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. 2011 Jul 25;16(7):295-302.
doi: 10.1186/2047-783x-16-7-295.

Development of a novel monoclonal antibody to B7-H4: characterization and biological activity

Y Qian  1 , L Shen, C Xu, Z Wu, N H Brockmeyer, P Altmeyer, N Wu, Hang-Ping Yao
Affiliations

Development of a novel monoclonal antibody to B7-H4: characterization and biological activity

Y Qian (V体育官网入口) et al. Eur J Med Res. .

Abstract (V体育官网入口)

Objective: B7-H4, a member of the B7 family of immunoregulatory receptors, may participate in the negative regulation of cell-mediated immunity. Aberrant B7-H4 expression is detected in some tumors and it plays a role in the occurrence and development of tumors. The aim of this study was to elucidate the functional and structural properties of B7-H4. VSports手机版.

Methods: We developed a monoclonal antibody (mAb) against the extracellular domain of B7-H4 through immunization of Balb/c mice with 3T3-mB7-H4 cells which expressed extrinsic B7-H4. A stable hybridoma cell line was established. Then, we analysed the characterization of the mAb through Enzyme linked immunosorbent assay (ELISA), Immunoprecipitation (IP), western blotting, Immunohistochemical (IHC), and tested the biological activity of the mAb V体育安卓版. .

Results: ELISA, IP, and western blotting analyses indicated that the mAb specifically recognized B7-H4. In addition, flow cytometry demonstrated that the mAb exhibits excellent reactivity when applied to leukemic cells. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The preliminary results of the mAb's biological activity showed that the mAb could effectively inhibit the function of B7-H4 in the inhibition of T cell, while promoting the growth of T cells and the secretion of Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-10 (IL-10) and Interferon-. (IFN-γ) V体育ios版. .

Conclusion: This mAb will be a valuable tool for the further investigation of B7-H4 function VSports最新版本. .

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"VSports app下载" Figures

Figure 1
Figure 1
The isotype of mouse monoclonal antibody against B7-H4. - The isotype of the 3E8 mAb, determined using a mouse monoclonal isotyping kit, was an IgM with a κ chain.
Figure 2
Figure 2
ELISA analysis of anti-B7-H4 mAb. - The lysate of 3T3-mB7-H4 cells, diluted to 10 μg/mL in 0.01 M coating solution concentrate, was coated onto high-binding polystyrene plates at 100 μL per well, and incubated at 4°C overnight. The lysate of 3T3 cells not transfected with B7-H4 was used as a control. The titer of 3E8 (at an original concentration of 1.5 mg/mL) was determined by ELISA and read at A450.
Figure 3
Figure 3
IP and western blotting analysis with 3E8 mAb. - A: Western blotting was carried out for B7-H4 from lysates of 3T3-mB7-H4 or 3T3 cells using 3E8 mAb, followed by goat anti-mouse IgM antibody coupled with HRP. B: Western blotting was carried out for B7-H4 from lysates of 3T3-mB7-H4 or 3T3 cells using goat anti-B7-H4 polyclonal antibody, followed by rabbit anti-goat IgG antibody coupled with HRP. 3E8 mAb (2 μ/sample). Western blotting was carried out for B7-H4 using goat anti-B7-H4 polyclonal antibody, followed by rabbit anti-goat IgG antibody coupled with HRP. Normal mouse IgM was used as an antibody control. Lane 1: lysate from normal 3T3 cells; Lane 2: lysate from 3T3-mB7-H4 cells; Lane 3: B7-H4 unrelated antibody control.
Figure 4
Figure 4
Flow cytometry analysis with 3E8 mAb. - U937, THP-1, HL60, and MM1R cells (2 × 106 cells per sample in PBS) were incubated with 3E8 mAb or goat anti-B7-H4 polyclonal antibody for 30 min at 4°C. Normal mouse IgM was used as an antibody control. Cells were washed and resuspended in a solution of goat antimouse IgM coupled with FITC or donkey anti-goat IgG coupled with FITC. Finally, cells were washed twice and samples were analyzed using a FACScan flow cytometer.
Figure 5
Figure 5
Expression of B7-H4 in normal tissues and corresponding cancer samples from breast, uterus, and ovary. - Human normal and cancerous samples in tissue arrays were subjected to IHC staining using mAb 3E8. The reactions were visualized using reagents from a DAKO Envision System kit. Normal mouse IgM was used as an antibody control (data not shown). Samples were photographed and staining intensity (0 to 3, least intense to most intense) and the proportion of stained cells (0 to 4, no cells stained to more than 70% cells stained) were semi-quantitatively determined. A combined score of ≥6 was considered to indicate overexpression.
Figure 6
Figure 6
Spleen cell growth and cytokines secretion assays. - A: Spleen cell growth determined by MTS. Spleen cells from normal Balb/c mouse (2 × 105 cells per well) were seeded in triplicate in a 96-well plate which was coated with anti-mouse CD3 mAb. Then, the cells were incubated with the purified recombinant B7-H4 protein, 3E8 mAb, or their admixture which was premixed for 30min. Normal mouse IgM was used as an antibody control. After incubating the wells for 72 hours at 37°C the wells were added with MTS. After incubating the wells for 4 hours at 37 OQ the OD of each well was measured using a Model 680 microplate reader at a wavelength of 490nm. The numbers of living cells were presented by OD490. B: Cytokines secretion determined by ELISA. Spleen cells were treated as above. The Cell culture supernatant was collected respectively after incubating the wells for 48 or 72 hours at 37°C The expression of IL-2, IL-4, IL-10 in 48h supernatant, and IFN-γ in 72h supernatant were determined with mouse cytokine ELISA kit. The expression levels of cytokines were presented by OD450. *: vs Lane 33 and Lane 5, P <0.05.
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V体育2025版 - References

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