Goat Anti-mouse IgG Antibody Research Articles

Highly sensitive lateral flow immunoassays based on Ag@Au nanoflowers with marine medaka (Oryzias melastigm) vitellogenin as a target analyte

In order to improve the sensitivity of lateral flow immunoassays (LFIAs) for the detection of piscine vitellogenin (Vtg), a well-established biomarker for environmental estrogens, Au coated Ag nanoflowers (Ag@Au NFs) were used as labeling probes to develop a LFIA for marine medaka Vtg. The synthesized Ag@Au NFs with good monodispersity had an average diameter of 44.1 nm and absorbance peak of 524 nm. When the concentration of goat anti-mouse IgG and anti-Vtg polyclonal antibody (anti-Vtg PAbs) were 1.3 and 0.4 mg/mL, respectively, the detection range of the LFIA was 0.19–25 ng/mL, and the visual detection limit was 0.1 ng/mL, which was approximately 80 times lower than that of LFIAs based on other nanoparticles (Au NPs, Ag NPs, Au NFs, and FM). After evaluation of its specificity and robustness, the usefulness of Ag@Au NFs labeled LFIA was validated by measuring Vtg induction in the plasma of marine medaka exposed to bisphenol A, a weak estrogenic chemical. This highly sensitive lateral flow immunoassay could detect Vtg biomarker within 15 min without the need of expensive and complicated instruments, and thus offered an ultrasensitive and robust on-site detection method for estrogenic activity in field environment.

Establishment of 11-dehydro-thromboxane B2 time-resolved immunoassay and application in membranous nephropathy

Background11-Dehydro-thromboxane B2 (11-dehydro-TXB2) is the final stable metabolite of thromboxane A2 (TXA2) and is involved in thrombus formation. Patients with membranous nephropathy (MN) are prone to thromboembolism events. MethodsTime-resolved fluorescence immunoassay (TRFIA) for 11-dehydro-TXB2 was established by indirect competitive method. The coated 11-dehydro-TXB2-BSA conjugate was used to bind the 11-dehydro-TXB2 antibody competitively to the 11-dehydro-TXB2 antigen in the samples, followed by Eu3+-labeled goat anti-mouse IgG antibody, to detect 11-dehydro-TXB2. This study measured 11-dehydro-TXB2 concentrations in serum samples from healthy individuals and patients with MN. ResultsThe linear range of TRFIA was 16.38–2000 pg/mL, the sensitivity was 4.70 pg/mL, the average coefficients of variation from intra-assay and inter-assay were 3.50% and 4.95%, respectively, and the recovery was 99.38%. The serum level of 11-dehydro-TXB2 in patients with MN was significantly higher than that in healthy subjects (P < 0.05). The serum 11-dehydro-TXB2 concentration detected by TRFIA was highly consistent with that by ELISA (ρ = 0.900). DiscussionThis study successfully established a new highly sensitive method for the detection of 11-dehydro-TXB2 in serum. 11-Dehydro-TXB2 has great potential in evaluating the risk of thromboembolic events in patients with MN and is expected to be applied to other thromboembolic-related diseases.

An integrated immunochromatographic device for C-reactive protein detection using hierarchical dendritic gold nanostructure films

Immunochromatographic test strips typically consist of sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. Even minute variations in the assembly of these components can lead to inconsistent sample-reagent interactions, thereby reducing reproducibility. In addition, the nitrocellulose membrane is susceptible to damage during assembly and handling. To address this issue, we propose to replace the sample pad, conjugate pad, and nitrocellulose membrane with hierarchical dendritic gold nanostructure (HD-nanoAu) films to develop a compact integrated immunochromatographic strip. The strip uses quantum dots as a background fluorescence signal and employs fluorescence quenching to detect C-reactive protein (CRP) in human serum. A 5.9 μm thick HD-nanoAu film was electrodeposited on an ITO conductive glass by the constant potential method. The wicking kinetics of the HD-nanoAu film was thoroughly investigated, and the results indicated that the film exhibited favorable wicking properties, with a wicking coefficient of 0.72 μm ms−0.5. The immunochromatographic device was fabricated by etching three interconnected rings on HD-nanoAu/ITO to designate sample/conjugate (S/C), test (T), and control (C) regions. The S/C region was immobilized with mouse anti-human CRP antibody (Ab1) labeled with gold nanoparticles (AuNPs), while the T region was preloaded with polystyrene microspheres decorated with CdSe@ZnS quantum dots (QDs) as background fluorescent material, followed by mouse anti-human CRP antibody (Ab2). The C region was immobilized with goat anti-mouse IgG antibody. After the samples were added to the S/C region, the excellent wicking properties of the HD-nanoAu film facilitated the lateral flow of the CRP-containing sample toward the T and C regions after binding to AuNPs labeled with CRP Ab1. In the T region, CRP-AuNPs-Ab1 formed sandwich immunocomplexes with Ab2, and the fluorescence of QDs was quenched by AuNPs. The ratio of fluorescence intensity in the T region to that in the C region was used to quantify CRP. The T/C fluorescence intensity ratio was negatively correlated with the CRP concentration in the range of 26.67–853.33 ng mL−1 (corresponding to 300-fold diluted human serum), with a correlation coefficient (R2) of 0.98. The limit of detection was 15.0 ng mL−1 (corresponding to 300-fold diluted human serum), and the range of relative standard deviation: 4.48–5.31%, with a recovery rate of 98.22–108.33%. Common interfering substances did not cause significant interference, and the range of relative standard deviation: 1.96–5.51%. This device integrates multiple components of conventional immunochromatographic strips onto a single HD-nanoAu film, resulting in a more compact structure that improves the reproducibility and robustness of detection, making it promising for point-of-care testing applications.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

A centrifugal microfluidic chip for point-of-care testing of staphylococcal enterotoxin B in complex matrices.

Staphylococcal enterotoxin B (SEB) is a typical biological toxin that causes food poisoning. Currently reported SEB detection methods have the drawbacks of sophisticated sample preparation and being time-consuming and labor-intensive. Herein, we propose a strategy based on an immune sandwich structure operating on a centrifugal microfluidic chip for point-of-care testing (POCT) of SEB. The fluorescent microparticle-labeled primary antibody (CM-EUs-Ab1), capture antibody (CAb), and goat anti-mouse IgG antibody (SAb) were modified on the bond area, T-area, and C-area, respectively. When SEB was added, it first reacted with the CM-EUs-Ab1 through the specific recognition between SEB and the Ab1. Then, under capillarity, the conjugates of SEB and the CM-EUs-Ab1 were captured by the CAb when they flowed to the T-area, and the remaining CM-EUs-Ab1 bound with the SAb in the C-area. Finally, this chip was put into a dry fluorescence detection analyzer for centrifugation and on-site detection of SEB. The fluorescence intensity ratio of the T-area to the C-area was positively correlated with the concentration of SEB. The resulting linear range was 0.1-250 ng mL-1, and the limit of detection (3σ/k) was 68 pg mL-1. This POCT platform only needs 20 μL of sample and can realize the full process of detection within 12 min. This chip also exhibits good stability for 35 days. Additionally, the proposed method has been successfully utilized for the detection of SEB in urine, milk, and juice without any pre-treatment of the samples. Thus, this platform is expected to be applied to food safety testing and clinical diagnosis.

Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses

BackgroundH7N9 avian influenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection.MethodsThe H7N9 AIV was inactivated and purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared.ResultsThe analysis of ELISA and virus neutralization test showed that the obtained McAbs specifically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log10EID50/0.1 mL for chicken swab samples.ConclusionThe McAbs were specific for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.

Development of a Colloidal Gold Immunochromatographic Strip for the Rapid Detection of Channel Catfish Virus.

Channel catfish virus disease (CCVD) has resulted in great economic losses and has restricted the development of fisheries. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of CCVD. A colloidal gold immunochromatographic strip has been developed for the detection of CCVD. In this study, a colloidal gold immunochromatographic strip for channel catfish virus (CCV) detection was developed using the monoclonal antibody 8B6 conjugated with colloidal gold as the detector antibody. A rabbit anti-CCV antibody was used as the capture complex at the test line, and a goat anti-mouse IgG antibody was used as the capture antibody at the control line. The strip was characterized in its specificity, sensitivity, and stability. In addition, an infection experiment was performed to test the applicability of the test strip. The strip was able to detect concentrations of the virus (104 tissue culture infective dose (TCID50)/mL) and showed analytical specificity when tested against other viral pathogens. The strips were still usable after 30 days of storage at 60°C. It was possible to detect CCV experimentally in infected fish within 10-15 min of using the strip. The strip can be used as a rapid and convenient tool for on-site diagnosis to control outbreaks and the spread of CCVD. The immunochromatographic strip was the first to be developed and applied for the detection of CCVD.

Development of a quantum dot nanobead-based fluorescent strip immunosensor for on-site detection of aflatoxin B1 in lotus seeds

Owing to the serious threat of aflatoxin B1 (AFB1) to public health, development of a reliable method for accurate determination of it is extremely necessary and urgent. In this study, a simple, rapid and highly-sensitive quantum dot nanobeads (QBs) based lateral flow fluorescent strip immunosensor was developed for on-site detection of AFB1 in edible and medicinal lotus seeds. Carboxylated QBs were used as the fluorescent markers to prepare the fluorescent probe through coupling QBs with anti-AFB1 antibodies. Bovine serum albumin (BSA)-AFB1 antigens and goat anti-mouse IgG antibodies were coated on the nitrocellulose (NC) membrane to prepare the test (T) and control (C) lines, respectively. Qualitative analysis of AFB1 was realized by naked eye, and the quantitative determination was achieved with a portable strip reader. Results showed that the newly-developed test strip sensor could achieve rapid detection of AFB1 within 15 min, allowing a limit of detection (LOD) of 1 ng/mL (2 μg/kg) and a linear range of 1–19 ng/mL (2–38 μg/kg). Recovery rates from the fortified lotus seeds with low, medium and high spiking concentrations (2.5, 5 and 10 μg/kg) ranged from 94.0% to 116.0% with relative standard deviations less than 10%. All the results were confirmed by a standard LC-MS/MS method. The QBs-based fluorescent strip immunosensor with high sensitivity, easy operation, and low cost provided a preferred solution for rapid, on-site screening and highly-sensitive quantitation of AFB1 in a large number of lotus seed samples.

Development and evaluation of colloidal gold immunochromatography test strip for rapid diagnosis of nervous necrosis virus in golden grey mullet (Chelon aurata)

A lateral flow immunochromatography strip test, based on antibody-gold nanoparticles specific for nervous necrosis virus (NNV), was developed for rapid, on-site detection of the virus in fish stocks. A monoclonal antibody against NNV was conjugated with colloidal gold as the detector antibody. A rabbit anti-NNV polyclonal antibody and goat anti-mouse IgG antibody were blotted onto the nitrocellulose membrane as the capture antibodies on the test line and control line, respectively. The reaction could be seen by the eye within 15min and did not cross-react with the other viruses tested. The detection limit of the strip was approximately 103 TCID50 /ml and had good stability after storage at 4°C for 8months. When brains of 70 naturally infected golden grey mullet, Chelon aurata, were tested with the strip test, the diagnostic specificity and sensitivity of the test compared to real-time RT-PCR were 100% and 74%, respectively. Therefore, the one-step test strip developed here had high specificity, reproducibility, and stability. This, together with its simplicity to use and rapid detection, without the requirement of sophisticated equipment or specialized skills, makes the strip suitable for pond-side detection of NNV in farmed fish.

Opsonization of multiple drug resistant (MDR)-bacteria by antimicrobial peptide fused hepatitis B virus surface antigen (HBsAg) in vaccinated individuals

Vaccination evoking immunity in susceptible individuals has become the most effective solution to combat infectious diseases. The surface antigen of hepatitis B virus (HBsAg) is a mandatory vaccine for children in China. Herein, we designed an antimicrobial protein consisting of an antimicrobial peptide Thanatin at the N-terminus fused with the HBsAg at the C-terminus. The expressed Thanatin-GFP-HBsAg (TGH) quantitively bound with the anti-HBsAg antibody by ELISA, and after exposure to TGH, Gram-negative E. coli cells became fluorescencent indicating the binding of TGH with the bacterial cells. We also demonstrated that TGH could intercalated into the lipid bilayer as shown by the quartz crystal microbalance with dissipation (QCM-D) and TEM. Moreover, the TGH bound E. coli cells attracted anti-HBsAg IgG as shown by the experiments that in turn treated the E. coli cells with TGF, anti-HBsAg serum and PE labelled goat anti-mouse IgG antibodies. After supplementation with serum from HBsAg vaccinated individuals, TGH showed improved bactericidal effect in vitro. In vivo experiments showed that the mice receiving TGH vaccination show quicker clearance of MDR E. coli pretreated with TGH and better survive in comparison with groups treated with piperacillin plus subatan. In addition, anti-HBsAg serum supplementation also improved the endocytosis of TGH decorated bacteria by leukocytes. This study reported a novel solution to combat infectious pathogens based on the membrane penetrating effect of antimicrobial peptides.

Development of a colloidal gold immunochromatographic strip for rapid detection of Riemerella anatipestifer in ducks

Riemerella anatipestifer is one of the major bacterial pathogens of ducks and causes significant economic losses in poultry agriculture. Usually, methods for detecting R. anatipestifer infection need specialized equipment and highly skilled personnel. In this study, a novel colloidal gold immunochromatographic strip was developed for rapid detection of R. anatipestifer in ducks. The monoclonal antibodies 2D5 and 2A6 against R. anatipestifer were used as colloidal gold-labeled protein and capture protein, respectively, to recognize the bacteria in tryptic soy broth medium culture and in hearts of infected ducks. The goat anti-mouse IgG antibody was labeled on nitrocellulose membrane as a control for C line. The labeling pH was optimized as 10.0, and the concentration of 2D5 labeled to colloidal gold particles was optimized as 18 μg/mL. The strip specifically detected serotypes 1, 2, and 10 R. anatipestifer strains and showed no cross-reaction with Escherichia coli, Salmonella enterica, and Pasteurella multocida strains. The sensitivity of the strip for detecting R. anatipestifer was 1.0 × 106 colony forming unit. The strips remained stable for up to 8 mo at 4°C, and the detection can be completed within 15 min. The strip can detect R. anatipestifer in hearts of the ducks experimentally infected with R. anatipestifer but not infected with E. coli, which were also confirmed with bacterial isolation followed by multiplex polymerase chain reaction. These results suggested that the strips are reliable methods for identification of R. anatipestifer in laboratories and in duck farms.

Functionalization of silicon dioxide and silicon nitride surfaces with aminosilanes for optical biosensing applications

AbstractThe development of optical biosensors based on silicon dioxide or silicon nitride transducers requires the chemical activation of their surface to achieve stable, repeatable and homogeneous binding of biomolecules. In the present study, the chemical activation of silicon dioxide and silicon nitride surfaces with 3‐aminopropyl‐triethoxysilane (APTES) was optimized so as to enable the immobilization of biomolecules by adsorption or covalent bonding. Chemical activation was performed with either aqueous or organic solution of APTES, and the surfaces were used to immobilize directly protein molecules by physical adsorption or further modified with glutaraldehyde to allow covalent binding of protein molecules. The protein immobilization capacity of the chemically activated silicon dioxide and silicon nitride surfaces was evaluated through incubation with mouse γ‐globulins and reaction with a fluorescently labelled goat antimouse IgG antibody. By determining the surface fluorescence signal intensity, it was found that modification with 5% (v/v) APTES solution in ethanol followed by modification with glutaraldehyde provided 30% higher fluorescence signals than all the other protocols tested. In addition, this method provided the lower signal variation between different chips. To test the possible advantages of the chemical activation protocols for optical biosensing applications, they were also applied to a label‐free white light interference spectroscopy sensor and evaluated through (a) real‐time monitoring of the reaction between immobilized on the sensor surface mouse γ‐globulins with an unlabelled goat antimouse IgG antibody and (b) a non‐competitive immunoassay for the determination of C‐reactive protein. The results showed that in case of antibody, physical absorption provided marginally higher binding capacity to covalent bonding.

Development and application of a colloidal gold test strip for the rapid detection of the infectious laryngotracheitis virus

Infectious laryngotracheitis disease is an acute, highly contagious viral disease seriously affecting poultry industry worldwide. In this study, a rapid and simple immune colloidal gold test strip for detecting infectious laryngotracheitis virus (ILTV) was developed based on membrane chromatography with monoclonal antibodies (mAbs) against gJ protein of ILTV and systematically evaluated for the detection of ILTV from clinical samples. mAb 2D4 1D7 was conjugated with colloidal gold as the detector antibody on the test strip. Another mAb, 1D8 1G3, was used as the capture complex at the test line (T-line), and goat antimouse IgG antibody was used as the capture antibody at the control line (C-line). The colloidal gold test strip showed high specificity in the detection of ILTV, with no cross-reaction with other avian pathogens, including infectious bronchitis virus, infectious bursal disease virus, avian influenza virus, Newcastle disease virus, fowl adenoviruses, and Marek's disease virus. Besides, the detection limit of this method was as low as 60 ELD50/mL for the ILTV Wanggang strain. Furthermore, we evaluated its application in 260 clinical samples suspected of infection with ILTV. Results from the strip test were nearly identical with those from real-time PCR (coincidence rate 99.6%) and showed higher sensitivity than conventional PCR. All the results obtained in this study indicated that the colloidal gold test strip can be applied as a simple, rapid, sensitive, and specific diagnostic tool for the detection of ILTV, especially in resource-limited areas.

Generation and evaluation of anti-mouse IgG IgY as secondary antibody

In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37 °C for 84 h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.

Ultrasensitive detection of praziquantel in sea bass (Lateolabrax japonicus) using a lateral flow immunochromatographic assay

ABSTRACT Praziquantel (PZQ) is a compound that usually used as insecticide, drug residue could be harm to human health. In this study, we have developed a simple, sensitive, and rapid lateral flow immunochromatographic assay (LFIA) for the detection of PZQ in fish (Lateolabrax Japonicus). We produced a sensitive mAb using enzyme-linked immunosorbent assay with half-maximal inhibitory concentration (2A11, IC50) of 2.06 ng/mL. The coating antigen (PZQ-HS-GA-OVA) and goat anti-mouse IgG antibody were sprayed onto a nitrocellulose membrane as the test and control lines, respectively. Under optimal conditions, the cut-off limits for PZQ detection on the test strips were determined to be 5 ng/mL in both 0.01 M phosphate buffered saline and fish samples, and results were obtained within 5 min. Our data suggests that this LFIA can be used for the sensitive, rapid, and specific on-site screening for PZQ in fish.

Detection of aminophylline in serum using an immunochromatographic strip test

ABSTRACT Aminophylline (AMP), a bronchodilator drug, is a modified form of theophylline (the pharmacological active ingredient). AMP is often given to treat respiratory diseases, including bronchial asthma, blocked emphysema, panting bronchitis, and cardiogenic asthma. In this study, we developed a rapid and sensitive method to determine AMP concentration in mouse serum. A sensitive monoclonal antibody against AMP was produced by immunizing BALB/c mice with a well-characterized AMP2-keyhole limpet hemocyanin and coating antigen (hapten-AMP1-OVA). The coating antigen and goat anti-mouse IgG antibody were sprayed onto a nitrocellulose membrane representing the test and control lines, respectively. Under optimized conditions, the cut-off limits of the test strip were 50 ng/mL in 0.01 M phosphate buffered saline (pH 7.4) and 250 ng/mL in serum. The results were obtained within 10 min and revealed that this immunochromatographic strip test is a sensitive, rapid, and simple tool for the detection of AMP in serum.

Development of an immunochromatographic strip test for rapid detection of sodium nifurstyrenate in fish

ABSTRACT A rapid, specific and sensitive method based an immunochromatographic strip test has been developed for the determination of sodium nifurstyrenate (NFS) residues in fish. Sample preparation procedure includes ultrasonic bath extraction with acetonitrile, sample centrifugation at 8000 rpm for 10 min, filtration through the 0.22 µm membrane filter, and drying over nitrogen (60°C) and res-suspension. The antigen (NFS-ovalbumin) and a goat anti-mouse IgG antibody were drawn onto a nitrocellulose membrane as the test line and control line, respectively. Under optimized conditions, the NFS cut-off limits for test strips was determined as 5 ng/mL in 0.01 M phosphate buffered saline and 10 ng/mL in fish. Results were obtained within 5 min. Our data suggests this immunochromatographic assay can be used for the sensitive, rapid, and specific on-site screening for NFS in fish.

Rapid detection of iprodione in cucumber and apple using an immunochromatographic strip test

ABSTRACT In recent years, fungicides have been frequently detected in soil, water, and air because of their extensive use, with detrimental effects on the environment. Therefore, the screening of foods for iprodione (IPRO) has become mandatory. A rapid, specific and sensitive method based on an immunochromatographic strip test has been developed for the determination of IPRO residues in cucumber and apple. The antigen IPRO-ovalbumin and a goat anti-mouse IgG antibody were drawn onto a nitrocellulose membrane as the test and control lines, respectively. Under optimal conditions, the cut-off limits of the semi-quantitative strip test for IPRO was as low as 10 ng/mL in 0.01 M PBS pH 7.4, 20 ng/mL in cucumber juice, and 25 ng/mL in apple juice and the results were obtained within 5 min. Our data suggests that this immunochromatographic assay can be used for the sensitive, rapid, and specific on-site screening of IPRO in cucumber and apple.

Detection of clothianidin residues in cucumber and apple juice using lateral-flow immunochromatographic assay

ABSTRACT Clothianidin (CTA), third generation neonicotinoid insecticide that has been widely used for the long-term control of a wide variety of pests. Therefore, we developed immunochromatographic assay for the detection of CTA residues in cucumber and apple juice. The antigen HCTA-EDC-OVA and the goat anti-mouse IgG antibody were added to a nitrocellulose membrane as the test and control lines, respectively. Under optimal conditions, the cut-off limits of the semi-quantitative strip test for CTA were 1 ng/mL in 0.01 M PBS (pH 7.4), 4 ng/mL in cucumber juice, and 5 ng/mL in apple juice, and the results were obtained within 5 min. The immunochromatographic assay can be used for the sensitive, rapid, and specific on-site screening of CTA residues in cucumber and apple juice.

Determination of 3.5-dinitro-N’-(5-nitrofurfurylidene) salicylic acid hydrazide in fish using immunochromatographic strip tests

ABSTRACT The nitrofurans were recently banned from use in livestock production due to their potential carcinogenicity in humans. Therefore, the screening of foods from animal origins for nitrofurans became mandatory. A rapid, specific and sensitive method based on immunochromatographic strip test has been developed for the determination of 3.5-dinitro-N’-(5-nitrofurfurylidene) salicylic acid hydrazide (DNSH) residues in fish. Fish samples were prepared by ultrasonic bath extraction, centrifugation, filtration, drying and res-suspension. The antigen (DNSH-glutaraldehyde-ovalbumin) and a goat anti-mouse IgG antibody were drawn onto a nitrocellulose membrane as the test and control line, respectively. Under optimal conditions, the DNSH cut-off limits for the test strips were determined as 10 ng/mL in 0.01 M phosphate buffered saline and 25 ng/mL in fish samples, with results obtained within 5min. Our data suggests this immunochromatographic assay can be used for the sensitive, rapid, and specific on-site screening of DNSH in fish products.