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 [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
ABSTRACT
Demyelination of CNS axons occurs under pathological conditions such as multiple sclerosis and spinal cord injuries, but can be repaired by cell therapy. Within the CNS remyelination can be achieved by transplantation of neural stem cells [NSCs]. NSCs are self-renewing cells that maintain the capacity to differentiate into CNS-specific cell types and can differentiate into the three main neural phenotypes: astroglia, oligodendroglia and neurons. They may also replace or repair diseased CNS tissue. Bone marrow stromal cells [BMSCs] were aseptically isolated from the tibia and femurs of young adult Sprague Dawley rats. BMSCs were evaluated by fibronectin and CD31 markers. BMSC-derived NSCs were evaluated by nestin and NF-68. An ethidium bromide-induced demyelinated dorsal column lesion was produced in young adult rats. Transplanting NSCs derived-BMSCs into demyelinated lesions after 3 days in adult rat spinal cords was done. Three weeks after transplantation of NSCs, the spinal cords were processed to evaluate remyelination by Luxol fast blue staining. After passage 3, BMSCs were evaluated and the result, showed the percentage of immunoreactive cells to fibronectin [94.7 +/- 2.65], however BMSC-derived NSCs expressed nestin [86.15 +/- 0.64] and NF-68 [84.55 +/- 0.94] which correlated with fibronectin down regulation. Histologically, the lesions showed slightly irregular elongated areas and had an average length of 1336.36 +/- 39.43 microm. Transplanted NSCs were capable of eliciting remyelination. These data support the conclusion that transplantation of NSCs results in functional remyelination of a dorsal column lesion and have valuable applications in the treatment of neurodegenerative diseases such as spinal cord injuries