The Protein A-mediated binding of Staphylococcus to antibodies in flow cytometric assays and its reduction using FcR blocking reagent

The Protein A-mediated binding of Staphylococcus to antibodies in flow cytometric assays and its reduction using FcR blocking reagent

Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through the expression of protein A (SpA). This species has consequently become a source of false positive signals in antibody-based test designed to detect bacteria other targets. In this work, flow cytometry was used to study the effect of a number of factors in SpA single-cell-mediated binding to anti-human IgG antibody, including: tension; heat kills; Overnight storage; growth phase; cell physiology; surface adhesion; and growth in food model systems. Co-staining cells through antibody-stained with a dye permeability, propidium iodide and Calcein Violet AM, the physiological status of cells associated with antibody-mediated binding SpA.

Generally, cells lacking permeabilised esterase activity is not strong antibody binding. Binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. also marked. Not all species of SpA-expressing strong showing binding of mouse IgG, and one species is not known to express a strong showing SpA binding. Most SpA-expressing strain rabbit IgG antibody bound to a certain extent, whereas only one strain bound goat IgG. To reduce or eliminate the binding of IgG-mediated SpA, the following products are evaluated as a blocking reagent and applied before staining with primary or secondary antibody: normal rabbit serum, IgG isotype control, goat IgG and commercial FCR blocking reagent.

Only FCR blocking reagent consistently reduce SpA-mediated binding of Staphylococcus spp. for antibodies to other species and can be recommended as a blocking reagent in an immunoassay designed to detect non-Staphylococcus species.Importance This study characterizes a widespread problem, but little studied associated with antibody-based detection of microbes – Staphylococcus Protein A (SpA) -mediated binding of IgG antibodies – and offered a solution: the commercial use FCR blocking reagent.

A common source of false positive signals in the detection of microbes in clinical, food or environmental samples can be removed by following the findings of the authors. Using flow cytometry, the author shows the degree of heterogeneity in this culture SpA-mediated antibody binding and that the level of antibody-mediated binding SpA strain, phase- and matrix dependent growth of food and affected by simulated food processing care and adherence of cells.

In addition, studies SpA-mediated binding of Staphylococcus spp. for antibodies against other bacterial species produce highly nuanced images, leading the authors to recommend testing against multiple strains of S. aureus and S. hyicus all the antibodies to be incorporated into any immunoassay designed to detect non-Staphylococcus spp.

The Protein A-mediated binding of Staphylococcus to antibodies in flow cytometric assays and its reduction using FcR blocking reagent
The Protein A-mediated binding of Staphylococcus to antibodies in flow cytometric assays and its reduction using FcR blocking reagent

ETS transcription factor ESE-1 / Elf3 an independent prognostic factor of survival in breast cancer patients with HR + HER2 +

Objective: ETS transcription factor ESE-1 has been shown to be important in breast cancer HER2 + and ESE-1 mRNA expression has been shown to associate with the results prognosis in HER2 + subtypes, as well as in ER +, HER2 + patients with luminal B. However, the clinical significance ESE-1 protein expression is still unknown. The purpose of this current exploratory study was to evaluate the prognostic value of ESE-1 protein expression in breast cancer molecular subtypes with particular emphasis on HER2 + hormone receptor positive (HR + HER2 +) and HER2 positive (HER2 + -only) breast cancer patients.

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Methods: We developed a mouse monoclonal anti-ESE-1 antibody, verified the specificity, epitope, and used immunohistochemical staining to assess ESE-1 expression in IBC approved records of 957 breast tumor samples.

Nathaniel

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