Abstract
Objective: This study attempted to generate monospecific antibodies through immunization with recombinant proteins and subsequent purification by synthetic peptides (the PrIPeP model).
Methods: The SRY gene was cloned on a pet-28a vector and the recombinant protein was expressed in the Escherichia coli (E.coli) BL21 strain. The purified antigen was emulsified in Freund’s adjuvant and injected into rabbits according to a standard time table. Then, a specific peptide was designed, synthesized, and conjugated to sepharose 4B to generate an affinity purification column. As a control, the peptide was conjugated to KLH and used for immunization, as above. Antisera against the conjugated peptide (Pep-antisera) and SRY recombinant protein (Pro-antisera) were evaluated by ELISA and subsequently subjected to the affinity purification column. Sensitivity and specificity of the purified antibodies against SRY recombinant protein as well as negative controls (recombinant HSFY, RBMY, and RPSFY) were assessed by Western blot analysis.
Results: Titration by ELISA confirmed proper immunization and specificity of both antigens. Western blot analysis validated the specificity and sensitivity of the IgG class purified antibodies.
Conclusion: By applying the PrIPeP model, it is possible to develop antibodies against the native structure of a protein whilst avoiding challenges of peptide-carrier protein conjugation.
Methods: The SRY gene was cloned on a pet-28a vector and the recombinant protein was expressed in the Escherichia coli (E.coli) BL21 strain. The purified antigen was emulsified in Freund’s adjuvant and injected into rabbits according to a standard time table. Then, a specific peptide was designed, synthesized, and conjugated to sepharose 4B to generate an affinity purification column. As a control, the peptide was conjugated to KLH and used for immunization, as above. Antisera against the conjugated peptide (Pep-antisera) and SRY recombinant protein (Pro-antisera) were evaluated by ELISA and subsequently subjected to the affinity purification column. Sensitivity and specificity of the purified antibodies against SRY recombinant protein as well as negative controls (recombinant HSFY, RBMY, and RPSFY) were assessed by Western blot analysis.
Results: Titration by ELISA confirmed proper immunization and specificity of both antigens. Western blot analysis validated the specificity and sensitivity of the IgG class purified antibodies.
Conclusion: By applying the PrIPeP model, it is possible to develop antibodies against the native structure of a protein whilst avoiding challenges of peptide-carrier protein conjugation.
| Translated title of the contribution | Introduction of a new method for mono-specific antibody production by sequential use of recombinant proteins and synthetic peptides (PrIPeP model) |
|---|---|
| Original language | Arabic |
| Pages (from-to) | 33-44 |
| Number of pages | 12 |
| Journal | Pathobiology Research |
| Volume | 18 |
| Issue number | 4 |
| Publication status | Published - 2016 |
| Externally published | Yes |