Detection and purification of two antibody-antigen complexes via selective adsorption on lowly activated anion exchangers.
Taken advantage of the mechanism of adsorption of macro-molecules on ionic exchangers, (a multipoint interaction between the protein and the support), it is possible to selectively adsorb large proteins leaving small ones in the supernatant. Associated proteins should present a significant difference in its size as compared to the non-associated forms. Thus, the protein complexes may have much larger surfaces to interact with the support. Here, by selecting the support with the highest activation degree that was unable to adsorb the non-associated proteins, we have shown the simple and selective adsorption of immuno complexes (as a model), while antibodies and antigens remained in the supernatant. Therefore, it was possible to selectively adsorb on lowly activated supports (e.g., agarose 4BCL having only 1 micromol of amino groups per g of support) rabbit IgG/anti-rabbit immunoglobulins (immuno complex), while these supports were unable to adsorb the individual immunoglobulines. Similarly, horseradish peroxidase (HRP)/anti-HRP were selectively adsorbed on lowly activated supports, while the individual proteins were not adsorbed at all. Afterwards, the adsorbed associated proteins (purified at least from the non-associated counterparts and concentrated by the adsorption on the support) may be cross-linked with aldehyde-dextran and be desorbed from the matrix for their analysis. This strategy may permit very simple experiments to detect the presence of protein-protein complexes. Finally, we have shown the advantages of this technique compared to the use of one of the proteins previous immobilized on a support.
Fuentes
Manuel
M
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Pessela
Benevides C C
BC
Mateo
Cesar
C
Munilla
Roberto
R
Guisán
Jose M
JM
Fernandez-Lafuente
Roberto
R
D000327:Adsorption; D000837:Anion Exchange Resins; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000302:isolation & purification; D002850:Chromatography, Gel
D000837:Anion Exchange Resins; D000936:Antigen-Antibody Complex