Utilizing ELISA to monitor protein-protein interaction.

Enzyme-linked immunosorbent assay (ELISA) is a commonly used method in analyzing biomolecular interactions. As a rapid, specific, and easy-to-operate method, ELISA has been used as a research tool as well as a widely adopted diagnostic method in clinical settings and for microbial testing in various industries. Inhibition ELISA is a one-site binding analysis method, which can monitor protein-protein interactions in solution as opposed to more commonly used sandwich ELISA in which the analyte capture step is required on a solid surface either through specific capture or through passive adsorption. Here, we introduce inhibition ELISA procedures, using a recombinant viral protein as an example, with emphasis on how inhibition ELISA could be used to probe subtle protein conformational changes in solution impacting protein-protein binding affinity. Inhibition ELISA is used to probe one binding site at a time for binding partners in solution with unrestricted conformation. The assay can be performed in a quantitative manner with a serially diluted analyte in solution for solution antigenicity or binding activity assessment.

Robust manufacturing and comprehensive characterization of recombinant hepatitis E virus-like particles in Hecolin(®).

The hepatitis E virus (HEV) vaccine, Hecolin(®), was licensed in China for the prevention of HEV infection and HEV-related diseases with demonstrated safety and efficacy [1,2]. The vaccine is composed of a truncated HEV capsid protein, p239, as the sole antigen encoded by open reading frame 2 and produced using Escherichia coli platform. The production of this virus-like particle (VLP) form of the antigen was successfully scaled up 50-fold from a bench scale to a manufacturing scale. Product consistency was demonstrated using a combination of biophysical, biochemical and immunochemical methods, which revealed comparable antigen characteristics among different batches. Particle size of the nanometer scale particulate antigen and presence of key epitopes on the particle surface are two prerequisites for an efficacious VLP-based vaccine. The particle size was monitored by several different methods, which showed diameters between 20 and 30nm for the p239 particles. The thermal stability and aggregation propensity of the antigen were assessed using differential scanning calorimetry and cloud point assay under heat stress conditions. Key epitopes on the particulate antigen were analyzed using a panel of murine anti-HEV monoclonal antibodies (mAbs). The immuno reactivity to the mAbs among the different antigen lots was highly consistent when analyzed quantitatively using a surface plasmon resonance technique. Using a sandwich ELISA to probe the integrity of two different epitopes in the antigen, the specific antigenicity of multiple batches was assessed to demonstrate consistency in these critical product attributes. Overall, our findings showed that the antigen production process is robust and scalable during the manufacturing of Hecolin(®).

Toward the development of monoclonal antibody-based assays to probe virion-like epitopes in hepatitis B vaccine antigen.

Prophylactic vaccines against hepatitis B Virus (HBV) infection were produced in different expression systems under different processing conditions. Since the recombinant HBV surface antigen (HBsAg) in these vaccines is a cysteine-rich protein with 14 cysteines among a total of 226 amino acids, the epitopes are dependent on the formation of intra- and intermolecular disulfide bonds. A panel of 22 monoclonal antibodies (mAbs) were developed and evaluated with respect to their sensitivity to disulfide reduction treatment of recombinant HBsAg. Not surprisingly, different mAbs showed different degree of sensitivity to controlled HBsAg disulfide reduction. With a view to exploring the functionality of anti-HBsAg mAbs to be used in HBsAg quality analysis, in vitro neutralization activity for the mAbs was assessed. One of the mAbs tested, 5F11, which showed high sensitivity to the disulfide integrity in HBsAg, was shown also to be highly effective in neutralizing HBV in vitro. Conversely, 42B6, while exhibiting similar neutralization activity, showed comparable binding HBsAg with or without reduction treatment. Based on these mAb characteristics, a sandwich ELISA with 42B6 being the capture Ab and detection Ab was developed to quantify HBsAg (like a "mass" assay) during antigen bioprocessing or in vaccine products. In parallel, when 5F11 was used as the detection Ab (with the same capture Ab), the assay can be used to probe disulfide-dependent and virion-like epitopes in intermediates or final products of hepatitis B vaccine, serving as a surrogate marker for vaccine efficacy to elicit neutralizing antibodies. This approach enables the comparative epitope specific antigenicity analysis of HBsAg antigen preparations from different sources.