Immunochromatographic Strip Test Research Articles

Development and application of a high-sensitivity immunochromatographic test strip for detecting pseudorabies virus

IntroductionPseudorabies (PR) is a multi-animal comorbid disease caused by pseudorabies virus (PRV), which are naturally found in pigs. At the end of 2011, the emergence of PRV variant strains in many provinces in China had caused huge economic losses to pig farms. Rapid detection diagnosis of pigs infected with the PRV variant helps prevent outbreaks of PR. The immunochromatography test strip with colloidal gold nanoparticles is often used in clinical testing due to its low cost and high throughput.MethodsThis study was designed to produce monoclonal antibodies targeting PRV through immunization of mice using the eukaryotic system to express the gE glycoprotein. Subsequently, paired monoclonal antibodies were screened based on their sensitivity and specificity for use in the preparation of test strips.Results and discussionThe strip prepared in this study was highly specific, only PRV was detected, and there was no cross-reactivity with glycoprotein gB, glycoprotein gC, glycoprotein gD, and glycoprotein gE of herpes simplex virus and varicellazoster virus, porcine epidemic diarrhea virus, Senecavirus A, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus. Moreover, it demonstrated high sensitivity with a detection limit of 1.336 × 103 copies/μL (the number of viral genome copies per microliter); the coincidence rate with the RT-PCR detection method was 96.4%. The strip developed by our laboratory provides an effective method for monitoring PRV infection and controlling of PR vaccine quality.

Rapid, on-site quantitative determination of mycotoxins in grains using a multiple time-resolved fluorescent microsphere immunochromatographic test strip

Rapid, on-site quantitative determination of mycotoxins in grains using a multiple time-resolved fluorescent microsphere immunochromatographic test strip

Development of an immunochromatographic test strip to detect pneumolysin by monoclonal antibody capture method

BackgroundThe currently available methods for the detection of Streptococcus pneumoniae are labour-intensive, and involve time-consuming steps needing skilled technicians to perform the assays. Therefore, we developed a rapid lateral flow based immunochromatographic test strip for detection of S. pneumoniae using pneumolysin using monoclonal antibody capture approach. Methodology − The strip was designed by assembling a sample pad, conjugation pad, nitrocellulose membrane and adsorbent pad supported by a plastic backing card. Optimization of control spot was done by using three different concentrations of monoclonal antibody (0.1 µg, 0.25 g and 0.5 µg). Monoclonal antibody coupled with gold nanoparticles was immobilized on conjugate pad. Recombinant pneumolysin at three different concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) was used. Diluted pneumolysin was poured onto the sample pad which migrated through the conjugate pad containing gold antibody conjugate and the antigen–antibody complex migrated to the test spot to bind to the polyclonal antibody. The remaining free antibody bound to the control spot to visualize a control spot. Results: Control spot was optimized at 0.5 µg of mouse IgG. Of three different concentrations of Pneumolysin, the spot was best visualized at 50 ng/ml Pneumolysin concentration. Conclusion: Our ICT based device could detect a commercially available recombinant Pneumolysin of S. pneumoniae. Future study is directed to validate the strip for detection of Pneumolysin using biological fluids of patients (particularly children) infected with S. pneumoniae.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

3D Printer-Controlled syringe pumps for Dual, Active, regulable and simultaneous dispensing of reagents. Manufacturing of immunochromatographic test strips

Lateral flow immunoassays (LFIA) are widely used worldwide for the detection of different analytes because they combine multiple advantages such as low production cost, simplicity, and portability. They allow biomarkers detection without requiring infrastructure or highly trained personnel. In this work we propose solutions for the manufacturing process of LFIA at laboratory-scale, particularly to the controlled and active dispensing of the reagents in the form the Test Lines (TL) and the Control Lines (CL). To accomplish this task, we adapted a 3D printer to also control Syringe Pumps (SP), since the proposed adaptation of a 3D printer is easy, free and many laboratories already have it within their infrastructure. In turn, the standard function of the 3D printer can be easily restored by disconnecting the SPs and reconnecting the extruder. Additionally, the unified control of the 3D printer enables dual, active, regulable and simultaneous dispensing, four features that are typically found only in certain high-cost commercial equipment. With the proposed setup, the challenge of dispensing simultaneously at least 2 lines (CL and TL) with SPs controlled by a 3D printer was addressed, including regulation in the width of dispensed lines within experimental limits. The construction of a LFIA for the detection of leptospirosis is shown as a practical example of the automatized reagent dispensing.

Functional Green-Emitting Mn2+-doped Zinc Germanate Persistent Luminescent Nanoparticles for Dual-Mode Immunochromatographic Detection.

Immunochromatography is a commonly used immediate detection technique, using signal labels to generate detection signals for rapid medical diagnosis. However, its detection sensitivity is affected by background fluorescence caused by the excitation light source. We have developed an immunochromatographic test strip using Zn2GeO4:Mn2+ (ZGM) persistent luminescent nanoparticles (PLNPs) for immediate fluorescence detection and highly sensitive persistent luminescence (PersL) detection without background fluorescence interference. ZGM emits a strong green light when exposed to ultraviolet (UV) excitation, and its green PersL can persist for over 30 min after the excitation light is turned off. We modified the surface of ZGM with heparin-binding protein (HBP) antibodies to create immunochromatographic test strips for the detection of HBP as the target analyte. Under UV excitation, the chromatography test paper can be visually observed at concentrations as low as 25 ng/mL. After the excitation light source is switched off, PersL can achieve a detection limit of 4.7 ng/mL without background interference. This dual-mode immunochromatographic detection, based on ZGM, shows great potential for in vitro diagnostic applications.

Development of a highly sensitive colloidal gold semiquantitative method for the determination of difenoconazole residues in citrus.

Difenoconazole (DIFE) is a common pesticide used in citrus cultivation; excessive intake can cause neurological damage to the organism, and the existing colloidal gold immunochromatographic test strips cannot meet the requirements for the detection of citrus samples. Difenoconazole test strip was prepared based on the colloidal gold immunochromatographic technique (GICT), and its application in citrus samples was investigated; with colloidal gold (CG) as the probe, the optimization of GICT parameters, and the determination of reaction method, the immunochromatographic test strips for the detection of DIFE in citrus was developed, and the limit of detection (LOD), specificity, accuracy, and stability of the test strips were verified. The results showed that the visual detection limit of the prepared colloidal gold immunochromatographic test strips was 0.2 mg/kg and the quantitative range was 0.06-0.6 mg/kg, and the test strips could specifically identify DIFE and have no cross-reaction with other common triazole pesticides. The detection method established in this study was verified by the GC-MS method, and the detection results achieved good consistency (R2 > 0.98). The test strips developed in this study have good performance and can be used for highly sensitive detection of citrus samples.

Development of Monoclonal Antibodies and Immunochromatographic Test Strips Applying Indirect Competitive Method for Daidzein Detection

Development of Monoclonal Antibodies and Immunochromatographic Test Strips Applying Indirect Competitive Method for Daidzein Detection

A rapid and sensitive time-resolved fluorescence microsphere immunochromatographic test strip for bongkrekic acid detection in edible fungi

Bongkrekic acid (BA) is a prevalent biological toxin in black fungus, tremella and fermented rice flour food. BA usually causes acute poisoning. Developing a rapid on-site detection method for BA is essential for timely prevention. This work proposed a time-resolved fluorescent microsphere immunochromatographic test strip (TRFM-ICTS) for the quantitative determination of BA in edible fungi. The process involved synthesizing BA artificial antigens and fluorescent probes. An independent quality control system was implemented. This system ensured the integrity of the control line (C line) even when the test line (T line) was present. Under the optimal conditions, the linear range of TRFM-ICTS was 0.1–10 ng/mL, and the half inhibitory concentration (IC50) was 0.264 ng/mL. The detection limit of the developed test strip (0.282 ng/mL) was 20 times lower than that of colloidal gold immunochromatographic test strip (5.7 ng/mL). Moreover, TRFM-ICTS showed good recovery in spiked samples, ranging from 81.22% to 119.48%, and the coefficient of variation (CV) was less than 7.76%. In comparison to conventional instrumental techniques, the implemented TRFM-ICTS demonstrated a highly sensitive, straightforward, expeditious, and feasible approach for the quantification of BA in food.

Development of highly sensitive dual-enhanced fluorescence quenching immunochromatographic test strips based on Pt nanoprobes

The fluorescence-quenching method is crucial in vitro analysis, particularly for immunochromatographic test strips (ICTs) using noble metal nanoparticles as probes. However, ICTs still fall short in meeting the requirements for the detection of traces biomarkers due to the noble metal nanoparticles can only quench fluorescence of the dyes within a confined distance. Interestingly, noble metal nanoparticles, such as Pt NPs cannot only perform fluorescence-quenching ability based on the Förster resonance energy transfer (FRET), but also show perfect oxidase-like catalytic performance on many kinds of substrates, such as 3,3’,5,5’ -tetramethylbenzidine (TMB). We observed that the oxTMB (the oxidation products of TMB) exhibited notable effectiveness in quenching Cy5 fluorescence by the strong inner filter effect (IFE), which obviously improved the fluorescence-quenching efficiency with extremely low background signal. Through the dual-enhanced fluorescence quenching mechanism, the fluorescence quenching constant (Kn) was 661.24-fold that of only Pt NPs on the NC membrane. To validate the feasibility of this technique, we employed two types of biomarkers, namely microRNA (miR-15a-5p) and the signature protein (PSA). The sensitivity of miR-15a-5p was 9.286 × 10−18 mol/L and 17.5-fold more than that based on Pt NPs. As for the PSA, the LOD (0.6265 pg/mL) was 15.5-fold enhancement more sensitive after catalysis. Overall, the dual-enhanced fluorescence quenching rFICTs could act as a practical detection for biomarker in real samples.

A fluorescent immunochromatographic strip based on CsPbBr3/CsPb2Br5@SiO2 facile encapsulation in pure water for the detection of human chorionic gonadotropin (HCG) antigen

Fluorescent immunochromatographic test (FICT) strips have received extensive attention due to their virtue of simple operation, low cost, and a point-of-care (POC) assay. In recent years, people are constantly seeking new labels to improve the sensitivity of immunosensors. Perovskite nanocrystals (PNCs) possess strong fluorescence and a narrow emission peak, making them an excellent choice as a new fluorescent signal label. Herein, we have developed a simple, fast, and environmentally friendly method to synthesize CsPbBr3/CsPb2Br5@SiO2 with the good water stability and high biocompatibility. These CsPbBr3/CsPb2Br5@SiO2 have been applied to construct FICT strips. To achieve the quantitative determination of human chorionic gonadotropin (HCG), a lab-made FICT strips detection system was established. The FICT strips had a good linear relationship to HCG in the range of 5–500 ng/mL, and the detection limit was 2.09 ng/mL. In addition, the recovery results of HCG detection in human urine samples also proved the feasibility of this FICT strips. CsPbBr3/CsPb2Br5@SiO2 can also be modified with different biomarkers for functionalization, which exhibits a promising application in the development of sensing platform of FICT strips.

Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a.

Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 102 copies/μl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas.

Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species

BackgroundTimely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results.MethodsColloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera.ResultsAMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%–23.4% of these samples were found to be positive for T. gondii.ConclusionsThis study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.Graphical

Development of a colloidal gold immunochromatographic strip for rapid detection of ribavirin in pig urine

ABSTRACT A specific and sensitive monoclonal antibody (mAb) for ribavirin (RBV) was generated by immunizing BALB/c mice with RBV – bovine serum albumin. Using the mAb, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold immunochromatographic (CGI) test strip were developed for detecting RBV in pig urine. Under optimum assay conditions, IC50 was 0.08 ng/mL in 0.05 M phosphate-buffered saline and the recovery rate in real-world samples ranged between 77.3% and 99%. The cut-off limit of the CGI test strip for RBV was 5 ng/mL in pig urine. The cross-reactivity rate of the test strip to the tested antiviral drugs including acyclovir and moroxydine hydrochloride and the metabolites 1,2,4-triazole-3-carboxamide and 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxylic acid was below 0.1%. The results indicate that ic-ELISA and the CGI test strip have high sensitivity and specificity and that these methods are suitable for the rapid detection of RBV in pig urine.

Development of Colloidal Gold-Based Immunochromatographic Strips for Rapid Detection and Surveillance of Japanese Encephalitis Virus in Dogs across Shanghai, China.

Japanese encephalitis virus (JEV) causes acute encephalitis in humans and is of major public health concern in most Asian regions. Dogs are suitable sentinels for assessing the risk of JEV infection in humans. A neutralization test (NT) or an enzyme-linked immunosorbent assay (ELISA) is used for the serological detection of JEV in dogs; however, these tests have several limitations, and, thus, a more convenient and reliable alternative test is needed. In this study, a colloidal gold immunochromatographic strip (ICS), using a purified recombinant EDIII protein, was established for the serological survey of JEV infection in dogs. The results show that the ICSs could specifically detect JEV antibodies within 10 min without cross-reactions with antibodies against other canine viruses. The test strips could detect anti-JEV in serum with dilution up to 640 times, showing high sensitivity. The coincidence rate with the NT test was higher than 96.6%. Among 586 serum samples from dogs in Shanghai examined using the ICS test, 179 (29.98%) were found to be positive for JEV antibodies, and the high seropositivity of JEV in dogs in China was significantly correlated with the season and living environment. In summary, we developed an accurate and economical ICS for the rapid detection of anti-JEV in dog serum samples with great potential for the surveillance of JEV in dogs.

Determination of Morphine Using Carboxylated Multiwalled Carbon Nanotubes (MWCNTs) as the Label for an Immunochromatographic Assay (ICA)

This study evaluated the application of carboxylated multi-walled carbon nanotubes (MWCNTs) as a label in immunochromatographic assay for morphine detection. MWCNTs were carboxylated using nitric acid and characterized by Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, field emission scanning microscopy (FESEM), and dynamic light scattering (DLS). The optimum concentration of polyvinyl pyrrolidone (PVP) as a surfactant for homogenous dispersion of MWCNTs was determined by ultraviolet-visible spectroscopy to be 0.5 mg/mL. The zeta potential of MWCNTs before adding PVP was −41.9 ± 1.02 mV, and after adding PVP, −54.1 ± 0.25 mV. On the surface of MWCNTs, the anti-morphine antibody (AM antibody) had a binding effectiveness ranging from 91.1% to 95.5%. The AM antibody-MWCNTs were conjugated to create immunochromatographic test strips. The visual limit of detection (vLOD) was 1.0 ng/mL and the cutoff concentration (COC) was 10.0 ng/mL. Compared to previous study, carboxylated-MWCNTs as a one-dimensional nanomaterial improved the sensitivity of immunochromatographic assay strips and may be utilized to develop the sensitive immunochromatographic assays.

Development and evaluation of a test strip for the rapid detection of antibody against equine infectious anemia virus

Equine infectious anemia (EIA) is a contagious disease of horses caused by the equine infectious anemia virus (EIAV). The clinical signs at the acute phase include intermittent high fever, thrombocytopenia, hemorrhage, edema, and anemia. The clinical signs at chronic and relapsing subclinical levels include emaciation and progressive weakness. Surviving horses become lifelong carriers because of the integration of the viral genome into that of the host, and these horses can produce and transmit the virus to other animals. This increases the difficulty of imposing practical control measures to prevent epidemics of this disease. Serological tests measuring the antibodies in equine sera are considered to be a reliable tool for the long-term monitoring of EIA. However, the standard serological tests for EIV either have low sensitivity (e.g., agar gel immunodiffusion test, AGID) or are time consuming to perform (e.g., ELISA and western blotting). The development of a rapid and simple method for detecting the disease is therefore critical to control the spread of EIA. In this study, we designed and developed a colloidal gold immunochromatographic (GICG) test strip to detect antibodies against EIAV based on the double-antigen sandwich. Both the p26 and gp45 proteins were used as the capture antigens, which may help to improve the positive detection rate of the strip. We found that the sensitivity of the test strip was 8 to 16 times higher than those of two commercially available ELISA tests and 128 to 256 times higher than AGID, but 8 to 16 times lower than that of western blotting. The strip has good specificity and stability. When serum samples from experimental horses immunized with the attenuated EIAV vaccine (n = 31) were tested, the results of the test strip showed 100% coincidence with those from NECVB-cELISA and 70.97% with AGID. When testing clinical serum samples (n = 1014), the test strip surprisingly provided greater sensitivity and a higher number of “true positive” results than other techniques. Therefore, we believe that the GICG test strip has demonstrated great potential in the field trials as a simple and effective tool for the detection of antibodies against EIAV.Key points• A colloidal gold immunochromatographic (GICG) fast test strip was developed with good specificity, sensitivity, stability, and repeatability• The test strip can be used in point-of-care testing for the primary screening of EIAV antibodies• Both the p26 and gp45 proteins were used as the capture antigens, giving a high positive detection rate in the testing of experimentally infected animal and field samples

Development and characterization of a lateral flow immunochromatographic assay test strip for rapid detection of rubella virus antigen

Rubella is still considered a dangerous infectious disease, especially for pregnant women. The symptoms of rubella can be similar to those of other illnesses. Therefore, an early and accurate diagnosis of the rubella virus will help doctors make a reasonable treatment plan. This study presents the development and characterization of a novel lateral flow immunochromatographic assay test strip for rapid recognition of rubella virus antigen. Rabbit polyclonal antibodies against the rubella virus were purified with a purity of 95%. Optimal conditions for the production of the main components of a test strip were determined. Anti-rubella virus polyclonal antibodies were physically adsorbed onto the star-shaped gold nanoparticles at the optimal concentration of 1.0 μg/ml at 25°C for 30 min. The amounts of antibody per conjugate pad and immobilized on the test line were 0.2 μg/pad and 4.5 μg/cm, respectively. The properties of the test strip were characterized by testing mock clinical specimens. Reproducibility and repeatability were achieved at 100%. The limit of detection of the test strip was determined to be as low as 5 × 105 virus particles/mL. The test strip did not cross-react with six different potential interfering pathogens. The sensitivity and specificity of the test strip were determined by testing with 400 mock clinical samples and reached 99.5% and 98.5%, respectively. The Kappa statistics (k coefficient = 0.98) showed very good agreement between the generated test strip and real-time PCR results. According to the findings, the test strip can be used as an additional rapid test tool for the detection of the rubella virus.

A visual, rapid, and sensitive detection platform for Vibrio parahaemolyticus based on RPA-CRISPR/Cas12a and an immunochromatographic test strip

Abstract Objectives Vibrio parahaemolyticus is the primary species that causes vibriosis. In this study, a point-of-care detection method was developed for V. parahaemolyticus. Materials and Methods The detection platform targeted the thermolabile haemolysin (tlh) gene of V. parahaemolyticus based on recombinant polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR/Cas) systems. The platform was combined with an immunochromatographic test strip (ICS) that enables low-cost, simple, visual detection of V. parahaemolyticus. Results The detection limit was 2.5×102 fg/µL for plasmids and 1.4×102 CFU/mL for V. parahaemolyticus. In addition, V. parahaemolyticus in salmon sashimi could be detected at a concentration of 154 CFU/g without enrichment, and the entire detection time was around 30 min. After enrichment for 6 h, 2 CFU/g V. parahaemolyticus could be detected. Conclusions Consequently, the proposed RPA-CRISPR/Cas12a-ICS platform could detect V. parahaemolyticus in seafood intuitively, quickly, and sensitively, leading to high practical application value.

Establishment and application of a gold nanoparticle-based immunochromatographic test strip for the detection of avian leukosis virus P27 antigen in egg white samples.

Avian leukemia is an infectious tumorous disease of chickens caused by subgroup A of the avian leukemia virus (ALV-A), which mainly causes long-term viremia, slow growth, immune suppression, decreased production performance, multi-tissue tumors, and even death. The infection rate of this disease is very high in chicken herds in China, causing huge economic losses to the poultry industry every year. We successfully expressed the specific antigen protein of ALV (P27) through recombinant protein technology and screened a pair of highly sensitive monoclonal antibodies (mAbs) through mouse immunity, cell fusion, and antibody pairing. Based on this pair of antibodies, we established a dual antibody sandwich ELISA and gold nanoparticle immunochromatographic strip (AuNP-ICS) detection method. In addition, the parameters of the dual antibody sandwich ELISA and AuNP-ICS were optimized under different reaction conditions, which resulted in the minimum detection limits of 0.2 ng mL-1 and 1.53 ng ml-1, respectively. Commonly available ELISA and AuNP-ICS products on the market were compared, and we found that our established immune rapid chromatography had higher sensitivity. This established AuNP-ICS had no cross-reactivity with Influenza A (H1N1), Influenza A (H9N2), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), Listeria monocytogenes listeriolysin (LLO), and Staphylococcal enterotoxin SED or SEC. Finally, the established AuNP-ICS was used to analyze 35 egg samples, and the results showed 5 positive samples and 30 negative samples. The AuNP-ICS rapid detection method established by our group had good specificity, high sensitivity, and convenience, and could be applied to the clinical sample detection of ALV-A.