Calibration-free concentration analysis of protein biomarkers in human serum using surface plasmon resonance

• Published in: Talanta (Oxford) Vol. 144; pp. 801 - 808
• Main Authors: Grover Shah, Veenita, Ray, Sandipan, Karlsson, Robert, Srivastava, Sanjeeva
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2015
• Subjects: Active concentration; amyloid; antibodies; beta 2-Microglobulin - blood; biomarkers; Biomarkers - blood; Blood Chemical Analysis - methods; blood serum; Calibration; Calibration-free concentration analysis; diffusivity; enzyme-linked immunosorbent assay; Humans; Label-free detection; monitoring; patients; proteomics; Protozoan Infections - blood; Serum; Serum amyloid A; Serum Amyloid A Protein - analysis; Surface plasmon resonance; Surface Plasmon Resonance - methods; therapeutics; Serum amyloid A; Serum; Surface plasmon resonance; Label-free detection; Active concentration; Calibration-free concentration analysis
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2015.06.074

In complex biological samples such as serum, determination of specific and active concentration of target proteins, independent of a calibration curve, will be valuable in many applications. Calibration-free concentration analysis (CFCA) is a surface plasmon resonance (SPR)-based label-free approach, which calculates active concentration of proteins using their known diffusion coefficient and observed changes in binding rates at different flow rates under diffusion-limited conditions. Here, for the first time we demonstrate the application of CFCA for determining protein biomarker abundance, specifically serum amyloid A (SAA), directly in the serum samples of patients suffering from different infectious and non-infectious diseases. The assay involves preparation of appropriate reaction surfaces by immobilizing antibodies on CM5 chips via amine coupling followed by serum sample preparation and injection over activated and reference surfaces at flow-rates of 5 and 100μL/min. The system was validated in healthy and diseased (infectious and non-infectious) serum samples by quantifying two different proteins: β2-microglobulin (β2M) and SAA. All concentration assays were performed for nearly 100 serum samples, which showed reliable quantification in unattended runs with high accuracy and sensitivity. The method could detect the serum β2M to as low as 13ng/mL in 1000-fold serum dilution, indicating the possible utility of this approach to detect low abundance protein biomarkers in body fluids. Applying the CFCA approach, significant difference in serum abundance of SAA was identified in diseased subjects as compared to the healthy controls, which correlated well with our previous proteomic investigations. Estimation of SAA concentration for a subset of healthy and diseased sera was also performed using ELISA, and the trend was observed to be similar in both SPR assay and ELISA. The reproducibility of CFCA in various serum samples made the interpretation of assay simple and reliable. This study illustrates a significant step forward in rapid monitoring of several protein markers in serum samples, with utility in biomarker validation and other therapeutic applications. [Display omitted] •Calibration-free quantification is presented for serum protein biomarker abundance.•Rapid approach for serum protein analysis without entailing protein purification.•Method was validated for two proteins from serum of healthy and diseased patients.•Assay could detect as low as 13ng/mL of serum protein in 1000-fold serum dilution.•Method has utility in clinical diagnostics, therapeutic and biomarker studies.