Novel and sensitive ELISA for the rapid quantification of recombinant p64K protein.

The antigenic P64k protein from the pathogenic bacterium Neisseria meningitidis has been used as an immunological carrier in several conjugated vaccines. The aim of this report was to develop and validate a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of recombinant p64k protein, to perform both manufacturing process and identification in different vaccine preparations. Validation studies were performed according to the guidelines of the International Conference of Harmonization (ICH). The reference curve showed to be precise and accurate over the entire linear range of 1.25 and 20ng/mL with a limit of quantification validated to 1.25ng/mL. The intra- and inter-assay coefficient of variation ranged from 0.35 to 6.65% and 4.70 to 10.63%, respectively. The ANOVA test used in the specificity/interference study revealed parallelism among curves (p>0.1), which indicates the lack of interference in the working range. Recovery obtained from the accuracy test, using three concentration levels, varied between 94 and 111%, confirming the assay's reliability. The short-term study shown the P64k is stable to −20°C up to 1-week. This ELISA was fully used to assess its manufacturing process and molecular interaction issues in several vaccine preparations. Thus, this immunoassay could be an excellent analytical choice to characterize the quality of that recombinant protein in several contexts as manufacturing process and molecular conjugates.