Generating monoclonal antibodies to neuronal antigens

Riggott, Marcia J.; Matthew, William Diller

doi:10.1016/0165-0270(96)00085-4

Cited by 6 publications

(2 citation statements)

References 42 publications

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“…As the means to detect potentially rare, localized, and unique antigens against a background of abundant, unlocalized antigens, subtractive immunization has been used successfully in several laboratories (for review see Zijlstra et al 2003). It has been applied to several different levels of cell system: the whole cell level (Hooper et al 2003;Sleeman et al 1999;Wulf et al 1996), wherein cell-surface antigens expressed specifically in tumor cells are detected; the subcellular localization level, wherein Xenopus egg proteins localized to the vegetal hemisphere but not in animal hemisphere or neuronal cell-specific antigens are detected (Denegre et al 1997;Riggott & Matthew *In condition 1, antibody was applied to egg samples before insemination (3-5 min prior to insemination). In condition 2, antibody was applied to egg samples after egg activation (25 -40 min after insemination).…”

Section: Discussionmentioning

confidence: 99%

“…1996), wherein cell‐surface antigens expressed specifically in tumor cells are detected; the subcellular localization level, wherein Xenopus egg proteins localized to the vegetal hemisphere but not in animal hemisphere or neuronal cell‐specific antigens are detected (Denegre et al . 1997; Riggott & Matthew 1996); and the single protein molecule level, wherein two proteins with more than 90% identity of the amino acid sequence are discriminated (Sleister & Rao 2001, 2002). In all cases, donor mice are immunized first with the control antigens, immediately followed by treatment with the immunosuppressant drug, cyclophosphamide.…”

Section: Discussionmentioning

confidence: 99%

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Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization

et al. 2005

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Here we report the generation and characterization of a monoclonal antibody, mAb 5H7-G1, which recognizes egg antigens in the animal cortex of fertilized, but not unfertilized, Xenopus eggs. The mAb 5H7-G1 was generated by subtractive immunization of mice: primary immunization with unfertilized egg extract followed by immunosuppression treatment with cyclophosphamide and repeated immunization with fertilized egg extract. In immunoblotting analysis, mAb 5H7-G1 recognizes multiple protein bands of fertilized (but not unfertilized or the ionophore-activated) Xenopus eggs. N-linked polysaccharide is most likely the target of mAb 5H7-G1 because immunoreactivity of mAb 5H7-G1 is effectively diminished when protein samples are treated with N -glycosidase F. Moreover, mAb 5H7-G1 recognizes some, but not all, tyrosine-phosphorylated proteins in eggs treated with H 2 O 2 , an artificial activator of the egg tyrosine kinase Src, suggesting that these proteins also contain N-linked sugars. When microinjected into fertilized Xenopus embryos, mAb 5H7-G1 causes a retardation or complete inhibition of first cell cleavage, suggesting that the mAb 5H7-G1-reactive antigens play an important role in this event. These results demonstrate that mAb 5H7-G1 is useful to analyze differential proteome display during fertilization and early development. More generally, subtractive immunization may work as a strategy to uncover cellular events that operate during different cellular conditions of interest.

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