Preparation of Antibody---Gold Complexes

Determine the minimum amount of protein A required to stabilize the colloidal gold sol being used. The colloidal suspension should be adjusted, if needed, with 0.1M K2CO3 to pH 6-7. Measure the pH of the sol using a gel-filled electrode. Determining the stabilization amount of protein A can be done according to the method described in Section 1, this chapter. 

Mix a stabilizing amount of protein A plus an additional 10 percent with the appropriate volume of colloidal gold. For example, Herbener (1989) mixed 10ml of a 14nm gold particle sol at pH 6.9 with 0.3 mg of protein A dissolved in 0.2 ml water. Mix well. 

After 1 minute, add 250pi of 1 percent polyethylene glycol (MW 20,000) per 10ml of gold sol used. The PEG helps to stabilize further the sol against aggregation. 

To remove excess protein A, centrifuge the preparation at a minimum of 50,000g for 30 minutes to several hours (4°C), depending on the size of the particles and the amount of solution. Discard the supernatant, and resuspend the protein A-gold pellet in 0.01M sodium phosphate, pH 7.4, containing 1 percent PEG. 

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It has been reported that some commercial preparations of colloidal gold-antibody complexes may contain free active antibody. Such free antibody will compete with antibody-colloidal gold particles for antigen binding sites and may reduce labeling intensity. The presence of free protein may be identified using a simple test procedure (20)... 

Kramarcy, N. R. and Sealock, R (1991) Commercial preparations of colloidal gold-antibody complexes frequently contain free active antibody. J Histochem. Cytochem 39, 37-39. 

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