Lateral Flow Immunochromatographic Strip Test Research Articles

Food safety and quality training: The use of a lateral flow immunochromatographic strip test for rapid detection of pesticide residue on leafy vegetables

Ensuring the safety and quality of leafy vegetables, especially by controlling any traces of pesticides, is crucial for maintaining human health. This international community service program was designed to expand the vocational knowledge and skills of the people of Timor-Leste in using lateral flow immunochromatographic strip tests for quickly detecting pesticide residue on leafy vegetables. The program was organized by UAD in partnership with PCIM Timor-Leste and the An-Nur Islamic Foundation. The program included several stages, including preparation, coordination, consolidation, implementation, monitoring, and evaluation. The participants’ knowledge of food safety and food hygiene increased significantly from 52% prior to training to 96% after the training. Moreover, the ability to detect pesticide contamination in vegetables using LFI technology improved by 84%, rising from just 8% before training to 92% after training. All participants expressed interest in the UAD in Timor-Leste's future continuation of this training program.

Development of Hemagglutinin-Neuraminidase Homologous Peptides as Novel Promising Therapeutic Agents Against Peste des Petits Ruminants Virus.

The lack of specific antiviral therapy and complications associated with the existing peste des petits ruminants (PPR) vaccines accentuates the search of novel antiviral blocking agents in order to curtail the PPR infection at initial level. The synthetic hemagglutinin-neuraminidase (HN) homologous peptides may compete with the natural HN protein of PPR virus for binding to signaling lymphocytic activation molecule (SLAM) receptor, consequently, may disrupt peste des petits ruminants virus (PPRV) at entry level. Therefore, insilico analysis, synthesis, purification and subsequent characterization of HN homologous peptides were conducted in this study. The HN homologous peptides were synthesized by means of solid phase chemistry and were purified by reversed-phase-high performance liquid chromatography. The mass as well as sequence of HN homologous peptides were assessed by mass spectroscopy while its secondary structure was elucidated by circular dichroism spectroscopy. The binding (interaction) efficacy of HN homologous peptides with PPRV antibodies was assessed via indirect enzyme linked immunosorbent assay, visual detection test (red wine to purple), bathochromic shift under UV-Vis spectrophotometry and lateral flow immunochromatographic strip test. The antiviral properties and cytotoxicity of these peptides were also assessed in B95a cell line with changes in cytopathic effect and titer of PPRV (Sungri/96). The presence of green fluorescein isothiocyanate over the B95a cell surface pointed towards the binding of HN homologous peptides with surface SLAM receptor. Moreover, the intact beta sheet configuration in water and lower cytotoxicity [cytotoxic concentration 50 (CC50) > 1000µg/ml] of these peptides signifies its in vivo use. Among HN homologous peptides, the binding efficacy and antiviral properties of pep A was relatively high in comparison to pep B and Pep ppr peptides. The prerequisite concentration of HN homologous peptides (pep A = 12.5µg/ml; pep B = 25µg/ml; pep ppr = 25µg/ml) to exemplify its antiviral effect was much lower than its CC50 level. Hence, this study signifies the therapeutic potential of synthetic HN homologous peptides.

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b

Recombinant human interferon α2b (rhIFNα2b) is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C. The current identification test for rhIFNα2b is complex. In this study, an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b. RhIFNα2b was used to immunize an alpaca, which established a phage nanobody library. After five steps of enrichment, the nanobody I22, which specifically bound rhIFNα2b, was isolated and inserted into the prokaryotic expression vector pET28a. After subsequent purification, the physicochemical properties of the nanobody were determined. A semiquantitative detection and rapid identification assay of rhIFNα2b was developed using this novel nanobody. To develop a rapid test, the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips. The developed rhIFNα2b detection assay had a limit of detection of 1 μg/mL. The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products. The principle of this novel assay is generally applicable for the rapid testing of other commercial products, with a great potential for routine use in detecting counterfeit recombinant protein products.

Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Lateral-Flow Immunochromatographic Strip for the Detection of Digoxin in Human Blood.

Mouse–mouse hybridoma cell lines producing stable, highly specific monoclonal antibodies with good affinity for the cardiac glycoside digoxin (DIG) were established to construct an indirect enzyme-linked immunosorbent assay and lateral-flow immunochromatographic strip to detect DIG in human blood. The hapten DIG was coupled to bovine serum albumin or chicken ovalbumin by sodium periodate oxidation. The highest sensitivity and specificity antibody had a median inhibitory concentration (IC50) of 0.45 ng/mL, a linear range of detection of 0.293–0.7 ng/mL, and low cross-reactivity with several DIG analogues. The cut-off value of the lateral-flow immunochromatographic strip was 5 ng/mL when the strip was tested with human blood. The immunochromatographic lateral flow strip test provides a quick and convenient method for determining DIG in plasma which can be visually observed in only 5 min to promote rational drug use.

Development of an immunochromatographic lateral flow strip test for the rapid detection of diclofenac in medicinal wine

ABSTRACT A rapid immunochromatographic lateral flow strip test (ILFST) of competitive format for the detection of diclofenac (DCF) in medicinal wine samples was developed. The half-inhibitory concentration (IC50) values of the ILFST for DCF and the limit of detection (LOD) in DCF-spiked samples were 0.66 and 0.15 ng/mL, respectively. Recoveries of DCF from spiked medicinal wine samples were from 84.4% to 85.9% within an assay (intra-assay) and from 83.7% to 88.4% between assays (inter-assay). The coefficients of variation (CV) for intra-assay and inter-assay were 4.10–7.56% and 4.71–8.94%, respectively. These results indicated that the ILFST was highly sensitive and re-producible. The same authentic medicinal wine samples were tested by both ILFST and high-performance liquid chromatography (HPLC) respectively. The results showed no significant differences between the two methods. Therefore, the ILFST can be widely used as a convenient tool for both qualitative and quantitative detection of DCF residues in medicinal wine samples.

Developing Gold Nanoparticles-Conjugated Aflatoxin B1 Antifungal Strips.

Lateral flow immunochromatographic assays are a powerful diagnostic tool for point-of-care tests, based on their simplicity, specificity, and sensitivity. In this study, a rapid and sensitive gold nanoparticle (AuNP) immunochromatographic strip is produced for detecting aflatoxin B1 (AFB1) in suspicious fungi-contaminated food samples. The 10 nm AuNPs were encompassed by bovine serum albumin (BSA) and AFB1 antibody. Thin-layer chromatography, gel electrophoresis and nuclear magnetic resonance spectroscopy were employed for analysing the chemical complexes. Various concentrations of AFB1 antigen (0–16 ng/mL) were tested with AFB1 antibody–BSA–AuNPs (conjugated AuNPs) and then analysed by scanning electron microscopy, ultraviolet–visible spectroscopy, and Zetasizer. The results showed that the AFB1 antibody was coupled to BSA by the N-hydroxysuccinimide ester method. The AuNPs application has the potential to contribute to AFB1 detection by monitoring a visible colour change from red to purple-blue, with a detection limit of 2 ng/mL in a 96-well plate. The lateral flow immunochromatographic strip tests are rapid, taking less than 10 min., and they have a detection capacity of 10 ng/g. The smartphone analysis of strips provided the results in 3 s, with a detection limit of 0.3 ng/g for AFB1 when the concentration was below 10 ng/g. Excellent agreement was found with AFB1 determination by high-performance liquid chromatography in the determination of AFB1 among 20 samples of peanuts, corn, rice, and bread.

Development and validation of a pen side test for Rift Valley fever.

BackgroundRift Valley fever (RVF) is one of the main vector borne zoonotic diseases that affects a wide range of ruminants and human beings in Africa and the Arabian Peninsula. A rapid and specific test for RVF diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures.Methodology/Principal findingsA first-line lateral flow immunochromatographic strip test (LFT) was developed for the detection of the nucleoprotein (N) of the RVF virus (RVFV). Its diagnostic performance characteristics were evaluated using reference stocks isolates recovered from different hosts and in geographic regions mimicking clinical specimens and from known RVF negative serum samples. A high level of diagnostic accuracy (DSe (35/35), DSp (167/169)) was observed, including the absence of cross-reactivity with viruses belonging to different genera.Conclusion/SignificanceThe fact no specialized reagents and laboratory equipment are needed, make this assay a valuable, first-line diagnostic tool in resource-poor diagnostic territories for on-site RVFV detection, however the staff require training.

An Immunochromatographic Lateral Flow Strip Test for the Rapid Detection of Danofloxacin in Milk

An immunochromatographic lateral flow strip test (ILFST) for the rapid detection of danofloxacin (DAN) in milk samples was developed based on colloidal gold-labeled anti-DAN monoclonal antibody 3E9 and a competitive format. Quantitative detections of DAN-spiked samples using the ILFST gave a half inhibitory concentration (IC50) of 0.513 ng/mL and a limit of detection (LOD) of 0.092 ng/mL. Recoveries of DAN from spiked milk samples were from 80.0 to 95.1% within an assay (intra-assay) and from 87.0 to 89.8% between assays (inter-assay). The coefficients of variation (CV) for intra-assay and inter-assay were 2.09–9.93% and 2.42–12.07%, respectively. These data indicated that the ILFST was highly sensitive and reproducible. Detections of authentic milk samples using ILFST and high-performance liquid chromatography (HPLC) showed no significant differences between the two methods. Hence, the ILFST can be widely used for both qualitative and quantitative detection of DAN residues in milk samples.

Generation of monoclonal antibodies against foot-and-mouth disease virus SAT 2 and the development of a lateral flow strip test for virus detection.

Foot-and-mouth disease (FMD) remains a major economic concern for the livestock productivity in many developing countries and a continued threat to countries that are disease free because of its potential devastating impact on agricultural, food chain and tourism sectors. FMD virus (FMDV) is recognized as having seven serotypes: O, A, C, Asia 1, South African Territories (SAT) 1, 2, 3 and multiple subtypes within each serotype. FMD outbreaks due to SAT 2 have been reported in many African countries. The development of a rapid and easily performed test for FMD detection is critical for controlling FMD outbreaks and containing its spread. The present project developed a lateral flow immunochromatographic (LFI) strip test for the rapid detection of FMDV SAT 2. A panel of monoclonal antibodies (mAbs) against FMDV serotype SAT 2 was produced and characterized. One mAb (#10) was selected as the capture mAb because it reacted to all 23 SAT 2 isolates archived at the National Center for Foreign Animal Disease. The LFI strip test was developed using biotin-conjugated mAb #10, and the colloid gold-conjugated FMDV serotype-independent mAb as the detection mAb. A generic Rapid Assay Device (gRAD) with one test line and a control line was used for the test. The LFI strip test detected all 23 tested SAT 2 isolates and recent outbreak strains. The results indicated that the diagnostic specificity and sensitivity of the LFI strip test were greater than the double antibody sandwich (DAS) DAS ELISA. The ability of the LFI strip test to produce rapid diagnostic results will be useful for early on-site diagnosis during FMD outbreaks.

A Lateral Flow Immunochromato-graphic Strip Test for Rapid Detection of Oseltamivir Phosphate in Egg and Chicken Meat

A lateral flow immunochromatographic strip test (LFIST) based on a competitive format was developed for rapid and sensitive on-site detection of oseltamivir phosphate (OP) residues in poultry product. The sensitivity (half inhibitory concentration, IC50) of the LFIST in the detection of egg and chicken meat samples was confirmed to be 2.56 and 2.63 µg/kg, and the limit detection (LOD) value were 0.43 and 0.42 µg/kg, respectively. For intra-assay and inter-assay reproducibility, recoveries of OP spiked samples ranged between 82.8% and 91.2% with coefficients of variations (CV) less than 5.67% (intra-assay) and 6.52% (inter-assay). The performance of LFIST was comparable to high-performance liquid chromatography (HPLC) in a parallel testing of egg samples and chicken samples. LFIST takes less than 5 minutes, eliminates the dependency on professional personnel, and thus can be used as a surveillance tool for on-site detection of OP residues.

A lateral flow immunochromatographic strip test for rapid detection of hexoestrol in fish samples.

A lateral flow immunochromatographic strip test was developed for rapid and sensitive on-site detection of hexoestrol (HES) residues in fish samples with colloidal gold labelling of the anti-HES monoclonal antibody. The strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad and a test membrane containing a control line and a test line. The sensitivity (half inhibitory concentration, IC50) of the strip in the detection of fish extract samples was confirmed to be 1.86 µg kg−1, and the limit of detection value was 0.62 µg kg−1. For intra-assay and inter-assay reproducibility, recoveries of HES-spiked samples ranged from 86.3% to 92.3% and 85.8% to 93.4%, coefficients of variation were 2.91–4.64% and 4.24–5.17%, respectively. High-performance liquid chromatography was employed to confirm the performance of the strip. The strip test takes less than 10 min, and thus provides a repaid method for on-site detection of HES residues.

Development of an antibody-based colloidal gold immunochromatographic lateral flow strip test for natamycin in milk and yoghurt samples

ABSTRACTAn immunochromatographic lateral flow strip test was developed for the detection of natamycin (Nata) residues in milk and cheese samples. Monoclonal antibody (mAb) against Nata was produced with a half maximal inhibitory concentration of 1.85 μg L−1. MAb conjugated with gold nanoparticles was the detection reagent. Nata conjugated with ovalbumin via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide method immobilized on a nitrocellulose membrane was the capture reagent. This semi-quantitative method required only 15 min to complete. The optimum concentrations of coating antigen and mAb were 0.15 µg mL−1 and 0.2 µg mL−1, respectively. Based on an optical density scanner, the visual limit of detection of Nata was 5 μg L−1 and 10 μg kg−1 in milk and yoghurt samples, respectively.

Development of a multiplex lateral flow strip test for foot-and-mouth disease virus detection using monoclonal antibodies

Foot-and-mouth disease (FMD) is one of the world's most highly contagious animal diseases with tremendous economic consequences. A rapid and specific test for FMD diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. This project developed a multiplex lateral flow immunochromatographic strip test (multiplex-LFI) for the rapid detection and serotyping of FMD viruses.The monoclonal antibodies (mAbs) against serotypes O, A, and Asia 1 were used as capture mAbs. The mAbs were conjugated with fluorescein, rhodamine or biotin for serotype O, A and Asia 1, respectively. The detection mAbs which consisted of a serotype-independent mAb in combination with one serotype A-specific mAb and one Asia 1-specific mAb, were each colloidal gold-conjugated. The strips used in this study contained one control line and three test lines, which corresponded to one of the three serotypes, O, A or Asia 1.The newly developed multiplex-LFI strip test specifically identified serotype O (n=46), A (n=45) and Asia 1 (n=17) in all tested field isolates. The sensitivity of this strip test was comparable to the double antibody sandwich ELISA for serotypes O and A, but lower than the ELISA for serotype Asia 1. The multiplex-LFI strip test identified all tissue suspensions from animals that were experimentally inoculated with serotypes O, A or Asia 1. FMD viruses were detected in 38% and 50% of the swab samples from the lesion areas of experimentally inoculated sheep for serotypes O and A, respectively.The capability of the multiplex-LFI strip tests to produce rapid results with high specificity for FMD viruses of multiple serotypes makes this test a valuable tool to detect FMD viruses at outbreak sites.

Development of a colloidal gold-based strip test for the detection of chlorothalonil residues in cucumber

An immunochromatographic lateral flow strip test using the competitive format was developed for the determination of chlorothalonil (CTN) residues in cucumber. The limit of detection was less than 100 ng/mL by visual assessment and was found to be 91.78 ± 0.17 ng/mL for quantitative detection using a test strip reader. The recoveries of test samples were from 89.26% to 102.02% and the coefficient of variation (%) was less than 4.75%. Parallel analysis of cucumber samples with CTN showed comparable results from the strip test and high performance liquid chromatography. The strip test requires 5–8 min to complete and provides a very useful screening method for the quantitative and qualitative detection of CTN residues in cucumber.

Evaluation of Rapid Stain IDentification (RSID™) Reader System for Analysis and Documentation of RSID™ Tests

The ability to detect the presence of body fluids is a crucial first step in documenting and processing forensic evidence. The Rapid Stain IDentification (RSID™) tests for blood, saliva, semen and urine are lateral flow immunochromatographic strip tests specifically designed for forensic use. Like most lateral flow strips, the membrane components of the test are enclosed in a molded plastic cassette with a sample well and an observation window. No specialized equipment is required to use these tests or to score the results seen in the observation window; however, the utility of these tests can be enhanced if an electronic record of the test results can be obtained, preferably by a small hand-held device that could be used in the field under low light conditions. Such a device should also be able to read the lateral flow strips and accurately record the results of the test as either positive, i.e., the body fluid was detected, or negative, i.e., the body fluid was not detected. Here we describe the RSID™ Reader System—a ruggedized strip test reader unit that allows analysis and documentation of RSID™ lateral flow strip tests using pre-configured settings, and show that the RSID™ Reader can accurately and reproducibly report and record correct results from RSID™ blood, saliva, semen, and urine tests.

Development of a quick and simple detection methodology for foot-and-mouth disease virus serotypes O, A and Asia 1 using a generic RapidAssay Device

BackgroundOutbreaks of Foot-and-mouth disease (FMD) have resulted in tremendous economic losses. Thus, the development of a rapid and easily performed test for FMD detection is important for controlling a FMD outbreak and containing its spread. The purpose of this project is to develop a lateral flow immunochromatographic (LFI) strip test for rapid detection of FMD virus serotypes O, A and Asia 1.MethodsSpecific monoclonal antibodies (mAbs) against each serotype were produced and used as the capture mAbs. A serotype independent mAb was selected and used as the detection mAb with the aim of subsequently developing a multi-serotype strip test. A new generation of the generic RapidAssay Device (gRAD) was used for the test.ResultEach strip test can specifically detect the FMDV O, A or Asia 1 viruses, but not other vesicular disease viruses. The LFI strip tests for serotypes A and Asia 1 were able to identify all tested serotype A (n= 39) and Asia 1 field isolates (n=17). Whereas the test for serotype O detected 45 out of 46 field isolates. The sensitivity of this strip test was comparable with the double antibody sandwich ELISA for viral antigen detection. All vesicular fluid and epithelium samples collected from experimentally infected animals with serotype O, A and Asia 1 were identified as positive by the LFI strip test. Swab samples (n=11) collected over the lesion area from experimentally inoculated animals (serotype A) were examined. All of them demonstrated positive results using the LFI serotype A strip test and double antibody sandwich (DAS) ELISA.ConclusionsThe ability of strip tests to produce rapid results and high specificity makes it a valuable tool for early detection of FMDV O, A and Asia 1 in the field.

Extract-free immunochromatographic assay for on-site tests of aflatoxin M1 in milk

Lateral flow immunochromatographic strip tests (IST) are very promising for use as on-site test diagnostic tools in developing countries. In this work, a fabrication process simplified, lateral flow immunochromatographic test strip for aflatoxin M1 (AFM1) detection in milk samples has been developed. The immunostrip was specific to AFM1 showing no cross-reactivity with other major aflatoxins. The visual detection limit (VDL) and threshold level for AFM1 was 0.3 and 4.0 ng mL−1, respectively. Evaluation results of the IST revealed good accuracy and repeatability. In the detection of AFM1 contamination level in milk samples, visual results of the IST are in good correlation with those of a quantitative enzyme-linked immunosorbent assay (ELISA). This AFM1–IST, employing a one-step method, was environmentally friendly without the use of any organic reagents or buffers and additional equipment, and could serve as an on-site and home-use device for AFM1 tests in milk, and be helpful to people's health care.

Developmental Validation of RSID™‐Semen: A Lateral Flow Immunochromatographic Strip Test for the Forensic Detection of Human Semen*

Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate-specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID™-Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID™-Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross-react with other human or nonhuman tissues tested. RSID™-Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA-based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID™-Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.

Developmental Validation of RSID™‐Saliva: A Lateral Flow Immunochromatographic Strip Test for the Forensic Detection of Saliva

Current methods for forensic identification of saliva generally assay for the enzymatic activity of alpha-amylase, an enzyme long associated with human saliva. Here, we describe the Rapid Stain IDentification (RSID-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID-Saliva detects less than 1 microL of saliva. The sensitivity of RSID-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.

Validation studies of rapid stain identification-blood (RSID-blood) kit in forensic caseworks

When bloodstains are detected at crime scene using presumptive tests (e.g. luminol, phenolphthalein, leuchomalachite green), it is important to establish the real human nature of each stain. This is possible using confirmatory tests. One of these is rapid stain identification-blood (RISD-blood) a lateral flow immuno-chromatographic strip test format which allows the identification of human blood by detection of glycophorin A, a red blood cell membrane antigen, using two anti-human glycophorin A (GPA) monoclonal antibodies. The aim of this study is to assess the sensitivity of RSID-blood test in old, degraded bloodstains and in some bloodstains previously treated with BlueStar Forensic, a presumptive test which is often used in crime scene investigations to detect latent bloodstains. The genetic analysis of all bloodstains of confirmed human nature was subsequently performed using the AmpF1STR Identifiler PCR Amplification Kit (Applied Biosystems), to validate the possibility of obtain a consistent and reliable DNA typing results.