A NOVEL TECHNIQUE FOR PRODUCING ANTIBODY-COATED MICROPROBES USING A THIOL-TERMINAL SILANE AND A HETEROBIFUNCTIONAL CROSS-LINKER

Antibody-coated microprobes are used to measure neuropeptide release i n the central nervous system. Although they are nor quantitative, they provide the most precise spatial resolution of the location of in viv o release of any currently available method. Previous methods of coati ng antibody microprobes are difficult and time-consuming. Moreover, us ing these methods we were unable to produce evenly coated antibody mic roprobes. This paper describes a novel method for the production of an tibody microprobes using thiol-terminal silanes and the heterobifuncti onal crosslinker, 4-(4-N-maleimidophenyl)butyric acid hydrazide HCl 1/ 2 dioxane (MPBH). Following silation, glass micropipettes are incubate d with antibody to substance P (SP) that has been conjugated to MPBH. This method results in a dense, even coating of antibody without decre asing the biological activity of the antibody. Additionally, this meth od takes considerably less time than previously described methods with out sacrificing the use of antibody microprobes as micropipettes. The sensitivity of the microprobes for SP is in the picomolar range, and t here is a linear correlation between the log of SP concentration (M) a nd B/B-0 (r(2) = 0.98). The microprobes are stable for up to 3 weeks w hen stored in 0.1 M sodium phosphate buffer with 50 mM NaCl (pH 7.4) a t 5 degrees C. Finally, insertion into the exposed spinal cord of an a nesthetized rat for 15 min produces no damage to the antibody coating. Published by Elsevier Science B.V.