Abstract
Antigen Used for Immunization
The carboxyl terminal processing protease of D1 protein (CtpA) gene of spinach was amplified by standard PCR method from spinach cDNA and constructed into pET-28a vector to give a recombinant expression plasmid. Recombinant CtpA fusion protein with His-tag was expressed as soluble protein in Escherichia coli BL21(DE3) after induction with 0.1 mmol/L IPTG at 8°C for 72 h. The protein was purified by Ni-NTA affinity chromatography and Superdex 75 gel filtration chromatography and verified by SDS-PAGE and Western blot analysis with anti-His antibody. Recombinant CtpA was used as antigen to immunize BALB/c mice for the production of monoclonal antibody.
Method of Immunization
Six-week-old BALB/c female mice were injected intraperitoneally three times with 0.2 μg rCtpA at 2 weekly intervals. Complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO) was used in the first immunization and incomplete Freund's adjuvant was used in the subsequent 2 × booster shots. A final immunization with 0.2 μg rCtpA only was given intravenously 3 days before euthanasia.
Parental Cell Line Used for Fusion
Hybridoma was prepared by fusion of immunized mouse splenocytes with mouse myeloma cells SP2/0 with ethylene glycol∼4000.
Selection and Cloning Procedure
ELISA was performed to screen out one line of positive hybridomas. Positive clones were cloned three times by limiting dilution using spleen feeders as filter cells. Positive clone was propagated in vitro and in vivo. After i.p. injection with the hybridomas, ascites were gathered and ELISA was performed to measure its antibody titer.
Heavy and Light Chains of Immunoglobulin
After multiple-step purification, the monoclonal antibody F4 pretreated with 2.5% SDS and 10% β-mercaptoethanol appears only as a band of light chain (26,000 ± 1,000) and a band of heavy chain (50,000 ± 1,000) on 15% SDS-PAGE electrophoresis pattern.
Specificity
Monoclonal antibody specificity to CtpA was evaluated by ELISA with titers ranging between 1 × 104 and 1 × 105. The specificity of the MAbs obtained was further investigated by Western blot. The Western blot analyses revealed that the MAbs were directed against the rCtpA and PDZ domains, while they had no reactivity with the BL21(DE3) cell lysate containing pET-28a(+). Signals at approximately 45 kDa and 15 kDa were observed, consistent with the expected molecular weight of the recombinant proteins of CtpA and PDZ. The results also showed that the activity with rCtpA was stronger than that with the PDZ domain. The affinity of MAbs to rCtpA was measured by the ELISA described using serial dilutions of MAbs. The results showed that the purified MAbs had a high titer with the proteins, consistent with the results of Western blot. The results of ELISA showed that the MAbs had a much stronger degree of binding to rCtpA than to the PDZ domain with the same molar concentration.
Specific Antigen Identified
The antigen is not evaluated yet; parts of the antigen determinant of CtpA may be located in the PDZ domain.