Ilse Bartke

Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies

Mouse monoclonal antibodies were raised against bacterially expressed protein sequences of the NR2A, NR2B, NR2C and NR2D subunits of the rat NMDA receptor. From immunoblots of rat brain proteins, the apparent molecular weights of these subunits were 165, 170, 135 and 145 kDa, respectively. Proteins of similar masses were observed on immunoblots of specifically transfected HEK293 cells. Deglycosylation with endoglycosidase F reduced the mass of each endogenous NR2 subunit by approximately 10 kDa. In distribution studies, NR2A-immunoreactive protein (IRP) was located throughout the adult rat brain, NR2B-IRP was primarily in the forebrain, NR2C-IRP was predominantly in the cerebellum and NR2D-IRP was mainly found in the thalamus, midbrain and brainstem. Whereas NR2A- and NR2C-IRPs increased during rat brain post-natal development, NR2B- and NR2D-IRPs were abundant at birth and declined with age, especially in cerebellum. NR2-IRPs of mouse, rabbit, frog and human brain were of sizes similar to those of the corresponding rat subunits and were similarly distributed. In summary, NR2 subunits are large glycoproteins whose specific expression profiles in the brain are developmentally and regionally regulated and which are similarly expressed in a variety of species.

Characterization of Mono‐ and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Abstract: 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 immu‐nosorbent 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 IgGl 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 immu‐nohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.