Production and characterization of antibodies against crosslinked gelatin nanoparticles and first steps toward developing an ELISA screening kit.

Nanotechnologies are finding a growing range of applications in the food sector. Nanoparticles are used notably to add vitamins and other nutrients to foods and beverages without affecting taste and color. They are also used to develop new tastes, preserve food texture, control the release of flavors, improve the bioavailability of compounds such as antioxidants and vitamins, and monitor freshness with nanosensors. Crosslinked gelatin nanoparticles are a component of nano-sized carriers for nutrient and supplement delivery in foods and related products. This paper describes the production and characterization of polyclonal antibodies against gelatin nanoparticles. Two immunization schemes were investigated: subcutaneous injection with and without a first intravenous injection. Two enzyme-linked immunosorbent assay formats were used to characterize the antibodies: an inhibition format with an antigen-coated plate for detection of the immune response and a sandwich format for development of the method. The antibodies showed good sensitivity with an IC50 equal to 0.11 ng mL(-1) using indirect ELISA format and a good specificity for the nanomaterials, without significant cross-reactivity against native gelatin. The limit of detection was determined-0.42, 0.27, 0.26, and 0.24 µg mL(-1) for apple, orange juice, milk, and soft drink matrices, respectively. ELISA technology offers rapid, low-cost assays for screening foods, feeds, and beverages. We have studied a prototype ELISA for detection of gelatin-based nanocarrier systems. Fruit juices, milk, and a soft drink were the matrices selected for assay development.

Thermodynamic benchmark study using Biacore technology.

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.

Deglycosylation of anti-beta amyloid antibodies inhibits microglia activation in BV-2 cellular model.

Immunotherapy has become a strategy for treatment of Alzheimer's disease, by inducing antibody response to amyloid-beta peptide (AbetaP) or by passive administration of anti-AbetaP antibodies. Clearance of amyloid plaques involves interaction of immunoglobulin Fc receptor (FcR)-expressing microglia and antibodyopsonized Abeta deposits, stimulating phagocytosis but may promote neuroinflammation. Carbohydrate moiety of Fc of the immunoglobulin G molecule plays a significant role in modulating binding to FcR and its effector functions. Here, we enzymatically removed Fc glycan from monoclonal antibody 196 raised against AbetaP Antigen binding ability and in vitro stability of deglycosylated antibody were unaffected by deglycosylation. Moreover, the deglycosylated antibody exhibits low affinity to FcR on microglial BV-2 cells and has limited ability to mediate microglial chemotaxis and antibodydependent cytotoxicity compared to native antibody. These data suggest that deglycosylation of anti-Abeta antibodies before in vivo administration might prevent microglial overactivation, thus reducing the risk of neuroinflammatory response during passive immunization.