Detection of glycoprotein receptors on blotting membranes by binding of live bacteria and amplification by growth

Anal Biochem. 1995 Jan 1;224(1):390-4. doi: 10.1006/abio.1995.1055.

Abstract

Conditions have been adapted for detecting bacteria bound to glycoprotein receptors on blotting membranes using a self-enhancing detection method based on bacterial growth. Neutrophil plasma membrane proteins, mediating adherence of mannose-binding type-1-fimbriated Escherichia coli and concanavalin A (Con A) to intact human neutrophils, were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was immersed in a suspension of mannose-binding type-1-fimbriated E. coli, and after repeated washings, bound bacteria were allowed to multiply into bacterial colonies by placing the membrane on a solid nutrient substratum. About one major and eight minor glycoproteins, some of which also were detected by Con A, selectively induced colony formation in a mannose-inhibitable fashion. Binding of [35S]methionine metabolically labeled E. coli to PVDF membranes produced a virtually identical binding pattern, demonstrating further the accuracy of this self-enhancing detection method which is rapid, simple, and sensitive and avoids radioisotopes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Adhesion*
  • Concanavalin A / metabolism
  • Escherichia coli / growth & development
  • Humans
  • Membrane Glycoproteins / metabolism
  • Neutrophils / microbiology
  • Platelet Glycoprotein GPIb-IX Complex*
  • Platelet Membrane Glycoproteins*
  • Polyvinyls
  • Receptors, Cell Surface / analysis*

Substances

  • Membrane Glycoproteins
  • Platelet Glycoprotein GPIb-IX Complex
  • Platelet Membrane Glycoproteins
  • Polyvinyls
  • Receptors, Cell Surface
  • glycoprotein receptor GPIb-IX
  • Concanavalin A
  • polyvinylidene fluoride