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Microbes & Immunity Anti-mouse CXCR5 monoclonal antibody

CHO/mCCR2, CHO/mCCR3, CHO/PA-mCCR4, optical density at 655 nm using the ELISA POD Substrate
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CHO/mCCR5, CHO/PA-mCCR6, CHO/mCCR7, TMB Kit (Nacalai Tesque, Inc., Kyoto, Japan).
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CHO/mCCR8, CHO/mCCR9, CHO/mCXCR1, CHO/
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mCXCR3, CHO/mCXCR4, and CHO/mCXCR6. 2.6. Flow cytometry analysis
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These transfectants were detected by the following mAbs: Cells were washed with PBS containing 0.1% BSA
anti-CCR1 mAb (clone S15040E; BioLegend, San Diego, (blocking buffer) and treated with 10, 1, 0.1, or 0.01 μg/
CA, USA), anti-CCR2 mAb (clone EPR20844; Abcam, mL of Cx Mab-3 or L138D7 for 30 min at 4°C. For the
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Cambridge, MA, USA), anti-CCR3 mAb (clone J073E5; peptide inhibition assay, Cx Mab-3 (0.1 μg/mL) or L138D7
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BioLegend, San Diego, CA, USA), anti-CCR5 mAb (clone (0.1 μg/mL) were pre-incubated with 1 μg/mL of mCXCR5
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C Mab-2 ), anti-CCR7 mAb (clone 4B12; BioLegend, San peptide or dimethyl sulfoxide for 30 min at 4°C, and further
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Diego, CA, USA), anti-CCR8 mAb (clone C Mab-2 ), incubated with CHO/mCXCR5 for 30 min at 4°C. The cells
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anti-CCR9 mAb (clone CW-1.2; BioLegend, San Diego, were then treated with anti-rat immunoglobulin G (IgG)
CA, USA), anti-CXCR1 mAb (clone 1122A; R&D Systems conjugated with Alexa Fluor 488 (Cell Signaling Technology,
Inc., Minneapolis, MN, USA), anti-CXCR2 mAb, anti- Inc., Danvers, MA, USA). Anti-mouse IgG conjugated with
CXCR3 mAb (clone CXCR3-173; BioLegend, San Diego, Alexa Fluor 488 and anti-rabbit IgG conjugated with Alexa
CA, USA), anti-CXCR4 mAb (clone L276F12; BioLegend, Fluor 488 (Cell Signaling Technology, Inc., Danvers, MA,
San Diego, CA, USA), anti-CXCR6 mAb (clone SA051D1; USA) were also used to detect the primary mAbs. The
BioLegend, San Diego, CA, USA), anti-XCR1 mAb, anti- fluorescence data were collected using the SA3800 Cell
CX3CR1 mAb, and NZ-1. Analyzer (Sony Corp., Tokyo, Japan). Cells were gated on
the dot plot (SSC vs. FSC), and the fluorescence intensity
2.4. Development of mCXCR5-producing was analyzed using FlowJo software (BD Biosciences,
hybridomas Franklin Lakes, NJ, USA).
A rat was immunized intraperitoneally with 100 μg of 2.7. Determination of dissociation constant (K ) by
KLH-conjugated mCXCR5 peptide (mCXCR5-KLH) with flow cytometry D
Alhydrogel adjuvant 2% (InvivoGen, San Diego, CA, USA).
The procedure included three additional weekly injections The K was determined by fitting saturation binding curves
D
(100 μg/rat), followed by a final booster dose (100 μg/rat) to the built-in one-site binding models in GraphPad PRISM
2 days before harvesting spleen cells. The harvested spleen 6 (GraphPad Software, Inc., La Jolla, CA, USA). This
cells were subsequently fused with P3U1 cells, using analysis was performed after the flow cytometry analysis of
PEG1500 (Roche Diagnostics, Indianapolis, IN, CHO/mCXCR5 cells treated with serially diluted Cx Mab-3
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USA). The resulting hybridomas were grown in RPMI or L138D7, followed by the incubation with anti-rat IgG
medium supplemented with 10% FBS, 100 units/mL of conjugated with Alexa Fluor 488 (1:200 dilution).
penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of 3. Results
amphotericin B. For hybridoma selection, hypoxanthine,
aminopterin, and thymidine (Thermo Fisher Scientific 3.1. Development of anti-mCXCR5 mAbs using
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Inc., Waltham, MA, USA) were added to the medium. The N-terminal peptide immunization
supernatants were subsequently screened using enzyme- To develop anti-mCXCR5 mAbs, one rat was immunized
linked immunosorbent assay (ELISA) with the mCXCR5 with the KLH-conjugated mCXCR5 peptide (Figure 1A).
peptide, followed by flow cytometry using CHO/mCXCR5 Splenocytes from the immunized rat were fused with
and CHO-K1 cells. myeloma P3U1 cells (Figure 1B). Positive wells for the
naked mCXCR5 peptide were identified using ELISA;
2.5. ELISA
further selection was conducted using flow cytometry
The synthesized peptide (MNYPLTLDMGSITYNMDDLC) to identify supernatants that were reactive to CHO/
was immobilized onto immunoplates. After blocking with mCXCR5 cells but non-reactive to CHO-K1 cells
1% bovine serum albumin (BSA)-containing 0.05% Tween20 (Figure 1C). The ELISA assay identified 74 out of 1342 wells
(PBST; Nacalai Tesque, Inc., Kyoto, Japan), the immunoplates (5.5%), which strongly reacted with the naked mCXCR5
were incubated with the hybridoma supernatants, followed by peptide. The flow cytometry analyses identified 18 out of
peroxidase-conjugated anti-rat immunoglobulins (1:20000 the 74 wells (24.3%), which exhibited strong reactivity to
dilution; Sigma-Aldrich Corp., St. Louis, MO, USA). The CHO/mCXCR5 cells while showing no reactivity to CHO-
enzymatic reactions were determined by measuring the K1 cells. After the limiting dilution and several additional

Volume 2 Issue 1 (2025) 103 doi: 10.36922/mi.5664