Reference measurement procedure development for C-reactive protein in human serum.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Accurate quantification of human C-reactive protein (CRP) is essential for clinical diagnostics and reference material certification.
• Existing methods may face challenges with bias in purification and digestion steps, particularly for low-abundance proteins.
• A lack of suitable internal standards complicates precise CRP measurement.

Purpose of the Study:

• To develop and validate a reference measurement procedure for quantifying human C-reactive protein (CRP) in serum.
• To address the challenge of measurement bias in affinity purification and enzymatic digestion steps.
• To enable the accurate certification of reference materials for CRP in clinically relevant concentrations.

Main Methods:

• Development of a reference measurement procedure combining affinity purification with anti-CRP monoclonal antibodies and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• Utilized the method of standard addition to overcome the absence of an internal standard for CRP quantification.
• Employed trypsin digestion of affinity-purified CRP followed by LC-MS/MS analysis of specific tryptic peptides.

Main Results:

• The developed method successfully quantified CRP in serum using affinity purification and LC-MS/MS.
• The standard addition approach effectively mitigated potential measurement bias.
• Measurements of CRP in control materials showed values comparable to expected concentrations, validated by external calibration.

Conclusions:

• Affinity purification coupled with LC-MS/MS is a feasible approach for developing reference measurement procedures for low-abundance serum proteins like CRP.
• The standard addition method provides a robust solution for quantification in the absence of internal standards.
• This work supports the accurate certification of reference materials for CRP, enhancing clinical diagnostic reliability.