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

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 [Pepantisera] 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

Expression analysis of RNA-binding motif gene on Y chromosome [RBMY] protein isoforms in testis tissue and a testicular germ cell cancer-derived cell line [NT2]

Background: RNA-binding motif gene on Y chromosome [RBMY], a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages

Methods: full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms

Results: selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate

Conclusion: the results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility. Iran. Biomed. J. 17[2]: 54-61, 2013