A Novel Method for Reproducible Fluorescent Labeling of Small Amounts of Antibodies on Solid Phase

J Immunol Methods. 2007 Apr 30;322(1-2):40-9. doi: 10.1016/j.jim.2007.01.023. Epub 2007 Feb 28.

Abstract

Fluorescently labeled antibodies are very important tools in cell biology, providing for specific and quantitative detection of antigens. To date, fluorophore labeling of antibodies has been performed in solution and has been limited by low-throughput methods requiring a substantial amount of pure antibody sample at a high concentration. We have developed a novel solid-phase labeling protocol for small amounts (i.e. micrograms) of antibodies with fluorescent dyes. Protein A affinity medium was used as solid support in a micropipette tip format. This solid-phase approach, including the advantage of the strong and specific interaction between Protein A and antibodies, allows for simultaneous purification, labeling and concentration of the antibody sample, making it possible to start with unpure antibody samples at low concentrations. We have optimized the protocol with regard to reaction pH, time, temperature and amount of amine reactive dye. In addition, we have evaluated the stability and activity of the labeled antibodies. To evaluate the reproducibility and robustness of this method we labeled eight antibodies with amine reactive fluorescent dyes followed by evaluation of antibody specificity on protein arrays. Interestingly, this gave an extremely high conformity in the degree of labeling, showing the robustness of the method. The solid-phase method also gave predictable and reproducible results and by varying the amount of reactive dye, the desired degree of labeling can easily be achieved. Antibodies labeled using this solid-phase method were similar in stability and activity to antibodies labeled in solution. This novel solid-phase antibody labeling method may also be applicable for other conjugation chemistries and labels, and has potential for high-throughput applications.

MeSH terms

  • Antibodies / chemistry*
  • Cyclic AMP / chemistry*
  • Fluorescent Dyes / chemistry*
  • Humans
  • Hydrogen-Ion Concentration
  • Immunohistochemistry
  • Protein Array Analysis
  • Reproducibility of Results
  • Staining and Labeling / methods*

Substances

  • Antibodies
  • Fluorescent Dyes
  • Cyclic AMP