Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of “superiority” of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development

A Cyclic Undecamer Peptide Mimics a Turn in Folded Alzheimer Amyloid β and Elicits Antibodies against Oligomeric and Fibrillar Amyloid and Plaques

The 39- to 42-residue amyloid β (Aβ) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aβ immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aβ, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aβ-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aβ(22–28)-YNGK′ elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound—although more weakly—to Aβ(1–42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aβ deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice

Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Immunoassays are used for routine potency assessment of several vaccines, in some cases having been specifically developed as alternatives to in vivo potency tests. These methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against diphtheria with a view to select antibodies that can be used for development of an in vitro potency immunoassay for diphtheria vaccines. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined by a cell-based toxin neutralisation test and diphtheria toxin-domain recognition was determined by Western blotting. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of “superiority” of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay&nbsp;development.</p

Antigenic fingerprinting of diphtheria toxoid adsorbed to aluminium phosphate

Drug Delivery Technolog

Trypsin diminishes the rat potency of polio serotype 3

Drug Delivery Technolog

Immunochemical and biophysical characterization of inactivated Sabin poliovirus products: insights into rapid quality assessment tools.

The aim of this study was to demonstrate the strength of combining immunochemical and biophysical analysis tools for assessing the quality of Sabin inactivated poliovirus vaccine (Sabin-IPV) bulk products. We assessed Sabin-IPV serotypes 1, 2 and 3 from six different manufacturers and evaluated their comparability through biosensor analysis and biophysical characterization methods, including tryptophan fluorescence and asymmetrical flow field-flow fractionation - multi-angle light scattering analysis. These methods enabled us to assess antigenic as well as conformational and structural integrity profiles, respectively. Based on Sabin-IPV samples that were subjected to accelerated storage conditions, we revealed that existing immunochemical methods exhibit remarkably similar trends to the results obtained by the biophysical characterization methods. While the results underpin that the comparability of Sabin-IPV bulk products of different manufacturers is weak, information about their quality can rapidly be obtained by using both immunochemical and biophysical methods. Furthermore, the study highlights that quality assessment of Sabin-IPV can be obtained through biophysical techniques can complement the assessments performed with monoclonal antibodies and suggests that similar techniques could be employed to characterize other enteroviruses.Drug Delivery Technolog