Avoiding nonspecific interactions in studies of the plasma proteome: practical solutions to prevention of nonspecific interactions for label-free detection of low-abundance plasma proteins.
The molecular constitution of blood can be highly representative of the physiological state of an individual and offers an ideal target for studies of biomarkers. High-abundance plasma proteins, particularly albumin, dominate the plasma proteome, but it is the low-abundance proteins (such as cytokines) that are commonly associated with many pathophysiological states. Several detection strategies, and particularly those that involve label-free detection, are available for low-abundance protein detection in plasma, but all can be severely compromised by the high-abundance of serum albumin. In the present study, we examine the effect of albumin interference on accurate label-free detection by protein microarrays. Albumin was found to disrupt specific antigen-antibody binding interactions of low-abundance proteins. In clinical analysis, where it is imperative to preserve the integrity of samples, depletion of albumin may further undermine quantitative measurements. We have optimized procedures that permit accurate analysis to be undertaken without the need for prior treatment of samples. The emphasis is placed on disrupting nonspecific interactions including both electrostatic (i.e., Colulombic) and electrodynamic (hydrophobic and other nonpolar based) interactions. These protocols appear to be generic with potential applications in several areas of analytical biotechnology.
Richens
Joanna L
JL
Cell Biophysics Group, School of Biology, University of Nottingham, Nottingham, England, NG7 2RD, UK.
Lunt
Elizabeth A M
EA
Sanger
Daniel
D
McKenzie
Graeme
G
O'Shea
Paul
P
D001798:Blood Proteins / Q000032:analysis*; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D010949:Plasma / Q000737:chemistry*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods; D011485:Protein Binding; D020543:Proteome / Q000032:analysis*; D012680:Sensitivity and Specificity; D013861:Thiocyanates / Q000737:chemistry
D001798:Blood Proteins; D020543:Proteome; D013861:Thiocyanates