Detecting Receptor Tyrosine Kinase ROR1 Using a Developed Anti-ROR1 Polyclonal Antibody.

Receptor tyrosine kinase ROR1 has been introduced as an interesting prognostic cancer marker in histopathology. The aim of this study was to produce a polyclonal antibody (PAb) against recombinant human ROR1 protein to be used as a tool for investigation of ROR1 expression in human cancer tissue blocks. The extracellular part of human ROR1 recombinant protein was expressed using pET-28b(+) plasmid in Escherichia coli Bl21(DE3) host. The recombinant ROR1, as a candidate immunogen, was purified and injected to a New Zealand rabbit. Followed by raising the titration of antibody, polyclonal anti-ROR1 antibody was purified through affinity chromatography column. After determining the purity of PAb anti-ROR1, its specific reactivity was assessed through various assessments. Flow cytometry analysis showed that PAb anti-ROR1 specifically recognizes ROR1 molecule in a number of positive and negative cell lines. Results obtained from detection of ROR1 in paraffin-embedded breast adenocarcinoma tissue blocks (n = 11) also demonstrated that PAb anti-ROR1 can effectively be used in immunohistochemistry. In conclusion, the developed anti-ROR1 PAb can be used as a tool for determining the prognostic value of ROR1 in histopathology of cancer tissues.

Antibody Response to Human Extracellular HER2 Subdomain Proteins in Mice.

BACKGROUND: In addition to passive immunotherapy using anti-HER2 monoclonal antibodies, active immunotherapy via HER2 targeting is an interesting approach to inducing specific anti-tumor immune responses. We have recently reported the immunogenicity of HER2 subdomains following DNA immunization and HER2 protein boosting. In the present study, we evaluated the immunogenicity of different HER2 extracellular subdomains for the induction of anti-HER2 antibody response in BALB/c mice. OBJECTIVE: To investigate and characterize antibody responses to human recombinant proteins of HER2 extracellular subdomains in immunized mice. METHODS: Four subdomains of HER2 extracellular domain were expressed in E.coli; subsequently, purified recombinant proteins were intraperitoneally injected in BALB/c mice with Freund's adjuvant. The anti-HER2 antibody response was detected by ELISA, immunoblotting and flow cytometry. RESULTS: All the four HER2 subdomains along with the full extracellular domain (fECD) were able to induce specific anti-HER2 antibodies. Although anti-HER2 subdomains antibodies could not react with eukaryotic recombinant fECD protein by ELISA, they were able to recognize this protein by immunoblotting under both reduced and non-reduced conditions. Furthermore, only the sera of mice immunized with fECD protein could recognize native HER2 on HER2 overexpressing tumor cells (>99%) by flow cytometry. Moreover, fECD immunized mice sera inhibited the proliferation of tumor cells by XTT assay. CONCLUSION: The prokaryotic recombinant proteins of HER2 extracellular subdomains are immunogenic, yet the induced specific antibodies do not react with the native HER2 protein due to the paucity of post-translation modifications and /or distortion of the native conformation of isolated HER2 extracellular subdomains which might be potentially effective for induction of cell mediated immune response against HER2.