A new immunochromatographic assay for on-site detection of porcine epidemic diarrhea virus based on monoclonal antibodies prepared by using cell surface fluorescence immunosorbent assay.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly effective pathogen that can cause death of new-born piglet, resulting in big economical loss in pig farming industry. For rapid detection of PEDV, a new immunochromatographic assay (ICA) based on monoclonal antibodies (mAbs) was developed in this study. RESULTS: The mAbs were prepared by using PEDV positive hybridoma cells that were selected by using cell surface fluorescence immunosorbent assay (CSFIA). Fourteen mAbs against PEDV strain isolated from south of China were prepared. The optimal mAb 4A11 was coated on NC membrane as the capturing reagent and the mAb A11H7 was coupled to gold nanoparticles (AuNPs) as detection reagent for the new ICA. The new ICA was used to measure PEDV in phosphate buffer containing tween-20. Results indicated that the limit of detection (LOD) of the new ICA was 0.47 µg/mL (5.9 × 103 TCID50/mL) and the liner detection range of the ICA was 0.625-10 µg/mL (7.8 × 103-105 TCID50/mL). The specificity analysis results showed that this new ICA had no cross reaction in the presence of other porcine viruses. The ICA was also validated for the detection of PEDV in swine stool samples with little interference from swine stool. To compare its accuracy to other traditional detection methods, 27 swine stool samples from south of China were investigated with the new developed ICA, commercial strip and RT-PCR. Results showed that the new ICA was more comparable to RT-PCR than commercial test strip. CONCLUSIONS: A new ICA based on mAbs prepared by CSFIA was developed in this study. It was a sensitive, specific and rapid method that could be used for on-site detection of PEDV and therefore was useful for the diagnosis and prevention of PED.

Practical immune-barometer sensor for trivalent chromium ion detection using gold core platinum shell nanoparticle probes.

The technology progress of biosensors has markedly improved healthcare, disease diagnosis, environment monitoring, and food safety control over the past few decades. However, development of sensitive, robust, low-cost and portable assays for on-site bioanalysis is still a great challenge. In this study, we described a portable, feasible and miniaturized immune-barometer sensor (IBS), which can be used to sensitively measure the changes in a pressure signal, and we applied this IBS in the detection of Cr(iii). In this system, a competitive immunoassay was incorporated as a signaling technique for Cr(iii) detection. To generate a signal of pressure changes (ΔP), Au@PtNPs (gold core platinum shell nanoparticles) were prepared for decomposing H2O2 to generate O2 in a sealed chamber. The expansion of gas volume was accurately detected using a sensitive barometer in the sealed reaction chamber. The ΔP correlated well with Cr(iii) concentrations ranging from 0.39 to 25 ng mL-1. The limit of detection (LOD) of the IBS was estimated to be as low as 0.35 ng mL-1. Furthermore, the IBS has high specificity and high recovery for Cr(iii) detection in tap water samples (97.5%-108.7%) and in the Pearl River water samples (95.6%-110.2%). Compared with the traditional enzyme-linked immunosorbent assay (ELISA), the IBS was observed to be more sensitive, of low-cost and portable for the on-site detection of Cr(iii). Therefore, the IBS is a promising potential method for the detection of heavy metals in aqueous solutions and many other fields.

A Rapid Method for Antigen-Specific Hybridoma Clone Isolation.

Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating >2-fold more hybridoma clones in <25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.

A simple and compact smartphone-based device for the quantitative readout of colloidal gold lateral flow immunoassay strips.

Colloidal gold lateral flow immunoassay strips (AuNPs-LFIS) have been widely applied as qualitative diagnostic tools for point-of-care tests (POCT). If strip readers were incorporated, their use could be extended to quantitative analysis. However, their cost and non-portability render commercial strip readers unavailable for use in either home testing, community or rural hospital diagnosis. This is particularly true for on-site testing. Here, a smartphone-based reader was designed and 3D-printed for quantitatively assess AuNPs-LFIS. The basic principle of the devise was relying on a smartphone's ambient light sensor (SPALS). This sensor was harnessed to measure the transmitted light intensities originating from the T-lines on the strips, the transmitted light intensities vary with concentration of AuNP on the T-lines. To validate this approach, our newly developed smartphone's ambient light sensor-based reader (SPALS-reader) was used to readout AuNPs-LFIS of three analytical targets: cadmium ion (Cd2+; limit of detection (LOD) was 0.16 ng/mL), clenbuterol (CL; LOD was 0.046 ng/mL), and porcine epidemic diarrhea virus (PEDV; LOD was 0.055 µg/mL). The result showed good consistency with the results of conventional image analysis approaches, indicating that the smartphone-based device is appropriate for use in AuNPs-LFIS readouts. Compared with the traditional analysis method, the developed AuNPs-LFIS reader is easier operated, lower cost and more portable, which provided an on-site quantitative analysis tool for AuNPs-LFIS and enhances the applied range of AuNPs-LFIS.

A Portable Smart-Phone Readout Device for the Detection of Mercury Contamination Based on an Aptamer-Assay Nanosensor.

The detection of environmental mercury (Hg) contamination requires complex and expensive instruments and professional technicians. We present a simple, sensitive, and portable Hg2+ detection system based on a smartphone and colorimetric aptamer nanosensor. A smartphone equipped with a light meter app was used to detect, record, and process signals from a smartphone-based microwell reader (MR S-phone), which is composed of a simple light source and a miniaturized assay platform. The colorimetric readout of the aptamer nanosensor is based on a specific interaction between the selected aptamer and Hg2+, which leads to a color change in the reaction solution due to an aggregation of gold nanoparticles (AuNPs). The MR S-phone-based AuNPs-aptamer colorimetric sensor system could reliably detect Hg2+ in both tap water and Pearl River water samples and produced a linear colorimetric readout of Hg2+ concentration in the range of 1 ng/mL-32 ng/mL with a correlation of 0.991, and a limit of detection (LOD) of 0.28 ng/mL for Hg2+. The detection could be quickly completed in only 20 min. Our novel mercury detection assay is simple, rapid, and sensitive, and it provides new strategies for the on-site detection of mercury contamination in any environment.