Surface analysis of pure and complex mucin coatings on a real-type substrate using individual and combined mBCA, ELLA, and ELISA.
In the past, we introduced the idea of using mucin coatings to improve biomaterials performance. Here, we evaluate non-radioactive methods for the analysis of pure and human host protein-containing (complex) mucin coatings on a real-type substrate (Thermanox). A common protein quantification assay (mBCA) was combined with mass-calibrated, enzyme-amplified assays based on lectin (ELLA) and antibody (ELISA) recognition, to determine the total and specific amounts of surface-associated proteins. Model studies showed the mBCA assay to be of limited use at low mass loads, and steric effects to influence the ELLA at high surface layer densities. Non-specific responses due to substrate interaction were low for the ELLA and ELISAs. Cross-reactions were observed during ELLA analysis of analytes sharing high degree of O-glycosylation. Combined mBCA-ELLA-ELISA analysis suggested that mucin desorption was low upon protein addition and that low concentrations of ELISA-determined protein for the complex coatings could be explained in terms of low accessibility of proteins to the bulk environment. Specifically, a methodology is presented for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating. Finally, X-ray photoelectron spectroscopy and infrared reflectance spectroscopy combined with multivariate data analysis were proven useful in the evaluation of mucin-based coatings.
Sandberg
Tomas
T
Department of Physical and Analytical Chemistry, Division of Surface Biotechnology, BMC, Uppsala University, SE-751 23 Uppsala, Sweden.
Mellin
Lisa
L
Gelius
Ulrik
U
Caldwell
Karin D
KD
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D009077:Mucins / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D013499:Surface Properties
D037102:Lectins; D009077:Mucins; D011506:Proteins