The Preparation of Chromatographically Purified Anti-Smallpox Immunoglobulin from Equine Blood Serum
The aim of the work was to obtain chromatographically purified anti-smallpox immunoglobulin from equine blood serum and evaluate its properties. Materials and methods . The horse blood serum containing antibodies to the vaccine virus, L-IVP strain, was used. Сhromatographic purification of serum was carried out on Akta Avant preparative chromatograph (GE HealthCare, Sweden), molecular parameter control at the stages of immunoglobulin production was conducted on the LC-20ad HPLC system (Shimadzu, Japan). The biological properties of immunoglobulin were evaluated in rabbits, white mice, and guinea pigs. The level of specific virus neutralizing antibodies was determined in the neutralization reaction on the chorionallantoic membrane (CAМ) of 12-day chicken embryos. Results and discussion . The technology for obtaining chromatographically purified anti-smallpox immunoglobulin from equine blood serum has been developed. At the initial stage, the immune blood serum of horses was purified by filtration through glass fiber cassette-type filters from fibrin sediment, residual quantity of lipids and lipoproteins, and then the immunoglobulin fraction was precipitated with a saturated solution of ammonium sulfate. At the next stage, ammonium salts, low molecular weight fractions, residual quantity of albumins and euglobulins were removed using ion exchange chromatographic purification. At the final stage, dimers, polymers, and aggregates of immunoglobulins were eliminated using gel filtration. The experimental series of immunoglobulin prepared using the developed technology is characterized by a high degree of purification. The monomer content in the finished product is at least 99 %, and the specific activity by titer of specific neutralizing antibodies – at least 1:16000.