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

Abstract

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.

A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

“…For example, Sakakibara et al . [7] found that the mAb 5H7-G1 generated by S.I. procedure could recognize egg antigens in the animal cortex of fertilized, but not unfertilized, Xenopus eggs.…”

“…method has achieved the desired results in the production of mAbs of different cells and proteins in the past decade [6], [7], [8], [9], [19], [20], [21], [22], [23]. First, S.I.…”

“…This procedure not only eliminates the production of undesired mAbs, but also achieves a highly specific antibody and increases the likelihood of generating mAbs against rare or weakly immunogenic epitopes in complex biological mixtures such as intact cells or tissues. There are several unique reactive antibodies targeting special protein or cells prepared successfully by this technique [6], [7], [8], [9]. However, to the best of our knowledge, there is so far no report on the preparation of discriminatory mAbs for bacteria by this procedure.…”

Focused Proteomics on Egg Membrane Microdomains to Elucidate the Cellular and Molecular Mechanisms of Fertilization in the African Clawed Frog Xenopus laevis

“…In Xenopus , some egg surface-interacting substances have been reported to act as an egg activator: examples include disintegrin peptides (Iwao and Fujimura 1996 ;Shilling et al 1998 ) and cathepsin B (Mizote et al 1999 ). As we mentioned earlier, an antibody that recognizes the extracellular domain of UPIII has been shown to inhibit normal fertilization (Sakakibara et al 2005a ). Therefore, we expect that an antibody(s) of the library, which can bind to the surface (in other words, some known or unknown components essential for fertilization) of unfertilized Xenopus eggs, would be able to activate or inhibit egg functions.…”

“…The fi rst subproject is to identify novel fertilization-related components by characterization of MDs-associated molecules. A major achievement in this subproject is the identifi cation of a type I transmembrane protein uroplakin III (UPIII) that is thought to be involved in gamete interaction, In this project, iEMD are subjected to in vitro treatment with sperm, cathepsin B, Src, or some others, and analyzed for their biochemical (e.g., tyrosine phosphorylation of MDsassociated proteins) as well as cell biological responses (e.g., ability of the sperm to fertilize eggs) which may involve the action of sperm-derived protease, and regulation of xSrc activity, in that UPIII may be involved in the negative regulation of xSrc in unfertilized eggs (Mahbub Hasan et al 2005, 2007Mammadova et al 2009 ;Sakakibara et al 2005a ) (Fig. 14.1 ).…”