Lateral Flow Immunochromatographic Assay Research Articles

Development and evaluation of a lateral flow immunochromatographic assay for the rapid detection of donkey meat in beef as a tool for meat adulteration identification

Development and evaluation of a lateral flow immunochromatographic assay for the rapid detection of donkey meat in beef as a tool for meat adulteration identification

Human Papillomaviruses 16 and 18 E6 Oncoprotein Detection Test in Primary Oropharyngeal Carcinomas and Metastatic Lymph Nodes: A Cross-Sectional Study.

Accuracy in the diagnosis of HPV-associated oropharyngeal carcinoma (OPSCC) of a rapid, low-cost lateral flow immunochromatographic assay for detecting E6 oncoprotein of HPV-16 and HPV-18 was previously evaluated in a small pilot study. This cross-sectional study aimed to assess on a large case series the sensitivity and specificity of E6 oncoprotein as a diagnostic marker for HPV-associated carcinogenesis in OPSCC. 137 consecutive patients with histologically confirmed OPSCC were enrolled in two hospitals in Northeast Italy. HPV status was determined by PCR for HPV DNA and p16INK4a immunohistochemistry on primary tumor biopsies. An OPSCC was defined as HPV-associated when double positive for high-risk HPV-DNA and p16INK4a overexpression in primary lesion. Cytological samples from primary tumors and metastatic lymph nodes were obtained and tested for HPV16/18 E6 oncoproteins using the lateral flow immunochromatographic assay, which requires between 90 and 120 min to provide a result. Diagnostic performances were calculated as percentage with confidence intervals (CI). Of the 137 OPSCC cases, 68 (49.6%) were HPV-associated, testing positive for both high-risk HPV-DNA and p16INK4a, with HPV16 predominating (82.4%). An average waiting time of 22 days was observed to obtain the results of p16INK4a and HPV-DNA after primary lesions biopsy. In patients with HPV16/18-associated OPSCC, the HPV16/18 E6 oncoprotein was detected in 59 out of 60 cytological samples from the primary lesion (sensitivity: 98.3%; 95% CI: 91.1-100%) and in 45 out of 51 cytological samples from lymph node metastases (sensitivity: 88.2%; 95% CI: 76.1-95.6%). The E6 oncoprotein assay showed a specificity of 100% in both primary tumors and lymph node metastases. The low-cost lateral flow immunochromatographic assay for detecting HPV16/18 E6 oncoproteins confirmed high accuracy for identifying HPV-associated OPSCC, particularly in primary tumors, suggesting its potential as a valuable diagnostic tool in clinical practice. Its rapid diagnostic capability could significantly accelerate the process of treatment decision-making, enhancing the timely management of patients.

Highly sensitive time-resolved fluorescent microspheres lateral flow immunoassay for the quantitative detection of triadimefon and its metabolite residues in fruits and vegetables.

Ageneral one-step lateral flow immunochromatographic assay (LFIA) for the quantitative detection of triadimefon (TDF) and triadimenol(TDN) in fruit and vegetable samples was developedusing time-resolved fluorescence microspheres (TRFM) as labels. A specific anti-triadimefon monoclonal antibody (mAb) was conjugated with TRFM to fabricate LFIA test strips. A time-resolved fluorometer as an LFIA reader was applied to obtain quantitative results and assess risk ranges for the LFIA test strips. Under the optimized experimental conditions, the limits of detection (LODs) in buffer/cucumbers/tomatoes/oranges were 0.046ng/mL, 0.135µg/kg, 1.047µg/kg, and 5.811µg/kg, respectively, which are ca. 1000 times lower than that of colloidal gold-labeled strips. The recovery in cucumber/tomato/orange samples was 109.4-116.7%, 87.7-110.9%, and 88.0-111.9%, respectively, indicating that the test strips had good reliability. Coupled with the easily customizable pretreatment procedures for various samples, the LFIA results were obtained within 18min without the need for professional personnel or complicated equipment. TRFM-LFIA for TDF and TDN also shows remarkable specificity and precision. The test strips were also low-cost, portable, and convenientto use. These results indicate the test strips could be utilized as a novel strategy for on-site detection of TDF and TDN, which has the potential to expand and detect other pesticide or insecticide residues in food.

B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae

Abstract Background In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.). Methods We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA). Results The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1). Conclusions The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.

A-240 Validation of the OSOM Flu SARS-CoV-2 Combo Home Test

Abstract Background COVID-19 and Influenza respiratory infections often cause similar symptoms, however, treatment is different for each infection. Delay in diagnosis can be fatal in some cases, therefore rapid detection and differentiation are critical for appropriate medical treatment. The OSOM Flu SARS-CoV-2 Combo Home Test is a lateral flow immunoassay intended for the qualitative and differential detection of nucleocapsid protein from SARS-CoV-2 and influenza A/B proteins using self-collected anterior nasal swab samples (self-collection is only recommended for those 14 years of age and older). An interpretation card, included with the test, is used to reliably distinguish among possible testing results. Methods A prospective study was conducted at multiple geographically distinct sites in the U.S during the 2023-2024 season for the validation of the OSOM Flu SARS-CoV-2 Combo Home Test to detect SARS-CoV-2/Flu A/Flu B in self-collected anterior nasal swab samples. The study evaluated the performance in symptomatic individuals only. A total of 703 prospective samples were evaluable for SARS. For Flu A/B, four additional subject samples were unevaluable due to comparator results not being available and a total of 699 samples were included in the data analysis. The comparator tests were FDA 510(k)-cleared molecular assays. Results Influenza A was detected with a sensitivity of 93.1% (95% CI: 84.8%-97.0%) and a specificity of 99.5% (95% CI: 98.6% - 99.8%). Influenza B was detected with a sensitivity of 89.1% (95% CI: 77.0% - 95.3%) and a specificity of 99.7% (95% CI: 98.9% - 99.9%). SARS CoV-2 was detected with a sensitivity of 87.0% (95% CI 78.6% - 92.4%) and a specificity of 99.1% (95% CI: 97.9% - 99.6%) when Ct values are standardized and 10% of samples are considered low positives, having a Ct value of ≥ 30. Conclusions With respiratory infections often showing indistinguishable symptoms and the possibility of co infection, lowering the testing hurdles for individuals to be able to perform a rapid differential test at home without the aid from a healthcare worker is the most effective way to mitigate spread and patient management from a public health perspective. Comparing the OSOM Flu SARS-CoV-2 Combo Home test to the FDA cleared comparators in a clinical setting, the OSOM Flu SARS-CoV-2 Combo Home test retains acceptable sensitivity and specificity. Using self-collected anterior nasal swabs which are minimally invasive alongside the low cost of a lateral flow immunochromatographic assay remains the most effective way to perform the serial screening testing recommended by the CDC in situations where transmission and severe outcome risk is high.

Development and preliminary study of the rLiNTPDase2 rapid test: A lateral flow immunochromatographic assay for Canine Visceral Leishmaniasis

Canine Visceral Leishmaniasis (CVL) is the most fatal form of Leishmania infection in dogs and is caused by L. infantum in the Americas. This parasite follows a zoonotic life cycle, raising concerns within domestic households, where dogs act as the primary reservoir of the parasite. Accurately detecting infected dogs is vital for effective epidemiological control in both canine and human populations. However, existing diagnostic methods in Brazil have limitations, particularly in detecting asymptomatic and oligosymptomatic dogs, leading to ineffective disease control. To address this challenge, we evaluated a novel recombinant antigen from L. infantum, the rLiNTPDase2. Previous studies have confirmed its high performance via ELISA, leading us to assess its suitability for a Lateral Flow Immunochromatographic Assay (LFIA), which is ideal for point-of-care testing. Standardization of the assay involved testing two nitrocellulose membranes (HF135 and HF120, Millipore), three blocking protocols, and five sample dilutions (1:10, 1:20, 1:40, 1:80, and 1:160). Following the chosen conditions (HF120 membrane, 1-minute blocking protocol, and 1:80 sample dilution), we validated our assay with a sample size of 78 dogs, comprising 32 negatives and 46 positives, including symptomatic (n=23), oligosymptomatic (n=17), and asymptomatic (n=6) cases. The results revealed a sensitivity of 86.9 %, specificity of 62.5 %, and accuracy of 76.9 %, which is consistent with ELISA performance for the same samples. Compared to DPP-LVC, our assay demonstrated promising results in detecting asymptomatic and oligosymptomatic cases. This study underscores the suitability of the rLiNTPDase2 antigen for the LFIA format, suggesting its potential as a novel point-of-care diagnostic test for CVL.

Phenylboronic Acid-Modified Membrane-Like Magnetic Quantum Dots Enable the Ultrasensitive and Broad-Spectrum Detection of Viruses by Lateral Flow Immunoassay.

Although lateral flow immunochromatographic assay (LFIA) is an effective point-of-care testing technology, it still cannot achieve broad-spectrum and ultrasensitive detection of viruses. Herein, we propose a multiplex LFIA platform using a two-dimensional graphene oxide (GO)-based magnetic fluorescent nanofilm (GF@DQD) as a multifunctional probe and 4-aminophenylboronic acid (APBA) as a broad-spectrum recognition molecule for viral glycoprotein detection. GF@DQD-APBA with enhanced magnetic/fluorescence properties and universal capture ability for multiple viruses was easily prepared through the electrostatic adsorption of one layer of density-controlled Fe3O4 nanoparticles (NPs) and thousands of small CdSe/ZnS-MPA quantum dots (QDs) on a monolayer GO sheet followed by chemical coupling with APBA on the QD surface. The GF@DQD-APBA probe enabled the universal capture and specific determination of different target viruses on the test strip through an arbitrary combination with the antibody-modified LFIA strip, thus greatly improving detection efficiency and reducing the cost and difficulty of multiplex LFIA for viruses. The proposed technique can simultaneously and sensitively diagnose three newly emerged viruses within 20 min with detection limits down to the pg/mL level. The excellent practicability of GF@DQD-APBA-LFIA was also demonstrated in the detection of 34 clinical specimens positive for SARS-CoV-2, revealing its potential for epidemic control and on-site viral detection.

The Developments on Lateral Flow Immunochromatographic Assay for Food Safety in Recent 10 Years: A Review

Food safety inspections are an essential aspect of food safety monitoring. Rapid, accurate, and low-cost food analysis can considerably increase the efficiency of food safety inspections. The lateral flow immunochromatographic assay (LFIA) technique has recently grown in popularity due to its ease of use and high efficiency. It is currently commonly utilized in food inspection. In this review, we briefly introduce the principle and classification of LFIA, critically discuss the recent application status of LFIA in food contaminantion detection, and finally propose that artificial intelligence and information technology will further advance the development of LFIA in the field of food safety monitoring.

Characteristics and Immunogenicity of Gluten Peptides in Enzyme-Treated and -Untreated Beers for Celiac Patients

The peptidomes from the literature of 24 prolyl-endopeptidase-treated beers during fermentation, declared gluten-free, and 13 untreated beers have been characterised and subjected to an extensive study to investigate their safety for celiac patients. The analysis contains 1996 gluten peptides, ascribed to the treated beers, and 1804 to the untreated beers. The prolyl-endopeptidase-untreated malt beers are hazardous for celiac patients. Peptides of most of these beers showed matches with complete celiac immunogenic motifs, and an additional 28% of the peptides have partial matches with complete immunogenic motifs. On the other hand, after the enzyme treatment during fermentation no celiac hazardous gluten peptides are identified in the treated beers. Due to partial matches with complete celiac immunogenic motifs, 11% potentially hazardous gluten peptides are still identified in the treated beers. Only a maximum of 17% of these peptides can be detected by ELISA analysis. A mass spectrometry analysis or the recently developed method based on G12/A1 monoclonal antibody lateral flow immunochromatographic assay seems necessary to thoroughly reveal the potential risk of the treated beers. The actual immune response of treated beer, described in the literature by the response of the serum antibodies of celiac disease (CD)-active patients and by in vitro immune response, could not be related to the presence of known (partial) CD-immunogenic motifs in the gluten peptides.

Assessment of an in-house IgG ELISA targeting SARS-CoV-2 RBD: Applications in infected and vaccinated individuals

The study evoluated an in-house Spike Receptor Binding Domain Enzyme-Linked Immunosorbent Assay (RBD-IgG-ELISA) for detecting SARS-CoV-2 IgG antibodies in infected and vaccinated individuals. The assay demonstrated a sensitivity of 91%, specificity of 99.25%, and accuracy of 95.13%. Precision and reproducibility were highly consistent. The RBD-IgG-ELISA was able to detect 96.25% of Polymerase chain reaction (PCR) confirmed cases for SARS-CoV-2 infection, demonstrating positive and negative predictive values of 99,18% and 91,69%, respectively. In an epidemiological survey, ELISA, lateral flow immunochromatographic assay (LFIA), and electrochemiluminescence immunoassay (ECLIA) exhibited diagnostic sensitivities of 68.29%, 63.41%, and 70.73%, respectively, along with specificities of 82.93%, 80.49%, and 80.49%, respectively. Agreement between RBD-IgG-ELISA/PCR was moderate (k index 0.512). However, good agreement between different assays (RBD-IgG-ELISA/LFIA k index 0.875, RBD-IgG-ELISA/ECLIA k index 0.901). Test performance on individuals' samples were inferior due to seroconversion time and chronicity. The IgG-RBD-ELISA assay demonstrated its effectiveness in monitoring antibody levels among healthcare professionals, revealing significant differences both before and after the administration of the third vaccine dose, with heightened protection levels observed following the third dose in five Coronavirus disease (COVID-19) vaccine regimens. In conclusion, the RBD-IgG-ELISA exhibits high reproducibility, specificity, and sensitivity, making it a suitable assay validated for serosurveillance and for obtaining information about COVID-19 infections or vaccinations.

Review of Detection Limits for Various Techniques for Bacterial Detection in Food Samples.

Foodborne illnesses can be infectious and dangerous, and most of them are caused by bacteria. Some common food-related bacteria species exist widely in nature and pose a serious threat to both humans and animals; they can cause poisoning, diseases, disabilities and even death. Rapid, reliable and cost-effective methods for bacterial detection are of paramount importance in food safety and environmental monitoring. Polymerase chain reaction (PCR), lateral flow immunochromatographic assay (LFIA) and electrochemical methods have been widely used in food safety and environmental monitoring. In this paper, the recent developments (2013-2023) covering PCR, LFIA and electrochemical methods for various bacterial species (Salmonella, Listeria, Campylobacter, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli)), considering different food sample types, analytical performances and the reported limit of detection (LOD), are discussed. It was found that the bacteria species and food sample type contributed significantly to the analytical performance and LOD. Detection via LFIA has a higher average LOD (24 CFU/mL) than detection via electrochemical methods (12 CFU/mL) and PCR (6 CFU/mL). Salmonella and E. coli in the Pseudomonadota domain usually have low LODs. LODs are usually lower for detection in fish and eggs. Gold and iron nanoparticles were the most studied in the reported articles for LFIA, and average LODs were 26 CFU/mL and 12 CFU/mL, respectively. The electrochemical method revealed that the average LOD was highest for cyclic voltammetry (CV) at 18 CFU/mL, followed by electrochemical impedance spectroscopy (EIS) at 12 CFU/mL and differential pulse voltammetry (DPV) at 8 CFU/mL. LOD usually decreases when the sample number increases until it remains unchanged. Exponential relations (R2 > 0.95) between LODs of Listeria in milk via LFIA and via the electrochemical method with sample numbers have been obtained. Finally, the review discusses challenges and future perspectives (including the role of nanomaterials/advanced materials) to improve analytical performance for bacterial detection.

Validation of the AgraStrip® Pro Total Aflatoxin WATEX® Method for Detection of Total Aflatoxins in Corn and Peanut Paste: AOAC Performance Tested MethodSM 032402.

To protect public and animal health against risks provoked by aflatoxins contained therein, maximum limits for aflatoxins are defined. Limit values vary depending on the intended use and regulatory authority, therefore quantitative detection is essential. Validation of a one-step competitive lateral flow immunochromatographic assay for quantitative screening of total aflatoxin (B1, B2, G1, and G2) in corn and peanut paste for the high-sensitivity range (0-50 µg/kg). Corn or peanut paste test portions are water-based extracted and prepared for testing within 15 min. The AgraStrip® Pro Total Aflatoxin WATEX® test method quantifies the concentration of aflatoxins in the sample. Selectivity, robustness, product consistency, and stability testing were performed in addition to matrix testing. No cross-reactivity was detected against possible interferants. Corn resulted in a LOD and LOQ of 0.9 and 2.8 µg/kg and overall recoveries between 74 and 108%. Peanut paste resulted internally in a LOD and LOQ of 0.8 and 2.3 µg/kg and recoveries between 86 and 98%. Stability testing showed no influence of the age of the respective lot on the result. Robustness testing demonstrated that varying the amount of water used for extraction, extraction time, and delay between extract dilution and analysis did not significantly affect the result. Due to supply chain issues, a change to the outer cartridge required an increase in the test aliquot size, which had no effect on method performance. The test kit was validated for the determination of total aflatoxins in corn and peanut paste. Recovery and precision met the requirements laid down in Codex Alimentarius CXG 71-2009 and acceptable robustness, selectivity, and product consistency and stability were demonstrated. The AgraStrip Pro Total Aflatoxin WATEX test kit in the high sensitivity range (0-50 µg/kg) was approved by the AOAC AOAC Research Institute (PTM number 032402).

Rapid and sensitive visual detection of avian leukosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow immunochromatographic strip assay.

Considering that avian leukosis virus (ALV) infection has inflicted massive economic losses on the poultry breeding industry in most countries, its early diagnosis remains an important measure for timely treatment and control of the disease, for which a rapid and sensitive point-of-care test is required. We established a user-friendly, economical, and rapid visualization method for ALV amplification products based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with an immunochromatographic strip in a lateral flow device (LFD). Using the ALVp27 gene as the target, five RT-LAMP primers and one fluorescein-isothiocyanate-labeled probe were designed. After 60 min of RT-LAMP amplification at 64 °C, the products could be visualized directly using the LFD. The detection limit of this assay for ALV detection was 102 RNA copies/μL, and the sensitivity was 100 times that of reverse transcription polymerase chain reaction (RT-PCR), showing high specificity and sensitivity. To verify the clinical practicality of this assay for detecting ALV, the gold standard RT-PCR method was used for comparison, and consistent results were obtained with both assays. Thus, the assay described here can be used for rapid detection of ALV in resource-limited environments.

Development of a lateral flow immunochromatography assay for the detection of fluoroacetamide in blood samples

Fluoroacetamide (FAM) has been employed as a rodenticide for an extended duration, leading to a multitude of incidents involving human ingestion poisoning. Currently, FAMs have been prohibited by nations globally; however, there are still instances of their illegal usage. Conventional instrument methods are characterized by their time-consuming nature and complex operational procedures, rendering them inadequate for meeting urgent diagnostic needs in patients with acute FAM poisoning. Therefore, there is an immediate need to develop a prompt, user-friendly, and precise immunoassay method for the diagnosis of acute poisoning induced by FAM. A lateral flow immunochromatography assay (LFIA) was developed in this study for the visual detection of FAMs in blood samples, representing the first report of such an approach. The method exhibited a cut-off value of 0.5 mg/mL under the optimized conditions, enabling the entire FAM detection process in blood samples to be completed within a mere 8 min without any pretreatment requirements. Notably, the results were easily discernible by visual inspection alone. These results indicate that the developed LFIA holds great promise as a convenient and rapid diagnostic tool for FAM poisoning diagnosis, thereby offering valuable support for subsequent treatment strategies.

Scalable synthesis of Au@CeO2 nanozyme for development of colorimetric lateral flow immunochromatographic assay to sensitively detect heart-type fatty acid binding protein

Nanozymes with core@shell nanostructure are considered promising biolabeling materials for their multifunctional properties. In this work, a simple one-pot strategy has been proposed for scalable synthesis of gold@cerium dioxide core@shell nanoparticles (Au@CeO2 NPs) with strong localized surface plasmon resonance (LSPR) absorption and high peroxidase-like catalytic activity by redox reactions of Ce3+ ions and AuCl4− ions in diluted ammonia solution under room temperature. A colorimetric lateral flow immunochromatographic assay (LFIA) has been successfully fabricated for sensitive detection of heart-type fatty acid binding protein (H-FABP, an early cardiac biomarker) by using the Au@CeO2 NPs as reporters. The as-developed LFIA with Au@CeO2 NP reporter (termed as Au@CeO2-LFIA) exhibits a dynamic range of nearly two orders of magnitude, and a limit of detection (LOD) as low as 0.35 ng mL−1 H-FABP with nanozyme-triggered 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric amplification. Furthermore, the practicality of Au@CeO2-LFIA has been demonstrated by profiling the concentrations of H-FABP in 156 blood samples of acute myocardial infarction (AMI) patients, and satisfactory results are obtained.

Assessment of aflatoxin levels in whole and peeled ginger marketed in Dar-Es-Salaam, Tanzania

Aflatoxins are naturally occurring toxins, predominantly produced by the fungi Aspergillus flavusand Aspergillus parasiticus. These toxins are often found in numerous agricultural and food products worldwide. The current study investigated the natural occurrence of total aflatoxins in dried split ginger purchased from different local markets in the Dar-Es-Salaam region, the eastern zone of Tanzania. Using a cross-sectional sampling design, a total of 50 ginger samples were analyzed in two preparations as a whole and peeled ginger based on Q+ Aflatoxin single-step lateral flow immuno-chromatographic assay. The results revealed that all ginger samples analyzed were positive for aflatoxins with levels ranging from 5.7–28.0 mg/kg and 3.1–21.5 mg/kg for whole and peeled ginger respectively. Forty (80%) and fifteen (30%) of the samples for whole and peeled ginge respectively were above the Tanzanian legally permissible limit of 10 mg/kg. Interestingly, aflatoxins level of contamination in whole ginger were significantly higher than in peeled ginger (p<0.0001). This is the first report on the natural occurrence of aflatoxins in whole and peeled ginger samples from Tanzania. The findings of this study indicated that ginger peels are relatively prone to aflatoxin contamination.

Sensitivity and specificity of LDBio Aspergillus ICT lateral flow assay for diagnosing allergic bronchopulmonary aspergillosis in adult asthmatics.

Aspergillus fumigatus-specific IgG estimation is crucial for diagnosing allergic bronchopulmonary aspergillosis (ABPA). A point-of-care LDBio immunochromatographic lateral flow assay (LFA) had 0%-90% sensitivity to detect IgG/IgM antibodies against A. fumigatus. To assess the accuracy of LDBio-LFA in diagnosing ABPA, using the modified ISHAM-ABPA working group criteria as the reference standard. The secondary objective was to compare the diagnostic performance between LDBio-LFA and A. fumigatus-specific IgG (cut-offs, 27 and 40 mgA/L), using a multidisciplinary team (blinded to A. fumigatus-IgG and LDBio-LFA results) diagnosis of ABPA as the reference standard. We prospectively enrolled adult subjects with asthma and ABPA. We performed the LDBio-LFA per the manufacturer's recommendations. We used the commercially available automated fluorescent enzyme immunoassay for measuring serum A. fumigatus-specific IgG. We used the same serum sample to perform both index tests. The tests were performed by technicians blinded to the results of other tests and clinical diagnoses. We included 123 asthmatic and 166 ABPA subjects, with a mean ± SD age of 37.4 ± 14.4 years. Bronchiectasis and high-attenuation mucus were seen in 93.6% (146/156) and 24.3% (38/156) of the ABPA subjects. The sensitivity and specificity of LDBio-LFA in diagnosing ABPA were 84.9% and 82.9%, respectively. The sensitivity of serum A. fumigatus-specific IgG ≥27 mgA/L was 13% better than LDBio-LFA, with no difference in specificity. There was no significant difference in sensitivity and specificity between LDBio-LFA and serum A. fumigatus-IgG ≥40 mgA/L. LDBio-LFA is a valuable test for diagnosing ABPA. However, a negative test should be confirmed using an enzyme immunoassay.

A rapid monoclonal antibody-based direct immunoassay and a lateral-flow device for on-site sensitive analysis of the mycotoxin alternariol

Alternariol is an emerging mycotoxin that may be present in many foods at relatively high concentrations. Therefore, sensitive analytical methods are needed to reduce human exposure to this secondary fungal metabolite. The aim of the present study was to develop alternative immunochemical methods for the rapid analysis of alternariol using novel homemade monoclonal antibodies. In this study, a direct competitive enzyme-linked immunosorbent assay for alternariol is reported for the first time, with an IC50 value and a detection limit of 0.020 ng/mL and 0.004 ng/mL, respectively. Under the optimized immunoassay conditions, alternariol could be accurately and precisely quantified in fruit juice samples in only 25 min. In addition, a nanoparticle-based lateral-flow immunochromatography assay with an IC50 value and a visual detection limit of 0.24 ng/mL and 1 ng/mL, respectively, was developed. This immunoassay was validated according to European guidelines for portable semiquantitative analytical methods. Thus, juice samples with alternariol levels above 4 ng/mL were detected by this rapid method in only 15 min, with a negligible false suspect rate.

Lateral Flow Immunochromatographic Assay for Competitive Detection of Crustacean Allergen Tropomyosin Using Phage-Displayed Shark Single-Domain Antibody.

The common food allergy crustacean tropomyosin (TM) poses a significant food safety challenge, which requires rapid and sensitive methods for screening TM in food. Herein, the variable new antigen receptor (VNAR) single-domain antibodies specific for the crustacean TM were isolated from a naïve phage-displayed shark VNAR library. Subsequently, a lateral flow immunochromatographic assay (LFIA) based on the gold nanoparticle-labeled phage-displayed shark VNAR (AuNPs@PSV) probe was developed for the detection of TM in food. The AuNPs@PSV-LFIA took 15 min for one test and had a visual limit of detection (vLOD) of 0.1 μg/mL and an instrumental LOD of 0.02 μg/mL. Good selectivity, accuracy, precision, and stability were confirmed for the AuNPs@PSV-LFIA. Moreover, the test results of 21 commercially available food products consisted of the allergen labels and were validated by a commercial ELISA kit. Therefore, this work demonstrated the great potential of VNAR for detecting TM in food by LFIA.

A novel lateral flow immunochromatographic assay using a recombinant VP2 antigen for total antibody detection of canine parvovirus-2.

Canine parvovirus-2 (CPV-2) is a viral disease of dogs causing acute hemorrhagic gastroenteritis and myocarditis with high morbidity and mortality rates. The infection is still widespread all over the world. Vaccines developed against infection have great importance in preventing infection. However, it is difficult to recommend a practical vaccination program without knowing the antibody level of a puppy. Despite widespread vaccination, difficulties in detecting the maternal antibodies in puppies remain the main cause of vaccination failure. The hemagglutination inhibition (HAI) test is the gold standard to determine the immune status of dogs for canine parvovirus 2, but the HAI test has several disadvantages such as the need for fresh porcine blood, well-equipped laboratory, and long incubation periods. In this study, for the first time we developed a colloidal gold-based competitive lateral flow assay (cLFA) system for the rapid detection of total antibodies in canine serum using CPV-2b-VP2 derived from field isolates. The recombinantly expressed capsid protein of CPV-2 in the prokaryotic expression system was used as a labeled molecule in cLFA. We carried out studies on our cLFA system using the standard antibody solution and the clinical samples from vaccinated puppy serum. We compared the results of the LFAs with the HAI test. Competitive lateral flow assay results showed good correlation with the gold standard method, the HAI test. In the developed platform, the limit of detection of the standard antibody was determined to be 375 ng mL-1, while the cut-off level of antibodies was observed to be 1 : 40 HAI titer in clinical samples. Our reported system will be a strong alternative for CPV-2 antibody-based detection applications.