Fluorescence Immunochromatography Research Articles

The performance of a new rapid interferon gamma release assay based on fluorescence immunochromatography for Mycobacterium tuberculosis infection testing in village doctors in China.

The performance of a new rapid interferon gamma release assay based on fluorescence immunochromatography for Mycobacterium tuberculosis infection testing in village doctors in China.

Rapid and sensitive detection of staphylococcal enterotoxin E using a time-resolved fluorescence immunochromatography assay

Staphylococcal food poisoning is a significant foodborne illness caused by staphylococcal enterotoxins (SEs). Immunoassays have become the primary method for rapidly detecting harmful bacteria and toxins because of their excellent sensitivity and specificity. However, these assays have limitations in that they cannot differentiate between types of SEs and do not provide rapid, on-site, quantitative testing. In this study, a time-resolved fluorescence immunochromatography assay (TRFICA) was developed specifically for detecting staphylococcal enterotoxin E (SEE), which is commonly found in dairy products. Compared with a double antibody sandwich enzyme-linked immunosorbent assay, which had a detection limit of 0.028 ng/mL, TRFICA demonstrated comparable sensitivity, enabling SEE quantification with a detection limit as low as 0.081 ng/mL in infant formula. Validation by spiking infant formula samples confirmed no cross-reactivity with analogs (recoveries ranged from 93.17% to 128.77%). Furthermore, with an 8-min reaction time and interpretation delivered by a portable TRFICA strip reader, our method demonstrates potential for use in mobile and on-site detection. This study describes a rapid, easy, and reliable method for detecting trace levels of SEE in infant formula, which could serve as an early screening tool toward preventing food poisoning in infants and children.

A sensitive immunochromatography assay based on quantum dot nanobeads for determination of acid orange II

Acid orange II (AO2) is a synthetic azo dye commonly used illegally in food production and processing. The development of a fast and effective analytical method is crucial for controlling the unlawful use of AO2 in food, especially in seasoning. Here, an antibody specific for AO2 was generated, with 1.01 ng/mL IC50 and minimal cross-reactivity with other analogs (<1 %, including sunset yellow, tartrazine, para red, chromotrope FB, Sudan Ⅰ, rhodamine B, 2-naphthol, amaranth, ponceau 4 R and allura red AC). Furthermore, a highly sensitive quantum dot beads-based fluorescence immunochromatography assay (QBs-FICA) was established for AO2 determination. The assay had a working range of 23–287 μg/kg with a 11 μg/kg limit of detection (LOD) in chili powder and hotpot seasoning samples. Using spiked samples, the results of QBs-FICA were found to be consistent with those of liquid chromatography-tandem mass spectrometry. This indicates that QBs-FICA is a reliable, rapid, and portable tool for testing of AO2.

Etiological surveillance of viral diarrhea from 2017 to 2019 in Zhangzhou city, Fujian province, China.

Viral diarrhea is one of the major causes of morbidity and mortality in children. This study aimed to conduct etiological surveillance of viral diarrhea in Zhangzhou city, Fujian province, China, from 2017 to 2019 to identify the prevalence, distribution, and characteristics of viral pathogens causing gastrointestinal infections in the region. Stool samples were collected from patients with acute diarrhea in Zhangzhou city, Fujian province, China, from 2017 to 2019. Rotavirus, norovirus, astrovirus, and adenovirus were detected using fluorescence immunochromatography assay. Of the total 5,627 samples that were collected, at least one of the viruses (rotavirus, norovirus, astrovirus and adenovirus) was found to be positive in 1,422 samples. Rotavirus, norovirus, astrovirus, and adenovirus, were detected in 53.73, 16.68, 15.52, and 14.97%, respectively. Mixed infections were determined in 17.65% of the positive samples. The predominant mixed infections observed were a combination of norovirus and astrovirus, followed by rotavirus and norovirus, and rotavirus and astrovirus. The highest positive rate was observed in the 12-23-month group for rotavirus and adenovirus, while a significantly higher positive rate was observed for norovirus and astrovirus in the 6-11-month group. These findings from this etiological surveillance highlight the significant burden of viral diarrhea in Zhangzhou city, with rotavirus being the predominant pathogen. The identification of common mixed infections provides insights into the complex nature of viral diarrhea transmission. Target interventions and public health strategies should be implemented, particularly during the winter and spring seasons, to prevent and control the spread of viral pathogens causing gastrointestinal infections in this region.

Quantitative detection of mpox antigen using time-resolved fluorescence immunochromatography.

Recently, concern has been raised about the spread of human mpox virus, and the demand for rapid detection reagents for mpox virus has increased. This study aims to establish a time-resolved fluorescence immunochromatography (TRFICO) method for qualitative/quantitative detection of mpox virus. A double-antibody sandwich TRFICO method was optimized and established using mpox recombinant fusion antigen and its paired monoclonal antibody. The test performance of the method was evaluated using mpox fusion antigen and control serum, including sensitivity, linearity range, specificity, precision, and reference interval. We successfully established a TRFICO method for qualitative/quantitative detection of mpox antigen, its linearity range 0-100ng/mL, analytical sensitivity 0.017ng/mL, and reference intervals greater than 0.045ng/mL. No cross-reaction was detected with various poxvirus and clinical negative controls, with good specificity. All average recoveries of the intra- and inter-batch ranged from 81.33% to 97.83%, and all CVs were below 10%. Additionally, the TRFICO strips can be stably stored at 37°C for 7days without significant changes in the fluorescence intensity. This TRFICO method, with high sensitivity, linearity range, specificity, precision, and stability with 16-min detection time, provides a new option for qualitative/quantitative and convenient testing of mpox virus.

Development of time-resolved fluorescence immunochromatography and ic-ELISA based on monoclonal antibodies specifically recognizing fumonisin B1

Fumonisins (FBs) are mycotoxins primarily synthesized by Fusarium moniliformis. Among these, FB1 exhibits not only high toxicity towards humans and animals but also carcinogenic properties. The global prevalence of FB1 contamination in cereals and related products, particularly maize, is alarmingly significant. Consequently, the accurate determination of FB1 levels in cereals holds immense importance. In this study, highly sensitive monoclonal antibodies specifically targeting FB1 were prepared and utilized for the establishment of a time-resolved fluorescence immunochromatographic assay (TRFIC) to detect FB1. The parameters of antibody labeling with time-resolved fluorescent microspheres were optimized. The detection time was significantly reduced to 6 min. The limits of detection (LOD) for corn, rice, and feed were determined as 0.496–0.844 μg/kg, and the quantification (LOQ) was 0.788–1.322 μg/kg. In addition, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was successfully developed. Under optimized conditions, the half inhibitory concentration (IC50) value for FB1 was determined as 2.137 μg/L. A strong correlation between the results obtained from these two methods and HPLC-MS/MS analysis was observed in the same samples tested. In conclusion, both immunological methods developed in this work are highly suitable for rapid FB1 detection in real field samples.

Designing a size exclusion-based hapten and the development of a quantitative and visual time-resolved fluorescence immunochromatography assay strip for detecting dimethomorph and flumorph in a group-specific manner

Based on the size and surface properties of dimethomorph and flumorph, we used a computer simulation-assisted size exclusion hapten design strategy to develop group-specific monoclonal antibodies that can simultaneously recognize dimethomorph and flumorph. For this, we performed quantitative and visual semi-quantitative time-resolved fluorescence immunochromatography (TRFICA) to simultaneously detect dimethomorph and flumorph in potatoes and apples. In potato samples, the visual limit of detection (vLOD) for dimethomorph and flumorph was 4 ng/mL and 8 ng/mL, respectively, whereas the quantitative limit of detection (qLOD) for dimethomorph and flumorph was 0.26 and 0.33 ng/mL, respectively. The vLOD of dimethomorph and flumorph in apple samples was 8 ng/mL, whereas the qLOD of dimethomorph and flumorph was 0.17 and 0.38 ng/mL, respectively. The average recovery of potato and apple samples ranged from 77.5% to 121.7%, which indicated that the method can be used to rapidly detect dimethomorph and flumorph in food samples.

Usefulness of a new immunochromatographic assay using fluorescent silica nanoparticles for serodiagnosis of Thai patients with amebiasis.

A fluorescence immunochromatography (FIC) kit was developed recently using fluorescent silica nanoparticles coated with a recombinant C-terminal fragment of the surface lectin intermediate subunit (C-Igl) of Entamoeba histolytica to establish rapid serodiagnosis of amebiasis. We further evaluated the system using serum samples from 52 Thai patients with amebiasis. Of the patients, 50 (96%) tested positive using FIC. The samples were also tested using enzyme-linked immunosorbent assay (ELISA) with C-Igl as the antigen. Two samples were negative on ELISA but positive on FIC. The correlation coefficient between the fluorescence intensity using FIC and the optical density value using ELISA was 0.5390, indicating a moderate correlation between the two tests. Serum samples from 20 patients with malaria and 22 patients with Clostridioides difficile infection were also tested using FIC. The false-positive rates were 4/20 (20%) and 1/22 (4%) in patients with malaria and C. difficile infection, respectively. Combining the data from the present study with our previous study, the sensitivity and specificity of FIC were determined to be 98.5% and 95.2%, respectively. The results of the 50 samples were studied using a fluorescence scope and a fluorescence intensity reader, and the findings were compared. Disagreements were found in only two samples showing near-borderline fluorescence intensity, indicating that the use of scope was adequate for judging the results. These results demonstrate that FIC is a simple and rapid test for the serodiagnosis of amebiasis.

Comparison of Plasma Concentration of Linezolid's Detection by FICA and LC-MS/MS.

Linezolid was the first totally synthetic oxazolidinone antibiotic that was approved for clinical use against Mycobacterium tuberculosis. Our study investigated the correlation and consistency of fluorescence immunochromatography assay (FICA) and high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in measuring linezolid levels in the plasma. Plasma samples were collected from 80 to 100 tuberculosis patients. The samples were analyzed by FICA and LC-MS/MS, and linezolid levels measured by the two methods were compared by outlier test, correlation coefficient, Bland-Altman scatter plot, Deming regression analysis and bias evaluation. A total of 83 plasma samples were collected, all of which were non-outlier. The correlation coefficient of FICA and LC-MS/MS results was 0.9845. Deming regression analysis showed that the regression equation of FICA and LC-MS/MS was y = 0.2229 + 1.0076x. The medical decision levels of 2 and 7μg/mL were taken into the regression equation, and the 95% CI of 2- and 7-μg/mL bias were both within the allowed error range. The findings indicated a strong correlation between the results obtained from FICA and LC-MS/MS were consistent showed good correlation, demonstrating their equivalence in measurement.

Rapid determination of furazolidone residues in animal foods by time-resolved fluorescence immunochromatography

Although furazolidone (FZD) was completely banned from livestock production in many countries many years ago due to its mutagenicity and carcinogenicity, the abuse of FZD is still common today. Accurate and rapid detection of FZD residues in animal-derived food products is highly important for human health. Here, a time-resolved fluorescence immunochromatography (TRFI) test strip for rapid and quantitative detection of 3-amino-2-oxazolidinone (AOZ) residues in animal foods was developed and validated. Its limit of detection and limit of quantification were 0.05 and 0.14 μg/kg, respectively. The typical recovery rates were 95–105 % in chicken breast samples spiked with the AOZ standard substance at concentrations of 0.05–2 μg/kg, with a coefficient of variation value ≤8.5 %. The cross-reaction rates of the TRFI-AOZ test strips with 3-amino-5-morpholinomethyl-2-oxazolidone, semicarbazide, and 1-amino-imidazolidin-2,4-dione were less than 1 %. The newly developed TRFI test strip has high sensitivity, high specificity, cost effectiveness and user-friendly control, and is suitable for the rapid and large-scale screening of AOZ residues in animal foods.

Development of a fluorescence immunochromatography method for quantitative measurement of matrix metalloproteinase-9

ObjectiveAbnormal serum matrix metalloproteinase-9 (MMP-9) levels are closely related to the occurrence and development of many diseases. This study aimed to establish a fluorescence immunochromatography (FIC) method using the lanthanide fluorescent element europium(III) (Eu3+) for the quantitative measurement of MMP-9 in serum. Design & MethodsThe FIC method for quantifying MMP-9 was optimized and established, and the FIC test strips (FICTS) were assembled and subsequently evaluated for sensitivity, specificity and precision. Furthermore, the reference interval and clinical sensitivity/specificity were estimated using clinical healthy/positive serum samples, and a commercial ELISA was used for comparison. ResultsWe successfully established an FIC method and prepared FICTS. The analytical sensitivity of the FICTS was 0.92 ng/mL, with a linearity range of 0–1000 ng/mL. The cross-reactivity of the 7 common serum interferents was less than 1.56%. All recoveries of the intra-array and inter-array samples ranged from 102.50% to 110.99%, and all CVs were less than 5%. The reference interval of the FICTS was >161.15 ng/mL. The clinical sensitivity was 96.00%, and the specificity was 97.5%. The results of 270 clinical serum samples were highly coincident with the clinical diagnostic results. Pearson correlation analysis and Bland‒Altman plots indicated that the FICTS and commercial ELISA results were consistent with the quantitative MMP-9 concentration. ConclusionsThe designed FIC method and test strips may be suitable for point-of-care quantitative measurement of MMP-9, which provides a new method for screening for atherosclerosis, xerophthalmia, etc.

Design of POCT Testing Device for Fluorescence Immunochromatography

Design of POCT Testing Device for Fluorescence Immunochromatography

A Fluorescent Immunochromatography Test Strip for the Rapid Identification of SVV and FMDV

Seneca Valley virus (SVV) and foot‐and‐mouth disease virus (FMDV) belong to the Picornaviridae family, which can cause similar symptoms. After infection, pigs will develop fever; loss of appetite; blister lesions on the skin and mucous membrane of the mouth, nose, and hoof; and other similar diseases, and the spread is very fast, causing major economic losses to the pig industry. Therefore, a rapid, accurate, and sensitive diagnostic method is necessary to enable rapid prevention and control measures for preventing the spread of these diseases. Here, a fluorescent immunochromatography test strip, using Eu‐doped fluorescence beads and monoclonal antibody, was developed for the simultaneous determination of FMDV and SVV. The test process for the assay could be completed in 12 min, which avoided the time cost of the current methods for FMDV/SVV detection. Under optimized conditions, the limit of detection of SVV is 5 × 104 PFU/mL, and that of FMDV is 5 × 104 PFU/mL under the Fluorescence Immunoassay Analyzer. Our assay results showed a good linear correlation with RT‐PCR installed in the clinical laboratory. The species design has a promising application prospect in the surveillance and control of the outbreak of idiopathic blister.

Deep learning assisted quantitative detection of cardiac troponin I in hierarchical dendritic copper-nickel nanostructure lateral flow immunoassay.

The rising demand for point-of-care testing (POCT) in disease diagnosis has made LFIA sensors based on dendritic metal thin film (HD-nanometal) and background fluorescence technology essential for rapid and accurate disease marker detection, thanks to their integrated design, high sensitivity, and cost-effectiveness. However, their unique 3D nanostructures cause significant fluorescence variation, challenging traditional image processing methods in segmenting weak fluorescence regions. This paper develops a deep learning method to efficiently segment target regions in HD-nanometal LFIA sensor images, improving quantitative detection accuracy. We propose an improved UNet++ network with attention and residual modules, accurately segmenting varying fluorescence intensities, especially weak ones. We evaluated the method using IoU and Dice coefficients, comparing it with UNet, Deeplabv3, and UNet++. We used an HD-nanoCu-Ni LFIA sensor for cardiac troponin I (cTnI) as a case study to validate the method's practicality. The proposed method achieved a 96.3% IoU, outperforming other networks. The R2 between characteristic quantity and cTnI concentration reached 0.994, confirming the method's accuracy and reliability. This enhances POCT accuracy and provides a reference for future fluorescence immunochromatography expansion.

Determination of serum CA724 levels using fluorescence immunochromatography

BackgroundCarbohydrate antigen 724 (CA724) is a sensitive and specific indicator for multiple malignant tumors. The aim of this study was to establish a Eu-time resolved fluorescence immunochromatography (Eu-TRFICO) method for quantitative detection of CA724 in serum.MethodsEu-TRFICO strips were optimized and assembled. The sensitivity, specificity and precision were evaluated using CA724 standard dilutions and matrix serum. Meanwhile, the reference interval, comparison, and sensitivity/specificity were performed using clinical negative/positive gastric cancer serum samples.ResultsThe standard curve equation was y = 9.869 x − 154.12 (R2 = 0.993), and the sensitivity was 0.42 U/mL. The common interferents in serum could not affect the quantitative results with low cross-reactivities (all no more than 1.09%). All average recoveries of the intra- and interbatch ranged from 102.38 to 106.40%, and all CVs were below 10%. The reference interval of the healthy subjects was < 4.68 U/mL and the reference interval of the subjects with grade I/II gastric cancer was > 9.54 U/mL. Additionally, a high Pearson r (0.9503) and sensitivity/specificity (92.86%/94.20%) were obtained.ConclusionThis study prepared Eu-TRFICO strips with high sensitivity, specificity, precision and satisfactory clinical testing performance, which provides more options for clinical quantitative and convenient testing of CA724.

Performance evaluation of SARS-CoV-2 antigen detection in the post-pandemic era: multi-laboratory assessment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen detection is an indispensable tool for epidemic surveillance in the post-pandemic era. Faced with irregular performance, a comprehensive external quality assessment (EQA) scheme was conducted by the National Center for Clinical Laboratories (NCCL) to evaluate the analytical performance and status of SARS-CoV-2 antigen tests. The EQA panel included ten lyophilized samples containing serial 5-fold dilutions of inactivated SARS-CoV-2-positive supernatants of the Omicron BA.1 and BA.5 strains and negative samples, which were classified into "validating" samples and "educational" samples. Data were analyzed according to qualitative results for each sample. A total of 339 laboratories in China participated in this EQA scheme, and 378 effective results were collected. All validating samples were correctly reported by 90.56 % (307/339) of the participants and 90.21 % (341/378) of the datasets. The positive percent agreement (PPA) was >99 % for samples with concentrations of 2×107 copies/mL but was 92.20 % (697/756) for 4×106 copies/mL and 25.26 % (382/1,512) for 8×105 copies/mL samples. Colloidal gold was the most frequently used (84.66 %, 320/378) but showed the lowest PPAs (57.11 %, 1,462/2,560) for positive samples compared with fluorescence immunochromatography (90 %, 36/40) and latex chromatography (79.01 %, 335/424). Among 11 assays used in more than 10 clinical laboratories, ACON showed a higher sensitivity than other assays. The EQA study can help to validate whether it's necessary to update antigen detection assays for manufacturers and provide participants with information about the performance of assays to take the first step toward routine post-market surveillance.

Paper-based biosensors relying on core biological immune scaffolds for the detection of procymidone in vegetables

In order to achieve a highly sensitive detection of procymidone in vegetables, three paper-based biosensors based on a core biological immune scaffold (CBIS) were developed, which were time-resolved fluorescence immunochromatography strips with Europium (III) oxide (Eu-TRFICS). Goat anti-mouse IgG and europium oxide time-resolved fluorescent microspheres formed secondary fluorescent probes. CBIS was formed by secondary fluorescent probes and procymidone monoclonal antibody (PCM-Ab). The first type of Eu-TRFICS (Eu-TRFICS-(1)) fixed secondary fluorescent probes on a conjugate pad, and PCM-Ab was mixed with a sample solution. The second type of Eu-TRFICS (Eu-TRFICS-(2)) fixed CBIS on the conjugate pad. The third type of Eu-TRFICS (Eu-TRFICS-(3)) was directly mixed CBIS with the sample solution. They solved the problems of steric hindrance of antibody labeling, insufficient exposure of antigen recognition region and easy loss of activity in traditional methods. They realized multi-dimensional labeling and directional coupling. They replaced the loss of antibody activity. And the three types of Eu-TRFICS were compared, among which Eu-TRFICS-(1) was the best detection choice. Antibody usage was reduced by 25% and sensitivity was increased by 3 times. Its detection range was 1–800 ng/mL, the limit of detection (LOD) was 0.12 ng/mL with the visible LOD (vLOD) of 5 ng/mL.

Fluorescent Immunochromatography of Vancomycin in Plasma: A Rapid and Sensitive Quantitative Analysis.

Vancomycin (VAN) has a narrow therapeutic window and variable pharmacokinetic properties, which require timely monitoring of plasma concentration to adjust the dosage for better effectiveness. A sensitive and quantitative immunochromatographic method was developed to detect vancomycin in plasma. The linear response range of the method to detect plasma vancomycin was 1.25-50 μg/mL. The intra-assay and inter-assay coefficients were 4.68% and 8.81%, respectively, while the recovery was 89.10-98.32%. The clinical comparative experiment results demonstrated a good correlation (r>0.90) between the fluorescent immunochromatographic strip test and the mass spectrum. In this study, a simple, rapid, and high-sensitivity immunochromatographic method was established for detecting vancomycin, which helped establish the basis for further application of monitoring plasma concentration.

A helper antibody based competitive fluorescence immunochromatographic assay for quantitative detection of florfenicol in poultry eggs.

Florfenicol (FF) is a chloramphenicol analogue used in animals, and florfenicol amine (FFA) is the main metabolite of FF. However, their residues in agricultural products are harmful to human health. A highly specific and sensitive assay for FF/FFA detection needs to be developed since the traditional detection methods are low sensitive. In this study, a new method for rapid quantification of FF/FFA in poultry eggs by fluorescent immunochromatographic assay (HAFIA) was established. The triple antibodies including a primary monoclonal antibody (mAb) specific to the targets FF and FFA, a secondary polyclonal antibody (pAb) labeled with Europium nanoparticles (EuNPs) and a helper monoclonal antibody (hAb) reacting with pAb but not react with mAb or target antigen are designed, which can form structural aggregation complexes in microwells with a single step of reactions. By loading the reaction sample solution, the triple-antibodies (mAb-pAb-hAb)-EuNPs complexes migrate to the test (T) line on nitrocellulose membrane of testing strip, which are competitively captured by the immobilized FF-BSA conjugates on the membrane and the FF/FFA targets in sample solution. Fluorescence on T line is read by a portable fluorescent strip reader in 10 min, and the result is given by the ratio of fluorescent intensity on T line than control (C) line. This new fluorescent testing strip with triple-antibody complexes' amplification signal has 50-fold higher sensitivity than conventional CG-LFIAs, and can detect as low as 0.01 ng/mL florfenicol and 0.1 ng/mL florfenicol amine targets from egg samples. The developed competitive fluorescent immunochromatography method based on auxiliary antibodies has the advantages of high sensitivity and specificity for the rapid and quantitative detection of FF/FFA in poultry eggs.

Comparison of a rapid fluorescence immunochromatographic test with an enzyme-linked immunosorbent assay for measurement of SARS-CoV-2 spike protein antibody neutralizing activity

BackgroundSARS-CoV-2 Spike protein Receptor Binding Domain neutralizing antibodies (NAbs-RBD) inhibit the viral binding to angiotensin-converting enzyme 2 (ACE2) receptors. We compared an ELISA and a fluorescence immunochromatography (FIC) method in NAbs-RBD detection after COVID-19 immunization. MethodSerum samples from healthcare workers (HCWs) vaccinated with BNT162b2 were collected one and four months after the second dose. NAbs-RBD (%) detection was performed using ELISA cPass™ (FDA approved) and FIC n-AbCOVID-19® assays. ResultsSamples from 200 HCWs [median age (IQR): 45(35−53)] were tested with both assays. There was a good qualitative agreement between the two methods [AUC: 0.92(95%C.I.: 0.89–0.94, P-value:0.007)]. NAbs-RBD (%), one and four months after immunization, were significantly lower with FIC compared to ELISA for all age groups (P-value<0.0001). The quantitative comparison between FIC and ELISA detected slight agreement one month after the second dose [(Lin’s Concordance Correlation Coefficient (CCC): 0.21(95%CI: 0.15–0.27)] which improved four months after the second dose [CCC: 0.6(95%CI: 0.54–0.66)]. ConclusionFIC had good qualitative agreement with ELISA in the detection of positive NAbs-RBD (%) and could be an alternative for rapid NAbs-RBD (%) testing.