Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization.
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 H2O2, 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.
Sakakibara
Keiichi
K
Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan.
Sato
Ken-ichi
K
Iwasaki
Tetsushi
T
Kitamura
Kazuyuki
K
Fukami
Yasuo
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D004527:Egg Proteins / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes; D005260:Female; D005306:Fertilization; D007114:Immunization; D051379:Mice; D010766:Phosphorylation; D011134:Polysaccharides / Q000276:immunology / Q000378:metabolism; D020543:Proteome / Q000276:immunology / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014982:Xenopus laevis; D015053:Zygote / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004527:Egg Proteins; D000939:Epitopes; D011134:Polysaccharides; D020543:Proteome; D014443:Tyrosine