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Microbes & Immunity A novel anti-EphB2 monoclonal antibody for flow cytometry

G418. This process resulted in the establishment of the 2.4. Flow cytometric analysis
Eph receptors-overexpressed CHO-K1 (e.g., CHO/EphB2) Cells were harvested following brief exposure to 0.25%
clones. In addition, the CHO/PA16-EphB4 cell line had trypsin and 1 mM ethylenediaminetetraacetic acid
been previously established. 43
(Nacalai Tesque, Inc., Japan). After trypsinization, the
CHO-K1 cells, Eph receptors-overexpressed cells were washed with 0.1% bovine serum albumin in
CHO-K1 cells, and P3U1 cells were cultured in a PBS (blocking buffer) and treated with 0.01, 0.1, 1, and
Roswell Park Memorial Institute (RPMI)-1640 medium 10 μg/mL of primary mAbs for 30 min at 4°C. Subsequently,
(Nacalai Tesque, Inc., Japan), supplemented with 10% the cells were treated with Alexa Fluor 488-conjugated
heat-inactivated fetal bovine serum (FBS; Thermo anti-mouse IgG (1:2,000). In addition, the cells were
Fisher Scientific Inc., USA), 100 units/mL of penicillin, suspended in 0.01, 0.1, 1, and 10 μg/mL concentrations
100 μg/mL of streptomycin, and 0.25 μg/mL of of the 2H9. Fluorescence data were collected using the
amphotericin B (Nacalai Tesque, Inc., Japan). LS174T, SA3800 Cell Analyzer (Sony Corp). Cells were gated on
LN229, and EphB2-overexpressed LN229 (LN229/EphB2) the dot plot based on side scatter and forward scatter, and
cells were cultured in Dulbecco’s Modified Eagle Medium the fluorescence intensity was determined using FlowJo
(Nacalai Tesque, Inc., Japan), supplemented with 10% software (BD Biosciences, USA).
FBS, 100 units/mL of penicillin, 100 μg/mL streptomycin,
and 0.25 μg/mL amphotericin B. All cells were grown in 2.5. Determination of dissociation constant (K ) by
D
a humidified incubator at 37°C with an atmosphere of 5% flow cytometry
CO and 95% air. CHO/EphB2 and LS174T cells were suspended in serially-
2
diluted Eb Mabs for 30 min at 4°C. The cells were treated
2.3. Development of hybridomas 2
with Alexa Fluor 488-conjugated anti-mouse IgG (1:200).
The animals were housed under specific pathogen-free The cells were suspended in serially diluted 2H9 conjugated
conditions. All animal experiments were approved by the with RB545. Fluorescence data were collected using the BD
Animal Care and Use Committee of Tohoku University FACSLyric (BD Biosciences, USA). The K was calculated
D
(Permit number: 2022MdA-001). by fitting saturation binding curves to the built-in one-
Two 5-week-old BALB/cAJcl mice were purchased site binding models in GraphPad PRISM 6 (GraphPad
from CLEA Japan (Japan) to develop mAbs against EphB2. Software, USA).
The immunization protocol involved intraperitoneal 3. Results
administration of LN229/EphB2 (1 × 10 cells) mixed
8
with 2% Alhydrogel adjuvant (InvivoGen, USA). This 3.1. Development of anti-EphB2 mAbs using the
initial immunization was followed by 3 additional weekly CBIS method
injections (1 × 10 cells/mouse), culminating in a final Mice were immunized with LN229/EphB2 cells to develop
8
booster intraperitoneal injection (1 × 10 cells/mouse) anti-EphB2 mAb (Figure 1A). The spleen was excised,
8
2 days before harvesting spleen cells. The harvested and splenocytes were fused with myeloma P3U1 cells
spleen cells were subsequently fused with P3U1 cells (Figure 1B). The developed hybridomas were subsequently
using PEG1500 (Roche Diagnostics, USA). Hybridomas seeded into ten 96-well plates and cultivated for 6 days.
were cultured in the RPMI-1640 medium with 10% The positive wells were screened by selecting CHO/
FBS, 5% BriClone (NICB, Ireland), 100 units/mL of EphB2-reactive and CHO-K1-non-reactive supernatants
penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of using flow cytometry (Figure 1C). A total of 133 positive
amphotericin B. For hybridoma selection, hypoxanthine, wells (13.9%) out of 956 wells were obtained. After limiting
aminopterin, and thymidine (Thermo Fisher Scientific the dilution of the positive wells and several additional
Inc., USA) were added to the medium. The supernatants screenings, 12 clones were finally established (Figure 1D).
were subsequently screened using flow cytometry with
CHO/EphB2 and CHO-K1 cells. 3.2. Flow cytometric analysis using anti-EphB2
The cultured supernatant of Eb Mab hybridomas was mAbs
2
then applied to 1 mL of Ab-Capcher (ProteNova, Japan). Eight mouse IgG clones (Eb Mab-1, 2, 3, 4, 7, 8, 10, and
1
2
After washing with phosphate-buffered saline (PBS), the 12) were selected, and the mAbs were purified from the
antibodies were eluted with an IgG elution buffer (Thermo supernatants (Table 1). The specificity of Eb Mabs and 2H9
2
Fisher Scientific Inc., USA). Finally, the eluates were in 14 Eph receptor tyrosine kinases-expressed CHO-K1
concentrated, and the elution buffer was replaced with PBS was were investigated. 2H9 exhibited reactivity to not only
using Amicon Ultra (Merck KGaA, Germany). CHO/EphB2 but also to CHO/EphA4, CHO/EphB1, and

Volume 2 Issue 3 (2025) 170 doi: 10.36922/mi.5728