Microparticle-enhanced nephelometric immunoassay for human C-reactive protein

Area of Science:

• Biochemistry
• Immunology
• Materials Science

Background:

• C-reactive protein (CRP) is a key biomarker for inflammation.
• Accurate and sensitive CRP quantification is crucial for clinical diagnostics.
• Existing immunoassay methods can be complex and require sample pretreatment.

Purpose of the Study:

• To develop a novel microparticle-enhanced nephelometric immunoassay for human CRP.
• To utilize polyfunctional hydrophilic microspheres for CRP detection.
• To establish a sensitive, reliable, and accurate assay for CRP quantitation.

Main Methods:

• Copolymerization of acrylic monomers to create 125-nm hydrophilic microspheres.
• Covalent binding of purified CRP to the microspheres to form a conjugate.
• Microparticle-enhanced nephelometric immunoassay using the CRP conjugate and anti-CRP antiserum.
• Measurement of light scattering by aggregates formed during immunoagglutination.
• Quantification of CRP via sensitive inhibition of agglutination by free CRP.

Main Results:

• The assay demonstrated high sensitivity, detecting CRP at 6 ng/ml.
• CRP could be quantified in highly diluted serum samples (1/500-1/2,000) without interference or pretreatment.
• The assay exhibited excellent reliability (CVs 1.3%-9.3%) and accuracy (mean recovery 104%).
• High correlation (r ≥ 0.97) was observed with accepted analytical methods over a wide concentration range.
• The inhibition mode prevented errors in the antigen excess zone and ensured accuracy at high CRP concentrations.

Conclusions:

• The developed microsphere-based immunoassay provides a sensitive, reliable, and accurate method for human CRP quantification.
• This novel assay simplifies sample handling by eliminating the need for washing or phase separation.
• The assay's robustness, particularly in the presence of high antigen concentrations, makes it suitable for routine clinical diagnostics.