W-Acetylaspartate (NAA) and W-acetylaspartylglutamate (NAAG) are highly concentrated acetylated compounds found predominantly in the nervous system of vertebrates and invertebrates.1-3 Their high concentrations make them good candidates for localization by immunohistochemistry. The two compounds are assumed to be related to one another in terms of biosynthesis. NAA is thought to be a direct precursor for NAAG biosynthesis,4 despite numerous failures in several laboratories to isolate or characterize a NAAG synthase enzyme capable of coupling NAA to glutamate. NAAG and NAA are found primarily in neurons,5-11 although much lower levels may be present in some glial cells,1213 and in somatic tissues.2 Because of their predominant neuronal localization, and the fact that the two molecules provide strong acetate signals in water-suppressed proton magnetic resonance spectra, reductions in their acetate signals have been used as a non-invasive diagnostic marker for neuronal loss or dysfunction.14-17 The N-terminal acetyl groups of NAAG and NAA make their localization by immunohistochemistry problematic, because both molecules lack an amine group, and thus lack a reactive group that would permit standard fixation coupling with glutaraldehyde. This problem has been examined in detail previously.10

The only chemically reactive groups available on NAAG and NAA for coupling to proteins are carboxyl groups; NAAG has three carboxyl groups, and NAA has two. The most effective reagents for inducing peptide bond formation between carboxyl-containing compounds and proteins are known as carbodiimides. Historically, carbodiimides were first used for tissue fixation for immunohistochemistry in the early 1970's.18,19 We have used the water-soluble carbodiimide, EDAC (1-ethyl-3 [3-dimethylaminopropyl] carbodiimide hydrochloride), for the immunohistochemical localization of NAAG and

* Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd, Bethesda MD, 20814, USA, email; [email protected].

NAA. EDAC-based fixation for immunohistochemistry is complicated by the fact that this coupling reagent is water soluble, but does not penetrate lipid-rich tissues, such as white matter in brain. To overcome this problem, we found that 5% DMSO could be used in the fixative solution to increase penetration of EDAC into white matter and other lipid-rich tissues. DMSO was found to be critical for uniform labeling of NAAG and NAA in nervous system white matter, but was also found to destroy the internal ultrastructure of neurons, making its use unsuitable for electron microscopy. Other improvements to carbodiimide fixations for light microscopy were achieved by increasing the temperature of the fixative solution to 37oC, and using the carbodiimide stabilizing agent; N-hydroxysuccinimide (1mM), to reduce non-productive hydrolysis of EDAC in solution.10 Another significant problem associated with small-molecule immunohistochemistry in general is cross-reactivity of the antibodies with non-specific epitopes in fixed tissue sections. The problems of cross-reactivity and high background staining in tissue sections were pronounced with affinity-purified NAAG and NAA antibodies. Solid phase immunoassays showed that affinity purified, polyclonal antibodies required an additional purification step to remove cross-reactive antibodies.11 This was accomplished by the use of nitrocellulose-immobilized hapten-protein conjugates produced with EDAC. Structurally related compounds such as aspartate and aspartylglutamate were coupled to bovine serum albumin with EDAC, and then adsorbed to nitrocellulose strips. The nitrocellulose strips were then incubated first with the crude antisera to remove most of the cross-reactive antibodies. Then these partially purified anti-NAAG and NAA antibodies were affinity purified on a NAAG-coupled or NAA-coupled aminoalkyl-agarose gels respectively. Finally, the antibody solutions were incubated again with new nitrocellulose sheets containing related EDAC conjugates to eliminate remaining cross-reactivity. In the case of NAAG antibodies, NAA-BSA was the most cross-reactive conjugate, whereas in the case of NAA antibodies, NAAG-BSA was the most cross-reactive conjugate. This three-step purification process produced highly specific anti-NAAG and anti-NAA antibodies with less than 1% cross-reactivity to all related protein-hapten conjugates.

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