Detection Of Avian Influenza Virus Research Articles

Development and validation of a blocking ELISA test for the detection of avian influenza antibodies in poultry species

Avian influenza (AI) is an infectious viral disease usually asymptomatic in wild birds that can spread to domestic poultry and cause large-scale outbreaks. Some of the AI viruses (AIV) can cross the species barrier and induce fatal disease to humans, a matter of great concern worldwide. Early detection of AIV is of major importance for disease control, since prompt implementation of adequate measures can prevent spread of the virus and therefore further outbreaks. This paper reports the development and validation of a blocking ELISA using a monoclonal antibody against a conserved structural protein for the serologic diagnosis of all AI virus subtypes from domestic bird species, allowing the quick, easily automated and low-cost screening of a high number of farms. The test will be of great value not only for surveillance, but also for monitoring the efficiency of vaccination programs. Cut-off values were established in 20% of inhibition for turkey sera and in 50% of inhibition for chicken and duck sera. Estimations of 100% specificity and 100% sensitivity were obtained based on the results of known AI positive (n=130) and negative (n=208) sera, including serum samples from birds infected with other common avian viral pathogens (n=7).ROC analysis showed an area under curve (AUC) of 1.0 for chicken, duck and turkey sera, indicating that the blocking ELISA was able to perfectly discriminate between negative and positive samples of any of the poultry species tested.Inter- and intra-assay coefficients of variation were above the acceptance threshold. Furthermore, the ELISA titers were similar to the known hemagglutination inhibition titers of three positive reference sera indicating sensitivity comparable with the golden standard HI method.The method here described is an economically attractive alternative to the commercial ELISAs currently available.

Ecology of Avian Influenza Virus in Wild Birds in Tropical Africa.

Several ecologic factors have been proposed to describe the mechanisms whereby host ecology and the environment influence the transmission of avian influenza viruses (AIVs) in wild birds, including bird's foraging behavior, migratory pattern, seasonal congregation, the rate of recruitment of juvenile birds, and abiotic factors. However, these ecologic factors are derived from studies that have been conducted in temperate or boreal regions of the Northern Hemisphere. These factors cannot be directly translated to tropical regions, where differences in host ecology and seasonality may produce different ecologic interactions between wild birds and AIV. An extensive dataset of AIV detection in wildfowl and shorebirds sampled across tropical Africa was used to analyze how the distinctive ecologic features of Afrotropical regions may influence the dynamics of AIV transmission in wild birds. The strong seasonality of rainfall and surface area of wetlands allows testing of how the seasonality of wildfowl ecology (reproduction phenology and congregation) is related to AIV seasonal dynamics. The diversity of the African wildfowl community provides the opportunity to investigate the respective influence of migratory behavior, foraging behavior, and phylogeny on species variation in infection rate. Large aggregation sites of shorebirds in Africa allow testing for the existence of AIV infection hot spots. We found that the processes whereby host ecology influence AIV transmission in wild birds in the Afrotropical context operate through ecologic factors (seasonal drying of wetlands and extended and nonsynchronized breeding periods) that are different than the one described in temperate regions, hence, resulting in different patterns of AIV infection dynamics.

A target-responsive and size-dependent hydrogel aptasensor embedded with QD fluorescent reporters for rapid detection of avian influenza virus H5N1

A target-responsive hydrogel based fluorescent aptasensor was developed for rapid detection of avian influenza virus (AIV) H5N1. A specific aptamer against H5N1 was selected and two single-stranded DNAs (ssDNA1 and ssDNA2) were designed to be partially complementary to two ends of the aptamer. Both aptamer and ssDNA1 were functionalized with acrydite at 5’-terminal for hydrogel synthesis by polymerization. Quantum dots (QDs), used as fluorescence reporters, were labeled at 5’-terminal of ssDNA2 and quenchers for QDs were conjugated at 3’-terminal of the aptamer. Hybridization between aptamer and ssDNAs formed the crosslinker to complete the QD-aptamer hydrogel. Initially, without targets, the prepared hydrogel remained in a shrunken state because of the crosslinking in the polymer network and the QD was quenched. With target AIVs, the crosslinking was dissociated due to the binding reaction between the aptamer and target, resulting in the abrupt swelling of the hydrogel and the release of aptamer-quencher and ssDNA2-QD. The response of aptamer upon target binding was demonstrated by quartz crystal microbalance (QCM) method and the microstructure of hydrogel was characterized by SEM. Spectrophotometric results indicated fluorescent changes at the emission peak were correlated to the titers of H5N1. The total detection time from sampling to results was 30min. The lowest detection limit was 0.4HAU with a detecting range of 2−1.2 to 26 HAU 20μL−1. The size-dependent property of this aptasensor was examined quantitatively and qualitatively. This simple, low-cost, specific, and label-free aptasensor has great potential for the in-field rapid detection of AIV H5N1.

A Portable Impedance Biosensing System for Rapid Detection of Avian Influenza Virus

Abstract. Avian influenza virus (AIV) H5N1 is a highly infectious pathogen that can be dangerous to both poultry and humans. A rapid and sensitive detection method is needed to help prevent the spread of AIV H5N1. In this study, a portable impedance biosensing system was designed to integrate a laptop computer with LabVIEW software, a data acquisition (DAQ) device, a sample delivery system, a micro-flow cell, and a gold interdigitated microelectrode (IDME) into an automatic instrument for rapid detection of AIV H5N1. Streptavidin was first immobilized onto the IDME surface, and then biotin-labeled H5N1-specific aptamer was immobilized on the IDME surface through streptavidin and biotin binding. Samples were delivered through the sample delivery system into the micro-flow cell, and AIV H5N1 was captured by the aptamer on the IDME, resulting in a change in the impedance. A virtual instrument (VI) was programmed with LabVIEW software to provide a platform for sample delivery, impedance measurement, data processing, and control. The audio card of the laptop was used as a function generator, while the DAQ device was used for data communication. The impedance measured by this biosensing system was compared with that measured by a BAS IM6 impedance analyzer, and the error was less than 5%. The results indicated that the developed system could detect AIV H5N1 at a detection limit of 0.84 HAU per 200 μL without interaction with three non-target AIV subtypes (H1N1, H5N2, and H5N3). The system was portable and automatic, and it showed great potential as a diagnostic and epidemiological tool for detection of AIV in the field.

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray.

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.

Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination.

Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

Reverse-transcription, loop-mediated isothermal amplification assay for the sensitive and rapid detection of H10 subtype avian influenza viruses.

BackgroundThe H10 subtype avian influenza viruses (H10N4, H10N5 and H10N7) have been reported to cause disease in mammals, and the first human case of H10N8 subtype avian influenza virus was reported in 2013. Recently, H10 subtype avian influenza viruses (AIVs) have been followed more closely, but routine diagnostic tests are tedious, less sensitive and time consuming, rapid molecular detection assays for H10 AIVs are not available.MethodsBased on conserved sequences within the HA gene of the H10 subtype AIVs, specific primer sets of H10 subtype of AIVs were designed and assay reaction conditions were optimized. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established for the rapid detection of H10 subtype AIVs. The specificity was validated using multiple subtypes of AIVs and other avian respiratory pathogens, and the limit of detection (LOD) was tested using concentration gradient of in vitro-transcribed RNA.ResultsThe established assay was performed in a water bath at 63 °C for 40 min, and the amplification result was visualized directly as well as under daylight reflections. The H10-RT-LAMP assay can specifically amplify H10 subtype AIVs and has no cross-reactivity with other subtypes AIVs or avian pathogens. The LOD of the H10-RT-LAMP assay was 10 copies per μL of in vitro-transcribed RNA.ConclusionsThe RT-LAMP method reported here is demonstrated to be a potentially valuable means for the detection of H10 subtype AIV and rapid clinical diagnosis, being fast, simple, and low in cost. Consequently, it will be a very useful screening assay for the surveillance of H10 subtype AIVs in underequipped laboratories as well as in field conditions.

Identification of a novel linear epitope on the NS1 protein of avian influenza virus.

BackgroundThe NS1 protein of avian influenza virus (AIV) is an important virulent factor of AIV. It has been shown to counteract host type I interferon response, to mediate host cell apoptosis, and to regulate the process of protein synthesis. The identification of AIV epitopes on NS1 protein is important for understanding influenza virus pathogenesis.ResultsIn this paper, we describe the generation, identification, and epitope mapping of a NS1 protein-specific monoclonal antibody (MAb) D9. First, to induce the production of MAbs, BALB/c mice were immunized with a purified recombinant NS1 expressed in E. coli. The spleen cells from the immunized mice were fused with myeloma cells SP2/0, and through screening via indirect ELISAs, a MAb, named D9, was identified. Western blot assay results showed that MAb D9 reacted strongly with the recombinant NS1. Confocal laser scanning microscopy showed that this MAb also reacts with NS1 expressed in 293T cells that had been transfected with eukaryotic recombinant plasmids. Results from screening a phage display random 7-mer peptide library with MAb D9 demonstrated that it recognizes phages displaying peptides with the consensus peptide WNLNTV--VS, which closely matches the 182WNDNTVRVS190 of AIV NS1. Further identification of the displayed epitope was performed with a set of truncated polypeptides expressed as glutathione S-transferase fusion proteins, and the motif 182WNDNT186 was defined as the minimal unit of the linear B cell epitope recognized by MAb D9 in western blot assays. Moreover, homology analysis showed that this epitope is a conserved motif among AIV.ConclusionsWe identified a conserved linear epitope, WNDNT, on the AIV NS1 protein that is recognized by MAb D9. This MAb and its epitope may facilitate future studies on NS1 function and aid the development of new diagnostic methods for AIV detection.

An Impedance Aptasensor with Microfluidic Chips for Specific Detection of H5N1 Avian Influenza Virus.

In this research a DNA aptamer, which was selected through SELEX (systematic evolution of ligands by exponential enrichment) to be specific against the H5N1 subtype of the avian influenza virus (AIV), was used as an alternative reagent to monoclonal antibodies in an impedance biosensor utilizing a microfluidics flow cell and an interdigitated microelectrode for the specific detection of H5N1 AIV. The gold surface of the interdigitated microelectrode embedded in a microfluidics flow cell was modified using streptavidin. The biotinylated aptamer against H5N1 was then immobilized on the electrode surface using biotin–streptavidin binding. The target virus was captured on the microelectrode surface, causing an increase in impedance magnitude. The aptasensor had a detection time of 30 min with a detection limit of 0.0128 hemagglutinin units (HAU). Scanning electron microscopy confirmed the binding of the target virus onto the electrode surface. The DNA aptamer was specific to H5N1 and had no cross-reaction to other subtypes of AIV (e.g., H1N1, H2N2, H7N2). The newly developed aptasensor offers a portable, rapid, low-cost alternative to current methods with the same sensitivity and specificity.

Comparison of three media for transport and storage of the samples collected for detection of avian influenza virus

Detection of avian influenza viruses (AIVs) is important for diagnosis, surveillance and control of avian influenza which is of great economic and public health significance. Proper transport and storage of samples is critical for the detection when the samples cannot be detected immediately. As recommended by some international or national authoritative entities and some publications, phosphate buffered saline (PBS), PBS–glycerol and brain heart infusion broth (BHIB) are frequently used for transport and storage of the samples collected for detection of AIVs worldwide. In this study, we compared these three media for transport and storage of simulated and authentic swab and feces samples collected for detection of AIVs using virus isolation and reverse transcription-PCR. The results suggest that PBS–glycerol is superior to PBS and BHIB as the sample transport and storage media. The results also suggest that the samples collected for detection of AIVs should be detected as soon as possible because the virus concentration of the samples may decline rapidly during storage within days at 4 or −20°C.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye.

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.

Seroprevalence and Detection of Avian Influenza Type A in Ducks at Nikli and Bajitpur Upazila of Bangladesh

Waterfowl are the natural reservoir of avian influenza viruses and ducks may play a role in the maintenance of avian influenza type A. The aim of the present study was to investigate the seroprevalence and detection of avian influenza virus (AIV) type A in duck. This study was carried out during July 2013 to December 2013 on AIV type A from semi-scavenging farm at Nikli and Bajitpur upazila of Kishoregonj district in Bangladesh. A total of 368 blood samples were collected from duck and tested by indirect ELISA for seroprevalence. For detection of AIV type A, The cloacal swabs were collected from 75 duck and subjected to RNA extraction and real time RT-PCR (rRT-PCR) with specific primer and probe for detection of matrix (M) gene. The average seroprevalance of AIV type A in seven different age groups was found to be 90.21%. The highest (25.81 %) seroprevalence was found in 5 months age of birds and the lowest (2.44 %) was found in 12 months age of birds. As regard to area distribution, the average degree of seroprevalence was 93.51% from Nikli had the highest order than Bajitpur (86.88%) upazila of Bangladesh. In case of cloacal sample by using rRT–PCR, out of 15 pooling cloacal samples, two pooling samples (13.33%) that contain 10 samples were positive and 13 pooling samples showed negative (86.67%) for AIV type A in duck. It can be concluded that the long distance movement of duck flocks, may influence outbreak of avian influenza virus (AIV) type A among different poultry species in Bangladesh. Therefore, it needs to develop control strategy for future dissemination of AIV in duck population.DOI: http://dx.doi.org/10.3329/bjvm.v13i1.23705Bangl. J. Vet. Med. (2015). 13 (1): 5-9

Light Weight and Flexible High‐Performance Diagnostic Platform

A flexible diagnostic platform is realized and its performance is demonstrated for early detection of avian influenza virus (AIV) subtype H1N1 DNA sequences. The key component of the platform is high-performance biosensors based on high output currents and low power dissipation Si nanowire field effect transistors (SiNW-FETs) fabricated on flexible 100 μm thick polyimide foils. The devices on a polymeric support are about ten times lighter compared to their rigid counterparts on Si wafers and can be prepared on large areas. While the latter potentially allows reducing the fabrication costs per device, the former makes them cost efficient for high-volume delivery to medical institutions in, e.g., developing countries. The flexible devices withstand bending down to a 7.5 mm radius and do not degrade in performance even after 1000 consecutive bending cycles. In addition to these remarkable mechanical properties, on the analytic side, the diagnostic platform allows fast detection of specific DNA sequences of AIV subtype H1N1 with a limit of detection of 40 × 10(-12) m within 30 min suggesting its suitability for early stage disease diagnosis.

Electrochemical biosensors for detection of avian influenza virus--current status and future trends.

Electrochemical biosensors have emerged as reliable analytical devices suitable for pathogen detection. Low cost, small sample requirement and possibility of miniaturization justifies their increasing development. Thus, we report in this review on the state of the art of avian influenza virus detection with genosensors and immunosensors working by an electrochemical mode. Their working principles focusing on the physical properties of the transducer, the immobilization chemistry, as well as new trends including incorporation of nanoparticles will be presented. Then, we critically review the detection of avian influenza virus in the complex matrices that use electrochemical biosensors and compare them with traditionally applied methods such as ELISA or Western blot.

Bio-nanogate controlled enzymatic reaction for virus sensing.

The objective of this study was to develop an aptamer-based bifunctional bio-nanogate, which could selectively respond to target molecules, and control enzymatic reaction for electrochemical measurements. It was successfully applied for sensitive, selective, rapid, quantitative, and label-free detection of avian influenza viruses (AIV) H5N1. A nanoporous gold film with pore size of ~20 nm was prepared by a metallic corrosion method, and the purity was checked by energy-dispersive X-ray spectroscopy (EDS) study. To improve the performance of the bio-nanogate biosensor, its main analytical parameters were studied and optimized. We demonstrated that the developed bio-nanogate was capable of controlling enzymatic reaction for AIV H5N1 sensing within 1h with a detection limit of 2(-9)HAU (hemagglutination units). The enzymatic reaction was able to cause significant current change due to the presence of target AIV. A linear relationship was found in the virus titer range of 2(-10)-2(2)HAU. No interference was observed from non-target AIV subtypes such as H1N1, H2N2, H4N8 and H7N2. The developed approach could be adopted for sensing of other viruses.

Upconversion Luminescence Resonance Energy Transfer (LRET)‐Based Biosensor for Rapid and Ultrasensitive Detection of Avian Influenza Virus H7 Subtype

Avian influenza viruses (AIV) with good adaptation and various mutations have threatened both human and animals' health. The H7 subtypes have the potential to cause pandemic threats to human health due to the highly pathogenic characteristics. Therefore, it is quite urgent to develop a novel biosensor for rapid and sensitive detection of H7 subtypes. In this work, a biosensor based on luminescence resonance energy transfer (LRET) from BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) to gold nanoparticles (AuNPs) has been developed for rapid and sensitive H7 subtypes detection. The amino modified capture oligonucleotide probes are covalently linked to poly(ethylenimine) (PEI) modified BaGdF5:Yb/Er UCNPs. The thiol modified oligonucleotides with H7 hemagglutinin gene sequence are conjugated to surfaces of AuNPs. The hybridization process between complementary strands of H7 Hemagglutinin gene and its probe brings the energy donor and acceptor into close proximity, leading to the quenching of fluorescence of UCNPs. A linear response is obtained ranging from 10 pm to 10 nm and the limit of detection (LOD) is around 7 pm with detection time around 2 hours. This biosensor is expected to be a valuable diagnostic tool for rapid and sensitive detection of AIV.

Surveillance on Avian Influenza H5N1 and H9N2 Subtypes In Egypt 2012-2013

Surveillance on Avian Influenza Virus in Egypt during 2012 - 2013 was undertaken to update the epidemiology of avian influenza virus infections among poultry flocks in Egypt. A total of 148 commercial chicken farms were tested by single-plex RT-PCR for direct detection of avian influenza viruses using type A specific nucleoprotein (NP) gene primer sets. The results revealed 5 positive chicken flocks infected with type-A AIV. These viruses were successfully isolated and propagated in SPF eggs. H5N1/HI test was positive only for three chicken flocks. Subtype specific (RTPCR) revealed 3 positive H5 chicken flocks (2%) and 2 positive H9 chicken flocks (1.35%). All examined chicken flocks were negative for H7 subtype. Multiplex RT-PCR using H5, H7 and H9 subtypes specific primers confirmed these results. This shows the continuous co-circulation of the AIV H5 and H9 in commercial chicken flocks in Egypt complicating the respiratory problems in affected flocks.

Rapid identification of H5 avian influenza virus in chicken throat swab specimens using microfluidic real-time RT-PCR

Avian influenza virus H5 is a widespread virus among humans and animals which has caused fatally systemic diseases through poultry-to-person transmission in the past few years. Reverse transcription polymerase chain reaction (RT-PCR) has been proved to be an effective approach for the identification and detection of avian influenza viruses. However, conventional tube RT-PCR is slow and reagent consuming and cannot meet the need for rapid and low cost detection of pathogenic bacteria and viruses. Microfluidic PCR is a burgeoning field among the techniques based on molecular analysis. In this paper, we reported a microfluidic PCR system that integrated RT-PCR and real time fluorescence detection for rapid identification of avian influenza virus H5. This microfluidic device mainly consisted of a thermal controlling unit providing actuation for the temperature cycling needed for amplification, an optical inspection system for online recording fluorescence and a microfluidic chip fabricated using polydimethylsiloxane (PDMS). In this study, influenza virus H5 from clinical chicken throat swab specimens was rapidly detected using the RT-PCR microfluidic system, which was consistent with the results of embryonated egg culture. Compared with a large-scale device, the integrated microfluidic system presented here can perform rapid nucleic acid amplification and analysis, possibly making it a crucial platform for pathogenic bacterium and virus detection in the future.

Production and Characterization of Monoclonal Antibodies Against Nucleoprotein of Avian Influenza Virus

The present study was carried out with an aim to develop anti-nucleoprotein (anti-NP) monoclonal antibodies (MAbs) for use in immunodiagnostic testing for detection of avian influenza virus (AIV) antigen or antibodies. The NP gene of AIV, cloned in pET vector, was expressed in Escherichia coli BL 21 strain to produce a 6x-His tagged recombinant NP (rNP) antigen of ∼61 kDa molecular weight as soluble fraction. The rNP antigen was detected in soluble fraction of bacterial cell lysate with anti-His HRPO conjugate and reacted with the reference AIV antibody positive serum in immunoblotting. The rNP was used to immunize BALB/c mice to produce hybridoma secreting anti-NP MAbs. Out of 11 anti-NP MAbs produced, 8D2-H5, 8D2-H9, and 6D11-A7 were of IgM isotype and 5D10-C9 and 5D10-F11 were of IgG2b type, while 3F3-D2, 7D2-C9, 7D2-G7, and 7D2-G8 were of IgG1 isotype. The MAbs 3F3-D2 and 7D2-G8 showed high intensity positive reaction with rNP and a low intensity reaction with H5N1 virus in Western blot analysis. The anti-NP MAbs produced in the present work may be valuable in developing a competitive ELISA or immunochromatographic strip test-based assays for the rapid diagnosis of avian influenza.

Label-free microcantilever-based immunosensors for highly sensitive determination of avian influenza virus H9

We report on label-free immunosensors for the highly sensitive detection of avian influenza virus. The method makes use of the microcantilevers of an atomic force microscope onto which monoclonal antibodies against avian influenza virus were covalently immobilized. The factors influencing the performance of the resulting immunosensors were optimized by measuring the deflections of the cantilever via optical reflection, and this resulted in low detection limits and a wide analytical range. The differential deflection signals revealed specific antigen binding and their intensity is proportional to the logarithm of the concentrations of the virus in solution. Under optimal conditions, the immunosensors exhibit a linear response in the 7.6 ng mL−1 to 76 μg mL−1 concentration range of avian influenza virus, and the detection limit is 1.9 ng mL−1. FigureLabel-free immunosensors based on microcantilevers of an atomic force microscope was fabricated by covalently immobilizing monoclonal antibodies to avian influenza virus onto the microcantilever. The performance and factors influencing the performance of the resulting immunosensors were investigated in detail by measuring the cantilever deflections using the optical reflection technique. Electronic supplementary materialThe online version of this article (doi:10.1007/s00604-013-1129-x) contains supplementary material, which is available to authorized users.