Purification and stability studies of immunoglobulins from Lachesis muta muta antivenom

Abstract

Immunoglobulins were isolated from hyperimmune horse plasma against Lachesis muta muta venom by ammonium sulfate precipitation and immunoaffinity technique (Sepharosevenom L. m. muta column). When necessary, limited proteolysis with pepsin was used to generate a bivalent antigen-binding fragment (F(ab′) 2). Solutions with immunoglobulins or F(ab′) 2 fragments were fractionated by molecular filtration chromatography (Superose 12) and the expected mol. wt species were observed. The L. m. muta venom shows caseinolytic and haemorrhagic activities. Incubation of the venom with these purified antibodies resulted in a decrease of both activities. High temperatures promote aggregation and the formation of protein precipitates. Sorbitol (1.0 M) was used as an osmolyte (a natural substance or an organic compound capable of stabilizing proteins) and decreased the formation of protein precipitates in solutions of antibodies, as judged by a spectrophotometric assay (280 nm), by nephelometry or when tested by enzyme-linked immunosorbent assay. Circular dichroism was used to study the spectra of antibodies in the presence of phosphate-buffered saline or sorbitol. Up to an osmolyte concentration of 1.0 M, there was no significant perturbation of the F(ab′) 2 fragments spectra in the amide region. However, whole immunoglobulins in the presence of 1.0 M sorbitol presented a small spectral perturbation, suggesting that the β-structure was reinforced. The effect of osmolyte on the affinity of antibodies was observed by enzyme-linked immunosorbent assay. There was no significant difference in the results when the antibodies were previously incubated with venom in phosphate-buffered saline or in the presence of 1.0 M sorbitol. In conclusion, an osmolyte (sorbitol) was shown to be capable of stabilizing antibodies at high temperatures, with no significant perturbation in the secondary structure or affinity to L. m. muta venom. These results point to the possibility of using sorbitol, or other osmolytes, as stabilizers of immunoglobulin preparations.