Anti-CD40LG monoclonal antibody, clone 35-42 [FITC]

Background: Platelets can activate B cells and stimulate them for the production of antibodies. Since platelet microparticles (PMPs) originate from platelets, they may have the same virtue. In the present study, the effect of PMPs was investigated on the production of human leukocyte antigen (HLA)-specific antibody from B cells in vitro. Materials and Methods: In this experimental study, HLA-DR antigen was solubilized from the immortalized B lymphocytes (Daudi cell line) and purified using the affinity chromatography. Antigen properties were determined by the ELISA technique. PMPs were isolated from platelet concentrate bags by centrifugation. Fresh blood products were prepared from the Innovation Center of Iranian Blood Transfusion Organization (IBTO) and B lymphocytes were purified by the MACS method. B cells were exposed with PMPs and HLA-DR antigens in the culture medium. On the third day of culture, the culture supernatant was examined in terms of antibody production using the ELISA test. The results were analyzed using paired sample T-test and P-value <0.05 was considered statistically significant. Results: The specificity of the purified HLA-DR antigen was confirmed using the anti-HLA-DR antibody and ELISA technique in the presence of appropriate controls. The results showed that PMPs could stimulate the production of antibodies from B cells. The difference between the case and control was significant (P-value=0.001). Although total immunoglobulin (IgG) was higher in HLA-DR-treated wells, HLA-DR-specific antibodies were not identified by ELISA technique. Conclusion: PMPs have the capability to induce IgG antibodies from B cells. In order to ensure the production of specific antibodies, further testing is required with high sensitivity.

Analytical techniques are essential in the process of standardizing and validating vaccines. In this study we described a methodology to establish an ELISA sandwich for the quantification of a new vaccine against avian influenza virus H5N1 based on the main antigenic determinant of the virus, the extracellular domain of the glycoprotein hemagglutinin (HA), fused to the extracellular domain of the chicken CD154 glycoprotein (HACD). The chimerical proteins HA and HACD were produced in SiHa cells and the experiments were performed by using three monoclonal antibodies (MAb-HA1, MAb-HA2 and MAb-HA3), alone or conjugated to horseradish peroxidase (HRP-HA1, HRP-HA2 and HRP-HA3). The hemagglutination inhibition assay was carried out with a negative and a positive H5N2 reference serum, together with the antigen H5N1 A/Mallard/Italy/3401/05, all purchased from the "Istituto Zooprofilattico delle Venezie", Italy. After demonstrating the similar recognition pattern between the HA and the HACD proteins, the MAb-HA2 at a concentration of 2,5 mu g/mL was selected as the capture antibody and the HRP-HA3 at a dilution of 1/20000 was selected as the detection antibody due to their optimal values of optical density at these conditions. The best dynamic range of the standard curve using the protein HACD was achieved at concentrations from 100 to 1,56 ng/mL. There were no significant differences when five batches of HACD were quantified by the ELISA sandwich and the bicinchoninic acid method linked to densitometry. In conclusion, the final parameters for the quantification of the chimeric protein HACD using an ELISA sandwich were described, which could contribute to develop a large-scale process for the final vaccine production.