Particulate nitrocellulose as a solid phase for protein immobilization in immuno-affinity chromatography

J Immunol Methods. 1993 Sep 27;165(1):59-66. doi: 10.1016/0022-1759(93)90106-h.

Abstract

The use of nitrocellulose paper as a solid phase matrix for protein immobilization and its application in immunoaffinity chromatography is described. Pieces of nitrocellulose paper were frozen in liquid nitrogen and ground to a powder (NCP) which was then fractionated according to particle size by repeated sedimentation/resuspension cycles in water. The flow properties of different NCP fractions were compared with those of Sephadex G-50. Protein binding capacity and binding dynamics were investigated in a model system with bovine serum albumin (BSA) in phosphate buffered saline. Applications of the material are illustrated by batch chromatography for the removal of anti-carrier protein antibodies from a hapten antiserum and by affinity purification of a hapten-specific antibody fraction using NaSCN elution from haptenated NCP. Furthermore, the applicability of the material in column chromatography is demonstrated by elution of a monospecific fraction of anti-fluorescein antibodies from a hapten/carrier mixed bed column by excess of soluble hapten. The results demonstrate that NCP chromatography appears to be a cheap and useful alternative to many other chromatographic media used for protein immobilization.

MeSH terms

  • Animals
  • Antibodies, Bacterial / isolation & purification
  • Chromatography, Affinity / methods*
  • Collodion*
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli / immunology
  • Immunoblotting
  • Immunoglobulins / isolation & purification
  • Ligands
  • Protein Binding
  • Rabbits

Substances

  • Antibodies, Bacterial
  • Immunoglobulins
  • Ligands
  • Collodion