Journal of immunological methods最新文献

Ancylostoma species are parasitic nematodes that release a multitude of proteins to manipulate host immune responses to facilitate their survival. Among the released proteins, Ancylostoma-secreted protein 5 (ASP5) plays a pivotal role in mediating host-parasite interactions, making it a promising target for interventions against canine hookworm infections caused by Ancylostoma species. Antibody phage display, a widely used method for generating human monoclonal antibodies was employed in this study. A bacterial expression system was used to produce ASP5 for biopanning. A single-chain fragment variable (scFv) monoclonal antibody against ASP5 was generated from the naïve Human AntibodY LibrarY (HAYLY). The resulting scFv antibody was characterized to elucidate its antigen-binding properties. The identified monoclonal antibody showed good specificity and binding characteristics which highlighting its potential for diagnostic applications in combating hookworm infections.

We have shown in previous studies that naive CD4⁺ T cells isolated from human peripheral blood are induced to proliferate by CD4^negCD11c⁺CD14^negCD16^neg dendritic cells presenting the superantigen SEE. Since this population is very poorly expressed in blood, we tried to find other antigen presenting cells (APCs) to induce this proliferation. The aim of the previous studies was to investigate the regulation of T cell proliferation in pediatric monogenic autoimmune diseases and the regulation of this proliferation by regulatory T cells (TREGs). Since the blood samples from pediatric patients were very small, it was important to study other APCs that are more commonly present in the blood. In this study we tested different APCs isolated from controls, CD19⁺ B cells, CD11c⁺CD14⁺ and CD11c⁺CD14^neg monocytes, CD11c⁺CD14^negCD16⁺ and CD16^neg dendritic cells. The different T cell populations, naive effector T cells and regulatory T cells were separated simultaneously from the same sample. We show in these studies that CD19⁺ B cells, CD14^neg and more specifically CD14^negCD16⁺, are also able to induce T cell proliferation as previously described with CD14^negCD16^neg DCs, but under different conditions. No proliferation was induced with CD14⁺ monocytes. However, these three APCs are less potent than CD16^neg and inhibition by TREG is more difficult to detect. In addition, when we test the role of CTLA-4 in the regulation of TEFF proliferation, we observe that for some APCs, the inhibition by CTLA-4 was quite different. No inhibition was observed with CD19⁺ B cells in contrast to CD11c⁺CD14^negCD16⁺ and CD11c⁺CD14^negCD16^neg .

Allergen-specific antibodies (Abs), IgE, and IgG4 increase during the early phase of oral immunotherapy (OIT) of allergen food in patients; subsequently, IgE levels decrease and specific IgG4 levels increase after successful OIT treatment. The detailed profile of these Abs during OIT remains largely unclear. We developed a diagnostic tool to assess the OIT efficacy and extent of responsiveness based on a profiling method by identifying epitopes recognized by the Ab classes of IgE or IgG4. A peptide microarray followed by microplate analysis using synthetic peptides was used to identify 14 epitopes widely recognized by IgE and/or IgG4 in the serum samples of patients with OIT among the amino acid sequences of five major cow's milk allergens. The set of defined 14 epitopes clarified different epitope profiles of allergen-specific IgE and IgG4 in each patient's serum samples. Moreover, the total signal of Abs recognizing all 14 epitopes was equal to the sum of all individual epitope-specific Abs. It was further observed that the quantitative value of IgE concentrations of 14 epitopes-ALL correlated with the ImmunoCAP IgE value. These findings strongly imply that the quantity of IgE and IgG4 recognizing epitopes-ALL may easily be used to measure allergy severity. To investigate this potential, we developed an immunochromatographic method that can detect IgE and IgG4 levels in patient samples. This study clearly demonstrated the usefulness of the defined 14 epitopes and their mixture, "epitopes-ALL," and that the simple and reliable methods of immunochromatography and microplate analyses demonstrating the epitope profile of allergen-specific Abs are applicable for diagnostic use at multiple disease stages and the OIT-treatment course in patients with cow's milk allergy.