Linkage of silver to antibodies through 2-imino thiolane

Earlier studies described the linkage of silver to antibodies using SH groups generated by the reduction of the SS groups using ascorbic acid (1) analogous to the Thakur and DeFulvio technique for linking technetium to antibodies. This work describes the linkage of silver to IgG after introducing SH groups by coupling the IgG to 2-imino thiolane. The protein was dissolved in sodium acetate buffer pH 4.5 containing 1 mM EDTA by dialysis/gel chromatography in a concentration of 20 mg/mL. 2-Imino thiolane dissolved in Tris-HCl acetate buffer, pH 8.2, 0.2M was added to give a final dilution of 0.2 mM 2-imino thiolane. The excess of 2-imino thiolane was removed by dialysis or G-25 Sephadex gel chromatography and then the protein was reacted with silver nitrate 0.1 mM. The unreacted SH groups were blocked by adding iodoacetamide to a concentration of 5 mM. The nonprotein reagents again were removed by dialysis or gel chromatography. The thiol groups were titrated using 1.5 mM 2 2-Py-SS-Py prior to and after addition of silver. It was observed that depending on the concentration of silver, 50-80% of the SH groups were coupled to silver. Higher concentrations of silver led to insoluble precipitates and should be avoided.