Dissociation constants

Smaller Kd = stronger affinity

Antibody/ReceptorAntigen/LigandKd
PirBBeta-amyloid oligomers180 nM
LilrB2Beta-amyloid oligomers206 nM
Gantenerumab

Beta-amyloid oligomers

1.2 nM

HyHEL-10Chicken lysozyme0.67 nM
VRC01gp1203.88 nM

 

Gantenerumab binding to other things:

Beta-amyloid oligomers

Beta-amyloid fibrils

Beta-amyloid monomers

1.2 nM

0.6 nM

17 nM

 

Detecting antigen binding

We can't directly label gp120 or chicken lysozyme with biotin or fluorophores because conjugation alters lysine residues, which are critical to binding between VRC01 and gp120 and between HyHEL-10 and chicken lysozyme.  This doesn't affect Gantenerumab's interaction with beta-amyloid oligomers because this interaction doesn't involve lysine residues (also, in this paper they use biotinylation to perform SPR between Gantenerumab and beta-amyloid oligomers).

Options are

  1. biotinylated antibodies (that we can use with the AF594-Streptavidin we already have)
  2. fluorophore-conjugated antibodies (preferably AF594)
  3. primary antibodies that we can combine with secondary antibodies we already have (see list here)

Gantenerumab:  Biotinylated beta-amyloid oligomers + Alexa fluor 594-conjugated Streptavidin

VRC01:

 

HyHEL-10: