Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies.

COVID-19 vaccines have been provided to the general public to build immunity since the 2019 coronavirus pandemic. Once vaccinated, SARS-CoV-2 neutralizing antibodies (NAbs-COVID-19) are needed for excellent protection against COVID-19. However, monitoring NAbs-COVID-19 is complicated and requires hospital visits. Moreover, the resulting NAbs-COVID-19 are effective against different strains of COVID-19 depending on the type of vaccine received. Here, an overlaid lateral flow immunoassay (O-LFIA) was developed for the simultaneous detection of two NAbs-COVID-19 against different virus strains, Delta and Omicron. The O-LFIA was visualized with two T-lines with a single device using competition between the free antigen and the antigen-binding antibody. Angiotensin-converting enzyme 2 (ACE2) immobilized on the T-line binds to the antigen remaining after antibody binding. Under the optimum conditions, the proposed device exhibited 50% inhibition concentrations (IC50 values) of 45.1 and 53.6 ng/mL for the Delta and Omicron variants, respectively. Additionally, the proposed platform was applied to real-world samples of animal and human serum, and the developed immunoassay provided results that were in good agreement with those obtained with the standard method. In conclusion, this developed O-LFIA can be used as an alternative method to detect NAbs-COVID-19 and can be enabled for future advancements toward commercialization.

Paper-based electrochemical immunosensor for highly sensitive detection of chicken anemia virus.

Chicken anemia virus (CAV) is one of the primary causes of morbidity and mortality in young chickens. Given the importance of timely detection for maintaining livestock quality, there is a pressing need for rapid and field-deployable diagnostic tools. This study introduces a highly sensitive paper-based electrochemical immunosensor (PEI) for the detection of the 60 amino acid N-terminally truncated viral protein 1 (Δ60VP1), a derivative of the CAV capsid (VP1). A custom antibody was produced for precise immunoassay detection, with results obtainable within 30 min using Square Wave Voltammetry (SWV). The underlying mechanism involves an immunocomplex in the sample zone that hinders the electron transfer of redox species, thereby reducing the current signal in proportion to the Δ60VP1 concentration. Under optimal conditions, the detection linearity for Δ60VP1 ranged from 80 to 2500 ng/mL, with a limit of detection (LoD) of 25 ng/mL. This device was then successfully applied to detect VP1 in 29 chicken serum samples, achieving 91.6% sensitivity and 94.1% selectivity. In conclusion, the PEI device presents a promising solution for rapid, sensitive, and disposable detection of chicken pathogens, potentially revolutionizing productivity and quality assurance in chicken farming.

Integration of a hamper pad on test strips for improved sensitivity of carbendazim detection.

Lateral flow immunoassays (LFIAs) are widely used to determine carbendazim (CBZ) residues in food products due to their advantages of low cost, ease and rapid use, on-site detection capability. However, conventional LFIAs have low detection sensitivity. Although improvements have been made to increase the sensitivity, it is not sufficient. Here, a hamper pad, polyvinyl alcohol coated on a nitrocellulose membrane, was integrated to enhance the sensitivity of LFIA for CBZ detection. The hamper pad was inserted between the conjugated and nitrocellulose pads to delay the flow rate, thereby increasing the possibility of the antibody and target analyte binding. This platform exhibited a fourfold sensitivity increase in CBZ detection compared with the conventional LFIA, and its limit of detection was 1.6 ng/mL. In addition, a single-step operation was successfully applied to detect CBZ in rice (white rice, brown rice, sticky rice, and paddy) and soybean samples, with acceptable recoveries of 93.6%-120.0%. This novel device was compared to the standard high-performance liquid chromatography method, which shows high accuracy with a Kappa coefficient of 0.91. Therefore, improved sensitivity with a rapid, simple, and inexpensive device could facilitate the detection of CBZ residues in agricultural products for on-field screening and improved user-friendliness.

Development of a lateral flow assay for rapid and accurate detection of chicken anemia virus.

Significant challenges to poultry health are posed by chicken anemia virus (CAV), which induces immunosuppression and causes increased susceptibility to secondary infections. The effective management and containment of CAV within poultry stocks require precise and prompt diagnosis. However, a deficiency persists in the availability of low-cost, rapid, and portable CAV detection devices. In this study, an immunochromatographic lateral-flow test strip-based assay was developed for CAV detection using in-house generated monoclonal antibodies (MABs) against CAV viral protein 1 (VP1). The recombinant truncated VP1 protein (Δ60VP1), with amino acid residues 1 to 60 of the native protein deleted, was produced via a prokaryotic expression system and utilized for immunizing BALB/c mice. Subsequently, high-affinity MABs against Δ60VP1 were generated and screened using conventional hybridoma technology combined with serial dilution assays. Two MABs, MAB1, and MAB3, both binding to distinct epitopes of Δ60VP1, were selected for the development of a lateral-flow assay. Sensitivity analysis demonstrated that the Δ60VP1 antigen could be detected by our homemade lateral-flow assay at concentrations as low as 625 ng/mL, and this sensitivity was maintained for at least 6 mo. The assay exhibited high specificity, as evidenced by its lack of reactivity with surrogate recombinant proteins and the absence of cross-reactivity with other chicken viruses and viral antigens. Comparative analysis with quantitative PCR data demonstrated substantial agreement, with a Kappa coefficient of 0.66, utilizing a sample set comprising 305 clinical chicken serum samples. In conclusion, the first lateral-flow assay for CAV detection was developed in this study, utilizing 2 specific anti-VP1 MABs. It is characterized by simplicity, rapidity, sensitivity, and specificity.

Acetylcholinesterase (AChE) monoclonal antibody generation and validation for use as a biomarker of glyphosate-based herbicide exposure in commercial freshwater fish.

Monoclonal antibody specific to acetylcholinesterase (AChE) was extracted from the brain of hybrid catfish after exposure to glyphosate-based herbicide for 24 h. AChE was partially purified using hydroxyapatite and chromatography columns. The specific characteristics of AChE were studied by western blot using commercial polyclonal antibody (Rabbit anti-Fish AChE). It was found that the protein band had a molecular weight of 71 kDa. After mice were injected with AChE 4 times, the spleen showed a response to the induction. Polyclonal B cells from the mouse's spleen were taken and fused with myeloma cells to produce hybrid cells. After two fusions were performed, the clones specific to AChE were selected by dot blot, ELISA, immunohistochemistry and western blot techniques. Two clones, ACHE 33 and ACHE 99, which had the isotype of IgM were found. These two produced monoclonal antibodies specific to AChE in both denatured and native forms. The ACHE 33 monoclonal antibody clone from hybrid catfish could be cross-react with two commercial freshwater fishes, Nile tilapia and climbing perch, based on dot blot, immunohistochemistry, and western blot techniques. Moreover, AChE in Nile tilapia and climbing perch with glyphosate- based herbicide exposure gave a positive result with ACHE 33 as protein with molecular weight of 66 kDa. Based on our results, the produced monoclonal antibody showed specificity and could be applied to test AChE expression to assess glyphosate-based herbicide contamination in hybrid catfish, Nile tilapia and climbing perch. It could be also be a useful tool in indicating the quality of water resources.

Acetylcholinesterase (AChE) polyclonal antibody from hybrid catfish (C. macrocephalus × C. gariepinus): Specification, sensitivity and cross reactivity.

AChE (acetylcholinesterase) is generally classified as a specific biomarker of pesticide exposure. The aim of this study was to produce AChE polyclonal antibody from hybrid catfish that were exposed to commercial glyphosate. The hybrid catfish was exposed to glyphosate (0.75 mL/L) for 24 h. After that, the fish brain was dissected, AChE was extracted and purified by hydroxyapatite column chromatography and eluted with 0.2 M potassium phosphate buffer pH 6.8. This protocol gave 70% yield. Then, the brain extract was characterized using 10% SDS-PAGE and Western blot probed with commercial polyclonal antibody specific to AChE (PAb-AChE). The protein, 71 kDa, was then used as an antigen to immunize mice for antibody production. The polyclonal antibody (PAb) was characterized using dot blot, Western blot and immunohistochemistry for immunolocalization of AChE in hybrid catfish exposed to glyphosate. We found that the appropriate dilution of antibody for both dot blot and Western blot was 1:3500, and 1:2500 for immunohistochemistry. Cross reactivity testing showed that PAb-AChE can be used with AChE from striped snakehead fish at the same dilution as used with AChE from hybrid catfish. It was concluded that PAb specific to hybrid catfish AChE from this work was highly specific and sensitive, and can cross-react with striped snakehead fish AChE. Thus, this polyclonal antibody may be used in monitoring glyphosate exposure in hybrid catfish and striped snakehead fish.

Novel ractopamine-protein carrier conjugation and its application to the lateral flow strip test for ractopamine detection in animal feed.

In this work, a novel conjugate of ractopamine and bovine serum albumin (RAC-BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC-BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC-BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection (LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075-0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%-103.7% with a relative standard deviation of 2.5%-3.6% for intra- and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC-BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.

Expression, purification and biological activity of monomeric insulin precursors from methylotrophic yeasts.

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been used for the production of recombinant monomeric insulin precursor (MIP). Recombinant plasmids with one, two and four cassettes of the MIP gene have been successfully constructed in the pPICZαA expression vector to study the effects of gene copy number on MIP production. The MIP protein can be detected by dot-blot analysis from the culture broth of P. pastoris KM71H 24 h after placement in MMH induction medium. The secretion levels of MIP protein in culture broth at 72 h after induction indicated that P. pastoris KM71H with one cassette of the MIP gene had highest MIP protein levels (4.19 ±â€¯0.96 mg L-1). The transcription levels of the MIP gene increased proportionately with copy number. However, the amount of secreted MIP protein showed no correlation. The MIP molecular mass was 5756.951 Da, as confirmed by typical MALDI-TOF mass spectrometry. The MIP protein in culture broth was purified by two steps purification including SP Sepharose Fast Flow chromatography followed by ultrafiltration (10 kDa MW cutoff). The percentage of MIP recovery after the two-step purification was 70%, with a single band in a native-PAGE. The biological activity of tryptic hydrolyzed MIP was determined via the expression of the glucose transporter 4 gene (GLUT4) in H9c2 (2-1) cell line by RT-qPCR, and the results demonstrated that the MIP protein can induce glucose uptake and upregulation of GLUT4 mRNA transcription at 3 h and that this activity was related to Humalog® insulin.

A monoclonal antibody against Lates calcarifer vitellogenin and a competitive ELISA to evaluate vitellogenin induction after exposure to xenoestrogen.

A monoclonal antibody specific to sea bass (Lates calcarifer) vitellogenin (VTG) was developed, for use as a tool for monitoring endocrine disrupting chemicals (EDCs). VTG was induced in sea bass by intramuscular injection of 17ß-estradiol (E2: 2 mg/kg) every three days. Blood was collected three days after the last injection. Plasma VTG was then purified by chromatography in hydroxyapatite and a sephacryl-S300 column. Characterizations of purified VTG were done by phospholipoglycoprotein staining on a native-PAGE with confirmation by mass spectrometry (LC-MS/MS). Antibody was raised in mice by injection of purified VTG. After monoclonal antibody production, the hybridoma clone No. 41 (MAb-sea bass VTG 41) was selected and developed for quantification of VTG by competitive enzyme-linked immunosorbent assay (ELISA). The ELISA method was sensitive with a detection limit of VTG 40 ng/mL. MAb-sea bass VTG 41 was specific to VTG from E2-treated sea bass and others EDCs (Nonylphenol, Benzo[a]pyrene and CdCl2). Moreover, cross-reactivity was also found in E2-treated coral grouper (Epinephelus corallicola). The ELISA method obtained from this work can be further applied for the assessment of EDCs in Thailand and Southeast Asia's aquatic environment.

Development of an automated wax-printed paper-based lateral flow device for alpha-fetoprotein enzyme-linked immunosorbent assay.

In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL-1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1-100ngmL-1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA.

Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp.

A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118% for an intra-assay and 96%-113% for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9% and 5.5%-14.9%, respectively.

Detection by using monoclonal antibodies of Yersinia enterocolitica in artificially-contaminated pork.

Monoclonal antibodies against Yersinia enterocolitica were produced by fusion of NS-1 mouse myeloma cells with spleen cells of ICR mice immunized with heat-killed and heat-killed plus SDS-mercaptoethanol treated forms of Y. enterocolitica ATCC 27729 alone or mixed with Y. enterocolitica MU. The twenty-five MAbs obtained from five fusions were divided into nine groups according to their specificities to different bacterial strains and species, as determined by dot blotting. The first five groups of MAbs were specific only to Y. enterocolitica, but did not recognize all of the isolates tested. MAbs in groups 6 and 7 reacted with all isolates of Y. enterocolitica tested but showed cross-reaction with some Yersinia spp. and Edwardsiella tarda, especially in the case of group 7. MAbs in groups 8 and 9 reacted with all isolates of Y. enterocolitica and Yersinia spp., as well as other Gram-negative bacteria that belong to the family Enterobacteriaceae. These MAbs recognized Y. enterocolitica antigens with apparent molecular weights ranging from 10-43 kDa by Western blotting, and could detect Y. enterocolitica from ∼10³-105 colony forming units (CFUs) by dot blotting. The hybridoma clone YE38 was selected for detection of Y. enterocolitica in pork samples which had been artificially-contaminated by inoculation with Y. enterocolitica ATCC 27729 at concentrations of ∼104-106 CFUs/g and incubation in peptone sorbitol bile broth at 4°C. Samples were collected and applied on a nitrocellulose membrane for dot blotting with trypticase soy and cefsulodin-Irgasan-novobiocin agars. After 48 hr of incubation, the detection limit was ∼10²-10³ CFU/g by dot blotting.