mL。已用于病人的血清样品分析。     

A novel mimetic enzymatic fluorescence immunoassay for hepatitis B surface antigen by using a thermal phase separating polymer

Iron tetrasulfonatophthalocyanine (FeTSPc), a peroxidase mimic, was used as a labeling reagent and poly(N-isopropylacrylamide) (PNIP) as the separation support of the immune complex for the mimetic-enzymatic immunoassay of hepatitis B surface antigen (HBsAg). PNIP was precipitated from aqueous solution when the ambient temperature was higher than its lower critical solution temperature of 31 degrees C. In a sandwich immunoassay, the antigen (HBsAg) first reacted with mouse anti-human HBsAg antibody immobilized on PNIP (PNIP-antibody) and then further reacted with FeTSPc-labeled mouse anti-HBsAg antibody (antibody-FeTSPc) at room temperature in a homogeneous format. After changing the temperature to separate the PNIP-antibody-HBsAg-antibody-FeTSPc conjugate moiety, it was re-dissolved and determined by coupling with the fluorogenic reaction of hydrogen peroxide and p-hydroxyphenylpropionic acid. The sensitivity of this method (3 ng mL-1) was close to that of the traditional ELISA using the same reactants. However, the assay was much faster (the assay time decreased from 100-120 to 45 min). This method was applied to determine HBsAg in human serum with satisfactory results.     

Expression and secretion of preS containing hepatitis B surface antigen in vaccinia virus system

The expression and secretion of preS containing hepatitis B surface antigen in vaccinia virus system was investigated. The human TK- 143 cells were infected with the recombinant vaccinia viruses vTMS-1 or vTLS-1. Cells infected with vTMS-1, which contains the preS2 + S gene, produced preS2 containing middle HBsAg proteins. Similarly, cells produced preS1 containing large HBsAg proteins upon infection with vTLS-1, which carries the preS1 + preS2 + S gene. The expression products could be secreted and form 22 nm particles. They reacted specifically with anti-preS1 and/or anti-preS2 monoclonal antibodies, and exhibited pHSA-receptor (for polymerized human serum albumin) activity. In addition, the major S components of hepatitis B surface antigen were also present in the products expressed by vTMS-1 and vTLS-1.     

Amplification immunoassay for the determination of Hepatitis B surface antigen

A sensitive sandwich immunoassay for the determination of Hepatitis B surface antigen (HBs) was developed, using a cascade system of Limulus amebocyte lysate as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to anti-HBs antibody. Anti-HBs antibody was adsorbed to polystyrene beads. First, HBs were reacted to solid phase anti-HBs antibody (a-HBs). After the reaction, the beads were rinsed, and were then reacted with a-HBs-LPS. Then, LPS activity specifically bound to the beads was measured. HBs could be measured in the range of 10(-10)-10(-12) g/mL.     

Gold nanoparticle-labeled detection antibodies for use in an enhanced electrochemical immunoassay of hepatitis B surface antigen in human serum

The application of gold nanoparticle-based electrochemical immunoassays have been extensively studied for the detection of hepatitis B surface antigen (HBsAg), but most often they exhibit low sensitivity. We describe the fabrication of a new electrochemical immunoassay for signal amplification of the antigen-antibody reaction combined with the nanogold-based bio-barcode technique. Hepatitis B surface antibody (HBsAb) was initially immobilized on a nanogold/thionine/DNA-modified gold electrode, and then a sandwich-type immunoassay format was employed for the detection of HBsAg using nanogold-codified horseradish peroxidase-HBsAb conjugates as secondary antibodies. Under optimal conditions, the current response of the sandwich-type immunocomplex relative to the H₂O₂ system was proportional to HBsAg concentration in the range from 0.5 to 650 ng·mL^?1 with a detection limit of 0.1 ng·mL^?1 (S/N?=?3). The precision, reproducibility and stability of the immunosensor were acceptable. Subsequently, the immunosensors were used to assay HBsAg in human serum specimens. Analytical results were in agreement with those obtained by the standard chemiluminescence enzyme-linked immunosorbent assay.     

Enhancement of sensitivity and specificity of the fluoroimmunoassay of Hepatitis B virus surface antigen through “flexible” coupling between quantum dots and antibody

Quantum dots (QDs) are widely used in the immune detection. Yet, the sensitivity and specificity of the immune detection are not satisfactory because the binding sites of QDs onto antibody (Ab) are often arbitrary and the influence of the large surface electronic potential energy of QDs on the directly conjugated Ab is nonnegligible. In this work, we provide a “flexible” coupling method, in which protein G (PG) is selected as the flexible bridge between the QDs and the Hepatitis B virus surface antibody (HBsAb), to improve the sensitivity and specificity of the fluoroimmunoassay compared to the directly covalent conjugation. Successful coupling of the HBsAb to our highly luminescent CdTe/CdS core/shell QDs is proven with Gel electrophoresis and atomic force microscopy (AFM). The assay results, based on the microelisa well plate as matrix to immobilize the sandwich structure, show that both sensitivity and specificity can be improved greatly through the flexible coupled QDs-PG-Ab conjugates.     

The isolation and purification of pre-S2 containing hepatitis B virus surface antigen by chemical affinity chromatography

A simple, rapid, and efficient method was developed to isolate and purify pre-S2 containing HBsAgs from the plasma of a single chronic carrier of HBsAg (adw) by ammonium sulfate fractionation, hydroxyapatite column chromatography, and polymerized human serum albumin-affinity column chromatography. About 500 μg of pre-S2 containing HBsAg was obtained from 140 mL of plasma containing 4,200 μg of HBsAg. Two purified pre-S2 containing HBsAgs were analyzed by SDS-polyacrylamide gel electrophoresis and their molecular weights were determined to be 31,000 and 68,000 respectively. No significant amount of HBsAg or its derivative was detected in the final product.     

Sensitive electrogravimetric immunoassay of hepatitis B surface antigen through hyperbranched polymer bridge linked to multiple secondary antibodies

A method for using a hyperbranched polymer (HBP) as a bridge to link multiple secondary antibodies at HBP branches to amplify the detection response signal on a quartz crystal microbalance (QCM)-based sandwich-type immunosensor is reported. Carboxyl groups were prepared at multiple branches of HBP to make possible chemical binding between HBP and secondary antibodies via the carboxyl-amine reaction. The total mass of HBP and its linked multiple secondary antibodies were used to enhance the signal on a QCM chip in comparison with a simple sandwich-type immune reaction. By contrast, the proposed method could cause one antigen to analogously react with multiple secondary antibodies as a result of the branch structure of HBP. The strategy of using HBP as a bridge to link multiple secondary antibodies succeeded in quantitatively detecting the hepatitis B surface antigen (HBsAg). By employing demonstrated HBP bridge-linking, the frequency shift on a QCM chip was approximately 5 times greater than conventional methods without modification at secondary antibodies. The limit of detection of HBsAg was achieved as 2.0 ng mL^?1, lower than most of the values recorded in the literature measured by the QCM technique. Taking into account the general chemical interaction of immunoreaction, this method has the potential to amplify the signal in sensing many other analytes of interest.     

The anti-HBsAg (human type B hepatitis, surface antigen) and anti-HBeAg (human type B hepatitis, e antigen) C18 dibenzocyclooctadiene lignans from Kadsura matsudai and Schizandra arisanensis

The C(18) dibenzocyclooctadiene lignans including three novel schizanrin F (1), G (2), H (3), along with the known kadsurarin (4), were isolated from Kadsura matsudai. A new C(19) homolignan named schiarisanrin E (5), together with the known C(18) lignans, gomisin B (6), G (7) and (+)-gomisin K(3) (8) were obtained from Schizandra arisanensis. Gomisin B, G and (+)-gomisin K(3) showed moderate to strong activity for antihepatitis in anti-HBsAg (human type B hepatitis, surface antigen) and/or anti-HBeAg (human type B hepatitis, e antigen) tests. The structural elucidations of new compounds 1-3 and 5 were based on two-dimensional (2D) NMR techniques including COSY, HMQC, HMBC, NOESY and CD spectra. Preliminary structure-activity relationship studies for these isolated lignans are also discussed.     

Docking of B-cell epitope antigen to specific hepatitis B antibody

The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.     

Disposable immunoassay for hepatitis B surface antigen based on a graphene paste electrode functionalized with gold nanoparticles and a Nafion-cysteine conjugate

We report on the modification of a graphene paste electrode with gold nanoparticles (AuNPs) and a Nafion-L-cysteine composite film, and how this electrode can serve as a platform for the construction of a novel electrochemical immunosensor for the detection of hepatitis B surface antigen (HBsAg). To obtain the immunosensor, an antibody against HBsAg was immobilized on the surface of the electrode, and this process was followed by cyclic voltammetry and electrochemical impedance spectroscopy. The peak currents of a hexacyanoferrate redox system decreased on formation of the antibody-antigen complex on the surface of the electrode. Then increased electrochemical response is thought to result from a combination of beneficial effects including the biocompatibility and large surface area of the AuNPs, the high conductivity of the graphene paste electrode, the synergistic effects of composite film, and the increased quantity of HBsAb adsorbed on the electrode surface. The differential pulse voltammetric responses of the hexacyanoferrate redox pair are proportional to the concentration of HBsAg in the range from 0.5–800?ng?mL^?1, and the detection limit is 0.1?ng?mL^?1 (at an S/N of 3). The immunosensor is sensitive and stable.

Electrochemical immunoassay of hepatitis B surface antigen by the amplification of gold nanoparticles based on the nanoporous gold electrode

We describe herein the combination of electrochemical immunoassay using nanoporous gold (NPG) electrode with horseradish peroxidase (HRP) labeled secondary antibody-gold nanoparticles (AuNPs) bioconjugates for highly sensitive detection of protein in serum. The electroactive product of o-phenylenediamine (OPD) oxidized with H₂O₂ catalyzed by HRP was reduced in the Britton-Robinson (BR) buffer and the peak current of which was used to determine the concentration of antigen (Ag) in the analyte. The active surface area of NPG electrode was larger than that of a bare flat one. The presence of AuNPs enhanced the immobilized amount of HRP labeled antibody (Ab), which improved the sensitivity of the immunoassay when used as the secondary antibodies. As a result of these two combined effects, the sensitivity of the immunoassay for the determination of target protein was increased significantly. Using hepatitis B surface antigen (HBsAg) as a model, we demonstrate a dose response in the range of 0.01-1.0 ng/mL with a detection limit of 2.3 pg/mL. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by enzyme-linked immune-absorbent assays (ELISA). In addition, the technique was about 100 times more sensitive in the detection of HBsAg than ELISA. All these demonstrated the feasibility of the present immunoassay method for clinical diagnosis.