Mouse Anti-human IL-18 Binding Protein Monoclonal Antibodies (Clones 18-7,29-6,38-3,and 78-4)

Antigen Used for Immunization

Recombinant human IL-18BP a and c proteins were expressed in Escherichia coli BL21 pLysE and in BL21 Codon+ cells that were cultured in LB broth containing 100 μg/mL ampicillin. The cultures were incubated at 37°C with constant shaking until the optical density at 600 nm reached 0.5∼0.6. Human recombinant protein was induced by adding IPTG (0.6 mM) and further cultured at 37°C with constant shaking for 3 h. The bacteria were harvested by centrifugation at 9500 g for 20 min.

The cell pellet was resuspended in 25 mL of buffer A (20 mM Tris–HCl [pH 8.0], 8 M urea) per gram of cells. The cells were disrupted by sonication and solubilized at room temperature (RT) with constant shaking for 1 h. The soluble proteins were harvested by centrifuging at 9500 g and RT for 20 min. The supernatant was dialyzed against buffer B (20 mM Tris–HCl, pH 8.0, filtered) using dialysis bags with a 6–8 kDa molecular weight cut-off. The dialyzed recombinant protein was centrifuged again at 4°C for 20 min in order to remove any aggregated proteins.

In the pPROEx HTa expression vector, the human IL-18BP a and c sequence is followed by the N-termini of six histidines and TEV protease cleavage sites for affinity purification using a Talon affinity chromatography. The supernatant was loaded onto a Talon column (Clontech Laboratories, Mountain View, CA), and the column was washed with 100 column volumes of buffer C (20 mM Tris–HCl [pH 8.0], 300 mM NaCl) containing 0.1% of TritonX-114 for endotoxin removal. Then, the His-tagged protein was eluted using buffer D (20 mM Tris–HCl [pH 8.0], 300 mM NaCl, 300 mM imidazole). The fractions containing the protein of interest were dialyzed against buffer B using the same dialysis bags. The N-termini of six histidines were removed by TEV protease.

The purification of recombinant was further performed by anion exchange chromatography (IEX) using HiTrap Q FF column (GE Healthcare, Pittsburgh, PA), or reverse-phase HPLC (high pressure liquid chromatography) using C4 column. The recombinant protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by mass spectrometry (data not shown).

Method of Immunization

Purified recombinant human IL-18BP a and c proteins were used to immunize female BALB/c mice (6 weeks of age) by subcutaneous injections. The mixtures of Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO) and recombinant proteins were used for the two immunizations with 2-week intervals. The mixtures of Freund's incomplete adjuvant and recombinant proteins were used for the third immunization. Mice were bled 10 days after each boost. The titer of mouse serum was determined by direct enzyme-linked immunosorbent assay (ELISA). The last boost of antigen was injected 3 days before a fusion.

Parental Cell Line Used for Fusion

BALB/c myeloma cell line FO was obtained from ATCC (Manassas, VA) and fused with splenocytes from immunized mice according to standard protocols.

Selection and Cloning Procedure

For screening of antibody producing hybridoma clones, recombinant human IL-18BP a and c were used as antigens with direct ELISA. The hybridoma cells of positive wells were cloned by limiting dilution in aminopterin-free selection Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. The single clone was screened by direct ELISA, dot blotting, and Western blotting for its specificity to each human IL-18BP isoform. A single hybridoma clone was expanded for further purification. Anti-human IL-18BP a and c IgGs were purified from hybridoma cell culture supernatants using protein G agarose (KPL, Gaithersburg, MD).

Heavy and Light Chains of Immunoglobulin

Not determined.

Specificity

Specific Antigen Identified

Not determined.

Availability