Cryoprotection of antibody by organic solutes and organic solute/divalent cation mixtures

Abstract

Antibodies are globular glycoproteins that protect animals from microbial and toxic insult. These proteins have proven to have substantial commercial and research value but are variably susceptible to freeze-thaw damage, thus limiting their usefulness. Several carbohydrates and divalent cations were examined alone and in combination to determine whether they could protect antibody from freeze-thaw damage. The amino acid proline was also tested. Two antibodies, derived from different sources and specific for different antigens, were tested by a direct enzyme-linked immunosorbent assay (ELISA). Confirmation of antibody freeze-lability was obtained by rocket electrophoresis and radial immunodiffusion tests. Neither carbohydrate nor divalent cation alone fully protected antibody activity from freeze-thaw damage. However, several combinations protected antibody activity completely when compared to their effect on antibody maintained at room temperature. In the case of affinity-purified antibody, full protection of antibody activity relative to an untreated control was obtained. In several instances, cryoprotection of antibody by solute-divalent cation combinations was synergistic and not an additive effect of each component. Alkaline phosphatase, an enzyme typically linked to antibody for an ELISA, was not freeze-thaw labile. These results indicate that antibody function can be fully protected from damage due to freeze-thaw treatment by organic solutes in combination with divalent cations.