Enhancing the immunofluorescent sensitivity for detection of Acidovorax citrulli using fluorescein isothiocyanate labeled antigen and antibody.

A rapid lateral flow immunochromatographic strip (ICS) using fluorescein isothiocyanate (FITC) labeled antigen and antibody was developed for the detection of Acidovorax citrulli (Ac) in melons and vegetable samples. In the ICS, signal amplification was realized based on antigen Ac and anti-Ac monoclonal antibody (McAb) 4F conjugated with FITC, respectively, which were forming two probes. The control line and the test line were obtained by immobilizing the goat anti-mouse IgG antibody and anti-Ac McAb 6D on both sides of the nitrocellulose membrane. The visual detection limit of the strip was 105 CFU/mL, which was 10-fold sensitive compared to the strip of FITC only labeling antigen or antibody. Signal amplification ICS was successfully applied to the detection of Ac in melon and vegetable samples with less detection time and operation procedures compared to the traditional enzyme-linked immunosorbent assay (ELISA) and PCR methods. This is the first report of using FITC labeled antigen and McAb as dual fluorescent probes to develop a direct-type immunofluorescence strip for the rapid and sensitive detection of Ac, which demonstrates a powerful tool for rapidly screening Ac in plant materials and other samples. Graphical abstract The schematic presentation of the test strip (a) and the positive result (b) or negative result

Fluorescein Isothiocyanate Labeling Antigen-Based Immunoassay Strip for Rapid Detection of Acidovorax citrulli.

A simple and fast immunoassay strip to detect Acidovorax citrulli (Ac) using fluorescein isothiocyanate as a marker was developed. Fluorescein isothiocyanate (FITC) was added to sample culture medium for bacteria incubation, and the bacteria could emit a yellow-green fluorescence under ultraviolet light and become a fluorescent probe. This immunofluorescence strip (IFS) was based on the binding between fluorescent bacteria and the unlabeled monoclonal antibody (McAb) immobilized on the test area in nitrocellulose membrane. The detection limit of the strip was 106 CFU/ml with a result that could be observed within 10 min. The IFS could detect eight strains of Ac and display no cross-reactions with 30 other pathogenic strains. The detection results would not be affected by impurities in plant or unknown microorganisms in natural field samples and were consistent with PCR results, indicating that the IFS has high accuracy. This is the first report of using only one unlabeled McAb to develop a direct-type immunofluorescence strip for the rapid detection of Ac. The IFS reduced detection time and simplified operation procedures compared with the traditional enzyme-linked immunosorbent assay (ELISA) and PCR methods. In addition, this simple and inexpensive method will play a significant role in monitoring plant pathogens on field detection.

Self-paired monoclonal antibody lateral flow immunoassay strip for rapid detection of Acidovorax avenae subsp. citrulli.

We screened a highly specific monoclonal antibody (McAb), named 6D, against Acidovorax avenae subsp. citrulli (Aac). Single McAb 6D was used as both nanogold-labeled antibody and test antibody to develop a single self-paired colloidal gold immunochromatographic test strip (Sa-GICS). The detection limit achieved using the Sa-GICS approach was 10(5) CFU/mL, with a result that can be observed by the naked eye within 10 min. Moreover, Sa-GICS can detect eight strains of Aac and display no cross-reactions with other pathogenic plant microorganisms. Artificial contamination experiments demonstrated that Sa-GICS would not be affected by impurities in the leaves or stems of the plants and were consistent with the PCR results. This is the first report on the development of a colloidal gold immunoassay strip with self-paired single McAb for the rapid detection of Aac. Graphical Abstract Schematic representation of the test strip.

Simultaneous quantification of Escherichia coli O157:H7 and Shigella boydii using a visual-antibody-macroarray.

Being high throughput, rapid, automated, economical, convenient to operate and highly sensitive, protein arrays have been widely used in the analysis of tumor markers and veterinary drug residues. Pathogenic microbes also can be detected qualitatively by DNA array or protein array; however, their high throughput detection and quantification remains a big obstacle. To evaluate the potentiality of protein arrays for multiple quantitative detection of microorganisms with naked eye examination without the help of any equipment, here we developed a visual-antibody-macroarray (VAMA) aiming at rapid and simultaneous quantification of Escherichia coli O157:H7 and Shigella boydii. The results show that this VAMA is highly specific and is able to distinguish mixed Escherichia coli O157:H7 and Shigella boydii synchronously. The detection limits are equivalent to 3.4 × 10(5) CFU mL(-1) and 3.2 × 10(5) CFU mL(-1), respectively, which conform to the results of plate counting and ELISA. Importantly, the examination can be solely performed with the naked eye. Therefore, we provide an easy, reliable and rapid method for quantitative analysis of microorganisms.

Inhibition of HDAC activity directly reprograms murine embryonic stem cells to trophoblast stem cells.

Embryonic stem cells (ESCs) can differentiate into all cell types of the embryonic germ layers. ESCs can also generate totipotent 2C-like cells and trophectodermal cells. However, these latter transitions occur at low frequency due to epigenetic barriers, the nature of which is not fully understood. Here, we show that treating mouse ESCs with sodium butyrate (NaB) increases the population of 2C-like cells and enables direct reprogramming of ESCs into trophoblast stem cells (TSCs) without a transition through a 2C-like state. Mechanistically, NaB inhibits histone deacetylase activities in the LSD1-HDAC1/2 corepressor complex. This increases acetylation levels in the regulatory regions of both 2C- and TSC-specific genes, promoting their expression. In addition, NaB-treated cells acquire the capacity to generate blastocyst-like structures that can develop beyond the implantation stage in vitro and form deciduae in vivo. These results identify how epigenetics restrict the totipotent and trophectoderm fate in mouse ESCs.

Highly efficient generation of blastocyst-like structures from spliceosomes-repressed mouse totipotent blastomere-like cells.

Mammalian embryogenesis begins with a totipotent zygote. Blastocyst-like structures can be captured by aggregated cells with extended pluripotent properties in a three-dimensional (3D) culture system. However, the efficiency of generating blastoids is low, and it remains unclear whether other reported totipotent-like stem cells retain a similar capacity. In this study, we demonstrated that spliceosomal repression-induced totipotent blastomere-like cells (TBLCs) form blastocyst-like structures within around 80% of all microwells. In addition, we generated blastoids initiating from a single TBLC. TBLC-blastoids express specific markers of constituent cell lineages of a blastocyst and resemble blastocyst in cell-lineage allocation. Moreover, single-cell RNA sequencing revealed that TBLC-blastoids share a similar transcriptional profile to natural embryos, albeit composed of fewer primitive endoderm-like cells. Furthermore, TBLC-blastoids can develop beyond the implantation stage in vitro and induce decidualization in vivo. In summary, our findings provided an alternative cell type to efficiently generate blastoids for the study of early mouse embryogenesis.

Development of a Bacterial Macroarray for the Rapid Screening of Targeted Antibody-Secreted Hybridomas.

Hybridoma screening is a key step for the successful generation of high-affinity analyte-specific monoclonal antibodies (MAbs). This work presents an innovative screening method, known as a bacterial macroarray, generated by contact printing of hybridoma cell supernatant samples on a nitrocellulose (NC) membrane initially coated with fluorescein isothiocyanate (FITC)-labeled bacteria. Given that bacterial fixation will be influenced by complex bacterial surface structures, we selected both gram-positive bacteria ( Staphylococcus aureus and Listeria monocytogenes) and gram-negative bacteria ( Escherichia coli O157:H7 and Cronobacter sakazakii) to optimize the fixation conditions for binding to the NC membrane, such as the aperture of the NC membrane, the concentration of bacteria, the dosage of glycerin in the spotting buffer, and the fixation time and temperature. As a result, we found that a better bacterial macroarray could be developed when the spotting buffer, containing 1011 CFU mL-1 of FITC-labeled bacteria and 15% (V/V) glycerol, was spotted onto a 0.45 µm NC membrane with an incubation of 2 h at 37 °C. Finally, we verified the stability and specificity of the prepared bacterial macroarray by detecting cell cultures with the addition of two MAbs ( Escherichia coli O157:H7 MAb E7 and Cronobacter sakazakii MAb 1E9) to simulate the screening experiments. Here, we describe a bacterial macroarray to efficiently screen the targeted antibody-secreted hybridomas.