Flow Immunoassay For Rapid Detection Research Articles

Novel triplex nucleic acid lateral flow immunoassay for rapid detection of Nipah virus, Middle East respiratory syndrome coronavirus and Reston ebolavirus

We report the development of a triplex nucleic acid lateral flow immunoassay (NALFIA) for the detection of the genomes of Nipah virus (NiV), Middle East respiratory syndrome coronavirus (MERS-CoV) and Reston ebolavirus (REBOV), which are intended for screening bats as well as other hosts and reservoirs of these three viruses. Our triplex NALFIA is a two-step assay format: the target nucleic acid in the sample is first amplified using tagged primers, and the tagged dsDNA amplicons are captured by antibodies immobilized on the NALFIA device, resulting in signal development from the binding of a streptavidin-colloidal gold conjugate to a biotin tag on the captured amplicons. Triplex amplification of the N gene of NiV, the UpE gene of MERS-CoV, and the Vp40 gene of REBOV was optimized, and three compatible combinations of hapten labels and antibodies were identified for end point detection. The lowest RNA copy numbers detected by the triplex NALFIA were 8.21e4 for the NiV N target, 7.09e1 for the MERS-CoV UpE target, and 1.83e4 for the REBOV Vp40 target. Using simulated samples, the sensitivity and specificity for MERS-CoV and REBOV targets were estimated to be 100%, while the sensitivity and specificity for the NiV target were 91% and 93.3%, respectively. The compliance rate between triplex NALFIA and real-time RT‒PCR was 92% for the NiV N target and 100% for the MERS-CoV UpE and REBOV Vp40 targets.

Evaluation of a Lateral Flow Immunoassay for Rapid Detection of CTX-M Producers from Blood Cultures

Bacteremia caused by extended-spectrum β-lactamases-producing Enterobacterales has increased rapidly and is mainly attributed to CTX-M enzymes. This study aimed to evaluate the NG-Test® CTX-M MULTI lateral flow assay (CTX-M LFA) for rapid detection of CTX-M producers in blood cultures (BCs) positive for Gram-negative bacilli in spiked and clinical BCs. Retrospective testing was performed on BC bottles spiked with a collection of well-characterized Enterobacterales isolates producing CTX-M (n = 15) and CTX-M-like (n = 27) β-lactamases. Prospective testing of clinical, non-duplicate BCs (n = 350) was performed in two hospital microbiology laboratories from April 2021 to March 2022 following detection of Gram-negative bacilli by microscopic examination. Results were compared against molecular testing as the reference. In the spiked BCs, the CTX-M LFA correctly detected all CTX-M producers including 5 isolates with hybrid CTX-M variants. However, false-positive results were observed for several CTX-M-like β-lactamases, including OXY-1-3, OXY-2-8, OXY-5-3, FONA-8, -9, -10, 11, 13 and SFO-1. In clinical BCs, the CTX-M LFA showed 100% (95% CI, 96.0-100%) sensitivity and 99.6% (97.9-100%) specificity. In conclusion, this study showed that rapid detection of CTX-M producers in BC broths can be reliably achieved using the CTX-M LFA, thus providing an opportunity for early optimization of antibiotics.

Quantitative lateral flow immunoassay for rapid detection of procollagen type I N-terminal propeptide in the monitoring of osteoporosis treatment

Appropriate follow-up after treatment initiation in patients with osteoporosis is challenging. Serum biomarkers may offer more efficient monitoring of bone mineral density (BMD) than the currently used dual X-ray absorptiometry; however, significant changes in BMD often occur over at least 12 months. During teriparatide treatment for osteoporosis, monitoring with markers such as procollagen type I propeptide (PINP), which is derived from osteoblasts, can provide clinically useful information for disease management. However, rapid and cost-effective methods for detecting serum PINP are lacking, necessitating a point-of-care test (POCT) for enhanced follow-up efficiency in osteoporosis management. For the quantitative detection of PINP, we developed a high-sensitivity lateral flow immunoassay with a stacking pad (sLFIA). We established a calibration equation based on the test line/control line ratio obtained from our PINP sLFIA results of various nonspiked serum samples to calculate the PINP concentrations in 40 serum samples and compared the result with those obtained using a fully automated electrochemiluminescence immunoassay. PINP concentrations between these two methods exhibited excellent correlation (R = 0.991). In addition, we assessed the serum PINP concentrations of patients with osteoporosis treated with teriparatide. At the 3-month follow-up, their PINP levels were nearly twice as high as those at baseline, thus implying that our method can be used for osteoporosis treatment monitoring. Our findings thus indicate that the PINP sLFIA can serve as a POCT for monitoring medication response and managing osteoporosis.

Clinical evaluation of cell-direct polymerase chain reaction-based nucleic acid lateral flow immunoassay for rapid detection of bacterial pathogens in clinically suspected sepsis: A multi-center study in Japan

Blood culture, a method for identifying causative agents of bacterial sepsis, requires several days. The combination of cell-direct polymerase chain reaction and nucleic acid lateral flow immunoassay (cdPCR-NALFIA) is a simple and sensitive detection method for identifying pathogenic bacteria. Furthermore, this assay, when applied directly to blood samples yields results within 4.5 h, without requiring culture. This study was performed at five hospitals in Japan between 2013 and 2016. Blood samples from 73 patients with clinically suspected sepsis yielded 18 positive blood cultures, and the isolated bacterial species were detectable using cdPCR-NALFIA in nine samples. Thirteen samples were positive on cdPCR-NALFIA. In total, 17 samples confirmed to have bacterial species were detectable using cdPCR-NALFIA and/or blood culture with a true positive rate of 76.5% and 64.7%, respectively. The combination of blood culture and cdPCR-NALFIA could improve the rate of detection of bacterial sepsis.

Quantitative lateral flow immunoassay for rapid detection and monitoring of cerebrospinal fluid leakage following incidental durotomy

Cerebrospinal fluid (CSF) leakage due to incidental durotomy is an inherent complication of spine surgery. With appropriate treatment, complications of CSF leakage, such as headache and even meningitis, can be reduced. CSF leakage could be detected on the basis of correlated clinical symptoms; diagnosis should be based on these symptoms and appropriate imaging studies. However, the diagnosis of CSF leakage remains a challenge, especially if incidental durotomy is unrecognized during surgery; even if incidental durotomy is detected and repaired intraoperatively, the severity of the leakage and quality of the primary dural repair are difficult to evaluate postoperatively. Rapid, inexpensive, and safe methods of detecting CSF-containing samples are currently lacking; hence, the development of a point-of-care test (POCT) method to improve diagnostic efficiency is necessary. We developed a high-sensitivity lateral flow immunoassay with a stacking pad (sLFIA) for quantitative detection of β-trace protein (BTP), a specific CSF marker. The BTP concentration in 39 clinical samples was calculated using a calibration equation for test-line intensity and evaluated by a standard laboratory method. To avoid the hook effect, we diluted each sample prior to testing. The correlation coefficient between the enzyme-linked immunosorbent assay and our BTP sLFIA method was 0.991 A 75-fold sample dilution was applied owing to the hook effect point, identified as 175 ng mL−1. We established an optimal sample-specific cutoff point at a value of 4.0 μg mL−1 for CSF leakage in subfascial drainage samples following spinal posterior decompression. The sensitivity and specificity of the BTP sLFIA method were 90% and 97%, respectively, according to a receiver operating characteristic curve analysis. In addition, clinical samples from patients who underwent primary dural repair intraoperatively were tested, and CSF leakage was successfully diagnosed using our method. Finally, the quantitation of BTP in samples collected daily provided an accurate assessment of the severity of the residual leakage. Our results demonstrate that the BTP sLFIA method possesses the potential to serve as a POCT method for screening and monitoring postoperative CSF leakage.

Development and validation of a lateral flow immunoassay for rapid detection of VanA-producing enterococci.

VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth. NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller-Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested. All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates. NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE.

Development of a Prototype Lateral Flow Immunoassay for Rapid Detection of Staphylococcal Protein A in Positive Blood Culture Samples.

Bloodstream infection (BSI) is a major cause of mortality in hospitalized patients worldwide. Staphylococcus aureus is one of the most common pathogens found in BSI. The conventional workflow is time consuming. Therefore, we developed a lateral flow immunoassay (LFIA) for rapid detection of S. aureus-protein A in positive blood culture samples. A total of 90 clinical isolates including 58 S. aureus and 32 non-S. aureus were spiked in simulated blood samples. The antigens were extracted by a simple boiling method and diluted before being tested using the developed LFIA strips. The results were readable by naked eye within 15 min. The sensitivity of the developed LFIA was 87.9% (51/58) and the specificity was 93.8% (30/32). When bacterial colonies were used in the test, the LFIA provided higher sensitivity and specificity (94.8% and 100%, respectively). The detection limit of the LFIA was 107 CFU/mL. Initial evaluation of the LFIA in 20 positive blood culture bottles from hospitals showed 95% agreement with the routine methods. The LFIA is a rapid, simple and highly sensitive method. No sophisticated equipment is required. It has potential for routine detection particularly in low resource settings, contributing an early diagnosis that facilitates effective treatment and reduces disease progression.

Silver nanoparticle-base lateral flow immunoassay for rapid detection of Staphylococcal enterotoxin B in milk and honey

A silver nanoparticle (AgNP)-based sandwich-type lateral flow immunoassay (LFIA) was evaluated for rapid detection of Staphylococcal enterotoxin B (SEB) in milk and honey. The role of trisodium citrate dihydrate (TSC) in the formation of Ag/TSC nanoparticles was established using UV–Vis spectroscopy. The association of silver with TSC in Ag/TSC nanoparticles was studied by the decrease in the intensity of anodic peak potential at 0.47 V and shift to 0.30 V in cyclic voltammetry (CV). The morphological, compositional and interaction studies of the AgNPs conjugated with the anti-SEB polyclonal antibody (Ag-sAb) was established using transmission electron microscopy (TEM) and X-ray photo electron spectroscopy (XPS) measurements. The visible detection limit and optical detection limit of the SEB test strip were 0.5 and 0.125 ppm, respectively, in SEB standard solution. This assay showed no cross-reaction with Staphylococcal enterotoxin A, Staphylococcal enterotoxin C or Salmonella typhi. Finally, the SEB test strip was effectively applied for the detection of SEB in spiked liquid milk and viscous honey, with optical detection limits of 0.25 and 0.5 ppm, respectively.

Functional nanozyme mediated multi-readout and label-free lateral flow immunoassay for rapid detection of Escherichia coli O157:H7.

To overcome the drawbacks of antibody labeling dependence and single-readout system in the conventional lateral flow immunoassays (LFIAs) as well as the non-targeted combination of new capture agents reported recently for pathogen detection, in this work, a multi-readout and label-free LFIA was proposed for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) based on a nanozyme-bacteria-antibody sandwich pattern. A type of functional nanozyme-mannose modified Prussian blue (man-PB), was introduced as the recognition agent as well as signal indicator. Apart from original signal intensity on the T-line, the peroxidase-like catalytic activity-driven generation of colorimetric signal could be used as another format of quantitation. Importantly, such LFIA could exhibit excellent performance for target pathogens detection separately with a quantitative range of 102-108 cfu·mL-1 and a low detection limit of 102 cfu·mL-1 based on different readout formats, indicating the application potential of the proposed LFIA in real samples.

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food.

Salmonella can cause serious foodborne diseases. We have developed a lateral flow immunoassay combined with recombinase polymerase amplification (LFD-RPA) for detection of Salmonella in food. The conserved fragment (fimY) was selected as the target gene. Under an optimal condition (37 °C, 10 min), the sensitivity was 12 colony-forming units (CFU)/mL in a pure culture. Testing with 16 non-Salmonella strains as controls revealed that LFD-RPA was specific to the fimY gene of Salmonella. The established assay could detect Salmonella at concentrations as low as 1.29 × 102 CFU/mL in artificially contaminated samples. This detection was at a slightly higher level than that for a pure bacterial culture. Combined with the test strip reader, the LFD-RPA is a feasible method for quantitative detection of Salmonella based on the test line intensity, which was the ratio for the test line and control line of the reflected light. The method could be a potential point-of-care test in limited resource areas and provides a new approach and technical support for the diagnosis of food safety.

An octuplex lateral flow immunoassay for rapid detection of antibiotic residues, aflatoxin M1 and melamine in milk

Antibiotic residues, aflatoxin M1 and melamine are three major types of chemical contaminants in milk. However, analytical approach for the simultaneous detection of these chemicals is currently not available. In this study, an octuplex lateral flow immunoassay was developed to address this problem. Specific monoclonal antibodies and receptor were prepared and were bound onto gold nanoparticles to form eight antibody/receptor-gold nanoparticle conjugates as detection probes. Eight corresponding hapten-protein conjugates were synthesized and immobilized on distinct test zones of nitrocellulose membrane as capture antigens. Thus octuplex immune-reactions can then simultaneously occur on a single strip. For qualitative analysis, the result can be visually judged by naked eyes and the cut-off values of quinolones, tetracyclines, β-lactams, sulfonamides, chloramphenicol, streptomycin, aflatoxin M1 and melamine were measured to be 12–80 ng/mL, 16–32 ng/mL, 2–80 ng/mL, 4–80 ng/mL, 1.2 ng/mL, 50 ng/mL, 0.5 ng/mL and 200 ng/mL, respectively. For semi-quantitative analysis, the test lines can be photographed by a built-in camera of smartphone and the line intensities can be analyzed by Image J software. The limits of detection for all the chemicals were far below the regulatory limits set by authorities. Furthermore, the detection time of this method was less than 20 min. Consequently, the developed immunoassay in this study is a promising approach for rapidly screening common chemical contaminants in milk.

Colloidal silver-based lateral flow immunoassay for rapid detection of melamine in milk and animal feed

An immunosilver chromatographic strip based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in milk and animal feed. Colloidal silver nanoparticles (AgNPs), of a diameter of approximately 10.4 nm, were surface-modified with trisodium citrate dehydrate (TSC) in order to improve their stability and dispersion. The anti-MEL monoclonal antibody (mAb) was successfully immobilized on the surface of AgNPs by ionic interactions and characterized using XRD, UV–Vis, TEM and XPS analyses. Surface modification of Ag-mAb conjugates with varying cross-reactivity and thermo-stability was employed to design and prepare labels for use in a lateral flow immunoassay (LFIA) to examine whether these factors affect the performance of the assay. The results showed that no cross-reactions with homologues cyanuric acid, ammeline or ammelide were found, and the test strip remained stable after storage for one year at room temperature. Under optimized conditions, a detection limit of 0.30 ppm for standard melamine was achieved, and limits of detection of 0.750 ppm and 0.875 ppm were obtained in milk and animal feed, respectively.

Aggregation-induced emission-based competitive lateral flow immunoassay for rapid detection of sulfamethazine in honey

ABSTRACTSulfamethazine (SMZ), widely used for disease prevention of bees, is harmful to consumers when excess SMZ residues are present in honey. In this study, a novel competitive lateral flow immunoassay (LFIA) was established by using aggregation-induced emission fluorescent microsphere (AIEFM) as label for the detection of SMZ. Results indicated that the limit of detection (LOD) for the developed competitive AIEFM-LFIA in honey was 0.028 ng/mL, and the corresponding linear range was 0.05−5 ng/mL. Recovery experiments showed that the average recovery range was 75.6–95.1% with coefficient variations of 5.7–12.9% in honey. The developed competitive AIEFM-LFIA was compatible with HPLC-MS/MS in detecting SMZ in 15 real samples from honey. In general, the proposed AIEFM-LFIA is well suited for the competitive format and has great potential for the detection of other small targets with single antigenic site in practical application.

Lateral Flow Immunoassay for Rapid Detection of Grapevine Leafroll-Associated Virus.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the main pathogens of grapes, causing a significant loss in yield and decrease in quality for this agricultural plant. For efficient widespread control of this infection, rapid and simple analytical techniques of on-site testing are requested as a complementary addition for the currently applied hybridization (PCR) and immunoenzyme (ELISA) approaches. The given paper presents development and approbation of the immunochromatographic assay (ICA) for rapid detection of GLRaV-3. The ICA realizes a sandwich immunoassay format with the obtaining complexes ((antibody immobilized on immunochromatographic membrane)–(virus in the sample)–(antibody immobilized on gold nanoparticles (GNP)) during sample flow along the membrane compounds of the test strip. Three preparations of GNPs were compared for detection of GLRaV-3 at different dilutions of virus-containing sample. The GNPs with maximal average diameters of 51.0 ± 7.9 nm provide GLRaV-3 detection for its maximal dilutions, being 4 times more than when using GNPs with a diameter of 28.3 ± 3.3 nm, and 8 times more than when using GNPs with a diameter of 18.5 ± 3.3 nm. Test strips have been manufactured using the largest GNPs conjugated with anti-GLRaV-3 antibodies at a ratio of 1070:1. When testing samples containing other grape wine viruses, the test strips have not demonstrated staining in the test zone, which confirms the ICA specificity. The approbation of the manufactured test strips indicated that when using ELISA as a reference method, the developed ICA is characterized by a sensitivity of 100% and a specificity of 92%. If PCR is considered as a reference method, then the sensitivity of ICA is 93% and the specificity is 92%. The proposed ICA can be implemented in one stage without the use of any additional reactants or devices. The testing results can be obtained in 10 min and detected visually. It provides significant improvement in GLRaV-3 detection, and the presented approach can be transferred for the development of test systems for other grape wine pathogens.

Development of a One-Step Lateral Flow Immunoassay for Rapid Detection of Icariin

Development of a One-Step Lateral Flow Immunoassay for Rapid Detection of Icariin

Lateral flow immunoassay for rapid detection of potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus

A lateral flow immunoassay for the rapid detection of Clavibacter michiganensis subsp. sepedonicus bacteria causing potato ring rot was developed. Multimembrane composites (test strips) containing poly� clonal antibodies against the bacteria and gold nanoparticleantibody conjugates were used for the analysis. The test strips are suitable for the analysis of potato tuber and leaf extracts within 10 min; the detection limit of bacteria is 4 × 10 5 cells/mL. No crossreactivity with strains of Clavibacter michiganensis subsp. michigan� ensis, Pectobacterium carotovorum subsp. carotovorum and saprophytes of healthy potato plants was detected. The results of analysis of 26 potato samples by the developed tests were compared with those obtained by the PCR method and using the commercial enzyme immunoassay kits. The results of lateral flow immunoassay were confirmed in 96.2% of cases, which supports the high correlation with other analytical approaches. The developed immunoassay may be considered as a promising means of phytosanitary control.