Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries

J Immunol Methods. 1988 Apr 6;108(1-2):115-22. doi: 10.1016/0022-1759(88)90409-7.

Abstract

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldose-Ketose Isomerases*
  • Animals
  • Antibodies / isolation & purification*
  • Antigens*
  • Brain Chemistry
  • Carbohydrate Epimerases / genetics
  • Cloning, Molecular / methods*
  • Collodion*
  • DNA / isolation & purification
  • Electrophoresis, Polyacrylamide Gel / methods*
  • Immune Sera / isolation & purification
  • Immunoassay / methods*
  • Intermediate Filament Proteins / genetics
  • Neurofilament Proteins
  • Paper
  • Rats

Substances

  • Antibodies
  • Antigens
  • Immune Sera
  • Intermediate Filament Proteins
  • Neurofilament Proteins
  • Collodion
  • DNA
  • Carbohydrate Epimerases
  • Aldose-Ketose Isomerases
  • xylose isomerase