Antibodies raised against synthetic peptides react with choline acetyltransferase in various immunoassays and in immunohistochemistry.

Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168-189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.

An efficient sandwich-ELISA for the determination of choline acetyltransferase.

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.

The basal forebrain cholinergic system in the raccoon.

The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the raccoon basal forebrain using a rabbit antiserum and a monoclonal antibody. Alternating sections were used for Nissl staining. ChAT-positive neurons were arranged in a continuous mass extending from the medial septum to the caudal pole of the pallidum. Based upon spatial relations to fibre tracts, the clustering of neuronal groups, and cytological criteria, the basal forebrain magnocellular complex can be subdivided into several distinct regions. Although clear nuclear boundaries were often absent, the ChAT-positive neurons were divided into: the nucleus tractus diagonalis (comprising pars septi medialis, pars verticalis and pars horizontalis); nucleus praeopticus magnocellularis; substantia innominata; and the nucleus basalis of Meynert. Comparison with Nissl-stained sections indicated the presence of varying proportions of non-cholinergic neurons clustered or arranged loosely within these basal forebrain subdivisions. These data provide a structural basis for studies concerned with the topographical and physiological aspects of the raccoon basal forebrain cholinergic projections and its comparison with the basal forebrains of other species.

Characterization of mono- and polyclonal antibodies against highly purified choline acetyltransferase: a monoclonal antibody shows reactivity in human brain.

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.