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Spatial and temporal immunoreactivity in the rat brain using an affinity purified polyclonal antibody to DNSP-11.

ABSTRACT: DNSP-11 antibody signal was investigated in perfusion fixated Fischer 344 rat brains by immunohistochemistry with a custom, affinity purified polyclonal antibody. The DNSP-11-antibody signal was differentially localized from the mature GDNF protein both spatially and temporally. In the mesencephalon of post-natal day 10 animals, when GDNF is maximally expressed, DNSP-11 and GDNF antibody immunoreactivities co-localize extensively but not exclusively. In adult 3-month-old animals, GDNF expression is markedly reduced while the DNSP-11 signal remains intense. DNSP-11-antibody signal was present in the 3-month-old rat brain with signal in the substantia nigra, ventral tegmental area, dentate gyrus of the hippocampus, with the strongest signal observed in the locus ceruleus where GDNF is not expressed. While amino acid sequence homologues such as NPY and Tfg do exist, binding patterns reported in the literature of do not recapitulate the immunoreactive patterns observed for the DNSP-11-antibody signal.

SUBMITTER: Sonne JWH

PROVIDER: S-EPMC6745710 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Spatial and temporal immunoreactivity in the rat brain using an affinity purified polyclonal antibody to DNSP-11.

Sonne James W H JWH Groshong Jason S JS Seavey Corey C Gash Don M DM

Journal of chemical neuroanatomy 20190805

DNSP-11 antibody signal was investigated in perfusion fixated Fischer 344 rat brains by immunohistochemistry with a custom, affinity purified polyclonal antibody. The DNSP-11-antibody signal was differentially localized from the mature GDNF protein both spatially and temporally. In the mesencephalon of post-natal day 10 animals, when GDNF is maximally expressed, DNSP-11 and GDNF antibody immunoreactivities co-localize extensively but not exclusively. In adult 3-month-old animals, GDNF expression ...[more]

PMID: 31394198

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Project description:We have performed a systematic variability study of polyclonal antibody catalysis by using five rabbits immunized with the same hapten. Important results from this work are the following. (1) Similarities were observed in the catalytic polyclonal antibodies derived from all five rabbits. Four of the five rabbits produced polyclonal samples that were nearly the same in terms of catalytic activity, whereas the fifth rabbit, designated as rabbit 2, displayed a somewhat higher level of catalytic activity. The catalytic activities (as kcat/kuncat) of these polyclonal samples were similar to that from the best murine monoclonal antibody that had been previously elicited by the same hapten. (2) Titre was not an accurate indicator of polyclonal antibody catalytic activity. (3) A mathematical analysis to describe a distribution of Michaelis-Menten catalysts was performed to help interpret our results. (4) Kinetic analysis indicated that the binding parameters of the different samples were remarkably homogeneous, because one or two components were all that were required to fit the on-rate and off-rate data satisfactorily. Interestingly, the most active catalytic polyclonal sample, that from rabbit 2, displayed the slowest off-rate (so slow it could not be measured) and thus the highest overall affinity. (5) Catalytic analysis of eluted fractions of antibody from a substrate column indicated that each polyclonal sample was also relatively homogeneous in terms of catalytic parameters. The main conclusion of our study is that for this hapten-animal system, the overall catalytic immune response is relatively consistent at two levels. Consistent catalytic activity was observed between the polyclonal samples elicited in the different animals, and the elicited hapten-specific polyclonal antibodies were relatively homogeneous in terms of binding and catalytic parameters within each immunized animal. The observed similarities of the catalytic activity in the different animals is surprising, because the immune response is based on specific binding of antibodies to hapten. There is no known selective pressure to maintain consistent levels of catalytic activity. Our results can therefore be interpreted as providing evidence that for this hapten there is a fixed relationship between hapten structure and catalytic activity and/or consistent genetic factors that dominate the catalytic immune response.

Dataset Information

Spatial and temporal immunoreactivity in the rat brain using an affinity purified polyclonal antibody to DNSP-11.

Publications

Spatial and temporal immunoreactivity in the rat brain using an affinity purified polyclonal antibody to DNSP-11.

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