Development of a Direct Alhydrogel Formulation Immunoassay (DAFIA)

Abstract

Alhydrogel® (aluminum hydroxide) is a widely used adjuvant in the US. Regulatory authorities require that vaccines be tested to determine the antigen content in the final vaccine product. The level of formulated antigen is currently determined in our laboratory by the o-Phthalaldehyde (OPA) fluorescent protein assay, and antigen identity and integrity are determined by Western blot and SDS-PAGE. However, OPA assay is non-specific and only limited to detection of total protein content, and it is often not sensitive enough to detect antigens in low dose formulations. Furthermore, antigens used in identity and integrity tests must be extracted from vaccines using an extraction procedure which is time-consuming and may not completely recover antigens for analysis or may alter the structures of antigens during extraction. The present study developed a Direct Alum Formulation Immunoassay (DAFIA) which was designed to directly (without antigen extraction), accurately, and sensitively determine the antigen content, identity and integrity on alum. The AMA1-C1/Alhydrogel formulation was used as a model vaccine in assay development and validation. The results showed that the DAFIA is highly antigen-specific, accurate (87-100%), sensitive (0.16 µg/ml), reproducible, and simple with a linear detection range of 0.16-10 µg/ml. These results demonstrate that DAFIA is an excellent assay to determine antigen content, identity and integrity of antigens bound to alum and may be used in routine vaccine quality control for testing antigens in Alhydrogel-based vaccines.

Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

“…To further explore structural changes/alterations of a specific region(s) or domains of rPA molecules adsorbed to Alhydrogel upon storage, we used a direct Alhydrogel formulation immunoassay (39). In this assay, an anti-rPA polyclonal antibody serum and several MAbs that recognize epitopes in different domains of rPA were used to directly probe the structure of rPA on the aluminum adjuvant.…”

“…A direct Alhydrogel formulation immunoassay, previously described by Zhu et al (39), was performed with freshly prepared or stored rPA-Alhydrogel formulations, with slight modifications, in an ELISA format. Briefly, the different rPAAlhydrogel formulations (100 l/well) were added to black, opaque, 96-well U-bottom plates (Corning Inc., Corning, NY).…”

Accelerated and long term stability study of Pfs25-EPA conjugates adjuvanted with Alhydrogel®

“…Pfs25-EPA content in the non-GMP and clinical grade Pfs25-EPA/Alhydrogel was determined by Direct Alhydrogel Formulation Immunoassay (DAFIA) (16) with anti-Pfs25 monoclonal antibody 4B7, which recognizes a partially concealed linear epitope of Pfs25 (14). Briefly, Alhydrogel formulation composed with standards or test samples (200 μl/well) were added to black, opaque, 96-well U-bottom plates and washed 3 times with phosphate-buffered saline (PBS, pH 7.4) by centrifugation at 1,000 g for 4 min.…”

Pharmaceutical Analysis and Quality Control of Vaccines

“…From a characterization point of view, we can foresee that assays based on the direct alhydrogel formulation immunoassay (DAFIA) or fl ow cytometry (Zhu et al 2009 ;Ugozzoli et al 2011 ) developed to exploit monoclonal antibodies may be of more immediate use than other techniques to shed light on the antigenic elements that do best stimulate antibody-mediated protection.…”