High-density immobilization of an antibody fragment to a carboxymethylated dextran-linked biosensor surface
✍ Scribed by Steve Howell; Mike Kenmore; Mark Kirkland; R. Andy Badley
- Book ID
- 101286038
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 110 KB
- Volume
- 11
- Category
- Article
- ISSN
- 0952-3499
No coin nor oath required. For personal study only.
✦ Synopsis
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'-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) and coupled an antibody fragment (scFv) to CM dextran at a very high density. Using an upgraded BIAlite 2 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.