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High‐density immobilization of an antibody fragment to a carboxymethylated dextran‐linked biosensor surface
Author(s) -
Howell Steve,
Kenmore Mike,
Kirkland Mark,
Badley R. Andy
Publication year - 1998
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/(sici)1099-1352(199812)11:1/6<200::aid-jmr423>3.0.co;2-7
Subject(s) - dextran , biosensor , chemistry , amine gas treating , covalent bond , conjugated system , molecule , antibody , chromatography , biochemistry , organic chemistry , polymer , biology , immunology
There are numerous chemical methods published that enable protein coupling to carboxymethyl (CM) dextran. Here we have taken traditional amine coupling using N ‐hydroxysuccinimide (NHS) and N ′‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC) and coupled an antibody fragment (scFv) to CM dextran at a very high density. Using an upgraded BIAlite™ from Biacore AB, more than 7000 RU of scFv was coupled to a CM dextran biosensor chip. In addition, scanning electron microscopy was performed on CM dextran biosensor chips following amine coupling of 30 nm gold anti‐IgG particles. This showed that amine coupling was uniform across the biosensor chip surface. Calculations show that 7620 RU of an scFv coupled to such a surface results in a mean distance between binding sites of 8.8 nm. This equates to a packing volume of approximately 20% of the available space occupied by the antibody fragment. Comparisons made with densities of covalently coupled IgG show that a greater number of antibody fragment molecules can be coupled per unit area. This is most likely due to the smaller size of an antibody fragment (scFv), which has a volume of less than 20% of an IgG molecule. The significance of these findings is discussed. Copyright © 1998 John Wiley & Sons, Ltd.