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

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Title Calibration-free concentration analysis of protein biomarkers in human serum using surface plasmon resonance
 
Creator SHAH, VG
RAY, S
KARLSSON, R
SRIVASTAVA, S
 
Subject OPTICAL BIOSENSOR IMMUNOASSAY
MASS-TRANSPORT LIMITATION
HIGH-THROUGHPUT
TECHNOLOGY
QUANTIFICATION
CANCER
ELISA
MILK
STANDARDIZATION
IMMUNOSENSOR
Surface plasmon resonance
Calibration-free concentration analysis
Label-free detection
Active concentration
Serum
Serum amyloid A
 
Description 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 noninfectious 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 100 mu L/min. The system was validated in healthy and diseased (infectious and non-infectious) serum samples by quantifying two different proteins: beta 2-microglobulin (beta 2M) 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 beta 2M to as low as 13 ng/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. (C) 2015 Elsevier B.V. All rights reserved.
 
Publisher ELSEVIER SCIENCE BV
 
Date 2016-01-15T05:49:29Z
2016-01-15T05:49:29Z
2015
 
Type Article
 
Identifier TALANTA, 144,801-808
0039-9140
1873-3573
http://dx.doi.org/10.1016/j.talanta.2015.06.074
http://dspace.library.iitb.ac.in/jspui/handle/100/17856
 
Language en