Vh. Routh et Cj. Helke, A NOVEL TECHNIQUE FOR PRODUCING ANTIBODY-COATED MICROPROBES USING A THIOL-TERMINAL SILANE AND A HETEROBIFUNCTIONAL CROSS-LINKER, Journal of neuroscience methods, 71(2), 1997, pp. 163-168
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.