Novel amperometric immunosensors based on iridium oxide matrices

Novel immunosensors based on antibodies immobilized in electrochemically grown iridium oxide (IrOx) thin film matrices have been developed. Antibody loading in the oxide was evaluated using a non-competitive electrochemical immunoassay for IgG. Anti-IgG loading in the oxide was found to be dependent on the concentration of anti-IgG present in the oxide growth step, with 400 microg/ml anti-IgG producing maximum amperometric responses.To study the potential analytical properties of the matrix, the dose-response behavior of the sensors was determined using optimized alkaline phosphatase-linked IgG immunoassay. Hydroquinone diphosphate (HQDP) was used as enzyme substrate and the oxidation of hydroquinone was detected amperometrically at +420 mV. The sensors displayed a linear dose-response behavior for IgG concentrations between 10 and 200 ng/ml, saturating above 600 ng/ml, and had a low detection limit of 8 ng/ml.Finally, the method was used to produce sensors containing immobilized anti-transferrin. Using a non-optimized electrochemical immunoassay for human transferrin (HT), dose-response behavior was observed for HT concentrations between 100 and 600 ng/ml.The results presented in this paper show that IrOx matrices represent a new method for immunosensor fabrication. The oxide acts as a hydrophilic, highly porous, three-dimensional matrix that can immobilize antibodies and retain their activity. The method is attractive because it offers the potential for high antibody loadings and is suitable for mass production of sensors in an easy and economical manner.     

Application of europium(III) chelate-dyed nanoparticle labels in a competitive atrazine fluoroimmunoassay on an ITO waveguide

We have demonstrated the use of an optical indium tin oxide (ITO) (quartz) waveguide as a new platform for immunosensors with fluorescent europium(III) chelate nanoparticle labels (Seradyn) in a competitive atrazine immunoassay. ITO as a solid surface facilitated the successful use of particulate labels in a competitive assay format. The limit of detection in the new nanoparticle assay was similar to a conventional ELISA. The effect of particle size on bioconjugate binding kinetics was studied using three sizes of bioconjugated particle labels (107, 304, and 396nm) and a rabbit IgG/anti-IgG system in a 96-well plate. A decrease in particle size resulted in faster binding but did not increase the assay sensitivity. Flux calculations based on the particle diffusivity prove that faster binding of the small particles in this study was primarily due to diffusion kinetics and not necessarily to a higher density of antibodies on the particle surface. The results suggest that ITO could make a good platform for an optical immunosensor using fluorescent nanoparticle labels in a competitive assay format for small molecule detection. However, when used in combination with fluorescent particulate labels, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size. Different regeneration methods tested in this study showed that repeated washings with 0.1 M glycine-HCl facilitated the reuse of the ITO waveguide.     

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.     

Ag(I)-cysteamine complex based electrochemical stripping immunoassay: ultrasensitive human IgG detection

An ultrasensitive electrochemical immunosensor for a protein using a Ag (I)-cysteamine complex (Ag-Cys) as a label was fabricated. The low detection of a protein was based on the electrochemical stripping of Ag from the adsorbed Ag-Cys complex on the gold nanoparticles (AuNPs) conjugated human immunoglobulin G (anti-IgG) antibody (AuNPs-anti-IgG). The electrochemical immunosensor was fabricated by immobilizing anti-IgG antibody on a poly-5,2':5',2'-terthiophene-3'-carboxylic acid (polyTTCA) film grown on the glassy carbon electrode through the covalent bond formation between amine groups of anti-IgG and carboxylic acid groups of polyTTCA. The target protein, IgG was sandwiched between the anti-IgG antibody that covalently attached onto the polyTTCA layer and AuNPs-anti-IgG. Using square wave voltammetry, well defined Ag stripping voltammograms were obtained for the each target concentration. Various experimental parameters were optimized and interference effects from other proteins were checked out. The immunosensor exhibited a wide dynamic range with the detection limit of 0.4 ± 0.05 fg/mL. To evaluate the analytical reliability, the proposed immunosensor was applied to human IgG spiked serum samples and acceptable results were obtained indicating that the method can be readily extended to other bioaffinity assays of clinical or environmental significance.     

Conductometric immunoassay for interleukin-6 in human serum based on organic/inorganic hybrid membrane-functionalized interface

Various sensor-based immunoassay methods have been extensively developed for the detection of interleukin-6 (IL6), but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. The aim of this work is to develop a simple and sensitive conductometric immunoassay for IL6 in human serum by using an organic/inorganic hybrid membrane-functionalized interface. Initially, thionine-bound 3,4,9,10-perylenetetracarboxylic acid was doped into colloidal alumina, then nanogold particles were immobilized onto the thionine surface, and then horseradish peroxidase-labeled anti-IL6 antibodies were conjugated on the nanogold surface. The organic/inorganic hybrid membrane provides a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The performance and factors influencing the performance of the immunosensor were evaluated. The detection is based on the change in local conductivity before and after the antigen-antibody interaction in 0.02 M phosphate buffer solution (pH 6.8) containing 50 μM H₂O₂, 0.01 M KI and 0.15 M NaC1. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 25 to 400 pg/ml towards IL6 with a relatively low detection limit of 5 pg/ml (S/N = 3). The stability, reproducibility and precision of the immunosensor were acceptable. 37 serum specimens were assayed by the developed immunosensor and standard enzyme-linked immunosorbent assay, respectively, and the results obtained were almost consistent. More importantly, the detection methodology provides a promising approach for other proteins or biosecuritys.     

Nanogold probe enhanced Surface Plasmon Resonance immunosensor for improved detection of antibiotic residues

An exhaustive study is reported on the effect that antibody nanogold probes produce on the performance of a Surface Plasmon Resonance (SPR) immunosensor. The paper studies the improvement that different nanogold probes prepared at different antibody:gold nanoparticle (IgG:AuNP) ratios and AuNP sizes produce on the maximum signal and detectability of a simple SPR immunosensor developed to analyze fluoroquinolone (FQ) antibiotic residues (SPReeta system). The investigation compares the features of sensor enhanced formats using both, secondary and primary nanogold probes (anti-IgG and IgG coupled to AuNP, on double and single-antibody immunochemical assay steps, respectively), in respect to the unenhanced format. For this purpose, a reproducible bioconjugation procedure for preparing gold biohybrid nanoparticles has been established, involving the formation of a mixed self-assembled monolayer (m-SAM) with PEGylated cross-linkers around the AuNP followed by the covalent attachment of the antibodies. The procedure allows controlling the IgG:AuNP ratio of the nanogold probes on a reproducible manner and the functionalized NPs have been found to be stable during assay and storage. Both formats, using secondary and primary nanogold probes, are excellent strategies to improve immunosensor detectability. Thus, using anti-IgG-AuNP, the detectability could be improved by a factor of 14 (LOD 0.07±0.01 μg L(-1) vs. 0.98±0.38 μg L(-1)) reducing at the same time the amount of primary antibody used (30,000 vs. 1000 dilution factor). Likewise, the format using IgG-AuNP also allows improving detectability (LOD 0.11±0.01 μg L(-1)), but reducing the number of needed steps.     

Effect of heterologous paralytic shellfish poisoning toxin-enzyme conjugates on the cross-reactivity of a saxitoxin enzyme immunoassay

A polyclonal antiserum against saxitoxin (STX) was used in a competitive enzyme immunoassay for the detection of paralytic shellfish poisoning toxins. The extent of cross-reactions was determined from the amounts of neoSTX, decarbamoylSTX and gonyautoxin 2/3 (GTX2/3) that gave 50% inhibition in the assay. Horseradish peroxidase (HRP) conjugates of the toxins and a bovine serum albumin conjugate of STX (STX-BSA) were used. When compared with STX-BSA and STX as standard, the extent varied to which heterologous conjugates affected the binding values of the other toxins to the antibodies. The antibodies did not bind GTX2/3-HRP. By use of neoSTX-HRP or decarbamoylSTX-HRP as the labelled antigen instead of STX-HRP, the detection limit for neoSTX was improved to 100 pg ml^-1.     

A rapid, simple immunofluorometric assay: development and characterization

A novel two-site immunofluorometric assay, which includes only one incubation step and one separation step, is described. The assay is based on using small-sized beads (0.5 mum diameter) as a soild support and measuring the unbound fraction of labeled antibody in the liquid. The use of a mixture of the solid-phase and labeled antibodies at different concentrations makes it possible to determine antigen concentration over a wide range, without an initial sample dilution. Two assays were developed: one using anti-IgG polyclonal antibodies and the other using antibovine serum albumin monoclonal antibodies. The detection range of the polyclonal antibody assay using 30-minute incubation was 0.2 to 40 mug/mL for human IgG standard. The detection range of the monoclonal antibody assay was 0.2 to 14 mug/mL for bovine serum albumin (BSA) standard with 2 minutes required for the incubation. The interassay variability for the BSA measurement was 1.9% at 4.0 mug/mL of BSA and intra-assay variability was 2.3% at 3.2 mug/mL of BSA. The principles of this assay can be applied in the measurement of any protein in a rapid and reproducible way. (c) 1992 John Wiley & Sons, Inc.     

Detection of human chorionic gonadotrophin hormone using a label-free epoxysilane-modified capacitive immunosensor

A new and label-free capacitive immunosensor based on antibody-functionalized epoxysilane on a glassy carbon electrode has been developed for quantitative detection of human chorionic gonadotropin (hCG). Monitoring the changes in the capacitance signals of antibodies before and after the binding of the antigen provides the basis for an immunoassay. The performance and factors influencing the immunosensor were also studied. Under the optimized conditions, the developed immunosensor quantitatively detected serum hCG in the range of 18–450 mIU/ml with a detection limit of 5.0 mIU/ml (at 3δ). Thirty-five patients’ sera were assayed by the proposed immunosensor, and the results agreed with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.998. Further research about the intrinsic electroactivity of antibodies and their target molecules would surely provide new and sensitive screening assays as well as extensive data regarding their interaction mechanisms.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

Determination of Schistosoma japonicum circulating antigens in dilution serum by piezoelectric immunosensor and S/N enhancement

A piezoelectric immunosensor assay was developed with immobilizing immunoglobulin G (IgG) as a probe to detect Schistosoma japonicum circulating antigens (SjCAg). Analytical strategy utilizes the polyclonal antibodies with broad-spectrum recognition to a complex target with high specificity. The immobilized antibodies were purified from immunized rabbit's sera (im-S) and infected rabbit's sera (inf-S) by S. japonicum. The detection capacities of antibodies were compared between the sera of different phenotypes and purified fractions. The sample dilution ratios were also evaluated and optimized. Additionally, the sera with a variety of infection degrees for validation could be discriminated quantitatively. The linear dose-response relationship indicates that the systematic sensitivity of this method is below 150Hz and the lowest limit of detectable range is above 500 cercariae of S. japonicum infection for 2 weeks. The novel immunosensor technique is well potential to determine the SjCAg in serum samples for clinical diagnosis of parasitosis in early stage.     

Ultrasensitive detection of adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified electrochemical immunosensors

This work reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropin hormone (ACTH). The immunoelectrode design involves the use of amino phenylboronic acid for the oriented immobilization of anti-ACTH antibodies onto screen-printed carbon modified electrode surfaces. A competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed. The electroanalytical response was generated by using alkaline phosphatase-labelled streptavidin and 1-naphtyl phosphate as the enzyme substrate. The electrochemical oxidation of the enzyme reaction product, 1-naphtol, measured by differential pulse voltammetry was employed to monitor the affinity reaction. Under the optimized working conditions, an extremely low detection limit of 18 pg/L was obtained. Cross-reactivity was evaluated against other hormones (cortisol, estradiol, testosterone, progesterone, hGH and prolactin) and the obtained results demonstrated an excellent selectivity. The developed immunosensor was applied to a human serum sample containing a certified amount of ACTH with good results.     

Multifunctional Fe₃O₄ core/Ni-Al layered double hydroxides shell nanospheres as labels for ultrasensitive electrochemical immunoassay of subgroup J of avian leukosis virus

A novel electrochemical immunosensor for ultrasensitive detection of subgroup J of avian leukosis virus (ALVs-J) was designed by using graphene sheets (GS)-layered double hydroxides (LDHs) composites modified electrode with multifunctional Fe(3)O(4) core/Ni-Al LDHs shell (LDHs@Fe(3)O(4)) nanospheres as labels. At first, the GS-LDHs were used for the immunosensor platform for improving the electronic transmission rate as well as increasing the surface area to capture a large amount of primary antibodies (Ab(1)). After that, ferrocene (Fc), secondary antibodies (Ab(2)) and horseradish peroxidase (HRP) multifunctional LDHs@Fe(3)O(4) nanospheres were used as labels with high load amount and good biological activity. Subsequently, in presence of H(2)O(2), amplified signals were obtained by an electrochemical sandwich immunoassay protocol. To embody the signal amplification property of the protocol, the analytical properties of various immunosensor platform and labels were compared in detail. Under optimal conditions, the reduction peak currents of the electrochemical immunosensor were proportional to the ALVs-J concentration over the range from 10(2.32) to 10(5.50) TCID(50)/mL with a low detection limit (180 TCID(50)/mL, S/N=3). The resulting immunosensor also displayed a good selectivity, reproducibility and stability.     

Label-free electrochemical immunosensor for the determination of fetoprotein based on core-shell-shell nanocomposite particles

A new approach toward the development of advanced immunosensors based on chemically functionalized core-shell-shell magnetic nanocomposite particles, and the preparation, characteristics, and measurement of relevant properties of the immunosensor useful for the detection of alpha-1-fetoprotein (AFP) in clinical immunoassays. The core-shell NiFe2O4/3-aminopropyltriethoxysilance (APTES) (NiFe2O4@APTES) was initially prepared by covalent conjugation, then gold nanoparticles were adsorbed onto the surface of NiFe2O4@APTES, and then anti-AFP molecules were conjugated on the gold nanoparticles. The core-shell-shell nanocomposite particles not only had the properties of magnetic nanoparticles, but also provided a good biocompatibility for the immobilization of biomolecules. The core-shell-shell nanostructure present good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. The analytical performance of the immunosensor was investigated by using an electrochemical method. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of AFP, and exhibits wide linear range from 0.9 to 110 ng/mL AFP with a detection limit of 0.5 ng/mL. Moreover, the proposed immunosensors were used to analyze AFP in human serum specimens. Analytical results, obtained for the clinical serum specimen by the developed immunosensor, were in accordance with those assayed by the standard ELISA. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

Gold nanoparticles-decorated amine-terminated poly(amidoamine) dendrimer for sensitive electrochemical immunoassay of brevetoxins in food samples

A sensitive electrochemical immunosensor for the fast screening of brevetoxin B (BTX-2) in food samples was developed by means of immobilizing BTX-2-bovine serum albumin conjugate (BTX-2-BSA) on the gold nanoparticles-decorated amine-terminated poly(amidoamine) dendrimers (AuNP-PAADs). The presence of gold nanoparticles greatly improved the conductivity of the PAADs, and three-dimensional PAADs increased the surface coverage of the biomolecules on the electrode. Under optimal conditions, three types of immunosensor, i.e. with AuNPs, PAADs, or AuNP-PAADs, were used for the determination of BTX-2 in a competitive-type immunoassay format using horseradish peroxidase-labeled anti-BTX antibodies (HRP-anti-BTX-2) as trace in the H(2)O(2)-o-phenylenediamine (o-PD) system. A low detection limit (LOD) of 0.01 ng/mL and a wide dynamic working linear range of 0.03-8 ng/mL BTX-2 using AuNP-PAADs as matrices were obtained in comparison with those of only using AuNP or PAADs. Intra-batch assay precision was substantially improved by resorting to the AuNP-PAADs manifold. The proposed method features unbiased identification of negative (blank) and positive samples. No significant differences were encountered in the analysis of the spiking real samples between the electrochemical immunosensor and liquid chromatography for the determination of BTX-2. Importantly, this method provided a biocompatible immobilization and a promising immunosensing platform for analytes with small molecules in the analysis and detection of food safety.     

Electrochemical immune-biosensor for immunoglobulin G based bioelectrocatalytic reaction on micro-comb electrodes

A new amplification strategy of electrochemical signaling from antigen-antibody interactions was proposed via back-filling immobilization of horseradish peroxidase (HRP), immunoglobulin G antibodies (anti-IgG) and gold nanoparticles onto a three-dimensional sol-gel (3DSG)-functionalized biorecognition interface. The 3DSG sol-gel network was employed not only as a building block for the surface modification but also as a matrix for ligand functionalization. The signal-amplification was based on the bioelectrocatalytic reaction of the back-filling immobilization of HRP to H(2)O(2). With the non-competitive format, the formation of the antigen-antibody complex by a simple one-step immunoreaction between the immobilized anti-IgG and IgG in sample solution inhibited partly the active center of HRP, and decreased the immobilized HRP towards H(2)O(2) reduction. Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to IgG in a dynamic range of 1.12-162 ng/mL with a detection limit of 0.56 ng/mL (at 3delta). Moreover, the precision, reproducibility and stability of the as-prepared immunosensor were acceptable. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of biomarkers and its metastasis.     

A novel disposable electrochemical immunosensor for phenyl urea herbicide diuron

A disposable electrochemical immunosensor has been developed for the determination of phenyl urea herbicide-diuron using a low cost laser ablated gold electrodes (LC-LAGE) fabricated on polystyrene substrate. The electrodes were electrochemically deposited with prussian blue-gold nanoparticle (PB-GNP) film, and a competitive inhibition immunoassay was performed on LC-LAGE by using a specific hapten-protein conjugate. The binding of available diuron specific antibody on conjugate coated electrode was detected using alkaline phosphatase rabbit anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of electrochemically active product, 1-naphthol, which was monitored using square wave voltammetry technique. The assay exhibited an excellent sensitivity and specificity showing the dynamic response range between 1 ppt and 10 ppm for diuron with detection limit around 1 ppt. This study provides insight into development of a rapid and high-throughput screening of pesticides in environmental samples at a very low cost.     

Ultrasensitive electrochemical immunosensor based on Au nanoparticles dotted carbon nanotube-graphene composite and functionalized mesoporous materials

A facile and sensitive electrochemical immunosensor for detection of human chorionic gonadotrophin (hCG) was designed by using functionalized mesoporous nanoparticles as bionanolabels. To construct high-performance electrochemical immunosensor, Au nanoparticles (AuNPs) dotted carbon nanotubes (MWCNTs)-graphene composite was immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies (Ab(1)) as well as improve the electronic transmission rate. The as-prepared bionanolabels. composed of mesoporous silica nanoparticles (MCM-41) coated with AuNPs through thionine linking, showed good adsorption of horseradish peroxidase-labeled secondary anti-hCG antibody. Interlayer thionine was not only a bridging agent between MCM-41 and AuNPs but also an excellent electron mediator. The approach provided a good linear response range from 0.005 to 500 mIU mL(-1) with a low detection limit of 0.0026 mIU mL(-1). The immunosensor showed good precision, acceptable stability and reproducibility. Satisfactory results were obtained for determination of hCG in human serum samples. The proposed method provides a new promising platform of clinical immunoassay for other biomolecules.     

Investigation of dual-layer membrane cloaking method by surface plasmon resonance for direct chronoamperometric immunoassay of serum sample

A "dual-layer membrane cloaking" (DLMC) method was developed to construct disposable electrochemical immunosensor for direct determination of serum sample. Mouse IgG (MIgG) molecules were firstly immobilized on a substrate. After the formation of a didodecyldimethylammonium bromide (DDAB) membrane on the MIgG modified substrate, an additional bovine serum albumin (BSA) thin layer was formed to build a BSA/DDAB dual-layer membrane (DLM). When alkaline phosphatase conjugated anti-mouse IgG antibodies (anti-MIgG-ALP) in human serum were incubated on the substrate, anti-MIgG-ALP was recognized specifically by the immobilized MIgG while all nonspecifically adsorbed proteins were selectively removed together with BSA/DDAB DLM by 5% Triton X-100 (v/v) before final measurements. The BSA/DDAB DLM was characterized and optimized by surface plasmon resonance (SPR) technique, and further employed in a disposable immunoassay based on an ITO chip. Under optimal conditions, MIgG in human serum was directly detected in the range of 2.0-18.0 ng mL(-1) without dilution or separation. A limit of detection as low as 0.922 ng mL(-1) (6.15 pM) was obtained. The proposed DLMC method can efficiently prevent the penetration of matrix proteins through single cloaking membrane and completely eliminate nonspecific adsorption. It has great potential in providing a versatile way for direct determination of serum sample with ultra-sensitivity.     

Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG,IgM and IgA using beads covalently coupled to the nucleocapsid protein

Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS-CoV-2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free assay efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5–96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry-based diagnosis.