Abstract
Monoclonal antibody MR6F3 (anti-rDKK-1 antibody). DKK-1 antigen______
Antigen Used for Immunization
A chimeric recombinant DKK-1 antigen was designed in silico, expressed and purified in pET-28a and pET-32a prokaryotic expression vectors, and employed for immunization of model animals. Escherichia coli Rosetta-Gami 2 (DE3) bacteria harboring pET-28a/rDKK1 and pET-32a/rDKK1 vectors expressed chimeric recombinant rDKK-1. A 31 and 45 kDa protein bands were observed in SDS-PAGE analysis, due to presence of the thioredoxin tag in pET-32 vector. Chimeric recombinant rDKK-1 (rDKK-1) was verified using anti-rDKK1 polyclonal antibody. The novel r-DKK-1 antigen consisted of the immunogenic part of DKK-1, soluble COMP domain, and p2 and p30 T cell epitopes of tetanus toxin. COMP domain was used to improve the solubility and enhance antigenic presentation, whereas p2 and p30 T cell epitopes were added to overcome the tolerance (Khalili et al., TMU, Tehran).
Method of Immunization
Immunization of animals was carried out in two groups with the 31 and 45 kDa chimeric recombinant DKK-1 antigen, expressed in pET-28a and pET-32a vectors, respectively. In brief, 8-week-old female BALB/c mice (Pasture Institute, Iran) were immunized intraperitoneally. In the first injection, 100 μL (50 μg) of the purified rDKK-1 was mixed with equal volume of Freund's complete adjuvant (Sigma) using an emulsifier syringe. Boosters were injected by a minimum interval of 2 weeks, with 25 μg of rDKK-1 emulsified with incomplete Freund's adjuvant. Noninjected BALB/c mice were used as normal control. The sera of animals were collected at 1 week after the final booster and stored at −20°C for enzyme-linked immunosorbent assay (ELISA) analysis.
Parental Cell Line Used for Fusion
SP2/0 mouse myeloma cell lines.
Selection and Cloning Procedure
Fused cells were added in Roswell Park Memorial Institute containing 1% hypoxanthine-aminopterin-thymidine, 20% FBS, and penicillin/streptomycin, and distributed in 96-well culture plates, so that there are ∼100,000 cells/well. Plates were kept in a humidified (92%–95% RH) CO2 incubator at 37°C for ∼10 days. Then, wells containing the clones were identified by microscopy and their culture medium was exchanged with the hypoxanthine-thymidine medium.
When hybridoma clones occupied about more than half of each well, supernatant was analyzed using ELISA. The optimum antigen concentration (10 μg/mL) was determined using checkerboard titrations. Seven clones with OD value 10 times higher than control were cloned by limiting dilution three times to obtain a homogeneous clone. Clones that produced stable antibodies were frozen with 10% v:v of dimethyl sulfoxide and stored in liquid nitrogen.
Heavy and Light Chains of Immunoglobulin
IgG2b was reported as an isotype for mAb MR6F3, possessing the Kappa (κ) light chain.
Specificity
To determine the specificity of monoclonal antibody MR6F3, recombinant rDKK-1, commercial DKK-2 (the protein most similar to the DKK-1), recombinant thyroid stimulating hormone,(1) and bovine serum albumin (BSA) were coated. Commercial DKK-1 expressed in the eukaryotic system (HEK-293 cells) was used as a positive control and indirect ELISA was performed, as previously described.
Specific Antigen Identified
The purified rDKK-1 protein was confirmed using commercial anti-DKK-1 polyclonal antibody (Elabscience) by Western blot analysis and ELISA. For Western blotting, rDKK-1 protein expressed in pET32a was subjected to 11% SDS-PAGE. Further steps were performed according to the standard procedures. Commercial active human DKK-1 protein (Abcam, United Kingdom), BSA, and recombinant DKK-1 purified through two distinct methods were coated (1 μg/well) onto the wells of polystyrene microtiter plates (Jet Biofil, China), and the absorbance was measured at 450 nm (Malaee et al., TMU, Tehran).