MAB5270-50UG Sigma-AldrichAnti-Choline Acetyltransferase Antibody, clone 1.B3.9B3

A specific, sensitive, and reliable sandwich-ELISA (enzyme-linked immunosorbent assay) has been established for the determination of choline acetyltransferase (CHAT) from porcine brain. The detection limit of the assay was 30 micrograms/l and the assay was linear up to 300 micrograms/l. The within-day and day-to-day coefficients of variation were found to be 3.3% and 4.7% respectively for low CHAT concentrations (30 micrograms/l) and 3.1% and 3.4% respectively for high levels of CHAT (300 micrograms/l). The immunoassay was more sensitive than the radiometric assay of Fonnum which is widely used for the measurement of enzyme activity. In the assay monoclonal antibody was adsorbed to the polystyrene surface of the immunoplate as the capture reagent. Using a standard peroxidase protocol the immobilized antigen was detected with a highly specific anti-CHAT antiserum raised in rabbits. Two monoclonal antibodies were available for antigen binding. One of the two--A10.29B4--reacted preferentially with the active enzyme the other one--B3.9B3--reacted only with a degraded form. The polyclonal antiserum recognized both native and denatured enzyme. The effectiveness of employing the two monoclonal antibodies separately or in combination was demonstrated by measurement of porcine CHAT diluted in human cerebrospinal fluid and serum. After some minor modifications the sandwich-ELISA could be used for the determination of CHAT from the central nervous system of the rat.

Choline acetyltransferase (ChAT) was purified by immunoaffinity chromatography using a covalently immobilized monoclonal antibody. In a two-step procedure, 10 kg porcine brain yielded 750 micrograms active enzyme of apparent homogeneity. This amount of ChAT was purified routinely. The purification factor was 18,000 and the yield of activity 4.3%. The affinity resin was stable under the experimental conditions applied and was used many times. The highly purified enzyme was subsequently employed to obtain a specific anti-ChAT antiserum of high titer.

Use of an ethylene glycol based cryoprotectant solution has been found to be effective for the long-term storage of brain tissue either in block form or as freely floating sections prior to immunocytochemical processing. Storage of tissue in the solution at -20 degrees C or 4 degrees C for up to 3 months produced no adverse effects upon tissue morphology, nor was LHRH immunoreactivity diminished or accompanied by elevated non-specific staining. Furthermore, ultrastructural analysis of cryoprotected tissue revealed excellent preservation of cellular morphology. It is anticipated that this method can find use when it is necessary or desirable for the investigator to retain tissue for later immunocytochemical or electron microscopic processing.

Choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, has been localized immunocytochemically with a monoclonal antibody in light and electron microscopic preparations of rat central nervous system (CNS). The antibody was an IgG1 subclass immunoglobulin that removed ChAT activity from solution. The specificity of the antibody and immunocytochemical methods has been confirmed by the demonstration of ChAT-positive neurons in a number of well-characterized cholinergic systems. For example, ChAT-positive reaction product was present in the cell bodies of spinal and cranial nerve motoneurons, as well as in their axons and terminations as motor end-plates in skeletal muscle. In addition, the somata of preganglionic sympathetic and parasympathetic neurons were ChAT-positive. The specificity of staining was further supported by a lack of reaction product in several groups of neurons thought to use neuroactive substances other than acetylcholine. No specific staining was observed in control specimens. The findings indicated that ChAT had an extensive intraneuronal distribution in many cholinergic neurons, being present in cell bodies, dendrites, axons and axon terminals. ChAT-positive somata were found in the medial septum and diagonal band, the medial habenula, and the basal nucleus of, the forebrain, 3 regions that are sources of cholinergic afferents to the hippocampus, interpeduncular nucleus and cerebral cortex, respectively. In addition, positively stained cell bodies were present within the cerebral cortex. ChAT-positive punctate structures were observed in the ventral horn of the spinal cord, where electron microscopic studies demonstrated that some of these structures were synaptic terminals. Other regions containing numerous ChAT-positive puncta included the hippocampus, the interpeduncular nucleus and the cerebral cortex. The light microscopic appearance of these putative cholinergic terminals varied among different brain regions. Large punctate structures related to well-defined post-synaptic elements were characteristic of some regions, such as the ventral horn of the spinal cord, while smaller punctate structures and varicose fibers with a diffuse pattern of organization distinguished other regions, such as the cerebral cortex.

Choline acetyltransferase (ChAT) has been purified from pig brain to greater than 95% homogeneity (purification factor: 646 000, specific activity of the purified enzyme: 128 mumol acetylcholine formed/min/mg). Gel electrophoresis of the purified enzyme in the presence of sodium dodecylsulphate and beta-mercaptoethanol revealed a single protein band at 68 000 daltons. Immunoprecipitation and double immunodiffusion tests showed that antisera raised against this protein specifically recognize ChAT. A monoclonal antibody prepared against the enzyme specifically binds a protein from crude pig brain supernatants which has a mol. wt. of 68 000 and a specific activity of 153 mumol/min/mg. This antibody shows no species cross-reactivity. The specificity of the immunohistochemical localization of ChAT has been established by comparing the labeling of pig retina using the antiserum with that obtained using the monoclonal antibody. Both probes specifically identify the same retinal structures: labeled cell bodies are found in the inner nuclear layer and the ganglion cell layer, while a double band is stained in the inner plexiform layer. In rat spinal cord, the antiserum labels the motoneurons and the preganglionic sympathetic neurons, located in the intermedio-lateral nucleus, the intercalated region, and the central autonomic area.