Colloidal Gold Immunochromatographic Assay Research Articles

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.

Ultra-rapid detection of nuclear protein of severe fever with thrombocytopenia syndrome virus by colloidal gold immunochromatography assay.

In 2009, severe fever with thrombocytopenia syndrome virus (SFTSV), also known as the Dabie bandavirus (DBV), was first discovered in Henan, China. It is a tick-borne zoonotic virus with a fatality rate ranging from 6% to 30%. Currently, we lack safe and effective vaccines or antiviral drugs to treat SFTSV infection. Therefore, the development of a specific, sensitive, and cost-effective detection method is crucial. Using inactivated SFTSV and recombinant SFTSV nucleocapsid protein (SFTSV-NP), we repeatedly immunized mice with different adjuvants and obtained two monoclonal antibodies against SFTSV-NP, which were used to develop a colloidal gold immunochromatographic assay (ICA) rapid test kit for SFTSV. Compared with nucleic acid testing (gold standard), the ICA test strips are 97.67% accurate in testing clinical serum samples (36 cases of clinical serum samples and seven cases of whole blood samples). The test kit was 100% accurate in detecting different SFTSV strains. No false-positive results were generated when detecting other arboviruses. Therefore, our developed SFTSV test kit conveniently, rapidly, and effectively detects SFTSV.

Rapid screening of prednisolone acetate adulterants in health foods using colloidal gold immunochromatographic assay

In this study, a rapid detection method utilizing colloidal gold immunochromatography (CG-ICA) was developed for the detection of illegally added prednisone acetate in health foods. Initially, the preparation conditions of colloidal gold solution were optimized. The optimal potassium carbonate dosage, antibody diluent type, antibody dosage, probe labeling time, blocking time and BSA dosage were determined. Technical analysis was performed to ensure that the established CG-ICA exhibited satisfactory color development and inhibition rates. Under optimized conditions, the cut-off value of CG-ICA was 250 μg/kg. The assay demonstrated a sensitivity of 100 %, a false positive rate of 8 %, and a false negative rate of 0, indicating high specificity for prednisone acetate. The results obtained from testing actual samples were consistent with those obtained using LC-MS/MS, thereby verifying the reliability of the developed method. This method offers robust support for the rapid detection of illegally added prednisone acetate in health foods.

Rapid and convenient screening method based on single-chain variable fragments for the detection of restricted monensin in chicken muscle

A colloidal gold immunochromatographic assay (CGIA) based on single-chain variable fragments (scFvs) has been successfully developed for the detection of monensin (MON). Colloidal gold probes were conjugated to anti-MON scFvs through electrostatic interaction, with the conjugated objects serving as the visual signals. The detection lines were formed by capturing the antibody with MON-OVA. This assay offers a rapid detection time of 15 min, a wide linear range from 2.19 to 10.76 ng mL−1, and boasts high accuracy, precision, and an absence of cross-reactivity. By homology modeling and molecular docking, we predicted the interaction patterns between the scFv and monensin, and the amino acid residues involved in the recognition of MON by the antibody were analyzed. These key amino acid sites are presumed integral to ligand recognition per current interaction models. This hypothesis was confirmed by computer-aided alanine scanning mutation, MM/P(G)BSA molecular dynamics simulation, and in vitro binding experiments. In this study, we successfully developed the scFvs-based CGIA system for rapid and easy quantification of monensin, providing a simple, efficient routine detection of chicken muscle samples.

Monoclonal antibody based colloidal gold immunochromatographic assay for the visual and rapid screening of profenofos

Profenofos (PFF) is a commonly used organophosphorus insecticide that requires strict monitoring due to its potential environmental, ecological, and human health risks originating from residues in soil and water systems, as well as accumulation in crops. In this study, a novel monoclonal antibody (mAb) specific to PFF was prepared for the first time and the recognition mechanism was investigated through molecular simulation. Subsequently, a mAb-based colloidal gold immunochromatographic assay (GICA) was developed for the rapid screening of PFF in fruit and vegetable samples. The mAb exhibited an IC50 value of 12.9 ng/mL, and limit of detection (LOD) of 4.6 ng/mL, respectively in indirect competitive immunosorbent enzyme-linked immunosorbent assay (ic-ELISA). After optimization, the developed GICA exhibited a visual limit of detection (vLOD) of 20 ng/mL and a quantitative of detection (qLOD) of 5.2 ng/mL, with a linear range from 10.0 to 83.8 ng/mL. Good correlation was observed between the results of GICA and standard Gas Chromatography-Tandem Mass Spectrometry (GC-MS/MS) in matrix and recovery test. The developed GICA can be used for rapid sample detection within 15 min, which is an excellent tool for screening PFF in foods and environmental samples.

An ultrasensitive strip sensor for rapid detection of African swine fever virus

African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) infecting pigs, which has caused huge economic losses in countries around the world. Currently, there is no effective vaccine, and the prevention and control of ASF is mainly through rapid detection, so it is particularly important to carry identify and develop rapid detection methods for ASFV. In this study, recombinant plasmid PET-28a(+)-p30 was constructed, and the recombinant protein was obtained by inducing expression and Ni+ resin affinity column purification. Mice were immunized with recombinant p30 protein, and after three immunizations, ten strains of hybridoma cells that stably secreted monoclonal antibodies (mAbs) against p30 protein were obtained by cell fusion and subcloning. A colloidal gold immunochromatography assay (GICA) based on double antibody sandwich technology was established to screen the paired antibodies, and the trapping and detecting antibodies were mAb-11F11 and mAb-7A8, respectively, with a detection limit of 1 ng/mL, which laid an important material foundation for the early detection of ASF in the future.

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.

Development of Monoclonal Antibody against PirB and Establishment of a Colloidal Gold Immunochromatographic Assay for the Rapid Detection of AHPND-Causing Vibrio.

Acute hepatopancreatic necrosis disease (AHPND) poses a significant threat to shrimp aquaculture worldwide, necessitating the accurate and rapid detection of the pathogens. However, the increasing number of Vibrio species that cause the disease makes diagnosis and control more difficult. This study focuses on developing a monoclonal antibody against the Photorhabdus insect-related (Pir) toxin B (PirB), a pivotal virulence factor in AHPND-causing Vibrio, and establishing a colloidal gold immunochromatographic assay for the enhanced early diagnosis and monitoring of AHPND. Monoclonal antibodies targeting PirB were developed and utilized in the preparation of colloidal-gold-labeled antibodies for the immunochromatographic assay. The specificity and sensitivity of the assay were evaluated through various tests, including antibody subclass detection, affinity detection, and optimal labeling efficiency assessment. The developed PirB immunochromatographic test strips exhibited a good specificity, as demonstrated by the positive detection of AHPND-causing Vibrio and negative results for non-AHPND-causing Vibrio. The study highlights the potential of the developed monoclonal antibody and immunochromatographic assay for the effective detection of AHPND-causing Vibrio. Further optimization is needed to enhance the sensitivity of the test strips for improved practical applications in disease prevention and control in shrimp aquaculture.

Nanobodies-based colloidal gold immunochromatographic assay for specific detection of parathion

Parathion is one of organophosphorus pesticide, which has been prohibited in agricultural products due to its high toxicity to human beings. However, there are still abuse cases for profit in agricultural production. Hence, we established nanobodies-based colloidal gold immunochromatographic assay (GICA) in which nanobodies (Nbs) as an excellent recognition element, greatly improving the stability and sensitivity of ICA. Under the optimal conditions, the developed Nbs-based GICA showed a cut-off value of 50 ng/mL for visual judgment and a half-inhibitory concentration (IC50) of 2.39 ng/mL for quantitative detection. The limit of detection (LOD) was as low as 0.15 ng/mL which was significantly 50-fold higher sensitivity than the commercial mAb-ICA. Additionally, this method exhibited good recoveries for the detection of cabbage, cucumber, and orange samples and excellent correlation with the UPLC-MS/MS method. The results showed that this method developed in this work based on nanobody can be used in practical detection of parathion in foods and nanobody is novel prospective antibody resource for immunoassays of chemical contaminants.

Characterization of viral infections in children with influenza-like-illness during December 2018-January 2019.

Respiratory viral infection (RVI) is of very concern after the outbreak of COVID-19, especially in pediatric departments. Learning pathogen spectrum of RVI in children previous the epidemic of COVID-19 could provide another perspective for understanding RVI under current situation and help to prepare for the post COVID-19 infection control. A nucleic acid sequence-based amplification (NASBA) assay, with 19 pairs of primers targeting various respiratory viruses, was used for multi-pathogen screening of viral infections in children presenting influenza-like illness (ILI) symptoms. Children with ILI at the outpatient department of Beijing Tsinghua Changgung Hospital during the influenza epidemic from 12/2018 to 01/2019 were included. Throat swabs were obtained for both the influenza rapid diagnostic test (IRDT) based on the colloidal gold immunochromatographic assay and the NASBA assay, targeting various respiratory viruses with an integrated chip technology. Of 519 patients, 430 (82.9%) were positive in the NASBA assay. The predominant viral pathogens were influenza A H1N1 pdm1/2009 (pH1N1) (48.4%) and influenza A (H3N2) (18.1%), followed by human metapneumovirus (hMPV) (8.8%) and respiratory syncytial virus (RSV) (6.1%). Of the 320 cases identified with influenza A by NASBA, only 128 (40.0%) were positive in the IRDT. The IRDT missed pH1N1 significantly more frequently than A (H3N2) (P<0.01). Influenza A pH1N1 and A (H3N2) were the major pathogens in <6 years and 6-15 years old individuals respectively (P<0.05). In summary, influenza viruses were the major pathogens in children with ILI during the 2018-2019 winter influenza epidemic, while hMPV and RSV were non-negligible. The coexistence of multiple pathogen leading to respiratory infections is the normalcy in winter ILI cases.

Rapid detection of the freshness of grass carp (Ctenopharygodon idella) fillets by colloidal gold immunochromatography assay

Consumers are greatly concerned about fish freshness. Therefore, this study was aimed to provide a colloidal gold immunochromatography assay (GICA) system for rapid detection of fish freshness. A suitable fish protein was chosen as an antigen for GICA; and the properties and effectiveness of the GICA system were investigated. Results showed that parvalbumin (PV) was suitable as a fish freshness indicator, proven by hierarchical cluster analysis and chosen as a GICA antigen. The GICA system revealed high specificity in the 27–212 μg/L detection range. The correlation coefficient R2 towards enzyme-linked immunosorbent assay reflected high reliability as 0.998. The GICA system was stable within 12 weeks at room temperature. The fitness for indicating freshness by GICA ranged from 87.27% to 111.72%. In short, the GICA system with PV as an antigen could be used as a simple, rapid, and accurate technique for the freshness detection of grass carp fillets.

A rapid colloidal gold immunochromatographic assay based on polyclonal antibodies against HtpsC protein for the detection of Streptococcus suis

An efficient and rapid immunochromatographic assay (ICA) has been engineered for the detection of Streptococcus suis (S. suis). The underpinning principle of this ICA test lies in the use of polyclonal antibodies (pAbs) decorated with colloidal gold, which are specific to S. suis. These pAbs were derived from rabbits immunized with type II histidine triad protein (HtpsC) and HtpsC-N of S. suis. The sensitivity of the ICA was noteworthy, identifying S. suis at bacterial concentrations as diminutive as 1.0 × 103 CFU/mL. Moreover, the assay demonstrated respectable specificity and did not indicate false positives for other bacterial species (Escherichia coli, Salmonella, Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus lactis, or Enterococcus faecalis). The assay was also capable of detecting multiple S. suis serotypes containing the htpsC gene, including serotypes 1–9, 12, 14, 16 and 23. Nonetheless, the detection of S. suis that lacks the htpsC gene remained beyond the capabilities of this assay. A simultaneous analysis of 16 samples utilizing PCR substantiated the reliability of the ICA test. The assay’s results can be procured within a 15-min window, making it a suitable option for field application. Broadly, this study underscores the potential of the HtpsC protein as a target antigen for the detection of S. suis, and proposes that the HtpsC protein be evaluated further in other detection assays specific for S. suis.

Generating Monoclonal Antibodies against Buprofezin and Developing Immunoassays for Its Residue Detection in Tea Samples.

The synthesis of a hapten and antigen for the preparation of a monoclonal antibody (mAb) for buprofezin is described. The recognition mechanism of hapten and buprofezin by monoclonal antibodies (mAb-19F2) is described. The effectiveness of the mAb-19F2 immunoassay technique was assessed, and the effective detection of buprofezin in tea samples was achieved through the establishment of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatography assay (GICA). The mAb-19F2 subtype was IgG1, with an IC50 of 1.8 ng/mL and a linear range (IC20-IC80) of 0.6-5.4 μg/L, and had a cross-reaction rate of less than 0.18% with 29 other pesticides (neonicotinoids and insect growth regulators). The study identified π-π stacking interactions between hapten and TYR-61 at the mAb-19F2 site and alkyl/phosphate interactions with TRP-105 and ARG-103. The ic-ELISA had an IC50 of 12.9 ng/mL in green tea and 5.65 ng/mL in black tea, with a recovery rate of 92.4%-101.0% and RSD of 2.1%-4.8%. The GICA had a limit of detection (LOD) was 500 ng/mL, with the complete disappearance of the test lines visible to the naked eye. The limit of quantitation (LOQ, IC20) was determined to be 16.8 ng/mL. Additionally, the developed GICA showed no cross-reactivity with neonicotinoid pesticides. The recovery rate of tea spiked recovered samples was 83.6%-92.2%, with an RSD of 5.3%-12.6%, and the results were consistent with the LC/MS method. This study is important for the real-time detection of buprofezin residues to ensure food safety and human health.

Development of multiplex immunochromatographic assay for the simultaneous detection of three polyether ionophore antibiotics in chicken muscle

ABSTRACT A rapid, reliable, sensitive, and multiplex colloidal gold immunochromatographic assay (CGIA) was developed for the simultaneous detection of three polyether ionophore antibiotics (PEs) in chicken muscle. The IC50 values of the optimized CGIA were 1.63, 1.94, and 1.18 ng/mL with a linear range of 0.87–3.04 ng/mL, 0.85–4.41 ng/mL, and 0.57–2.44 ng/mL for maduramicin (MAD), monensin (MON), and salinomycin (SAL), respectively. The cut-off values were 10, 5, and 5 ng/mL, respectively. The whole testing process was completed within 15 min. A parallel analysis in blind chicken muscle samples was conducted by liquid chromatography–tandem mass spectrometry (LC-MS/MS), the developed CGIA did not appear false-positive and false-negative. The results show that the established method could provide a rapid and effective approach for on-site screening and determination of PEs residues in chicken muscle samples.

Effects of rapid drug sensitivity testing for multidrug-resistant bacteria on the prognosis of patients with severe intra-abdominal infection

Objective: To examine the clinical value of rapid detection of drug-resistant bacteria by immunochromatography and the effects of rapid detection on the prognosis of patients with severe intra-abdominal infection complicated by carbapenem-resistant Enterobacteriaceae (CRE) bloodstream infection. Methods: This was a retrospective cohort study. We analyzed clinical data of 73 patients with severe abdominal infections with sepsis or septic shock complicated by CRE bloodstream infection admitted to the general surgery department of Jinling Hospital between February 2022 and February 2023. Patients were divided into a colloidal gold immunochromatographic assay (GICA) group (17 patients) and conventional testing group (56 patients) based on whether a GICA for CRE had been performed on the patients' first blood culture sample during the diagnosis and treatment process. There were no statistically significant differences between the GICA and conventional testing groups in age ([55.9±17.3] vs. [47.6±16.4] years), sex ([16 men vs. one woman ] vs. [41 men vs. 15 women]), median Charlson comorbidity index (3.0[2.0,4.0] vs. 3.0[2.0, 4.8]), septic shock (10 vs. 39), or acute kidney injury (8 vs. 40) (all P>0.05). Both groups routinely underwent traditional bacterial identification and drug susceptibility testing. Additionally, patients in the GICA group were tested directly for positive blood cultures using a GICA carbapenemase test kit. The main outcomes were mortality rates on Days 28 and 90 after the first identification of CRE bloodstream infection in both groups. We also compared the microbial clearance rate, duration of hospitalization and intensive care unit stay, and time from onset of CRE bloodstream infection to initiation of targeted and appropriate antibiotics between the two groups. Results: The rate of microbial clearance of bloodstream infection was significantly greater in the GICA group than in the conventional testing group (15/17 vs. 34/56 [60.7%], χ2=4.476, P=0.034), whereas the 28-day mortality tended to be lower in the GICA than conventional testing group [5/17 vs. 44.6% [25/56], χ2=1.250, P=0.264). The 90-day mortality (8/17 vs. 53.6% [30/56], χ2=0.222, P=0.638), median duration of hospitalization (37.0 [18.0, 46.5] days vs. 45.5 [32.2, 64.8] days, Z=-1.867, P=0.062), and median duration of intensive care unit stay (18.0 [6.5, 35.0] days vs. 32.0 [5.0, 51.8] days, Z=-1.251, P=0.209). The median time between the onset of bloodstream infection and administration of antibiotics was 49.0 (38.0, 69.0) hours in the GICA group, which is significantly shorter than the 163.0 (111.8, 190.0) hours in the conventional testing group (Z=-5.731, P<0.001). The median time between the onset of bloodstream infection and administration of appropriate antibiotics was 40.0 (34.0, 80.0) hours in the GICA group, which is shorter than in the conventional testing group (68.0 [38.2, 118.8]) hours; however, this difference is not statistically significant (Z=-1.686, P=0.093). Conclusions: GICA can provide information on carbapenemase- producing pathogens faster than traditional drug sensitivity testing, enabling early administration of the optimal antibiotics. The strategy of 'carbapenemase detection first' for managing bacterial infection has the potential to improve prognosis of patients and reduce mortality rate.

Molecular epidemiological characterization of hypervirulent carbapenem-resistant Klebsiella pneumoniae in a hospital in Henan Province from 2020 to 2022

Objective: The study investigated the clinical distribution, antimicrobial resistance and epidemiologic characteristics of hypervirulent Carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) in a hospital in Henan Province to provide a scientific basis for antibiotic use and nosocomial infection prevention and control. Methods: A retrospective analysis of the clinical data from the cases was carried out in this study. Clinical data of patients infected with the CRKP strain isolated from the clinical microbiology laboratory of Henan Provincial Hospital of Traditional Chinese Medicine from January 2020 to December 2022 were retrospectively analyzed. A string test, virulence gene screening, serum killing, and a G. mellonella infection model were used to screen hv-CRKP isolates. The clinical characteristics of hv-CRKP and the drug resistance rate of hv-CRKP to twenty-five antibiotics were analyzed using WHONET 5.6. Carbapenemase phenotypic characterization of the hv-CRKP was performed by colloidal gold immunochromatographic assay, and Carbapenemase genotyping, multi-locus sequence typing (MLST) and capsular serotyping of hv-CRKP isolates were performed by PCR and Sanger sequencing. Results: A total of non-duplicate 264 CRKP clinical isolates were detected in the hospital from 2020 to 2022, and 23 hv-CRKP isolates were detected, so the corresponding detection rate of hv-CRKP was 8.71% (23/264). The hv-CRKP isolates in this study were mainly from the intensive care unit (10/23) and neurosurgery department (8/23), and the main sources of hv-CRKP isolates were sputum (10/23) and bronchoalveolar lavage fluid (6/23). The hv-CRKP isolates in this study were highly resistant to β-lactam antibiotics, fluoroquinolones and aminoglycosides, and were only susceptible to colistin, tigecycline and ceftazidime/avibactam. The detection rate of the blaKPC-2 among 23 hv-CRKP isolates was 91.30% (21/23) and none of the class B and class D carbapenemases were detected. Results of MLST and capsular serotypes showed that ST11 type hv-CRKP was the dominant strain in the hospital, accounting for 56.52% (13/23), and K64 (9/13) and KL47 (4/13) were the major capsular serotypes. Conclusion: The hv-CRKP isolates from the hospital are mainly from lower respiratory tract specimens from patients admitted to the intensive care department and the drug resistance is relatively severe. The predominant strains with certain polymorphisms are mainly composed of the KPC-2-producing ST11-K64 and ST11-KL47 hv-CRKP isolates in the hospital.

A colloidal gold test strip based on catalytic hairpin assembly for the clinical detection of influenza a virus nucleic acid

Influenza A epidemics, which occur annually in varying degrees worldwide, is a global challenge to healthcare facilities owing to several limitations of the current detection methods. Therefore, the development of a rapid, convenient, and economical method for the early diagnosis of influenza A will aid clinical treatment and epidemic control. Currently, most of the commonly used clinical rapid tests utilize colloidal gold test strips that detect specific influenza virus antigens but are limited by low sensitivity. Therefore, this study combined catalytic hairpin assembly (CHA) with colloidal gold immunochromatographic assay (GICA) to develop a highly sensitive and visual CHA-GICA test strip. Clinical sample analysis revealed that the sensitivity of the assay was 81.8% and 74% under optimal (35 °C) and room temperature (25 °C) conditions, respectively. In conclusion, this study developed a rapid nucleic acid assay for detecting influenza A virus with high sensitivity and specificity, which can improve the clinical detection of influenza A.

Aflatoxin detection technologies: recent advances and future prospects.

Aflatoxins have posed serious threat to food safety and human health. Therefore, it is important to detect aflatoxins in samples rapidly and accurately. In this review, various technologies to detect aflatoxins in food are discussed, including conventional ones such as thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA), colloidal gold immunochromatographic assay (GICA), radioimmunoassay (RIA), fluorescence spectroscopy (FS), as well as emerging ones (e.g., biosensors, molecular imprinting technology, surface plasmon resonance). Critical challenges of these technologies include high cost, complex processing procedures and long processing time, low stability, low repeatability, low accuracy, poor portability, and so on. Critical discussion is provided on the trade-off relationship between detection speed and detection accuracy, as well as the application scenario and sustainability of different technologies. Especially, the prospect of combining different technologies is discussed. Future research is necessary to develop more convenient, more accurate, faster, and cost-effective technologies to detect aflatoxins.

Preparation of a broad-specificity antibody against zearalenone and its primary analogues and development of immunoassay of Coicis Semen and related products.

The purpose of this study was to prepare a highly sensitive and specific zearalenone (ZEN) monoclonal antibody, which was then used to develop an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold immunochromatographic assay (GICA). These techniques were used for the detection of Coicis Semen and related products (Coicis Semen flour, Yimigao, and Yishigao). Immunogens were synthesized by oxime active ester techniques and characterized via ultraviolet spectrophotometry. Immunogens were injected subcutaneously into the abdominal cavities and backs of mice. Using the prepared antibodies, we developed ic-ELISA and GICA rapid detection methods, which were then applied for the rapid detection of ZEN and its analogues from Coicis Semen and related products. For ic-ELISA, the half maximal inhibitory concentration (IC50 ) values for ZEN, α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL), and β-zearalanol (β-ZAL) were determined to be 1.13, 1.69, 2.06, 0.66, 1.20, and 0.94ng•mL-1 , respectively. For GICA, the cutoff values of ZEN, α-ZEL, β-ZEL, α-ZAL, and β-ZAL on test strips were 0.5ng•mL-1 in phosphate buffer saline (0.01M, pH 7.4), while ZAN was found to be 0.25ng•mL-1 . Furthermore, the cutoff values of test strips were between 10 and 20µg∙kg-1 in Coicis Semen and related products. The results of these two detection methods were in good agreement with results from liquid chromatography-tandem mass spectrometry. This study provides technical support for the preparation of broad-specificity monoclonal antibodies against ZEN and lays the foundation for the simultaneous detection of multiple mycotoxins from food and herbal medicines.

Development of colloidal gold immunochromatography and reverse transcription loop-mediated isothermal amplification assays to detect lychnis mottle virus.

Lychnis mottle virus (LycMoV; genus Unassigned, family Secoviridae) infection of Angelica sinensis produces mottle and mosaic symptoms, damaging the host. Early detection of relevant pathogens is the most critical step in preventing the potential transmission of infectious disease. Polyclonal antibodies (pAbs) with high potency and high specificity were prepared using the recombinant LycMoV capsid protein as an antigen. Here, we developed and optimized a rapid colloidal gold immunochromatography assay (GICA) detection system for LycMoV using this antibody. Under optimum conditions, GICA specifically detected (up to 10,000-fold) positive LycMoV samples. A real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) system was also established by selecting the primers with high sensitivity and specificity to LycMoV. The RT-LAMP detection threshold was 1.42 fg/μl (291 copies/μl). A GICA-RT-LAMP assay system was further established and optimized. The minimum GICA detection line was calculated at 1.52x10-2 ng/μl. Although GICA did not detect positive samples after capturing 2.53x10-3 ng/μl of virus, GICA-LAMP and GICA-RT-PCR did, whose sensitivity was comparatively greater than six-fold. This is the first report showing that GICA-RT-LAMP is a cost effective approach for use in detecting LycMoV without extracting nucleic acids. These sensitive assays will help improve virus disease management in A. sinensis crops.