A novel technique for producing antibody-coated microprobes using a thiol-terminal silane and a heterobifunctional crosslinker.
Antibody-coated microprobes are used to measure neuropeptide release in the central nervous system. Although they are not quantitative, they provide the most precise spatial resolution of the location of in vivo release of any currently available method. Previous methods of coating antibody microprobes are difficult and time-consuming. Moreover, using these methods we were unable to produce evenly coated antibody microprobes. This paper describes a novel method for the production of antibody microprobes using thiol-terminal silanes and the heterobifunctional crosslinker, 4-(4-N-maleimidophenyl)butyric acid hydrazide HCl 1/2 dioxane (MPBH). Following silation, glass micropipettes are incubated with antibody to substance P (SP) that has been conjugated to MPBH. This method results in a dense, even coating of antibody without decreasing the biological activity of the antibody. Additionally, this method takes considerably less time than previously described methods without sacrificing the use of antibody microprobes as micropipettes. The sensitivity of the microprobes for SP is in the picomolar range, and there is a linear correlation between the log of SP concentration (M) and B/B0 (r2 = 0.98). The microprobes are stable for up to 3 weeks when stored in 0.1 M sodium phosphate buffer with 50 mM NaCl (pH 7.4) at 5 degrees C. Finally, insertion into the exposed spinal cord of an anesthetized rat for 15 min produces no damage to the antibody coating.
Routh
V H
VH
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
Helke
C J
CJ
D000818:Animals; D000906:Antibodies; D001345:Autoradiography; D002490:Central Nervous System / Q000737:chemistry; D003432:Cross-Linking Reagents; D004853:Epoxy Resins; D005976:Glutaral; D008839:Microelectrodes; D051381:Rats; D012821:Silanes; D013373:Substance P / Q000032:analysis / Q000276:immunology; D013438:Sulfhydryl Compounds
D000906:Antibodies; D003432:Cross-Linking Reagents; D004853:Epoxy Resins; D012821:Silanes; D013438:Sulfhydryl Compounds; D013373:Substance P; D005976:Glutaral