Molecular studies of rheumatoid factor using pseudobioaffinity membrane chromatography.
Using pseudobioaffinity ligand L-histidine immobilized to poly(ethylene vinyl alcohol) hollow fiber membrane is an interesting approach for the purification of total IgG and its subclasses from untreated serum in a single step. Gentle adsorption and elution conditions of this chromatography system allow efficient recovery of the protein in its native form. This approach was employed for the recovery and molecular study of rheumatoid factor (RF), an anti-IgG autoantibody (AAb) that form immune complexes with autologous IgG Abs in the sera. The purity of the recovered molecule was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), revealed a 150-kDa IgG band and an additional approximately 300 kDa band which may be RF bound IgG complex. Since RF is an AAb, the purified protein was studied for its catalytic functions like peptide, DNA, and RNA hydrolyzing activities. The substrate Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) hydrolyzing activity by total IgG from different patient sera was found to be greater than healthy controls. In an effort to identify the subclass specificity for the proteolytic function, the pre-purified total IgG fractions from rheumatoid arthritis (RA) sera were subjected to rechromatography using a discriminating buffer. In this experiment, the activity was found in the non-retained fractions suggesting IgG2 specificity for the catalytic function. A comparative study between different catalytic functions was performed for IgG separated from individual patient.
Kamalanathan
A S
AS
Centre for Bioseparation Technology, VIT University, Vellore 632 014, Tamil Nadu, India.
Vijayalakshmi
M A
MA
D000888:Antibodies, Anti-Idiotypic / Q000276:immunology* / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D008566:Membranes / Q000737:chemistry*; D012313:RNA / Q000378:metabolism; D012217:Rheumatoid Factor / Q000276:immunology / Q000302:isolation & purification*; D012260:Ribonucleases / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D000936:Antigen-Antibody Complex; D001323:Autoantibodies; D007074:Immunoglobulin G; C048844:anti-IgG; D006639:Histidine; D012313:RNA; D004247:DNA; D012217:Rheumatoid Factor; D012260:Ribonucleases