Ovine anti-rabies antibody production and evaluation

In view of the disadvantages of human and equine rabies immunoglobulin still there is urgent needs for safe and cost-control anti-rabies immunoglobulins especially for person who have been severely exposed (categories III) to the virus. Our attempt to produce a less immunogenic and cheaper anti-rabies immunoglobulin affordable for those people living in developing countries, has been harnessed the ovine as a bioreactor instead the horse. The animals have been intramuscular immunized, and the plasma processed with 5% caprylic acid to yield IgG with purity of 95%. Moreover, antibody apparently indicated that the titer and neutralizing indexes were harmonized, especially at higher antibody dilution. The results showed that three immunized sheep were produced about 7000 IU of purified anti-rabies antibody. Sheep's IgG has low immunogenic effect than human and horse antibodies when injected into the mouse. Pure concentrated ovine antibody may serve as a possible alternative to currently available anti-rabies human or equine immunoglobulin.     

Production and purification of ovine anti-tetanus antibody

We used the ovine as bioreactor for the production and optimization of anti-tetanus toxin antibody. Four female sheep were immunized with human tetanus vaccine (TT-alum) every two weeks for 16 weeks, after which serum was collected and its titer was estimated by ELISA. The highest titer obtained was 39,000 IU ml-1. To optimize a purification protocol for ovine anti-tetanus toxin, we used four procedures; weak anion (DEAE-Sephadex), weak cation (CM-Sephadex), ammonium sulfate precipitation alone or in combination with caprylic acid. Fifty percent saturation with ammonium sulfate combined with caprylic acid gave us the highest yield of protein with specific activity and the purest Fab product.     

Development of Highly Selective Kv1.3-Blocking Peptides Based on the Sea Anemone Peptide ShK

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC₅₀s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.