Analysis of the physicochemical state of C-reactive protein in different preparations including 2 certified 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.
Rzychon
Malgorzata
M
European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium.
Zegers
Ingrid
I
Schimmel
Heinz
H
D015153:Blotting, Western; D002097:C-Reactive Protein / Q000737:chemistry* / Q000592:standards; D002122:Calcium Chloride / Q000737:chemistry; D002138:Calibration; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005612:Freeze Drying; D006801:Humans; D012015:Reference Standards
D002097:C-Reactive Protein; D002122:Calcium Chloride