Mouse Anti-NP Recombinant Antibody (clone N1G9) (CAT#: PABW-094)

Figure 1 NP-binding IgM Abs possess cross-reactivity to analogs of the original antigen.

(A) Binding of IgG mAbs to NP 26-BSA (open bars) and DNP 26-BSA (closed bars). IgG mAbs with different Ka values to NPs, namely, N1G9 (Ka = 5 × 10 5 M −1 ), B2 (Ka = 3.4 × 10 6 M −1 ), and E11 (Ka = 10 8 M −1 ), were used. (B) Binding of IgM mAbs to NP 26-BSA (open bars) and DNP 26-BSA (closed bars). Recombinant IgM mAbs whose V-D-J genes were from N1G9, B2 and E11 (N1G9-, B2-and E11-IgM, respectively) were used. (C) Binding of IgM mAbs secreted by hybridomas (1A350, 1A8, and 1A86, respectively) to NP 26-BSA (open bars) and DNP 26-BSA (closed bars). (D) Binding of IgM mAbs performed as in (B) in the presence of the indicated concentrations of NP-Cap. (E) Binding of IgM Abs to NP 2-, NP 26-, and DNP 26-BSA in serum collected from an individual mouse (n = 3) at 1 week after primary immunization (primary 1w) and secondary immunization (secondary 1w). The serum was diluted 200-fold, and binding was measured by ELISA. *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicates the s.d. Data are representative of three independent experiments per experiment (A-D) or four independent experiments with one mouse per experiment (C).

Tashiro, Y., Murakami, A., Hara, Y., Shimizu, T., Kubo, M., Goitsuka, R., ... & Azuma, T. (2018). High-affinity IgM+ memory B cells are defective in differentiation into IgM antibody-secreting cells by re-stimulation with a T cell-dependent antigen. Scientific reports, 8.

Figure 2 SPR measurement analysis of the interactions between NP –BSA – bio and anti-NP mAbs.

The ratios of RUeq (RU at t = 470 s) between NP0.5 –BSA – bio21 and NP7.4 –BSA – bio21 are shown.

Shimizu, T., Oda, M., & Azuma, T. (2003). Estimation of the relative affinity of B cell receptor by flow cytometry. Journal of immunological methods, 276(1-2), 33-44.

Figure 3 Expression of IgG1 BCR on the hybridoma surface.

Total RNA was prepared from NP-specific hybridomas as well as from a myeloma cell line, SP2/0. Expression of anti-NP BCR and mAb were analyzed by RT-PCR using primers specific for VH186.2 and a membrane form of (VH-mCg1) or a secreted form of IgG1 (VH-sCg1). Amplification of h-actin cDNA confirmed the quality of the prepared cDNA.

Shimizu, T., Oda, M., & Azuma, T. (2003). Estimation of the relative affinity of B cell receptor by flow cytometry. Journal of immunological methods, 276(1-2), 33-44.

Figure 4 Expression of IgG1 BCR on the hybridoma surface.

Hybridomas were stained with FITC-anti-IgG1 and analyzed by flow cytometry. Survival cells were gated. The numbers in each panel indicate MFI.

Shimizu, T., Oda, M., & Azuma, T. (2003). Estimation of the relative affinity of B cell receptor by flow cytometry. Journal of immunological methods, 276(1-2), 33-44.

Figure 5 Expression of IgG1 BCR on the hybridoma surface.

Western blotting. Proteins on the cell surface were biotinylated and precipitated with SA–agarose beads (B lanes). Samples of total cell lysates (L lanes) and culture supernatants (S lanes) were prepared by adding SDS-sample loading buffer to cell pellets or supernatants, respectively. These samples as well as the purified secreted form of C6 (P lanes) were separated by reduced SDS-PAGE, and IgG1 molecules were detected by peroxidase-labeled anti-IgG1 Ab.

Shimizu, T., Oda, M., & Azuma, T. (2003). Estimation of the relative affinity of B cell receptor by flow cytometry. Journal of immunological methods, 276(1-2), 33-44.