Affinity fractionation of lymphocytes using a monolithic cryogel.
A new type of continuous, supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing specific fractionation and separation of human peripheral blood lymphocytes in a chromatographic format. The affinity adsorbent was used to design a novel cell separation strategy, which was based on the interaction of protein A from Staphylococcus aureus with cells bearing IgG antibodies on the surface. After treating lymphocytes with goat anti-human IgG(H+L), the IgG-positive B-lymphocytes were efficiently separated from T-lymphocytes. Protein A covalently coupled to epoxy activated dimethylacrylamide (DMAA) cryogel matrix specifically bound IgG-bearing B-lymphocytes through the Fc region, while non-bound T-lymphocytes passed through the column. More than 90% of the B-lymphocytes were retained in the column while the cells in the breakthrough fraction were enriched in T-lymphocytes (81%). The viability of the T-lymphocytes isolated was greater than 90%. The bound lymphocytes released by human or dog IgG recovered 60-70% of the B-cells without significantly impairing the cell viability. The technique can be applied in general to cell separation systems where IgG antibodies against specific cell surface markers are available.
Kumar
Ashok
A
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.
Plieva
Fatima M
FM
Galaev
Igor Yu
IY
Mattiasson
Bo
B
D001798:Blood Proteins; D002458:Cell Fractionation / Q000379:methods*; D002846:Chromatography, Affinity; D059985:Cryogels; D005353:Fibronectins; D005434:Flow Cytometry; D006801:Humans; D020100:Hydrogels; D008214:Lymphocytes / Q000166:cytology*; D013205:Staphylococcal Protein A
D001798:Blood Proteins; D059985:Cryogels; D005353:Fibronectins; D020100:Hydrogels; D013205:Staphylococcal Protein A