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Microbes & Immunity A novel anti-EphA8 monoclonal antibody

Accession No.: NM_004438), EphA5 (Catalog No.: 2.2. Antibodies
RC213206, Accession No.: NM_004439), EphA6 (Catalog The Alexa Fluor 488-conjugated anti-mouse
No.: RC223510, Accession No.: NM_001080448), EphA7 immunoglobulin g (IgG) secondary antibody was
(Catalog No.: RC226293, Accession No.: NM_004440), purchased from Cell Signaling Technology Inc. (USA).
EphA10 (Catalog No.: RC218374, Accession No.:
NM_001099439) EphB1 (Catalog No.: RC214301, 2.3. Development of hybridomas
Accession No.: NM_004441), EphB2 (Catalog No.:
RC223882, Accession No.: NM_004442), EphB6 (Catalog To develop anti-EphA8 mAbs, two 6-week-old female
BALB/cAJcl mice purchased from CLEA Japan (Japan)
No.: RC229404, Accession No.: NM_004445), were were immunized intraperitoneally with 1 × 10 LN229/
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purchased from OriGene Technologies Inc. (USA). EphA2
(Catalog No.: HGY095959, Accession No.: NM_004431), PA16-EphA8 cells. The immunogen was harvested after
EphA3 (Catalog No.: HGY053437, Accession No.: a brief exposure to 1 mM ethylenediaminetetraacetic
acid (EDTA; Nacalai Tesque Inc., Japan). For the initial
NM_005233), and EphB3 (Catalog No.: HGX039581,
Accession No.: NM_004443) cDNAs were purchased from immunization, Alhydrogel adjuvant 2% (InvivoGen, USA)
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RIKEN DNA Bank (Japan). was added. Three additional injections of 1 × 10 LN229/
PA16-EphA8 cells were performed without an adjuvant
EphA2 and EphB3 cDNAs were cloned into a pCAGzeo addition every week. A final booster immunization
vector (FUJIFILM Wako Pure Chemical Corporation, was performed with 1 × 10 LN229/PA16-EphA8 cells
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Japan). EphB6 cDNA was cloned into a pCMV6 vector. intraperitoneally 2 days before harvesting splenocytes.
EphA1 cDNA was cloned into a pCAGzeo-ssnPA vector. Splenocytes from the immunized mice were fused with
EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, P3U1 myeloma cells using polyethylene glycol 1,500
and EphB1 cDNAs were cloned into a pCAGzeo_ssnPA16 (PEG1,500; Roche Diagnostics, USA) under warmed
vector. conditions.
The plasmids were also transfected into CHO-
K1 cells, and stable transfectants were established by Hybridomas were cultured in RPMI-1640 medium
staining with specific antibodies: an anti-EphA2 mAb supplemented as shown above, with additional
(clone SHM16; BioLegend, USA), an anti-EphB3 mAb supplementation of hypoxanthine, aminopterin, and
(clone 647354; R & D Systems Inc., USA), an anti-EphB6 thymidine (HAT) (HAT; Thermo Fisher Scientific
mAb (clone T49-25; BioLegend), and an anti-PA16 tag Inc., USA), 5% BriClone (NICB, Ireland), and 5 μg/
mAb (clone NZ-1 for EphA2, EphA3, EphA4, EphA5, mL of plasmocin (InvivoGen, USA). The hybridoma
EphA6, EphA7, EphA10, and EphB1), followed by supernatants were screened by flow cytometry using
sorting using the SH800 cell sorter. After sorting, cells CHO/EphA8 and parental CHO-K1 cells. The culture
were cultivated in a medium containing 0.5 mg/mL of supernatant from Ea Mab-9-producing hybridomas was
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Zeocin (InvivoGen, USA) or 0.5 mg/mL of G418. Eph filtrated and purified using Ab-Capcher Extra (ProteNova,
receptors-overexpressed CHO-K1 (e.g., CHO/EphA1) Japan).
clones were finally established. CHO/PA16-EphB4 cells
were described previously. 31 2.4. Flow cytometric analysis
CHO-K1, P3U1, and CHO-K1 cells overexpressing CHO-K1, CHO/EphA1, CHO/EphA2, CHO/EphA4,
each Eph receptor were cultured in Roswell Park Memorial CHO/EphA6, CHO/EphA7, CHO/EphA8, CHO/
Institute (RPMI)-1640 medium (Nacalai Tesque Inc., EphB1, CHO/EphB6, LN229, and LN229/EphA8 cells
Japan) supplemented with 10% heat-inactivated fetal were harvested after brief exposure to 1 mM EDTA
bovine serum (FBS; Thermo Fisher Scientific Inc., USA), (Nacalai Tesque Inc., Japan). CHO/EphA3, CHO/EphA5,
100 units/mL penicillin, 100 μg/mL streptomycin, and CHO/EphA10, CHO/EphB2, CHO/EphB3, and CHO/
0.25 μg/mL amphotericin B (Nacalai Tesque Inc., Japan). EphB4 cells were harvested after brief exposure to 0.25%
LN229 and LN229/EphA8 cells were cultured in Dulbecco’s trypsin and 1 mM EDTA (Nacalai Tesque Inc., Japan). All
Modified Eagle Medium (Nacalai Tesque Inc., Japan) cells were then washed with 0.1% bovine serum albumin
supplemented with 10% heat-inactivated FBS (Thermo (BSA) in phosphate-buffered saline (PBS), treated with the
Fisher Scientific Inc., USA), 100 units/mL penicillin, primary mAb for 30 min at 4°C, and subsequently treated
100 μg/mL streptomycin, and 0.25 μg/mL amphotericin with Alexa Fluor 488-conjugated anti-mouse IgG (1:1000).
B (Nacalai Tesque Inc., Japan). All cells were cultured in Fluorescence data were collected using the SA3800 Cell
a humidified incubator at 37°C with 5% CO and 95% air. Analyzer (Sony Corp., Japan).
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Volume 2 Issue 4 (2025) 152 doi: 10.36922/MI025060010