Colloidal Gold Immunochromatographic Strip Research Articles

A point-of-care testing strip based on a rationally designed antigen and antibody for fast and reliable Diquat poisoning detection: Ready for clinical application

AbstractDiquat (DQ) is a nonselective quick-acting herbicide, which can directly contaminate humans and the environment during improper use and production. The early diagnosis of DQ poisoning allows for patients to receive timely treatment. Here, a novel monoclonal antibody was screened based on the rational design and synthesis of a DQ hapten and complete antigen. With the custom-designed anti-DQ antibody, we developed a stable and specific immunochromatographic strip to recognize DQ within 10 ∼ 15 min in various biological samples. The test strips could recognize DQ at concentrations as low as 20 ng/mL and be read by the naked eye, reaching the detection level of liquid chromatography-mass spectrometry. Moreover, quantitative methods involving the use of immunochromatographic strips and ELISA were developed to continuously monitor DQ concentrations. DQ and paraquat (PQ) joint test strips were developed to simultaneously measure DQ and PQ. Notably, the clinical performance of the proposed test strips was evaluated in multiple hospitals and public health agencies. Taken together, our study provides the first clinical DQ-targeted colloidal gold immunochromatographic test strips with excellent performance, which are expected to be useful for clinical practice, environmental monitoring, food safety and scientific research.

Development of a colloidal gold immunochromatographic test strip for detecting the smooth Brucella

SummaryBrucellosis, caused by Gram-negative Brucella, spreads in human and animal populations through contact with infected animals and products. Developing a rapid and sensitive detection technology for pathogen is crucial to reduce the risk of this disease transmitting between animal populations and to humans. We produced a monoclonal antibody LPS-6B5, which shows high affinity to LPS and limited cross-reactivity with other bacteria. Based on LPS-6B5, a colloidal gold immunochromatographic assay (GICA) was developed which demonstrates high sensitivity and specificity in detecting cultured B. melitensis, B. abortus and B. suis. The Gold Immunochromatographic Assay (GICA) strips exhibited the most sensitive detection limits, with a value of 7.8125 × 105 CFU/mL for Brucella melitensis, surpassing the sensitivity levels observed for Brucella abortus and Brucella suis. It is also suitable for clinical and field samples, providing a cost-effective and user-friendly alternative to traditional methods.

Fluorescence immunochromatographic detection of antibodies to the p72 protein of African swine fever virus

The African swine fever virus (ASFV), a highly contagious pathogen responsible for African swine fever (ASF), causes significant economic losses in the global pork industry. Due to its large and complex structure, ASFV remains refractory to commercial vaccine development, necessitating the creation of rapid, sensitive, and specific diagnostic tools for disease control. In this study, quantum dots were conjugated to ASFV p72 protein to establish a fluorescent immunochromatographic assay for detecting ASFV-specific antibodies. The assay test strips contained four adjacent pads arranged sequentially: a sample-application pad, a pad containing mobile antigen-probe conjugate, a nitrocellulose readout pad featuring a test line containing immobilised staphylococcal protein A and a control line containing immobilised monoclonal antibodies against the ASFV p72 protein, and an absorbent pad driving the directional flow of liquid via capillary action. The resulting fluorescence immunochromatographic assay demonstrated highly sensitive and specific ASFV antibody detection in under 15 min. Specificity testing showed no cross-reactivity with serum antibodies against other viruses and sensitivity surpassing that of commercial ASFV antibody colloidal gold immunochromatographic test strips. This novel approach offers rapid detection, excellent specificity, and high sensitivity, and supports the future development of fluorescent immunochromatographic test strips for ASFV antibody detection.

Development of a Sensitive Monoclonal Antibody-Based Colloidal Gold Immunochromatographic Strip for Lomefloxacin Detection in Meat Products.

Lomefloxacin (LOM), an antibiotic crucial for preventing various animal diseases in animal husbandry, can pose serious health risks when found in excessive amounts in meat products. The development of highly specific and sensitive colloidal gold immunochromatographic test strips is essential for the accurate detection of this class of antibiotics. Our study utilized a monoclonal antibody (mAb) assay and immunochromatographic strips to detect lomefloxacin residues in meat products. The results showed minimal cross-reactivity with other structural analogs, with a maximum half inhibitory concentration (IC50) of 0.93 ng/mL and a linear range of 0.38 to 2.3 ng/mL for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). The recovery of LOM was 80% to 120%, with an average coefficient of variation below 5%. The immunochromatographic strip test results showed a visual detection limit of 2.5 ng/g, meeting the market requirements for the test. This study highlights the significance of specific and sensitive testing methods for detecting lomefloxacin, ensuring consumers' safety and health.

A colloidal gold immunochromatography-based method for detecting enramycin residues in feed

To achieve rapid detection of enramycin in feed, we employed the competitive inhibition method to develop a colloidal gold immunochromatographic test strip based on the anti-enramycin A monoclonal antibody (anti-Er.A-mAb). Colloidal gold probes were prepared with a laboratory-prepared high-purity anti-Er.A-mAb. The effects of pH, antibody titer, and antigen concentration (test line) on the test strip performance were investigated. The colloidal gold test strip prepared with 8 μL potassium carbonate addition, 4 µg/mL antibody, 1.0 mg/mL antigen (test line), and 3 μL gold-labeled antibody showed acceptable specificity and a low limit of detection. The test strip showed the detection limit of 25 ng/mL for enramycin A, with a linear range of 25-300 ng/mL. The experiments on the feed with positive sample addition proved that the test strip had good repeatability and was more sensitive than high-performance liquid chromatography, being applicable for the rapid detection of enramycin in large batches of feed samples.

Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus.

In recent years, porcine diarrhea-associated viruses have caused significant economic losses globally. These viruses present similar clinical symptoms, such as watery diarrhea, dehydration, and vomiting. Co-infections with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are common. For the rapid and on-site preliminary diagnosis on the pig farms, this study aimed to develop a colloidal gold immunochromatography assay (GICA) strip for the detection of PEDV and TGEV simultaneously. The GICA kit showed that there was no cross-reactivity with the other five common porcine viruses. With visual observation, the lower limits were approximately 104 TCID50/mL and 104 TCID50/mL for PEDV and TGEV, respectively. The GICA strip could be stored at 4°C or 25°C for 12 months without affecting its efficacy. To validate the GICA strip, 121 clinical samples were tested. The positive rates of PEDV and TGEV were 42.9 and 9.9%, respectively, and the co-infection rate of the two viruses was 5.8% based on the duplex GICA strip. Thus, the established GICA strip is a rapid, specific, and stable tool for on-site preliminary diagnosis of PEDV- and TGEV-associated diarrhea.

Computerized analysis of haptens for the ultrasensitive and specific detection of Pyriftalid

Pyriftalid (Pyr) is one of the most commonly used herbicides and due to its widespread and improper use, it has led to serious pollution of groundwater, soil and other ecosystems, threatening human health. A rapid method to detect Pyr was urgently needed. A high specific monoclonal antibody (mAb) against Pyr with IC50 values of 4.7 ng/mL was obtained by mAb screening technique and method with enhanced matrix effect. The study firstly proposed colloidal gold immunochromatographic test strips (CGIA) for Pyr, which enables rapid qualitative and quantitative determination of a large number of samples anytime and anywhere, so as to effectively monitor Pyr in environment and grain samples. Based on the properties of the desired Pyr antibody, the hapten Pyr-hapten-4 with high structural similarity to Pyr molecule, similar electrostatic potential distribution, and the ability to expose Pyr functional groups was screened out from five different Pyr haptens, which was consistent with mouse antiserum test. The CGIA quickly analyze the Pyr content in positive samples such as water samples, soil samples, paddy samples, brown rice samples within 10 min, the LOD for Pyr by CGIA as low as 1.84 ng/g, the v LOD value as low as 6 ng/g, and the extinction value as low as 25 ng/g. The content of positive samples detected by CGIA was consistent with the quantitative results of LC-MS/MS, the relative accuracy was within the range of 97–103 %. The recovery rate range for Pyr by CGIA was 92.0–99.7 %, and the coefficient of variation was between 1.30–8.56 %. It indicated Pyr-targeted CGIA test strip was an efficient and fast detection method to detect real environment and food samples.

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 a colloidal gold immunochromatographic strip for rapid detection of avian coronavirus infectious bronchitis virus

Avian infectious bronchitis virus (IBV) still causes serious economic losses in the poultry industry. Currently, there are multiple prevalent genotypes and serotypes of IBVs. It is imperative to develop a new diagnosis method that is fast, sensitive, specific, simple, and broad-spectrum. A monoclonal hybridoma cell, N2D5, against the IBV N protein was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The N2D5 monoclonal antibody (mAb) and the previously prepared mouse polyclonal antibody against the IBV N protein were used to target IBV as a colloidal gold–mAb conjugate and a captured antibody, respectively, in order to develop an immunochromatographic strip. The optimal pH and minimum antibody concentration in the reaction system for colloidal gold–mAb N2D5 conjugation were pH 6.5 and 30 μg/mL, respectively. Common avian pathogens were tested to evaluate the specificity of the strip and no cross-reaction was observed. The sensitivity of the strip for detecting IBV was 10−1.4522 EID50/mL. The strip showed a broad-spectrum cross-reactive capacity for detecting IBV antigens, including multiple IBV genotypes in China and all of the seven serotypes of IBV that are currently prevalent in southern China. Additionally, the result can be observed within 2 min without any equipment. The throat and cloacal swab samples of chickens that were artificially infected with three IBV strains were tested using the developed strip and the qPCR method; the strip test demonstrated a high consistency in detecting IBV via qPCR gene detection. In conclusion, the immunochromatographic strip that was established is rapid, sensitive, specific, simple, practical, and broad-spectrum; additionally, it has the potential to serve as an on-site rapid detection method of IBV and can facilitate the surveillance and control of the disease, especially in resource-limited 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.

Highly specific and super-sensitive Dot-ELISA and colloidal gold immunochromatographic strip for detecting Xanthomonas oryzae pv. oryzicola of rice bacterial leaf streak

Rice bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a worldwide destructive rice bacterial disease, and seriously affects the global rice industry. Effective integrated management measures for BLS depend on the timely and proper detection of Xoc. Here, two highly specific and super-sensitive monoclonal antibodies (MAbs) against Xoc were first prepared using the cultured Xoc RS105 as an immunogen. Then, two serological assays, Dot enzyme-linked immunosorbent assay (Dot-ELISA) and Colloidal gold immunochromatographic strip (CGICS), were developed for the super-sensitive and broad-spectrum detection of Xoc in homogenates prepared from field-collected rice plants. The newly created Dot-ELISA and CGICS assays can detect ten different Xoc strains from various provinces in China and other countries without cross-reactivity with the other ten tested plant bacteria. Furthermore, Dot-ELISA and CGICS can detect Xoc in bacterial suspensions diluted up to 9.78 × 103 CFU/mL and 4.88 × 103 CFU/mL, respectively, or in rice plant tissue homogenates diluted up to 1:51,200 (w/v, g/mL). Surprisingly, both Dot-ELISA and CGICS serological assays were more sensitive than the conventional PCR. Additionally, analysis results using field-collected rice samples showed that the newly created Dot-ELISA and CGICS were reliable in detecting Xoc in rice tissues. Thus, the two serological assays were highly valuable and effective for the diagnosis of rice bacterial leaf streak in rice-grown areas and for inspection and quarantine of Xoc.

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

Isolation and identification of BRV G6P[1] strain in Heilongjiang province, Northeast China.

Bovine rotavirus (BRV) is the main cause of acute gastroenteritis in calves, resulting in significant economic losses to the cattle industry worldwide. Additionally, BRV has multiple genotypes, which could enable cross-species transmission, thereby posing a significant risk to public health. However, there is a problem of multiple genotypes coexisting in BRV, and the cross-protection effect between different genotypes of rotavirus strains is not effective enough. Therefore, mastering clinical epidemic genotypes and using epidemic genotype strains for vaccine preparation is an effective means of preventing and controlling BRV. In this study, BRV strain DQ2020 in MA104 cells was identified by transmission electron microscopy (TEM), reverse transcription polymerase chain reaction (RT-PCR), and colloidal gold immunochromatographic test strips. The whole genome of BRV strain DQ2020 was sequenced and pathogenicity in suckling mice was assessed. The results showed that after 10 passages in MA104 cells, BRV strain DQ2020 induced cytopathic effects. Wheel-shaped virus particles (diameter, ~80 nm) were observed by TEM. A target band of 382 bp was detected by RT-PCR, a positive band was detected with the colloidal gold immunochromatographic test strips, and significant green fluorescence was observed by indirect immunofluorescence (IFA). The highest median tissue culture infectious dose of strain DQ2020 after 9 passages in MA104 cells was 10-4.81 viral particles/0.1 mL. Based on phylogenetic analysis of 11 gene fragments, the genotype of BRV strain DQ2020 was G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3, confirming transmission of the G6-P[1] genotype in Chinese cattle herds. Further analysis showed that the isolated strain was a reassortant of bovine (VP7, VP6, NSP3, and NSP5), human (VP4, VP1, VP2, VP3, NSP2, and NSP4), and ovine (NSP1) rotaviruses. BRV strain DQ2020 caused damage to the intestinal villi of suckling mice and diarrhea, confirming pathogenicity. In summary, this study identified a reassortant strain of bovine, human, and ovine rotavirus that is pathogenic to lactating mice, and conducted whole genome sequence analysis, providing valuable insights for the genetic evolution of the virus and the development of vaccines.

Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

Development and application of monoclonal antibody-based dot-ELISA and colloidal gold immunochromatographic strip for rapid, specific, and sensitive detection of tomato brown rugose fruit virus

Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus that has become a great concern to tomato production industry. Due to the lack of resistant cultivars, precise detection of ToBRFV is essential to prevent the spread of ToBRFV. In this study, we produced highly sensitive and specific monoclonal antibodies against ToBRFV and established dot-enzyme-linked immunosorbent assay (dot-ELISA) and colloidal gold immunochromatographic strip (CGICS)-based methods for ToBRFV detection. These two methods could specifically detect ToBRFV without cross-reaction with seven tested tobamoviruses and three frequently occurring tomato-infecting viruses. Sensitivity analysis showed that the limit of detection of the established dot-ELISA and CGICS methods reached up to 1:6400 and 1:10,000 (w/v, g/mL) dilution of ToBRFV-infected tomato tissue, respectively. Further analyses using field-collected tomato foliar and fruit samples showed that the results obtained by dot-ELISA and CGICS were consistent with those obtained by reverse transcription polymerase chain reaction. The established methods here allow for specific, sensitive, and robust detection of ToBRFV, and will be helpful for precise monitoring and early warning of ToBRFV.

Colloidal gold and fluorescent immunochromatographic test strips for canine parvovirus detection.

Canine parvovirus (CPV) is an acute and highly infectious virus causing disease in puppies and, thus, affecting the global dog industry. The current CPV detection methods are limited by their sensitivity and specificity. Hence, the current study sought to develop a rapid, sensitive, simple, and accurate immunochromatographic (ICS) test to detect and control the spread and prevalence of CPV infection. More specifically, 6A8, a monoclonal antibody (mAb) with high specificity and sensitivity, was obtained by preliminary screening. The 6A8 antibody was labelled with colloidal gold particles. Subsequently, 6A8 and goat anti-mouse antibodies were coated onto a nitrocellulose membrane (NC) as the test and control lines, respectively. Furthermore, 6A8 and rabbit IgG antibodies were labelled with fluorescent microspheres and evenly sprayed onto a glass fibre membrane. Both strips could be prepared in 15min with no noticeable cross-reactivity with other common canine intestinal pathogens. The strips were simultaneously used to detect CPV in 60 clinical samples using real-time quantitative PCR, hemagglutination, and hemagglutination inhibition assays. The colloidal gold (fluorescent) ICS test strip was stable for 6 (7) and 4 (5) months at 4 °C and room temperature (18-25 °C). Both test strips were easy to prepare and rapidly detected CPV with high sensitivity and specificity. Moreover, the results were easily interpretable. This study establishes a simple method for two CPV diseases, colloidal gold and fluorescent immunochromatographic (ICS) test strips. KEY POINTS: • CPV test strips do not exhibit cross-reactivity with other canine intestinal pathogens. • The strips are stable for months at 4 °C and at room temperature (18-25 °C). • These strips are a promising approach for the timely diagnosis and treatment of CPV.

Development of a colloidal gold immunochromatographic strip with enhanced signal for the detection of bovine parvovirus.

Bovine parvovirus (BPV) is a pathogen responsible for respiratory and digestive tract symptoms in calves and abortion and stillbirth in pregnant cows. In this study, we developed a colloidal gold immunochromatographic (GICG) strip with an enhanced signal for detecting BPV according to the double-antibody sandwich principle and an enzyme-based signal amplification system to amplify the signal. This system utilizes horseradish peroxidase reacting with a substrate solution containing 3,3',5,5'-tetramethylbenzidine and dextran sulfate to obtain insoluble blue products on the test and control lines. We optimized different reaction conditions, including the amount of monoclonal antibodies (mAbs), pH of the colloidal gold solution, coating solution, blocking solution, sample pad treatment solution, antibody concentration in the control line, and antibody concentration in the detection line. The sensitivity of the signal-enhanced GICG strip showed that the minimum amount for detecting BPV was 102 TCID50, 10 times higher than that of the traditional GICG strip. The results of the specificity test showed that the signal-enhanced GICG strip had no cross-reactivity with BRV, BVDV, or BRSV. The results of the repeatability test showed that the coefficient of variation between and within batches was less than 5%, showing good repeatability. Moreover, for validation, PCR and the signal-enhanced GICG strip were used to detect 280 clinical bovine fecal samples. The concordance rate compared with PCR was 99.29%. Hence, the developed strip exhibited high sensitivity and specificity for the detection of BPV. Therefore, this strip could be a rapid, convenient, and effective method for the diagnosis of BPV infection in the field.

Development of Indirect Competitive ELISA and Colloidal Gold Immunochromatographic Strip for Endosulfan Detection Based on a Monoclonal Antibody.

Endosulfan, as an effective broad-spectrum insecticide, has been banned in agricultural areas because of the potential harmful effects on human health. This study aimed to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatographic (ICA) strip based on a prepared monoclonal antibody (mAb) for quantitative and qualitative detection of endosulfan. A new mAb with high sensitivity and affinity was designed and screened. The ic-ELISA showed a 50% inhibition concentration (IC50) value of 5.16 ng/mL for endosulfan. Under optimum conditions, the limit of detection (LOD) was determined to be 1.14 ng/mL. The average recoveries of endosulfan in spiked pear and apple samples ranged from 91.48-113.45% and 92.39-106.12% with an average coefficient of variation (CV) of less than 7%, respectively. The analysis of colloidal gold ICA strip could be completed within 15 min by naked eye and the visual limit of detection (vLOD) was both 40 ng/mL in pear and apple samples. In conclusion, both developed immunological methods were suitable and reliable for the on-site detection of endosulfan in real samples at trace levels.