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Development and Application of Antibodies to Primary (DA, L-DOPA) and Secondary (cGMP) Messengers: A technical report

  • Chapter
Neurocytochemical Methods

Part of the book series: NATO ASI Series ((ASIH,volume 58))

Abstract

In the last decade antibodies have been raised against a large number of amine- and amino-acid neurotransmitters and candidates. These antibodies have been used in tracing neuronal connections in the central nervous system and locating the cell bodies and varicose fibers synthesizing these molecules. There are a number of specific difficulties inherent to the use of antibodies against these small, water soluble, molecules. To obtain these antibodies, the small haptens have to be coupled to a larger protein molecule to acquire an immunogenic conjugate. Two fixatives, formaldehyde and glutaraldehyde, have been used in most cases, and highly specific antibodies have been raised against conjugates prepared with both fixatives. In visualizing a molecule cross-linked to tissue constituents the fixative applied is probably the most important factor in combination with the antibody used. Ideally, the procedure for cross-linking small molecules to tissue constituents should be strictly identical with the procedure to prepare the immunogen against which the antibody is raised. The fixative might alter the structure of the hapten and the antibody against this molecule might not recognize the free, unmodified hapten in solution. Furthermore, in the cases of formaldehyde and glutaraldehyde, the fixatives are part of the antigenic determinant, together with an unknown part of the protein molecule to which the hapten is coupled. One more problem is the lack of knowledge about the amount of hapten actually retained by the tissue after fixation. The specificity of an antibody raised against an antigen is the most important factor in determining the usefulness in all its applications. A commonly applied testing criterion is the sensitivity and specificity of a particular antibody in a radioimmunoassay. However, in immunocytochemistry the situation is more complex because one has to deal with an antigen that might be modified in a decisive way by some chemical agent (the fixative) in a surrounding which is not defined (the cellular matrix).

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Authors and Affiliations

  1. Dept. Pharmacology, Faculty of Medicine, Free University, 1081 BT, Amsterdam, The Netherlands

    Harry W. M. Steinbusch, S. Peter Van Vliet, John G. J. M. Bol & Jan De Vente

Authors
  1. Harry W. M. Steinbusch
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  2. S. Peter Van Vliet
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  3. John G. J. M. Bol
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  4. Jan De Vente
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Editors and Affiliations

  1. Département de Cytologie Institut des Neurosciences, CNRS Université Pierre & Marie Curie, 7 Quai Saint-Bernard, 75252, Paris Cedex 05, France

    André Calas  & Daniel Eugène  & 

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Steinbusch, H.W.M., Van Vliet, S.P., Bol, J.G.J.M., De Vente, J. (1991). Development and Application of Antibodies to Primary (DA, L-DOPA) and Secondary (cGMP) Messengers: A technical report. In: Calas, A., Eugène, D. (eds) Neurocytochemical Methods. NATO ASI Series, vol 58. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-84298-6_1

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  • DOI: https://doi.org/10.1007/978-3-642-84298-6_1

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