Purification and characterization of Ki antigen and detection of anti‐Ki antibody by enzyme‐linked immunosorbent assay in patients with systemic lupus erythematosus

Abstract

Ki antigen from rabbit thymus extract was purified by ammonium sulfate precipitation, anti-Ki affinity chromatography, and high pressure liquid chromatography gel filtration. The purified Ki antigen gave a single polypeptide with an MW of 32,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it specifically reacted with anti-Ki antibody by immunoblotting. The isoelectric point of the Ki antigen was found by isoelectric focusing to be 4.3. An enzymelinked immunosorbent assay using the purified Ki antigen was established, and the clinical significance of the anti-Ki antibody in systemic lupus erythematosus (SLE) was studied. Thirty of 140 patients with SLE (21.4%) had anti-Ki antibody by enzyme-linked immunosorbent assay, whereas 11 (7.9%) were positive by double immunodiffusion. Analysis of clinical and serologic data on patients with SLE suggested a higher prevalence of central nervous system involvement in patients with anti-Ki antibody.Synthesis of autoantibodies against cellular components such as DNA, RNA, or proteins has been demonstrated in patients with autoimmune diseases

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

“…Next, an IgG fraction of serum AK, prepared by 33% ammonium sulfate fractionation and DE52 (Whatman, Clifton, NJ) ion exchange chromatography, was coupled to CNBr-activated Sepharose 4B (Pharmacia, Piscataway, NJ). This was followed by anti-PCNA affinity chromatography using 2 mg of mAb (TOB7, TO17, and TO30) per ml of Sepharose 4B gel to purify the PCNA (29). Anti-PCNA gels were packed into an EconoColumn (Bio-Rad, Richmond, CA), and PCNA-containing RTE was passed over the column at a rate of 5 ml/hour.…”

“…Purified PCNAs were transferred electrophoretically onto nitrocellulose filters (Bio-Rad), as previously described (2,29). For immunochemical detection of proteins, the filters were first blocked for 24 hours in 3% bovine serum albumin (BSA) in 0.1% Tween-PBS (T-PBS) and then incubated for 2 hours with anti-PCNA-positive mAb in T-PBS (2.5 mg/ml) or with sera obtained from patients (anti-PCNApositive sera, anti-Sm, and U1 RNP serum YT; anti-topo I serum YM) (1:200 dilution in T-PBS).…”

“…ELISAs. ELISAs were performed to test the reactions of the purified PCNAs to anti-PCNA-positive sera and mAb, and to antibodies to various proteins interacting with PCNA (29). Fifty microliters of the purified PCNA solution (5.0 g/ml) was added to the wells of Immunoplate II plates (Nunc, Roskilde, Denmark) and incubated overnight at 4°C.…”

Reactivity of Anti-Proliferating Cell Nuclear Antigen (PCNA) Murine Monoclonal Antibodies and Human Autoantibodies to the PCNA Multiprotein Complexes Involved in Cell Proliferation

“…An IgG fraction of serum AK, prepared by 33% ammonium sulfate fractionation and DE52 (Whatman, Clifton, NJ) ion exchange chromatography coupled to cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia, Piscataway, NJ), followed by anti-PCNA affinity chromatography using 2 mg of mAbs (TOB7, TO17, and TO30) per milliliter of Sepharose 4B gel, were conducted to purify PCNA (30). Anti-PCNA gels were packed into a Bio-Rad Econo-Column (Bio-Rad, Richmond, CA), and PCNA-containing RTE was passed over the column at 5 ml/h.…”

“…After washing with more than three column bed volumes of PBS and 1 M NaCl/ 0.01 M phosphate buffer (pH 7.4), the bound material was eluted with 3 M NaSCN and the eluate was dialyzed against PBS. Ki Ag was purified from RTE by affinity chromatography using IgG separated from anti-Ki serum UC (30) and used as a negative control in ELISA.…”