Development of a DNA immunoadsorbent: coupling DNA on sepharose 4FF by an efficient activation method.
To remove anti-DNA antibodies from a patient's plasma with systemic lupus erythematosus (SLE), a DNA immunoadsorbent was developed by covalently coupling calf thymus DNA on activated Sepharose 4FF. Sepharose 4FF was activated with 5-norbornene-2,3-dicarboximido carbonochloridate (Cl-CO-ONB), which was proven to be a very effective method for preparation of affinity chromatographic adsorbents. The activation was carried out in dry acetone using 4-(dimethylamine)pyridine (DMAP) and triethylamine (TEA) as catalysts at 4 degrees C or at room temperature. The coupling of DNA to the activated support was investigated as a function of pH, temperature, time, concentration of DNA, and activation level. It was found that the pH for optimal coupling is 3.0, and the amount of coupled DNA increases with an increase either in the concentration of DNA or the activation level. The maximum amount of coupled DNA could reach 1.0 mg DNA/ml support. The incubation of 5 to 20 ml of SLE plasma with 1.0 ml of adsorbent resulted in an 80 to 90% decline in the anti-DNA antibody level. Nonspecific adsorption for normal IgG and total protein is less than 15%.
Kong
D L
DL
R&D Center of Bioengineering, Rostock, Germany.
Schuett
W
W
Boeden
H F
HF
Kunkel
S
S
Holtz
M
M
Matic
G
G
Yamada
R
R
Yu
Y T
YT
Klinkmann
H
H
D000096:Acetone / Q000737:chemistry; D000327:Adsorption; D000818:Animals; D000974:Antibodies, Antinuclear / Q000097:blood; D001672:Biocompatible Materials / Q000737:chemistry*; D001798:Blood Proteins / Q000032:analysis; D002417:Cattle; D002846:Chromatography, Affinity / Q000295:instrumentation; D004247:DNA / Q000737:chemistry; D005021:Ethylamines / Q000737:chemistry; D005782:Gels; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000097:blood; D007164:Immunosorbents / Q000737:chemistry*; D008180:Lupus Erythematosus, Systemic / Q000097:blood; D009636:Norbornanes / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
C417980:4-(dimethylamine)pyridine; D000974:Antibodies, Antinuclear; D001672:Biocompatible Materials; D001798:Blood Proteins; D005021:Ethylamines; D005782:Gels; D007074:Immunoglobulin G; D007164:Immunosorbents; D009636:Norbornanes; D011725:Pyridines; D000096:Acetone; D004247:DNA; D012685:Sepharose; C069687:N-chlorocarbonyloxy-5-norborene-2,3,-dicarboximide; C016162:triethylamine