Troubleshooting immunoassays for adjuvanted vaccine products
The safety record, affordability, and compatibility of aluminum to many antigens makes them a primary choice of adjuvant. Aluminum adjuvant compounds (such as aluminum hydroxide (Al(OH)3) and aluminum phosphate (AlPO4)) help maintain the physical and chemical characteristics of antigens and increase their repository effect to the immune cells for long duration which maximizes the immunogenicity of the antigens.
Adsorption of antigens onto preformed aluminum gel is a widely used method for preparing adjuvanted vaccines. However, certain physical and chemical characteristics can impact the adsorption efficiency of antigens into aluminum gel; these include size, isoelectric point and adsorption capacity of the aluminum gel, aluminum to phosphate ratio, mixing speed and pH-values.
Antigen identity and integrity, which are determined by Western blot and SDS-PAGE, are among the tests required by regulatory authorities for final vaccine product. However, these assays may at times not be able to detect certain antigen because they cannot pass through the SDS-PAGE gel when attached to Alhydrogel (Fig. 1). To overcome such limitations samples need to be pre-treated with sodium citrate to desorb the antigens from the aluminum gel before running on SDS-PAGE and Western blot.
Alternatively, concentration of antigens can be determined by o-Phthalaldehyde (OPA) fluorescent protein assay and/or enzyme linked immunosorbent assay (ELISA). While OPA assay is non-specific and can only detect total protein content; ELISA is specific to each individual antigen therefore a more sensitive detection method.
When using ELISA pre-treatment of antigen samples is not necessary because Alhydrogel does not interfere with ELISA-based detection. ELISAs run with and without Alhydrogel result in standard curves that look very similar demonstrating Alhydrogel does not interfere in the ELISA assay (Fig. 2). This is due to the phosphate-based buffers used in ELISA which help to desorb the antigens. The phosphate tends to form a complex (insoluble precipitates or chelates) with metal ions like aluminum whereas Tris based buffers don’t complex with aluminum of Alhydrogel.
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