Lateral Flow Immunochromatographic Strip Research Articles

A Novel Immunochromatographic Test Strip Using Lanthanide-Labeled Fluorescent Nanoparticles for the Serological Detection of Toxoplasma gondii in Dogs and Cats.

Toxoplasma gondii (T. gondii) is an important zoonotic pathogen which induces both acute and chronic toxoplasmosis. Timely diagnosis of T. gondii is crucial for effective disease management. Here, we present a pioneering approach using europium (III)-chelated nanoparticles (EuNPs) in a rapid lateral flow immunochromatographic test strip (ICTS) for detecting T. gondii antibodies in serum samples. By conjugating EuNPs with Staphylococcus aureus protein A, we efficiently captured T. gondii-specific antibodies, which bound to T. gondii antigens on the test line (T-line), generating a distinct fluorescent signal. Employing this novel method, we conducted an extensive epidemiological investigation of T. gondii infections among dogs and cats in Shanghai, China. This innovative ICTS allows for rapid results within 25 min, which include a qualitative result through naked-eye observation under an ultraviolet lamp and a quantitative one derived using a strip reader. With a detection limit of 1:6400 for dog positive serum and no cross-reactivity with other canine and feline pathogens, the EuNPs-ICTS demonstrated excellent consistency with standard enzyme-linked immunosorbent assay results for dogs (κ = 0.91) and cats (κ = 0.92). In addition, 20.38% of 996 dog serum samples and 14.18% of 416 cat serum samples revealed T. gondii antibodies, highlighting the efficacy of this approach. Our study presents a rapid, sensitive, specific, and reproducible EuNPs-ICTS, serving as a promising tool for on-the-spot diagnosis of T. gondii infections in dogs and cats.

Establishment of highly sensitive lateral flow immunochromatographic strips for quinclorac detection utilizing signal amplification nanoparticles

Highly selective herbicide quinclorac (Qui) is a type of quinoline carboxylic acid hormone herbicide, which has the characteristics of long half-life and difficulty for degradation, causing high risk to the environmental safety. In this study, anti-Qui 8A3 monoclonal antibody (mAb) with good specificity and high affinity (3.89 × 109 L/mol) was prepared, and two kinds of lateral flow immunochromatographic strips (LFICS) including nano-flower nanoparticles (AuNF) - and latex microsphere (LM)- based LFICS were established based on the antibody and signal amplification. The linear range of the AuNF- and LM- based LFICS were 5.31–345.48 ng/mL and 2.52–257.92 ng/mL, respectively. The limit of detection (LOD) of the AuNF- and LM- based LFICS were determined to be 5.31 ng/mL and 2.52 ng/mL, respectively. In summary, the developed LFICS using AuNF and LM as signal amplification reporters exhibited excellent sensitivity and provided the rapid on-site screening of Qui and other analytes in food safety field.

Colorimetric Detection of Fentanyl Powder on Surfaces Using a Supramolecular Displacement Assay.

Fentanyl is a potent synthetic opioid with an alarmingly low lethal dosage of 2 mg. The equipment necessary to detect fentanyl in field settings (e.g., hand-held spectrometers) is restricted to highly trained, well-funded, and specialized personnel. Established point-of-need technologies, such as lateral flow immunochromatographic strips, are available; however, they often involve multiple contact-based steps (e.g., collection, mixing) that pose a higher risk to users handling unknown substances. Herein, we developed a colorimetric displacement assay capable of contactless detection of fentanyl in liquid or solid samples. The basis of our assay relies on the presence of fentanyl to displace a redox mediator, ferrocene carboxylic acid, inclusively bound in the cavity of a supramolecular host, CB[7]. The displacement is only possible in the presence of high affinity binding guests, like fentanyl (KA ∼ 106 M-1). The liberated redox guest can then react with indicator reagents that are free in solution, producing either: (i) a distinct blue color to indicate the presence of fentanyl or (ii) a pale blue tint in the absence of fentanyl. We demonstrate rapid and specific detection of fentanyl free base and fentanyl derivatives (e.g., acetyl fentanyl and furanyl fentanyl) against a panel of 9 other common drugs of abuse (e.g., morphine, cocaine, and heroin). Furthermore, we highlight the intended use of this assay by testing grains of fentanyl derivatives on a surface with a drop (i.e., 25 μL) of the assay reagent. We anticipate that this approach can be applied broadly to identify the presence of fentanyl at the point of need.

Nanoparticles-based lateral flow strip for halofuginone ultrasensitive detection in chicken and beef

Halofuginone (HFG) is a widely used anti-coccidiosis drug in animal husbandry. There is an urgent need for a rapid detection method to detect HFG residues in chicken and beef to ensure human health. In this study, suitable HFG haptens were selected using computer simulation, and a highly sensitive monoclonal antibody (mAb) was developed with the 50%-inhibitory concentration (IC50) of 0.66 ng/mL. Then, gold nanoparticle-based lateral flow immunochromatographic (ICA) strips for HFG detection were produced. Under optimization conditions, the ICA strips exhibited outstanding performance with a visual limit of detection (vLOD) of 50 μg/kg and 5 μg/kg, and cut-off values of 100 μg/kg and 10 μg/kg, for chicken and beef samples, respectively. In addition, the reliability of strips was verified by enzyme linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Therefore, the developed strip can be used as an effective tool for HFG residues screening in chicken and beef samples.

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

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

Development and evaluation of a time-resolved fluorescence labelled immunochromatographic strip assay for rapid and quantitative detection of bovine herpesvirus 1.

Bovine herpes virus 1 (BoHV-1) causes a wide variety of diseases in wild and domestic cattle. The most widely used method for viral identification is real-time PCR, which can only be performed in laboratories using sophisticated instruments by expert personnel. Herein, we developed an ultrasensitive time-resolved fluorescence lateral flow immunochromatographic strip (ICS) assay for detecting BoHV-1 in bovine samples using a monoclonal antibody against BoHV-1 labelled with fluorescent microspheres, which can be applied in any setting. The intact process from sample collection to final result can be achieved in 15 min. The limit of detection of the assay for BoHV-1 was 102 TCID50/100 μL. The coincidence rate of the ICS method and real-time PCR recommended by the World Organization for Animal Health (WOAH) was 100% for negative, 92.30% for positive, and 95.42% for total, as evaluated by the detection of 131 clinical samples. This detection method was specifically targeted to BoHV-1, not exhibiting cross-reactivity with other bovine pathogens including BoHV-5. We developed an ICS assay equipped with a portable instrument that offers a sensitive and specific platform for the rapid and reliable detection of BoHV-1 in the field. The Point-of-Care test of BoHV-1 is suitable for the screening and surveillance of BoHV-1 in dairy herds.

Barcode-style lateral flow immunochromatographic strip for the semi-quantitative detection of ochratoxin A in coffee samples

Ochratoxin A (OTA) is a mycotoxin contaminating agricultural products produced by fungi, associated with important toxic effects. Thus, the development of fast, sensitive, and economical approaches for OTA detection is crucial. In this study, a barcode-style lateral flow assay for the semi-quantitative detection of OTA in coffee samples was developed. To achieve this goal, a BSA-OTA complex was immobilized in three test zones to compete with OTA molecules in the sample for binding with anti-OTA antibodies labeled with gold nanoparticles. Different concentrations of OTA in the sample produced distinct colour patterns, allowing semi-quantification of the analyte. The assay exhibited high sensitivity, with a limit of detection of 2.5 µg.L−1, and high reproducibility, with variation coefficient values between 2% and 13%. Moreover, the colour patterns obtained in the analysis with coffee samples were similar to the results obtained with standard OTA solutions, demonstrating a reliable applicability in real samples.

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.

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 a Lateral-Flow Immunochromatographic Strip for the Detection of Oxytetracycline Residues in Biological Fluids.

Oxytetracycline (OTC) is extensively used in veterinary medicine and for growth promotion around the globe. The indiscriminate use of OTC in food-producing animals leaves residues in animal products. The presence of these residues in animal products causes economic losses and harmful effects on consumers. Different regulatory bodies set maximum residue limits (MRLs) for different tetracyclines. To avoid harmful effects, there is a need for a simple, fast, and economical method for the screening of animal products. In this study, a fast, economical, and user-friendly lateral-flow immunochromatographic (LFIC) assay based on gold nanoparticles (AuNPs) was developed to detect the presence of OTC residues in biological fluids. AuNPs provided visual results as red lines in 6-15 min. Polyclonal rabbit IgG antibodies were produced using the immunogen of OTC. These antibodies were purified by the combined ammonium sulfate-octanoic acid precipitation method. Antibodies were conjugated to AuNPs as recognition biomolecules. A LFIC strip was optimized using borate buffer spiked with different concentrations of the OTC. The visual limit of detection (LOD) in different biological samples (milk, serum, and urine) was determined using samples spiked with OTC. The LOD was found to be 15 μg/L, which is very low from the MRL (100 μg/L) set by different regulatory authorities. This LFIC strip can be used to detect OTC residues in biological fluids for point-of-care testing (POCT). These strips are easy to use, cost-effective, and portable and provide quick results without the use of laboratory instruments.

A lateral flow immunochromatographic strip based on RPA and quantum dot nanobeads for rapid identification of pathogenic Yersinia enterocolitica

To detect pathogenic Yersinia enterocolitica in meat, a lateral flow immunochromatographic strip targeting on virulence gene ail was developed, which was based on recombinase polymerase amplification (RPA) and quantum dot nanobeads (QDNBs). Under optimal conditions, the minimum detection limit of the strip was 9.6 × 103 CFU/mL, with a standard curve equation of y = 0.144x - 0.5098 (R2 = 0.9863), and the detection times including the RPA reaction at hand temperature (35–36.5 °C), could be controlled in 10 min. When testing spiked sample, the recoveries (%) of Yersinia enterocolitica at 9.6 × 104 CFU/mL were 44.27 ± 2.08 and 45.31 ± 3.12 in beef and pork samples and was 107.55 ± 1.30 in chicken samples. When testing 15 actual samples, only one pork sample was tested positive, consistent with polymerase chain reaction (PCR) results. The strips are body temperature-triggered and rapidly visualized, suitable for rapid field detection of pathogenic Yersinia enterocolitica.

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.

Multiplex lateral flow immunochromatographic assay for the qualitative and quantitative detection of six steroid hormone residues in chicken and pork

To ensure food safety from animal origins and to prevent potential hazards caused by steroid hormones, there is an urgent need to develop a rapid detection method for steroid hormones in foods from animal origins. Three highly sensitive monoclonal antibodies (mAb) against testosterone propionate (Tp), stazolol (STA) and estradiol benzoate (EB) were prepared, with IC50 values of 0.13, 0.14 and 0.19 ng/mL, respectively. Tp-mAb is a group-selective mAb that simultaneously detects Tp, testosterone (T), methyltestosterone (MT) and testosterone phenylpropionate (TPP), and their IC50 values were 0.13, 0.77, 0.90, and 1.25 ng/mL, respectively. A novel multiplex lateral flow immunochromatographic strip (ICS) was further prepared, which could simultaneously detect Tp, STA, and EB in chicken and pork samples with limits of quantification (LOQ) of 0.32, 0.51, 0.35 μg/kg and 0.36, 0.64, 0.43 μg/kg, respectively. Based on the broad spectrum of Tp-mAb, ICS has been further developed to detect Tp, T, MT and TPP simultaneously. This multiplex ICS rapid detection method was efficient, sensitive, and fast, for the simultaneous detection of multiple steroid hormones in actual samples. Its detection time is only 40 min, which it represents a favorable monitoring tool for the detection of steroid hormones in animal-derived foods from the marketplace.

Utilization of a lateral flow colloidal gold immunoassay strip based on surface-enhanced Raman spectroscopy for rapid detection of glycinin

Glycinin is an important storage protein in soybean, but it can also lead to allergic reactions in humans. In this study, based on a low-cost, simple, rapid and portable lateral flow immunoassay test strip, combined with high sensitivity surface enhanced Raman spectroscopy (SERS) technology, a sandwich lateral flow immunochromatographic test strip for rapid detection of soybean allergen glycinin is established. In the experiment, colloidal gold was covalently conjugated with rabbit-derived polyclonal antibodies of glycinin and Raman probe molecule 4-aminothiophenol(4-PATP) to prepare the immunoprobe. The respective optimal PATP and optimal antibody labeling amounts of colloidal gold solution were 1.05 × 10-2mol/L and 4.6 × 10-8mol/L. The detection limit of the test strip for glycinin was 4.87 ng/mL. The recovery rate ranged from 91 to 107 % and the CV was between 3 and 10 %. The test strip underwent no cross-reaction with β-conglycinin, sesame protein, peanut protein, wheat protein or whey protein. The results of the experiment showed that this method exhibits high sensitivity and specificity.

Rapid determination of serum amyloid A using an upconversion luminescent lateral flow immunochromatographic strip.

The early diagnosis and real-time prognosis of cardiovascular diseases (CVDs) at the bedside are important. However, real-time detection of myocardial infarction involves the use of large-scale instrumentation and long test times. Herein, a simple, rapid and sensitive lateral flow immunochromatographic strip (LFIS) based on Yb/Er co-doped NaYF4 upconversion nanoparticles (UCNPs) was demonstrated for use in the detection of myocardial infarction. First, through heavy Yb/Er doping and an inert NaYF4 shell coating on the nanoparticles, the surface-related luminescence quenching effect of UCNPs was eliminated to enhance the upconversion luminescence. Second, through uniform coating of a SiO2 layer on the UCNPs, the biological affinity was improved to couple UCNPs and antibody proteins. Finally, through modification and activation with a specific antibody protein (serum amyloid A (SAA)), the UCNPs exhibited intense upconversion luminescence and high specificity when applied as a lateral flow immunochromatographic strip (LFIS). The developed UC-LFIS was highly sensitive (0.1 μg mL-1) and specific for detecting SAA in only 10 μL of serum. The UC-LFIS holds great potential for the early diagnosis and prognosis of CVDs.

Reverse distal similarity of hapten structure enhancing antibody’s group-specificity: Development of an immunochromatographic strip for tylosin and tilmicosin in milk and water

The most common macrolide veterinary drugs used in animal husbandry are tylosin and tilmicosin. To protect human health, tylosin and tilmicosin residues in animal products and environmental water samples must be monitored on-site. A monoclonal antibody and gold nanoparticle-based lateral flow immunochromatographic strip (LFIS) were used in this study to detect tylosin and tilmicosin simultaneously, allowing for quantitative detection with a portable reader. The four-parameter equation fit produced a standard sigmoid curve under optimized conditions, with IC50 of 17.69 ng/mL and LOD(IC10) of 1.72 ng/mL for tylosin and IC50 of 18.95 ng/mL and LOD(IC10) of 7.07 ng/mL for tilmicosin. Additionally, good linearity results were obtained in the actual concentration range of 3.25–50 ng/mL, with R squares of 0.9946 for tilmicosin and 0.9953 for tylosin. The degree of cross-reactivity with other analogs was less than 0.1%. The average recoveries for tylosin and tilmicosin ranged from 77.5% to 119.7% and from 80% to 126.1% in milk, from 81.1% to 108.0% and from 98.3% to 106.8% in environmental water, respectively. The results could be obtained in 5–8 min. Therefore, the developed lateral flow immunochromatographic strip was met for the rapid detection of tylosin and tilmicosin residues in milk and environmental water samples using group screening. Furthermore, we discovered that the high matching of three dimensional structure at the distal structural fragments of the hapten arm site, as well as the consistency of the frontier orbital distribution, are very critical factors for the analytical performance of antibody against target molecules and should be considered as a universal theoretical guide for hapten design.

Tetrodotoxin and Its Analogues in Food: Recent Updates on Sample Preparation and Analytical Methods Since 2012.

Tetrodotoxin (TTX), found in various organisms including pufferfish, is an extremely potent marine toxin responsible for numerous food poisoning accidents. Due to its serious toxicity and public health threat, detecting TTX and its analogues in diverse food matrices with a simple, fast, efficient method has become a worldwide concern. This review summarizes the advances in sample preparation and analytical methods for the determination of TTX and its analogues, focusing on the latest development over the past five years. Current state-of-the-art technologies, such as solid-phase microextraction, online technology, novel injection technology, two-dimensional liquid chromatography, high-resolution mass spectrometry, newly developed lateral flow immunochromatographic strips, immunosensors, dual-mode aptasensors, and nanomaterials-based approaches, are thoroughly discussed. The advantages and limitations of different techniques, critical comments, and future perspectives are also proposed. This review is expected to provide rewarding insights to the future development and broad application of pretreatment and detection methods for TTX and its analogues.

A colloidal gold immunochromatography for the detection of flumioxazin residues in fruits.

In this study, an excellent monoclonal antibody (mAb) was produced that could sensitively and specifically recognize Flu in fruit samples. The mAb belonged to the IgG2 subclass and had a half-inhibitory concentration (IC50 ) of 0.59ng/ml and a limit of detection (LOD) of 0.13ng/ml. A cross-reactivity test revealed that the mAb had good specificity for Flu. A gold nanoparticle-based lateral-flow immunochromatographic (ICA) strip was used for the rapid screening of Flu in fruit samples. The method had a visual cut-off value of 10µg/kg for Flu in three different fruit samples when evaluated with the naked eye. The LODs were 5.25, 3.13, and 4.64µg/kg for Flu in apples, grapes, and oranges, respectively, as obtained with the aid of a strip scan reader. Therefore, this ICA strip assay represented a potential tool for the detection of Flu in fruit samples.

Limits of Detection of Mycotoxins by Laminar Flow Strips: A Review

Mycotoxins are secondary metabolic products of fungi. They are poisonous, carcinogenic, and mutagenic in nature and pose a serious health threat to both humans and animals, causing severe illnesses and even death. Rapid, simple and low-cost methods of detection of mycotoxins are of immense importance and in great demand in the food and beverage industry, as well as in agriculture and environmental monitoring, and, for this purpose, lateral flow immunochromatographic strips (ICSTs) have been widely used in food safety and environmental monitoring. The literature to date describing the development of ICSTs for the detection of different types of mycotoxins using different nanomaterials, nanoparticle size, and replicates was reviewed in an attempt to identify the most important determinants of the limit of detection (LOD). It is found that the particle size and type of materials contribute significantly to determining the LOD. The nanoparticle sizes used in most studies have been in the range 15–45 nm and gold nanoparticle-based ICSTs have been shown to exhibit the lowest LOD. Perspectives for potential future development to reduce the LODs of ICSTs are also discussed.

Development and evaluation of a rapid lateral flow immunochromatographic strip assay for detection of 17β-estradiol in sheep sera

Development and evaluation of a rapid lateral flow immunochromatographic strip assay for detection of 17β-estradiol in sheep sera