Coupling to hydrazide particles

The following protocol describes the oxidation of carbohydrate (glycans) on antibody molecules to form aldehydes and the subsequent coupling to hydrazide particles. 

Figure 14.19 Aldehyde-containing molecules, such as periodate-oxidized carbohydrates or glycoproteins, can be coupled to hydrazide-particles to form a hydrazone bond. This bond can be further stabilized by reduction...

Perhaps a better design for a bis-hydrazide compound to modify carboxylate particles would include a short PEG spacer arm between the two hydrazide groups. This type of linker would result in a hydrophilic surface due to the presence of the PEG spacers, while providing the terminal hydrazide functionality necessary for coupling to carbonyl compounds. Unfortunately, this type of compound is not currently available, so the aliphatic bis-hydrazides are the only choice. 

Figure 14.18 Carboxylate-particles or aldehyde-particles can be modified with the carbohydrazide in excess to create a hydrazide-particle that can be used to couple with aldehyde-containing molecules.

After the final wash, resuspend the particles at a concentration of 10 mg/ml in coupling buffer and add an appropriate amount of the solution from step 6, which contains the purified, oxidized antibody. The amount of oxidized antibody to add to the particles should be about 1-10 X over the amount of the calculated monolayer for the particle type used. (Note For 100 mg of 1 pm hydrazide particles, a monolayer equivalent of antibody will be about 1.5 mg, so the total amount added should be in the range of 1.5-15 mg for a 1-10 X excess). 

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