Dioxin immunosensor using anti-2,3,7,8-TCDD antibody which was produced with mono 6-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate as a hapten

To detect dioxin using a quartz crystal microbalance (QCM) immunosensor, anti-2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) monoclonal antibodies (MAbs) were produced as types of IgG1 and IgM, with mono 6-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate (as a hapten) conjugated with bovine serum albumin (dioxin-BSA). Furthermore, ScFv was generated from hybridoma-producing IgG1 MAb. Among these antibodies, ScFv showed excellent capability for dioxin detection using QCM immunosensors.     

Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.     

Investigation of osteoprotegerin interactions with ligands and antibodies using piezoelectric biosensors

Osteoprotegerin (OPG, osteoclastogenesis inhibitory factor) is a secretory glycoprotein involved as a soluble factor in the regulation of bone mass. OPG and its ligand (RANKL) levels in serum indicate the osteoclast formation activity. Alterations of the RANKL/OPG concentration ratio may be the cause of bone loss in many imbalances including osteoporosis, hypercalcaemia, metastatic osteolytic lesions and rheumatic bone degradation. The interactions of OPG with several antibodies were studied using the piezoelectric quartz crystal sensor. Monoclonal anti-OPG antibodies (5H3, 4E6H9 and OPG1.3) were immobilised on the sensing surface modified with covalently attached monolayer of protein A. Binding of both OPG standard and recombinant OPGFc chimeric protein was followed in real time. All antibodies were able to bind OPG and OPGFc, though in the case of MAb 4E6H9 the immunocomplexes dissociated quickly in the absence of OPG. Alternatively, biorecognition layers with RANKL were used. Two versions of the piezoelectric sensor for OPG were developed. The direct immunosensor was based on the antibody 5H3 and the affinity sensor employed the immobilised RANKL. The RANKL sensor exhibited poor reproducibility of results. For the immunosensor, the measuring range was 1.2-35 U/L of OPG. One analysis was completed within 15 min; the sensors were used repeatedly using regeneration with glycine buffer (pH 2.0). The developed immunosensor seems promising for rapid determination of osteoprotegerin in serum.     

Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts

Investigations into the development of a prototype electrochemical immunosensor for estradiol (E(2)) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E(2)) were immobilised by passive adsorption onto the surface of screen-printed carbon electrodes (SPCEs). A competitive immunoassay was then performed using an alkaline-phosphatase (ALP)-labelled E(2) conjugate. Calibration plots for E(2) buffer standards, performed colorimetrically on the SPCEs using a para-nitrophenyl phosphate substrate solution, were in good agreement with ELISA calibration plots. Electrochemical measurements were then performed using differential pulse voltammetry (DPV) following the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current versus E(2) concentration showed a measurable range of 25-500 pg/ml with a detection limit of 50 pg/ml. A coefficient of variation of between 13.0 and 15.6% was obtained for repeat measurements. The immunosensor was applied to the determination of E(2) in spiked serum, following an extraction step with diethyl ether. A mean recovery for the method of 102.5% was obtained with a CV of 19.1%. The options available for further development of the sensor regarding precision, limit of detection and direct sample analysis are discussed.     

On-line monitoring of monoclonal antibodies in animal cell culture using a grating coupler

A grating coupler was used for the on-line determination of monoclonal antibodies produced in perfused animal cell bioreactor. The device was connected with the culture vessel via a flow-injection analysis (FIA) system, which was controlled automatically. Specific antimouse lgG antibodies were immobilized on the surface of the sensor-chip. After injection of the sample, the binding of mouse lgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the on-line detection of produced monoclonal antibodies in the range of 10 to 150 mug/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off-line) and a competitive immunochemical assay using the grating coupler (off-line). (c) 1993 John Wiley & Sons, Inc.     

Development of quartz-crystal-microbalance-based immunosensor array for clinical immunophenotyping of acute leukemias

An integrated piezoelectric immunosensor array has been developed to immunophenotype acute leukemias in clinic. Each quartz crystal microbalance (QCM) was fabricated with plasma-polymerized film of n-butylamine, nanogold particles, and protein A (PA) to be used to immobilize antibodies in orientation. Leukemic lineage-associated monoclonal antibodies were separately immobilized onto the nanogold-PA-modified surface of the crystals, which were constructed by a 2 x 2 type of probes forming a QCM-based immunosensor array. The main detection conditions were investigated, including the immobilization amount of antibodies, pH, immunoreaction time, sample dilution ratio, etc. The immunophenotyping feasibility of the new technique was investigated through simultaneously analyzing Jurkat cells by the immunosensor array method, immunohistochemistry, and flow cytometry. It was found that the developed technique could readily identify leukemia samples in 5 min and might monitor dynamically the immunoreaction processes. Moreover, comparison studies were carried out for CD antigens expressed on the nucleated cells isolated from 96 acute leukemic patients and 24 normal subjects using the QCM-based immunosensor method and the fluoroimmunoassay. Results obtained by immunophenotyping patients' samples with the immunosensor-based method achieved the rate of 88.93% in 768 groups of numerical data, where no significant statistical difference was observed between the two methods when checked by chi2 analysis (chi2 = 3.4, p > 0.05). This new immunosensor array showed the merits of high sensitivity, high specificity, good reproducibility, easy operation, and low cost. The results of specimen evaluation indicated that it might be clinically suitable for quantifying human differentiated leukocytes and immunophenotyping of acute leukemias.     

Investigation of highly sensitive piezoelectric immunosensors for 2,4-dichlorophenoxyacetic acid

The improved highly sensitive piezoelectric immunosensor has been developed and evaluated using a model interaction of antibody with the model hapten-herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). For immobilization of 2,4-D, the self-assembled layers of cystamine, 4-aminothiophenol or 3,3'-dithio-bis(propionic acid N-hydroxysuccinimide ester) were formed on smooth and rough crystals coated with gold or silver electrodes. The immunochemical interactions performed well in all cases, the aminothiophenol on gold was chosen as the optimum with regard to regeneration of immunosensing surfaces. The kinetics of interaction of surface-bound 2,4-D with free antibody provided significantly higher kinetic parameters (kinetic association rate constant) when using optically smooth crystals compared to common rough crystal. Therefore, the smooth crystal should be preferred for future kinetic studies. The competitive assay of the herbicide 2,4-D achieved a limit of detection of 10 ng/l using the monoclonal anti-2,4-D antibody F6C10. Finally, a direct assay format has been evaluated using a thicker layer of glutaraldehyde-crosslinked antibody on the sensing surface. The direct binding of a small herbicide molecule was followed in real time. The detected concentration of 2,4-D (5 microg/l) was low enough for future direct monitoring of this herbicide in water.     

Multi-analyte capillary immunosensor for the determination of hormones in human serum samples

In this work we present the development of a multi-analyte immunosensor for the determination of follitropin, human chorionic gonadotropin and prolactin in human serum. The immunosensor is based on plastic capillaries. According to the methodology, discrete areas of the internal capillary surface are coated with different antibodies, which are highly specific for each one of the analytes to be determined. The sample that will be analyzed along with a mixture of analyte-specific biotinylated antibodies is introduced into the capillary. The coated and the detection antibodies react with different epitopes of the analytes in the sample to form a 'sandwich'. The detection is based on reaction of the immobilized biotinylated antibody with streptavidin labeled with R-phycoerythrin. The fluorescent areas formed were quantified by scanning the capillary with a light beam of appropriate wavelength. A light sensor placed at the end of the capillary detects the emitted photons, that are trapped and waveguided into the capillary walls. The multi-analyte immunosensor assays were characterized by high specificity and short analysis time. In addition, the results obtained by the multi-analyte optical capillary immunosensor were comparable to those obtained by immunofluorimetric assays performed in microtitration wells. Potential applications of the proposed immunosensor include determination of several analyte panels in a broad spectrum of disciplines such as endocrinology, hematology, and oncology.     

A novel piezoelectric quartz micro-array immunosensor based on self-assembled monolayer for determination of human chorionic gonadotropin

A novel multi-channel 2 x 5 model of piezoelectric quartz micro-array immunosensor has been developed for quantitative detection of human chorionic gonadotropin (hCG) in serum or urine samples. Every crystal unit of the fabricated piezoelectric hCG micro-array immunosensor can oscillate independently without interfering each other. A 2 x 5 model of micro-array immunosensor as compared with a one-channel immunosensor can provide eight times higher detection speeds for hCG assay. The anti-hCG antibody is deposited on the gold electrode's surface of 10 MHz quartz AT-cut crystal by self-assembled technique using sulfosuccinimidyl 6-[3'-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP), and serves as an antibody recognizing layer. The highly ordered self-assembled monolayers (SAM) ensure well-controlled surface structure and offer many advantages to the performance of the sensor. Compared with conventional antibody immobilization methods, the amount and the reaction activity of antibody monolayer coated by the SAM binding are bigger than those by the SPA method, and less non-specific binding caused by other analytes in sample is found. Under the optimized experimental conditions, the results showed that micro-array immunosensor quantitatively detected serum or urine hCG in the range of 2.5-500 mIU/ml with high precision (CV<5%); other hormones in human serum and urine did not interfere with the determination markedly. Serum and urine samples of 60 patients were detected by the micro-array immunosensor, and the results agreed well with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.92. After regeneration with urea solution the coated immunosensor can be reused five times without appreciable loss of activity.     

Analysis of carbohydrates using liquid chromatography--surface plasmon resonance immunosensing systems

An immunosensing system based on surface plasmon resonance (SPR) was used for on-line detection and characterization of carbohydrate molecules separated by high-performance liquid chromatography. These analytes, with or without serum, were continuously separated and analyzed in the combined liquid chromatography-surface plasmon resonance (LC-SPR) system. By using weak and readily reversible monoclonal antibodies, the SPR system allowed specific on-line monitoring of the substances. To increase the specificity of the immunosensor, nonrelevant antibodies were used as reference in a serial flow cell. The sensitivity of the LC-SPR system was dependent on molecular weight of the carbohydrate, affinity of binding, and design of the sensor.     

Circulating monoclonal IgM proteins in B cell chronic lymphocytic leukemia: their identification, characterization and relationship to membrane IgM

Accumulated evidence indicates that there is a circulating monoclonal Ig protein related to the leukemic cell-associated Ig in the majority of patients with B cell chronic lymphocytic leukemia (CLL) despite the failure to demonstrate such a protein by conventional serum electrophoresis. Methodology has been developed to reveal these hidden monoclonal bands and to show that they are related to the leukemia-associated membrane Ig (mIg). Of nine CLL cases with stainable mIgM and without discernable plasma Ig bands, marked hypogammaglobulinemia was evident in six. In the other three, a significant amount of protein was present in the gamma region. IgM was isolated from the plasma of these patients by affinity chromatography with Sepharose-4B, conjugated with affinity purified anti-human IgM antibodies. One to 3 mg were isolated from 20 to 40 ml of plasma. Agarose electrophoresis revealed a monoclonal Ig band in the isolated IgM in all cases. Eight of these IgM proteins were analyzed by high-pressure liquid chromatography. Five were found to be pentameric IgM. In the remaining three, various amounts of monomeric IgM were detected. Attempts to make anti-idiotypic antibodies to the isolated proteins have been successful. Thus far, a rabbit anti-idiotypic antiserum was obtained in one case and two mouse monoclonal anti-idiotypic antibodies in two additional cases. Immunofluorescence analysis revealed that plasma IgM and mIgM shared similar idiotypic determinants. One other monoclonal antibody was shown to be specific for a V region marker of a minor Ig population. These findings indicate that B leukemic lymphocytes do secrete a small amount of IgM and lend further support to the thesis that the maturation defect in CLL is incomplete. It is also feasible to isolate the secreted IgM and to produce anti-idiotypic antibodies to them. In view of the potential therapeutic effect of anti-idiotypic antibodies, this may offer an alternative and efficient approach to generate a large panel of anti-idiotypic antibodies for clinical trials. The possibility also exists that this approach is applicable to other B cell proliferative disorders such as the non-Hodgkin B cell lymphomas.     

Quartz crystal microbalance immunosensors for environmental monitoring

This paper presents discussion of quartz crystal microbalance (QCM) immunosensors for environmental monitoring. Factors limiting the practical application of antibodies to analytical problems are also presented. Among several candidates for the QCM immunosensor device, selected QCM devices and oscillating circuits were tested thoroughly and developed to obtain highly stable and sensitive frequency signals. The biointerface of QCM immunosensor was designed and controlled to immobilize antibody on the QCM surface, to reduce non-specific binding and to suppress denaturation of immobilizing antibody by self-assembled monolayer technique and artificial phospholipid (2-methacryloyloxyethyl phosphorylcholine (MPC)) polymer. MPC polymer as a antibody-stabilizing reagent was added to reduce non-specific binding of the antigen solution and stabilize the immunologic activity of the antibody-immobilized QCM. In addition, it provides examples for detection and quantitation of environmental samples using QCM immunosensors. The analytical results for fly ash extracted samples of dioxins using the QCM immunosensor indicated a good relationship with GC/MS methods. The integrating protocols of the competitive immunoassay and signal-enhancing step are for detecting low molecular analytes with extremely low detection limits using an QCM immunosensor. Furthermore, its detect limitation was extended from 0.1 to 0.01 ng/ml by the signal-enhancing step when the anti-bisphenol-A antibody conjugated MPC polymeric nanoparticles was used. The QCM immunosensor method has demonstrated its effectiveness as an alternative screening method for environmental monitoring because these results were compared with results obtained through environmental monitoring methods such as ELISA and GC/MS.     

Peptide-coated nanotube-based biosensor for the detection of disease-specific autoantibodies in human serum

We demonstrate a label-free peptide-coated carbon nanotube-based immunosensor for the direct assay of human serum. A rheumatoid arthritis (RA)-specific (cyclic citrulline-containing) peptide, was immobilized to functionalized single-walled carbon nanotubes deposited on a quartz crystal microbalance (QCM) sensing crystal. Serum from RA patients was used to probe these nanotube-based sensors, and antibody binding was detected by QCM sensing. Specific antibody binding was also determined by comparing the assay of two serum control groups (normal and diseased sera), and the native unmodified peptide. The sensitivity of the nanotube-based sensor (detection in the femtomol range) was higher than that of the established ELISA and recently described microarray assay systems, detecting 34.4 and 37.5% more RA patients with anti-citrullinated peptide antibodies than those found by ELISA and microarray, respectively. There was also an 18.4 and 19.6% greater chance of a negative test being a true indicator of a person not having RA than by either ELISA or microarray, respectively. The performance of our label-free biosensor enables its application in the direct assay of sera in research and diagnostics.     

Electrochemical immunosensor detection of antigliadin antibodies from real human serum

The determination of antigliadin antibodies from human serum samples is of vital importance for the diagnosis of an autoimmune disease such as celiac disease. An electrochemical immunosensor that mimics traditional ELISA type architecture has been constructed for the detection of antigliadin antibodies with control over the orientation and packing of gliadin antigen molecules on the surface of gold electrodes. The orientation of the antigen on the surface has been achieved using a carboxylic-ended bipodal alkanethiol that is covalently linked with amino groups of the antigen protein. The bipodal thiol presents a long poly(ethyleneglycol)-modified chain that acts as an excellent non-specific adsorption barrier. The bipodal nature of the thiol ensured a good spacing and hence good diffusion properties of electroactive species through the self-assembled monolayer, which is vital for the efficiency of the constructed electrochemical immunosensor. The electrochemical immunosensor was characterized using surface plasmon resonance as well as electrochemical impedance spectroscopy. Amperometric evaluation of the sensor with polyclonal antigliadin antibodies showed stable and reproducible low limits of detection (46 ng/mL; % RSD = 8.2, n = 5). The behaviour and performance of the electrochemical immunosensor with more complex matrixes such as reference serum solutions and real patient samples was evaluated and compared with commercial ELISA kits demonstrating an excellent degree of correlation in thirty minutes total assay time; the electrochemical immunosensor not only delivers a positive or negative result, it allows the estimation of semi-quantitative antibody contents based on the comparison against clinical reference solutions.     

Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays

Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.     

Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on surface plasmon resonance

A direct human ferritin immunosensor was developed using anti-human ferritin monoclonal antibodies (MAbs) immobilized on the gold surface of a self-assembled surface plasmon resonance (SPR) apparatus. A kind of self-assembled monolayer (SAM) prepared by cystamine-glutaraldehyde method was applied to immobilize the MAbs. The reusability of the sensor chip adopting the SAM was found to be better than the other immobilization methods including adsorption, protein A, concanavalin A method. Ten cycles of measurements could be performed on the same chip regenerated with a 0.1M HCl solution. A linear relationship existed between the angle shifts (millidegrees) and the log values of ferritin concentrations in the range from 0.2 to 200 ng/ml in buffer and human serum. When used for 15 days, the angle shifts were all >95% of those on the response at the first day. A 10 M NaOH solution was used for clearing nonspecific binding in human serum. Correlation coefficient was 0.991 between this SPR method and chemiluminescent immunoassay for determination of ferritin in clinical human serum samples. The SPR sensor offers advantages of simplicity of immobilization, high sensitivity, high specificity, low sample requirement, high reusability, no label and no pretreatment etc.     

玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立

玉米赤霉烯酮具有较强的生物毒性,检测谷物中的玉米赤霉烯酮在食品和饲料安全中具有重要的作用.将玉米赤霉烯酮与牛血清白蛋白的偶联物免疫BALB/c小鼠制备单克隆抗体,并建立基于单克隆抗体的酶联免疫法作为检测玉米赤霉烯酮的方法.结果共筛选到4株抗玉米赤霉烯酮单克隆抗体,3株抗体亚类为IgG1,1株为IgG2b.选择其中的一株杂交瘤细胞2C9制备小鼠腹水,纯化后测定了抗体效价为1/40 000.以此单抗建立的间接竞争ELISA方法,其半数抑制率(IC50)为1.90 ng/mL,检测限(IC10)为0.051 ng/mL,检测区间(IC20-IC80)为0.115-13.900 ng/mL;且对玉米赤霉烯酮有很好的特异性.回收率检测在样品含1.46-93.80 μg/kg时回收率为96.5％-113.0％.本实验建立的检测方法可用于多种谷物及饲料样本中玉米赤霉烯酮的检测.     

Novel potentiometric immunosensor for hepatitis B surface antigen using a gold nanoparticle-based biomolecular immobilization method

A novel potentiometric immunosensor for the detection of hepatitis B surface antigen has been developed by means of self-assembly to immobilize hepatitis B surface antibody on a platinum disk electrode based on gold nanoparticles, Nafion, and gelatin as matrices in this study. The modification procedure of the immunosensor was further characterized by using cyclic voltammetry and the enzyme-linked immunosorbent assay (ELISA) method. The detection is based on the change in the electric potential before and after the antigen-antibody reaction. In contrast to the commonly applied methods (e.g., the glutaraldehyde crosslinking procedure), this strategy could allow for antibodies immobilized with a higher loading amount and better retained immunoactivity, as demonstrated by the potentiometric measurements. A dynamic concentration range of 4-800 ng ml(-1) and a detection limit of 1.3 ng ml(-1) were observed. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by ELISA. In addition, the technique presents some distinct advantages over the traditional sandwich format in that the analyzing performances are direct, rapid, and simple without multiple separation and labeling steps.     

A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1

As a potential pandemic threat to human health, there has been an urgent need for rapid detection of the highly pathogenic avian influenza (AI) H5N1 virus. In this study, magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection. Polyclonal antibodies against AI H5N1 virus surface antigen HA (Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA). Target H5N1 viruses were then captured by the immobilized antibodies, resulting in a change in the frequency. Magnetic nanobeads (diameter, 30nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen (virus). Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy (ESEM). The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2h. The nanobeads amplification resulted in much better detection signal for target virus with lower titers. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. The immunosensor was evaluated using chicken tracheal swab samples. This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid, sensitive, and specific detection of AI virus H5N1 in agricultural, food, environmental and clinical samples.     

Conventional detection method of fibrinogen and fibrin degradation products using latex piezoelectric immunoassay

We developed a conventional immunosensor for fibrinogen and fibrin degradation products (FDP) to combine a quartz crystal microbalance (QCM) with the agglutination reaction of immunized latex beads. FDP induced an immunoreaction due to anti-FDP antibody immobilized latex particles. We successfully measured FDP concentration of in human serum within 10 min by QCM method. The detection range of QCM immunosensor is covered with screening concentration of FDP in serum (<10 microg/ml of FDP). The time course of latex agglutination obtained from QCM immunosensor is synchronized to that of latex photometric immunoassay. SEM was used to observe the surface of QCM that applied FDP serum. The size of latex particles agglutinated on the QCM electrode increased concomitant with FDP concentration. Frequency shift on immunoreaction explains the increased adsorption amount of agglutinated latex on QCM.