Analysis of the Physicochemical State of C-Reactive Protein in Different Preparations Including 2 Certified Reference Materials

Abstract

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.

Characterization of the New Serum Protein Reference Material ERM-DA470k/IFCC: Value Assignment by Immunoassay

“…It was later found that lyophilization can induce changes in the oligomeric structure of CRP (16 ). Therefore, CRP was not certified in ERM-DA470/IFCC, and a separate liquid frozen material was produced (ERM-DA472/IFCC) and found to be suitable.…”

Influence of freeze-drying on the recovery of the tumour invasion markers uPA and PAI-1 from prostate cancer resections

“…8 However, the recovery of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and C-reactive protein has been shown to be lower and in the region of 80%. 9,10 Differences in the recovery between target molecules may be attributable to differences in the handling of samples before freeze-drying, in particular temperature which promotes protein denaturation and irreversible adhesions. It is, therefore, imperative to ensure that sample processing is carried out in the mildest possible conditions.…”

Regulated conformation changes in C-reactive protein orchestrate its role in atherogenesis

“…Subsequent studies confirmed the enhanced interactions of mCRP with modified lipoproteins [61] and complement [62][63][64][65][66][67] and further revealed the important contributions of mCRP in promoting non-inflammatory clearance of apoptotic or necrotic cells by controlling complement activation on the cell surface through balanced recruitment of C1q, Factor H and C4bp [64][65][66]. Because inappropriate handling of CRP, including lyophilization [68], storage in the absence of calcium [69] and immobilization onto microtiter wells [60,70], leads to the disruption of the pentameric structure, the reported properties of CRP suffering these technical pitfalls should be re-evaluated with Factor H interaction as a prominent example [60,64,71].…”