Covalent coupling procedure : N-hydroxysuccinimide ester activation

Materials 

• Particles EM1-100 / 40- 10 % solid 250 µl
•  Water soluble carbodiimide
• [1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide] 30 mg
• N-hydroxysuccinimide 18 mg
• Buffer A : NaH2PO4 – 10 mM – pH 6.0 10 ml
• Buffer B : HCl – 2 mM 10 ml
• Buffer C : NaH2PO4 /Na2HPO4-20 mM – pH 7.5 10 ml 

Method

1°) Wash particles twice 1 ml of buffer A.
2°) Dissolve 30 mg of carbodiimide and 18 mg of NHS in 3 ml of buffer A, in a glass tube. Add
2.5 ml of this solution to the pellet of magnetic particles.
3°) Place the tube under agitation for 15 minutes at room temperature. Wash the particles with
1 ml of buffer B.
4°) Resuspend the pellet of particles with 1.480 ml of buffer C. Add immediately the proteins (*1)
and mix. Place the tube under agitation 2 hours at room temperature.
5°) Wash the coated particles three times with buffer C and resuspend in the appropriate buffer (*2).

Note
 

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

For IgG, 15 104 nmole / cm² can be tried as a basic example.
Concentration of IgG : 5 mg / ml. Volume to be added : 71µl.
The protein suspension 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 )