Conjugate Pad Research Articles

Detection strip of anti-NGAL antibody coupled with fluorescent microspheres-A novel tool for reliable and accurate prediction of renal injury

ObjectiveNeutrophil gelatinase-associated lipocalin (NGAL) has been identified by the International Nephrology Association (INA) as a promising biomarker for the early evaluation of renal injury. This study aimed to develop and evaluate NGAL test strips as a rapid, simple, and economical method for the early diagnosis of acute kidney injury (AKI). MethodsRecombinant prokaryotic expression vectors, purified NGAL protein, and anti-NGAL monoclonal antibodies were prepared. NGAL test strips were developed, and serum samples were collected from healthy individuals and patients with early-stage kidney injury at the Third Affiliated Hospital of Sun Yat-sen University between January 2023 and May 2024. Samples were tested using both the self-made strips and commercially available reagents. ResultsThe NGAL test strip comprised a conjugate pad containing 0.2 μL of fluorescent microspheres conjugated with anti-NGAL monoclonal antibody (McAb7#), a test line containing 1 mg/mL of a different anti-NGAL monoclonal antibody (McAb3#), and a control line containing 0.5 mg/mL of goat anti-mouse IgG. The test utilized 60 μL of sample (30 μL serum diluted with 30 μL of sample diluent) and was completed within 15 min at 25 °C and 35 %-85 % relative humidity. The developed strip accurately detected NGAL, demonstrating good linearity within the range of 0–160 ng/mL (R2 = 0.9943). The sensitivity and specificity of the NGAL strip for AKI diagnosis were 86.1 % and 78.8 %, respectively, comparable to the performance of commercially available testing reagents. ConclusionThe developed test strip, utilizing anti-NGAL antibodies coupled with fluorescent microspheres, effectively detected trace amounts of NGAL protein in serum samples.

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

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

Modified streptavidin–biotin based lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples

Compared to other infectious diseases, for which LFT development can take years, SARS-CoV-2 antigen LFTS were developed and deployed within months. LFTS for antigen detection were adopted on an unprecedented scale during the COVID-19 pandemic, but many of them lack the sensitivity especially for samples with low viral load. In our previous work, we developed an enhanced signal strip for detection of SARS CoV-2 SI antigens in saliva. Here we introduce some modification to improve the sensitivity, and specificity, and to lower the cost of the strip, by using biotin streptavidin (BS) system. In the modified BS strip, gold-streptavidin and biotinylated Nanobodies (Nbs) against S1 antigen were externally mixed with the tested samples (saliva or nasopharyngeal swab) before their application on the sample pad of the test strip containing angiotensin converting enzyme (ACE-2), as the capturing probe. The study included 320 individuals, with 180 being positively confirmed by RT-PCR and 140 confirmed negative, as well as, 45 health care workers, who were responsible for screening and handling of surgical cases in General Surgery Department and COVID clinic of TBRI. Our results proved that modified BS strip improved the overall sensitivity and specificity of S1antigen detection in saliva samples (95.21% and 99.29% respectively) compared to our previously developed enhanced LFTS (91.66% and 98.57% respectively). Also, the sensitivity of cases with Ct ≤ 30, Ct ≤\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\le$$\\end{document} 35, and Ct ≤\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\le$$\\end{document} 40 using the modified BS strip showed higher values (98.54%, 95.38%, and 88.89% respectively), compared to the corresponding results of our previously developed enhanced LFTS (95.86%, 92.31%, and 82.22% respectively). There were no cross-reactions with either Middle East respiratory syndrome corona virus MERS-CoV or SARS-CoV antigens. Furthermore, we found that the lower viral detection limit (LVD) of BS strip was obviously lower than our previous LVD limit of the enhanced LFTS (0.2 × 104 copies/ml vs. 0.4 × 104 copies/ml, respectively). Our developed BS strip showed that saliva samples gave better results than nasopharyngeal swabs of the same patients. The fact of using smaller amounts of Nbs, and ACE2, as well as the dispensing off of conjugate pad when applying BS strip modifications, justified the expected reduction in the costs of the strip. The implementation of BS strips on saliva samples of 45 health co-workers, who were tested 4 and 6 days after exposure to infection, showed an increase in the sensitivity, starting from the 4th day and reaching its highest level on the 6th day in both high risk and paramedic groups (90.9%, and 80.0%, respectively). This study provides evidence that employment of the modified BS system could increase the sensitivity of the strips, lower their cost, and render them an effective screening tool for early detection of the virus in saliva of suspected Covid-19 patients.

DEVELOPMENT OF THE RECYCLING PROCEDURE FOR RAPID ANTIGEN TESTS

The article presents the problem of rapid antigen tests when they become mass waste after use. Based on this, the hypothesis was made that rapid antigen tests can be recycled. Rapid antigen tests, which were used in the Covid-19 epidemic to quickly detect infections in the population or to confirm the presence of the Sars-Cov 2 virus in patients, were intended to limit the spread of the epidemic. To confirm the hypothesis of recycling for rapid antigen tests, the LFIA-REC ATP 150 project was prepared, which was selected for co-financing by the Norwegian Fund.
 Rapid antigen tests consist of a sample and conjugate pad, detectable or nitrocellulose membranes and absorbent pads and a plastic case. The function of the sample pad is to evenly absorb the sample (mucus, blood) and lead it to the conjugate pad with a steady flow. Gold nanoparticles (labels) are deposited on the conjugate pad. The key is that the gold nanoparticles are conjugated with capture molecules capable of binding to potentially present antibodies or virus in the sample.
 The scope of the research problem thus required the inclusion of various characterization techniques that must be applied to the individual material in the rapid antigen test to subsequently develop an efficient recycling process for the rapid antigen tests. The result of the research presented in this paper represents a newly developed algorithm of characterization techniques, which includes a recommended description of the preparation of samples of key materials from rapid antigen tests. This algorithm successfully achieved the characterization of gold nanoparticles from rapid antigen tests. Based on the developed algorithm, the final part of the project will validate the recycling process of rapid antigen tests, so that they can be recycled, i.e. gold nanoparticles or plastic used in new products. The paper presents the algorithm of characterization techniques with a description of the preparation of material samples from rapid antigen tests.

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

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

An Aptamer-Based Lateral Flow Biosensor for Low-Cost, Rapid and Instrument-Free Detection of Ochratoxin A in Food Samples.

In this work, a simple and cost-efficient aptasensor strip is developed for the rapid detection of OTA in food samples. The biosensor is based on the lateral flow assay concept using an OTA-specific aptamer for biorecognition of the target analyte. The strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane (NC) and an absorbent pad. The conjugate pad is loaded with the OTA-specific aptamer conjugated with gold nanoparticles (AuNPs). The test line of the NC membrane is loaded with a specific OTA-aptamer probe and the control line is loaded with a control probe. The assay is based on a competitive format, where the OTA present in the sample combines with the OTA aptamer-AuNP conjugate and prevents the interaction between the specific probe immobilized on the test line and the OTA aptamer-AuNP conjugates; therefore, the color intensity of the test line decreases as the concentration of OTA in the sample increases. Qualitative detection of OTA is performed visually, while quantification is performed by reflectance colorimetry using a commercial scanner and image analysis. All the parameters of the assay are investigated in detail and the analytical features are established. The visual limit of detection (LOD) of the strip is 0.05 ng mL-1, while the LOD for semi-quantitative detection using reflectance colorimetry is 0.02 ng mL-1. The lateral flow strip aptasensor is applied to the detection of OTA in wine, beer, apple juice and milk samples with recoveries in the range from 91 to 114%. The assay exhibits a satisfactory selectivity for OTA with respect to other mycotoxins and lasts 20 min. Therefore, the lateral flow strip aptasensor could be useful for the rapid, low-cost and fit-for-purpose on-site detection of OTA in food samples.

Serodiagnosis of multiple cancers using an extracellular protein kinase A autoantibody-based lateral flow platform

Extracellular protein kinase A autoantibody (ECPKA-AutoAb) has been suggested as a universal cancer biomarker due to its higher amounts in serum of several types of cancer patients than that of normal individuals. Herein, we first developed a lateral flow immunoassay (LFIA) tool, using a sandwich format, toward ECPKA-AutoAb in human serum. For this format, 3G2 as a capture antibody was identified using hybridoma technique and a series of screenings where it showed superior capacity to recognize Enzo PKA catalytic subunit alpha (Cα), compared to other PKA antibodies and antigens. Using these components, we performed sandwich ELISA toward a mimic and real sample of ECPKA-AutoAb. As per the results, limit of detection (LOD) was found to be 135 ng/mL and ECPKA-AutoAb levels were higher in various cancer patients than in normal individuals like previous studies. Based on these results, we applied this sandwich format into LFIA tool and found that the LOD of the fabricated LFIA tool showed about 3.8 ng/mL using spiked PKA-Ab, which is significantly improved compared to the LOD of sandwich ELISA. Also, the developed LFIA tool demonstrated a remarkable ability to detect significant differences in ECPKA-AutoAb levels between normal and cancer patients within 15 min, showing a potential for point-of-care (PoC) detection. One interesting point is that our LFIA strip contains an additional conjugation pad II, named because of its position behind the conjugation pad, in which PKA Cα is dried, enabling a sandwich format.

Recovery Study of Gold Nanoparticle Markers from Lateral Flow Immunoassays.

Lateral flow immunoassays (LFIAs) are a simple diagnostic device used to detect targeted analytes. Wasted and unused rapid antigen lateral flow immunoassays represent mass waste that needs to be broken down and recycled into new material components. The aim of this study was to recover gold nanoparticles that are used as markers in lateral flow immunoassays. For this purpose, a dissolution process with aqua regia was utilised, where gold nanoparticles were released from the lateral flow immunoassay conjugate pads. The obtained solution was then concentrated further with gold chloride salt (HAuCl4) so that it could be used for the synthesis of new gold nanoparticles in the process of ultrasonic spray pyrolysis (USP). Various characterisation methods including scanning electron microscopy, transmission electron microscopy, ultraviolet-visible spectroscopy and optical emission spectrometry with inductively coupled plasma were used during this study. The results of this study showed that the recovery of gold nanoparticles from lateral flow immunoassays is possible, and the newly synthesised gold nanoparticles represent the possibility for incorporation into new products.

Introduction of multilayered quantum dot nanobeads into competitive lateral flow assays for ultrasensitive and quantitative monitoring of pesticides in complex samples.

A competitive fluorescent lateral flow assay (CFLFA) is proposed for direct, ultrasensitive, quantitative detection of common pesticides imidacloprid (IMI) and carbendazim (CBZ) in complex food samples by using silica-core multilayered quantum dot nanobeads (SiO2-MQB) as liquid fluorescent tags. The SiO2-MQB nanostructure comprises a 200-nm SiO2 core and a shell of hundreds of carboxylated QDs (excitation/emission maxima ~365/631 nm), and can generate better stability, superior dispersibility, and higher luminescence than traditional fluorescent beads, greatly improving the sensitivity of current LFA methods for pesticides. Moreover, using liquid SiO2-MQB directly instead of via the conjugate pad both simplifies the structure of LFA system and improves the efficiency of immunobinding reactions between nanotags and the targets. Applying these methods, the established CFLFA realized the stable and accurate detection of IMI and CBZ in 12 min, with detection limits down to 1.94 and 14.79 pg/mL, respectively. The SiO2-MQB-CFLFA is practicable for application to real food samples (corn, apple, cucumber, and cabbage), and undoubtedly a promising and low-cost tool for on-site monitoring of trace pesticide residues.

Stepwise Au decoration-assisted double signal amplified lateral flow strip for ultrasensitive detection of morphine in fingerprint sweat

Point-of-care testing (POCT) of morphine (MOP) without invasion of privacy is of critical importance for law-enforcement departments to realize on-site rapid screening. In this study, ultrasensitive and non-invasive screening of MOP residues in the fingerprint sweat was easily realized by stepwise Au decoration-assisted double signal amplification and antibody-saving strategies on lateral flow strip (LFS). The construction of LFS was not intrinsically changed compared with traditional LFS except the labeling material on conjugation pad for enhanced signal reporting. The gold nanoparticle-seeded SiO2 was adopted as the labeling materials in place of traditional gold nanoparticles, which acted as the first-round signal amplification and ready for second-round gold deposition-assisted amplification. And the second-round amplification could be completed in just 10 s, which did not alter the intrinsic simplicity of LFS for rapid and on-site screening. With the designed signal amplification principle of LFS, target MOP in the fingerprint sweat can be effectively transferred to the LFS for analysis without invasion of privacy. As low as 0.5 pg MOP in fingerprint sweat can be visually judged with this double signal amplified LFS, the sensitivity of which has been improved at least 10-fold compared with traditional Au-labeled LFS, guaranteeing accurate screening of trace MOP in the fingerprint sweat. Of great importance, the consumption of valuable antibody can be reduced to just 1/20, which greatly reduces the cost of high-throughput screening. This stepwise Au decoration-assisted double signal amplified LFS holds great potential in the ultrasensitive screening of trace analytes in various fields and further widens the application scope of lateral flow strips.

Triplex-colored nucleic acid lateral flow strip and multiplex polymerase chain reaction coupled method for quantitative identification of beef, pork and chicken

Meat adulteration is one of the worldwide challenges for food quality control. Now, we developed a multiplex PCR and nucleic acid lateral flow strip (mPCR-NALFS) integrated method that employs triplex-color latex microspheres (LMs) as visual labels for simultaneous identification of beef, pork and chicken DNA. First, three primer pairs for beef, pork and chicken DNA are tailed with specific oligonucleotide sequences (anti-recognition/anti-detection/anti-control probes) as swing tags. Then, double-stranded amplicons obtained after mPCR amplification were flanked by tags. Finally, these amplicons can accurately hybridize with the pre-immobilized recognition probes on the conjugate pad, detection probes on the test lines and control probes on the control line, respectively. The LMs and recognition probe modified LMs were characterized by scanning electron microscopy, ultraviolet-visible spectroscopy and Fourier Transform Infrared spectroscopy. This strip can quantitatively detect beef, pork and chicken DNA within 10 min without compromising on accuracy compared to the electrophoresis. Specificity analysis revealed no cross-reactivity with meat derived from beef, pork, chicken, duck, mutton, rabbit, goose, ostrich, camel and horse meat. As low as 0.1 % (wt%) of beef, pork and chicken in the simulated adulterated meats were co-detected. This assay was successfully employed for adulteration identification in 28 commercial beef and lamb products.

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.

Spike- and nucleocapsid-based gold colloid assay toward the development of an adhesive bandage for rapid SARS-CoV-2 immune response detection and screening

Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies are important biomarkers used for the diagnosis and screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in both symptomatic and asymptomatic individuals. These antibodies are highly specific to the spike (S) and nucleocapsid (N) proteins of the SARS-CoV-2 virus. This paper outlines the development steps of a novel hybrid (vertical-lateral-vertical) flow assay in the form of a finger-stick point-of-care device, similar to an adhesive bandage, designed for the timely detection and screening of IgM and IgG immune responses to SARS-CoV-2 infections. The assay, comprising a vertically stacked plasma/serum separation membrane, conjugate pad, and detection (readout) zone, utilizes gold nanoparticles (AuNPs) conjugated with SARS-CoV-2 S and N proteins to effectively capture IgM and IgG antibodies from a pinprick (~15 µL) of blood in just one step and provides results of no immune IgM−/IgG−, early immune IgM+/IgG−, active immune IgM+/IgG+ or immune IgM−/IgG+ in a short amount of time (minutes). The adhesive bandage-like construction is an example of the design of rapid, low-cost, disposable, and easy-to-use tests for large-scale detection and screening in households. Furthermore, the bandage can be easily adjusted and optimized to detect different viral infections as they arise by simply selecting appropriate antigens related to pandemics and outbreaks.

Lateral Flow Assay for Hepatitis B Detection: A Review of Current and New Assays.

From acute to chronic hepatitis, cirrhosis, and hepatocellular cancer, hepatitis B infection causes a broad spectrum of liver diseases. Molecular and serological tests have been used to diagnose hepatitis B-related illnesses. Due to technology limitations, it is challenging to identify hepatitis B infection cases at an early stage, particularly in a low- and middle-income country with constrained resources. Generally, the gold-standard methods to detect hepatitis B virus (HBV) infection requires dedicated personnel, bulky, expensive equipment and reagents, and long processing times which delay the diagnosis of HBV. Thus, lateral flow assay (LFA), which is inexpensive, straightforward, portable, and operates reliably, has dominated point-of-care diagnostics. LFA consists of four parts: a sample pad where samples are dropped; a conjugate pad where labeled tags and biomarker components are combined; a nitrocellulose membrane with test and control lines for target DNA-probe DNA hybridization or antigen-antibody interaction; and a wicking pad where waste is stored. By modifying the pre-treatment during the sample preparation process or enhancing the signal of the biomarker probes on the membrane pad, the accuracy of the LFA for qualitative and quantitative analysis can be improved. In this review, we assembled the most recent developments in LFA technologies for the progress of hepatitis B infection detection. Prospects for ongoing development in this area are also covered.

An integrated immunochromatographic device for C-reactive protein detection using hierarchical dendritic gold nanostructure films

Immunochromatographic test strips typically consist of sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. Even minute variations in the assembly of these components can lead to inconsistent sample-reagent interactions, thereby reducing reproducibility. In addition, the nitrocellulose membrane is susceptible to damage during assembly and handling. To address this issue, we propose to replace the sample pad, conjugate pad, and nitrocellulose membrane with hierarchical dendritic gold nanostructure (HD-nanoAu) films to develop a compact integrated immunochromatographic strip. The strip uses quantum dots as a background fluorescence signal and employs fluorescence quenching to detect C-reactive protein (CRP) in human serum. A 5.9 μm thick HD-nanoAu film was electrodeposited on an ITO conductive glass by the constant potential method. The wicking kinetics of the HD-nanoAu film was thoroughly investigated, and the results indicated that the film exhibited favorable wicking properties, with a wicking coefficient of 0.72 μm ms−0.5. The immunochromatographic device was fabricated by etching three interconnected rings on HD-nanoAu/ITO to designate sample/conjugate (S/C), test (T), and control (C) regions. The S/C region was immobilized with mouse anti-human CRP antibody (Ab1) labeled with gold nanoparticles (AuNPs), while the T region was preloaded with polystyrene microspheres decorated with CdSe@ZnS quantum dots (QDs) as background fluorescent material, followed by mouse anti-human CRP antibody (Ab2). The C region was immobilized with goat anti-mouse IgG antibody. After the samples were added to the S/C region, the excellent wicking properties of the HD-nanoAu film facilitated the lateral flow of the CRP-containing sample toward the T and C regions after binding to AuNPs labeled with CRP Ab1. In the T region, CRP-AuNPs-Ab1 formed sandwich immunocomplexes with Ab2, and the fluorescence of QDs was quenched by AuNPs. The ratio of fluorescence intensity in the T region to that in the C region was used to quantify CRP. The T/C fluorescence intensity ratio was negatively correlated with the CRP concentration in the range of 26.67–853.33 ng mL−1 (corresponding to 300-fold diluted human serum), with a correlation coefficient (R2) of 0.98. The limit of detection was 15.0 ng mL−1 (corresponding to 300-fold diluted human serum), and the range of relative standard deviation: 4.48–5.31%, with a recovery rate of 98.22–108.33%. Common interfering substances did not cause significant interference, and the range of relative standard deviation: 1.96–5.51%. This device integrates multiple components of conventional immunochromatographic strips onto a single HD-nanoAu film, resulting in a more compact structure that improves the reproducibility and robustness of detection, making it promising for point-of-care testing applications.

Self-enhancement lateral flow immunoassay for COVID-19 diagnosis

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

Enhancing a SARS-CoV-2 nucleocapsid antigen test sensitivity with cost efficient strategy through a cotton intermembrane insertion

Lateral flow antigen tests have been widely used in the Covid-19 pandemic, allowing faster diagnostic test results and preventing further viral spread through isolation of infected individuals. Accomplishment of this screening must be performed with tests that show satisfactory sensitivity in order to successfully detect the target protein and avoid false negatives. The aim of this study was to create a lateral flow test that could detect SARS-CoV-2 nucleocapsid protein in low concentrations that were comparable to the limits of detection claimed by existing tests from the market. To do so, several adjustments were necessary during research and development of the prototypes until they were consistent with these criteria. The proposed alternatives of increasing the test line antibody concentration and addition of an intermembrane between the conjugate pad and the nitrocellulose membrane were able to increase the sensitivity four-fold and generate a new rapid test prototype called “lateral flow intermembrane immunoassay test” (LFIIT). This prototype showed an adequate limit of detection (2.0 ng mL−1) while maintaining affordability and simplicity in manufacturing processes.

Portable paper-based electrochemiluminescence test incorporating lateral-flow immunosensors for detection of interferon-γ levels.

Tuberculosis (TB) poses a serious threat to human health and social development. Accurate diagnosis of mycobacterium tuberculosis infection plays a critical role in the prevention and treatment of tuberculosis. Interferon-γ (INF-γ) release assay (IGRA) is currently the only quantitative tuberculosis infection diagnosis method. An accurate, fast, and easily handled INF-γ detection method is the key to obtaining accurate results. Herein, we report a novel paper-based electrochemiluminescence (ECL) method based on lateral flow immunosensors that combines the easy handling characteristics of immunochromatography and the high sensitivity of electrochemiluminescence to detect IFN-γ. To our knowledge this is the first INF-γ detection method that combines immunochromatography with electrochemiluminescence. The paper-based ECL-LFI test consists of a sample pad, conjugation pad (with binding antibody IFN-γ-Ab1 conjugated with ruthenium tripyridine), detection pad (with capture antibody IFN-γ-Ab2 immobilized on nanospheres), absorbent pad, and electrode for signal activation. The ECL signal is obtained by cyclic voltammetry scanning at a speed of 0.1V/s in the detection area of the paper-based ECL-LFI test. In our experiments, the paper-based ECL-LFI test exhibited a minimum detection limit of 2.57pg/mL within 12 min, and a broad detection range of 2.57-5,000pg/mL, with repeatability of 8.10% and stability of 4.97%. With the advantage of high accuracy and sensitivity, easy handling, and low user training requirements, this ECL-LFI test might be used as point-of-care testing (POCT) in the IGRA for tuberculosis diagnosis.

A Protocol for the Optimization of Lateral Flow Immunoassay Strip Development

Background: Lateral flow immunoassay (LFIA) is a well-known method because of its extensive application in many fields, such as medical, food safety, and environmental analysis. LFIA is also under development for pathogen or protein detection, where rapid detection is essential. However, there are few studies on the optimal process for LFIA production. Methods: This study aimed to evaluate the roles of the different materials that make up the LFIA strip, consisting of the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. Different membranes and pads were evaluate regarding membrane permeability, generation of the test line, capillary flow time, and result visibility. Results: The results indicated that the optimized LFIA strip is composed of four membranes and pads, including the CF3 sample pad, FF170HP nitrocellulose membrane immobilized with 1 mg of antibody, Standard 17 conjugate pad, and CF6 absorbent pad. The result was visually detectable within 15 minutes. Conclusion: This optimization protocol could be a reference for the development of new test strips.

A Monoclonal Antibody-Based Immunochromatographic Test Strip and Its Application in the Rapid Detection of Cucumber Green Mottle Mosaic Virus

Two specific monoclonal antibodies (mAbs) were screened, and an immunochromatographic strip (ICS) test for rapid and specific detection of cucumber green mottle mosaic virus (CGMMV) was developed. The coat protein of CGMMV was heterologously expressed as an immunogen, and specific capture mAb 2C9 and the detection mAb 4D4 were screened by an uncompetitive immunoassay. The test and control lines on the nitrocellulose membrane were coated with the purified 2C9 and a goat anti-mouse IgG, respectively, and a nanogold probe combined with 4D4 was applied to the conjugate pad. Using these mAbs, a rapid and sensitive ICS was developed. Within the sandwich mode of 2C9-CGMMV-4D4, the test line showed a corresponding positive relationship with CGMMV in infected samples. The ICS test had a detection limit of 1:5000 (w/v) for CGMMV in samples and was specific for CGMMV, with no observed cross-reaction with TMV or CMV.