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Immunocytochemical characterization of mouse monoclonal ACTH antibodies with a note on staining conditions and control procedures
Authors:L -I Larsson  L Scopsi  D Modena  G Racchetti  Y M Galante
Institution:(1) Unit of Histochemistry, University Institute of Pathological Anatomy, Copenhagen, Denmark;(2) Department of Molecular Cell Biology, State Serum Institute, Building 81, Amager Boulevard 80, DK-2300 Copenhagen S, Denmark;(3) Section of Biochemistry and Immunology, Recordati S.p.A., Milan, Italy;(4) Present address: Anatomia Patologica Istituto, Nazionale Tumori, Via G. Venezian, 1, I-20133 Milano, Italy
Abstract:Summary Mouse monoclonal antibodies (MAbs) have been produced against porcine ACTH and tested for their immunocytochemical utility. Ten out of 12 MAbs reacted with formaldehyde-fixed human ACTH1–39] and fragments thereof. Cytochemical fragment testing revealed that 6 of the 10 MAbs recognized epitopes in the vicinity of the region where porcine ACTH differs from mouse ACTH (amino acids 26, 29 and 31). Both tissue and cytochemical model data indicate that many of the MAbs detected porcine ACTH with somewhat higher potency than human and rat ACTH (rat ACTH1–39] is identical to mouse ACTH1–39]). MAbs Nos. 5, 8 and 12, in particular, revealed a highly satisfactory signal to noise ratio also in formalin-fixed, par-affin-embedded specimens. Most of the MAbs were potent in detecting both the high concentrations of ACTH congeners in corticotrophs and melanotrophs and the lower concentrations of such peptides in human antropyloric gastrin cells. Blocking of tissue endogenous peroxidase activity reduced reactivity towards the MAbs. This could be circumvented by use of biotinylated primary antibodies combined with avidin/streptavidin-alkaline phosphatase detection. Availability of MAbs and of the corresponding synthetic antigen also made some quantitative comparisons and analyses of appropriate control procedures possible.
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