title: Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein.
abstract: Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below ~150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20-150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between ~150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases.
authors: Department of Chemical and Biomolecular Engineering, Polytechnic Institute of New York University, 6 Metrotech Center, Brooklyn, NY 11201, USA.
authors: Han
authors: H
authors: Yuan
authors: Mihir
authors: M
authors: Tank
authors: Abeer
authors: A
authors: Alsofyani
authors: Naman
authors: N
authors: Shah
authors: Nishant
authors: N
authors: Talati
authors: Jaclyn C
authors: JC
authors: LoBello
authors: Jin Ryoun
authors: JR
authors: Kim
authors: Yoji
authors: Y
authors: Oonuki
authors: Carol A
authors: CA
authors: de la Motte
authors: Mary K
authors: MK
authors: Cowman
mesh_terms: D019921:Biotinylation; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry*; D006820:Hyaluronic Acid / Q000737:chemistry*; D015151:Immunoblotting; D008970:Molecular Weight
chemical_list: D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid
keywords: ELISA; blotting; detection; hyaluronan; quantification
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