[Preparation, characterization and potential application of monoclonal antibody 18A4 against AGR2]

To prepare mouse monoclonal antibodies (mAb) against AGR2 (human homolog of xenopus anterior gradient 2), to characterize these antibodies' properties, and to develop potential applications.

BALB/c mice were immunized with AGR2-MBP (maltose binding protein) fusion protein. The mAb was prepared by hybridoma technique and purified by protein G affinity chromatography. The titer and specificity of the mAb was determined by ELISA and Western blot respectively. The mAb was then further characterized by immunoprecipitation, immunofluorescent staining and tumor cell inhibition assay.

One clone of hybridoma, 18A4, secreting specific mAb against AGR2, was obtained. The Ig subclass of the mAb was IgG1 (kappa). The titer of the mAb was 1 x 10(-6);. Western blot analysis showed specific binding of 18A4 with both recombinant and native AGR2 in cell extract. Immunofluorescent staining using 18A4 mAb demonstrated specific staining of AGR2 in the cytoplasma of Breast cancer cell line MCF7. Immunoprecipitation assay confirmed that this mAb could specifically bind and effectively precipitate the native AGR2. mAb 18A4 could also inhibit the growth of breast cancer cell MCF7.

The mAb anti-AGR2 with high titer and specificity has been obtained. This mAb, 18A4, can be used in most molecular biology studies including Western blot, immunostaining, immunoprecipitation. It also inhibited the growth of cancer cells and therefore is a potential therapeutic starting point. It is a useful tool for the functional study of AGR2 and for the diagnosis and potential treatment of certain cancers.