HighlightsDifferent ELISA kits for measuring gluten yield different responses.The differences between pairs of kits can vary by a factor of 20.The current measurement system cannot fully support legislative gluten-free claims.Comparability of measurements is limited by poor correlation between kit results.Lack of correlation creates bias that cannot be removed using reference materials.
BACKGROUND
Standardization of clinical measurements of C-reactive protein (CRP) is based on the availability of reference materials. The serum protein reference material ERM-DA470 was used as a calibrant for various commercially available immunoassays but has now been exhausted. The recently released ERM-DA470k/IFCC was intended to fully replace ERM-DA470. However, the new material was not suited for the certification of CRP because of a bias introduced by the lyophilization process that caused loss of about 20% of CRP measurable by routine immunoassays, compared with the nonlyophilized material that was stored in a liquid frozen state.
METHODS
We investigated the physicochemical state of CRP in a set of 4 lyophilized and 2 nonlyophilized serum-based CRP-containing materials by semi-native gel electrophoresis, Western blotting, and gel filtration.
RESULTS
We detected a monomeric form of CRP (mCRP) in lyophilized materials at a concentration significantly higher than seen in the materials not subjected to lyophilization. Different reconstitution protocols led to variations of the monomeric CRP fraction found in reconstituted, previously lyophilized material.
CONCLUSIONS
Most of the 20% loss in measured CRP after lyophilization of the material can be accounted for by the dissociation of natively pentameric CRP into subunits. The observed dissociation results from lyophilization and subsequent reconstitution of the material at insufficient concentration levels of calcium ions. In the presence of various protein forms, differences in antibody specificity and reactivity between immunoassays and alterations of stoichiometry of antigen–antibody interactions can contribute to the divergence of the measured values.
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