A Mouse Monoclonal Antibody Specific for Calreticulin

Reagents and cell lines

Freund's adjuvant, bovine serum albumin (BSA), polyethylene glycol 4000 (PEG 4000, 50%w/v), 3,3’,5,5’-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (St.Louis, MO). Hypoxanthine/aminopterin/thymidine (HAT) and hypoxanthine/thymidine (HT) were from Roche (Indianapolis, IN), and DMEM with L-glutamine culture medium and fetal bovine serum were purchased from Gibco (Invitrogen, Carlsbad, CA). Peroxidase-labeled goat anti-mouse IgG(H+L) (HRP-IgG) was from Jackson Immunoresearch Laboratories (West Grove, PA).

Mouse melanoma B16-F1 cells, mouse lymphoma EL4 cells, and mouse myeloma cell line SP2/0 cells were cultured in RPMI-1640 medium (for B16-F1 and EL4 cells) or Dulbecco's modified Eagle's medium (for SP2/0 cells) supplemented with 20% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin in a humidified 5% CO₂ and 95% air incubator at 37°C.

Expression and purification of recombinant CRT protein

The construction of pET-28a(+)-dCRT plasmid has been described previously.⁽¹¹⁾ Escherichia coli BL21 (DE3) cells were transformed with pET-28a(+)-dCRT, and the recombinant protein was induced at 30°C for 12 h under 0.1 mM IPTG. The cells were harvested, resuspended in lysis buffer (PBS containing 0.5%Triton X-100 and 1 mM PMSF), and disrupted by sonication. The recombinant protein was purified by affinity chromatography using Ni-NTA agarose resin (Amersham Pharmacia Biotech, Piscataway, NJ) as recommended by the manufacturer. Protein concentrations were determined using a Bradford Assay kit, and purified proteins were stored at −20°C.

Immunization and preparation of monoclonal antibody

Anti-CRT mouse MAbs were generated based on the murine lymphocyte method. A 5-week-old female BALB/c mouse was injected via the hind footpads with 200 μL of an emulsion containing 100 μg of recombinant dCRT protein and Freund's complete adjuvant. Booster doses of dCRT protein (50 μg) in Freund's incomplete adjuvant were then injected into the above mouse once every 2 weeks for three immunizations. After the final booster immunization, the murine splenocytes immunized with an antigen were fused with murine myeloma Sp2/0 cells at a ratio of 5:1 in a 50% polyethylene glycol (PEG 4000 solution). The resulting hybridoma cells were plated onto 96-well plates and cultured in HAT selection medium. Hybridoma colonies were expanded in the HT medium at 10 days post-fusion. The hybridoma supernatants were screened by means of ELISA against the CRT-His recombinant protein. Positive clones were subcloned and rescreened by ELISA and immunoblotting. A total of ascitic fluid in BALB/c female mice was prepared for the interesting colonies.

ELISA screening

CRT protein (100 μL/well, 1.0 μg/mL) in 0.05 M carbonate buffer (pH 9.6) was coated on the surface of a 96-well flexible microplate (Nunc, Roskilde, Denmark) by overnight incubation at 4°C. To avoid unspecific binding, the plates were blocked with 1% bovine serum albumin (BSA) in PBST (a PBS buffer containing 0.05%Tween-20 [v/v]). Hybridoma supernatants (100 μL) were incubated for 30 min at 37°C and then washed with PBST three times. The plates were incubated for 30 min at room temperature with horseradish peroxidase (HRP)-conjugated anti-murine IgG antibody (diluted 1:3000). After washing with PBST three times, ELISAs were developed using 100 μL of TMB substrate, stopped with 50 μL of 2M H₂SO₄, and read at 450 nm.

Western blot analysis

The plasmid pcDNA3.1(+) or pcDNA3.1(+)-dCRT was transfected into B16-F1 cells with LipofectamineTM 2000 reagent, according to the manufacturer's instruction. B16-F1/pcDNA3.1(+) and B16-F1/pcDNA3.1(+)-dCRT cells were collected and lysed in SDS sample buffer. Lysates were boiled and equal amounts of proteins were subjected to 10% SDS-PAGE and electro-transferred onto a PVDF member, which was blocked with 5% non-fat milk in TBST overnight at 4°C. The membrane was then incubated at room temperature for 1 h with the prepared monoclonal 7C3; followed by HRP-labeled goat anti-mouse IgG for 1 h. The blot was developed using an ECL Plus Western Blotting Detection System (GE Healthcare, Pittsburgh, PA) and exposed to a piece of BioMax x-ray film (Kodak, New Haven, CT).

Immunofluorescence staining

For detection of CRT protein on the surface of drug-treated cells by immunofluorescence (CIF), B16-F1 cells were grown on cover slips in each culture medium and treated by the antitumor drug mitoxantrone (1 μmol/L) for 48 h. Then the cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and treated with a blocking solution (PBS containing 50 mM glycine and 1% BSA). The cells were incubated with monoclonal antibody 7C3 and detected with rhodamine-conjugated secondary antibody. 4′,6-Diamidino-2-phenylindole (DAPI) at 1 μg/mL was used to counterstain the nuclei. The samples were examined using an Eclipse TE2000-S inverted fluorescent microscope (Nikon, Tokyo, Japan).

Flow cytometry

CRT expression on the surface of the tumor cells was detected by flow cytometry (FCM) using the monoclonal antibody 7C3. The EL4 cells were treated by the antitumor drug mitoxantrone (1 μM/L) for 48 h, collected, and used for membrane CRT detection. The cells were incubated with the MAb 7C3 at room temperature for 30 min, followed by incubation with the FITC-conjugated anti-murine IgG antibody (1:1000) at room temperature for 1 h (avoid light). After washing, the cells were analyzed by EPICS XL-4 flow cytometer (Beckman-Coulter, Fullerton, CA) to identify CRT on the EL4 cell surface.

Expression and purification of recombinant protein CRT

In order to obtain the CRT antigen, we constructed pET28a(+)-dCRT and expressed and purified the dCRT protein. Figure 1 shows the SDS-PAGE results of purified 34 kDa dCRT, which was fused with a 6x His to the CRT C-terminal. The protein was identified by Western blot using a His antibody (Fig. 1B).

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FIG. 1.

Expression and purification of mouse recombinant calreticulin in prokaryotic cells. (A) Analysis of the purified dCRT protein by 10% SDS-PAGE and stained with Coomassie brilliant blue R250. Lane 1, protein molecular weight marker (in kDa); lane 2, dCRT protein expression without IPTG induction; lane 3, dCRT protein expression with IPTG induction; lanes 4 and 5, purified dCRT through affinity chromatography using Ni-NTA agarose resin. (B) dCRT protein was resolved by SDS-PAGE and immunoblotted for the presence of His epitope. Lane 1, dCRT protein expression without IPTG induction; lane 2, dCRT protein expression with IPTG induction; lane 3, purified dCRT. Prestained protein molecular weight marker was used in the immunoblotting.

Sensitivity and specificity determination of anti-CRT MAb

Monoclonal antibody 7C3 was prepared through standard immunization, cell fusion, and antibody screening. After purification and concentration, the interesting antibody 7C3 was evaluated for its affinity to CRT protein. Purified CRT antibody was diluted in a 2-fold series for sensitivity test by ELISA. The result showed that the sensitivity of MAb 7C3 was over 10⁵ dilution (Fig. 2A). We also evaluated the ability of MAb 7C3 to recognize endogenous and transfected CRT by Western blot. MAb 7C3 recognized a 46 kDa protein that exists in a variety of eukaryotic cells (Fig. 2B) and a recombinant dCRT that was expressed in the transfected cells (Fig. 2C). This means that MAb 7C3 appears to have an affinity for natured CRT.

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FIG. 2.

Evaluation of anti-CRT monoclonal antibody (MAb 7C3) to the endogenous and transfected CRT by ELISA and Western blot. (A) Sensitivity of anti-CRT MAb 7C3 was assayed by ELISA. Hybridoma ascites were prepared and tested at 2-fold series dilution in PBS+1% BSA on pre-blocked plates, coated with 250 ng per well of purified CRT. The antibody had very high affinity to the CRT and the dose-dependent manner was observed at 10⁴–10⁶ dilution of the antibody. (B, C) Specificity of anti-CRT MAb 7C3 was identified by Western blot. (B) We checked the endogenous CRT of COS-7 cells, which are derived from kidney cells of the African green monkey, and A-549 cells, which are adenocarcinomic human alveolar basal epithelial cells. (C) After transfection of pcDNA3.1(+)-dCRT (deleted C-terminal CRT) the recombinant dCRT protein was observed in transfected B16-F1 cells.

Measurement of CRT expression on membranes of B16-F1 and EL4 cells

These antibodies were suitable for recognizing the membrane CRT by flow cytometry and immunofluorescence. Red fluorescence on the membrane of B16-F1 and EL4 cells was observed by immunofluorescence using the antibody 7C3 (Fig. 3), which was consistent with the previous report. When B16-F1 and EL4 cells (Fig. 3A,B) were treated by the antitumor drug mitoxantrone, apoptosis⁽¹²⁾ was induced; at the same time, the CRT quickly relocated from the ER lumen onto the cell surface. In summary, we have generated a new monoclonal antibody, 7C3, that specifically recognizes CRT protein by Western blot analysis, flow cytometry, and immunofluorescence. Preparation of the 7C3 antibody will be helpful to elucidate CRT functions.

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FIG. 3.

Reactivity of MAb 7C3 to ecto-CRT by immunofluorescent staining and flow cytometry. (A) B16-F1 cells were grown on cover slips and treated with mitoxantrone (1 μmol/L) for 48 h. Then the cells were fixed with 4% paraformaldehyde, incubated with monoclonal antibody 7C3, and detected with rhodamine-conjugated secondary antibody. DAPI were used to stain cell nucleus. No special fluorescence was found on normal cell (400×). (B) EL4 cells were treated by antitumor drug mitoxantrone (1 μg/mL) for 48 h and used for membrane CRT detection. The MAb 7C3 was diluted at 2-fold series and FITC-conjugated anti-murine IgG antibody was used. The stained cells were analyzed by flow cytometer and gated according to isotype antibody. Meanwhile untreated EL4 cells were used as control. The results are representative of three independent experiments.