Covalent coupling procedure : use of soluble carbodiimide (CDI)

Materials 

• Particles at 10% solid
• Water soluble carbodiimide 250 µl
• [ 1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide] 40 mg
• Buffer A : NaH₂PO₄ – 10 mM pH 6.0

Method 

1°) Dialyse the protein 2 x 1h in buffer A.
2°) In a 5 ml clean glass tube, mix 1000 µl of Buffer A with 250 µl of particles at 10 %.
3°) Dissolve water soluble carbodiimide at a final concentration of 20 mg / ml in Buffer A.
4°) Add 125 µl of solubilised water soluble carbodiimide ( 3 ) to the diluted particle suspension ( 2 )
and mix.
5°) Add protein at the desired final coverage (see *1).
6°) Incubate for 2 h at room temperature or overnight at +4°C, under weak agitation.
7°) Wash the particles twice with Buffer A and resuspend in the appropriate buffer (see *2) at the
desired concentration.

Note 

*1. Most proteins bind to particles within the range [5-60] 10^-4 nmole/ cm².

For IgG, 15 10^-4 nmole / cm² can be tried as a basic example. Note that only the total surface of particle must be considered (do not consider the number of particles).
The protein buffer must be free of primary amines.

*2.Example of appropriate buffer :

• Na2HPO4-20 mM
• Glycin – 100 mM
• Adjust to pH 7.5 with NaH2PO4
• Bovine albumin – 1 to 10 g / l
• Preservative (avoid sodium azide for peroxidase based immunoassay)