Introduction of a new method for mono-specific antibody production by sequential use of recombinant proteins and synthetic peptides (PrIPeP model)

Achieving mono-specific antibodies through immunization with recombinant proteins and subsequent purification by synthetic peptides (PrIPeP model).
SRY gene was cloned on Pet-28a vector and the recombinant protein was expressed in E. Coli, BL21 strain. The purified antigen was emulsified in Freund adjuvant and injected to rabbits according to a standard time table. Then, a specific peptide was designed, synthsized and conjugated to sepharose 4B for making affinity purification column. As a control, the peptide was conjugated to KLH and used for immunization as above. Antisera against conjugated peptide (Pep-antisera) and SRY recombinant protein (Pro-antisera) were evaluated by ELISA and then subjected to 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.
Titration by ELISA experiment confirmed proper immunization and specificity of both antigens. Moreover, specificity and sensitivity of IgG class purified antibodies were validated by western blot analysis.
By applying PrIPeP model, it is possible to develop antibodies which were raised against native structure of a protein whilstavoiding challenges of peptide-career protein conjugation.