The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA.
Antibodies to nuclear antigens (antinuclear antibodies or ANAs) are the serological hallmark of systemic lupus erythematosus (SLE). These antibodies bind diverse nuclear antigens that include DNA, histones and non-histone proteins as well as complexes of proteins with DNA and RNA. Because of the frequency of ANA expression in SLE, testing is an important component of clinical evaluation as well as determination of eligibility for clinical trials or utilization of certain therapies. Immunofluorescence assays have been commonly used for this purpose although this approach can be limited by issues of throughput, variability and difficulty in determining positivity. ELISA and multiplex assays are also useful approaches although these assays may give an incomplete picture of antibodies present. To develop a sensitive and quantitative ANA assay, we have explored an ELISA platform in which plates are pre-coated with a positively charged nucleic acid binding polymer (NABP) to increase adherence of antigens containing DNA or RNA. As a source of antigens, we have used supernatants of Jurkat cells undergoing apoptosis in vitro. As results presented show, a poly-L-lysine (PLL) pre-coat significantly enhances detection of antibodies to DNA as well as antigens such as histones, SSA, SSB and RNP. Comparison of the ELISA assay with the PLL pre-coat with a multiplex assay using the BioPlex® 2200 system indicated good agreement in results for a panel of lupus sera. Together, these studies indicate that a pre-coat with a positively charged polymer can increase the sensitivity of an ANA ELISA using as antigens molecules released from dead and dying cells. This assay platform may facilitate ANA testing by providing an ensemble of antigens more similar in composition and structure with antigens present in vivo, with a NABP promoting adherence via charge-charge interactions.
Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
Nancy A
NA
Stearns
Bio-Rad Laboratories Clinical Diagnostic Group, 400 Alfred Nobel Drive, Hercules, California, United States of America.
Shuxia
S
Zhou
Division of Rheumatology, Johns Hopkins University School of Medicine, 1830 East Monument Street, Suite 7500, Baltimore, Maryland, United States of America.
Michelle
M
Petri
Bio-Rad Laboratories Clinical Diagnostic Group, 400 Alfred Nobel Drive, Hercules, California, United States of America.
Steven R
SR
Binder
Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.|Medical Research Service, VA Medical Center, Durham, North Carolina, United States of America.
David S
DS
Pisetsky
D000974:Antibodies, Antinuclear / Q000737:chemistry*; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D004247:DNA / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D019169:Jurkat Cells; D009707:Nucleosomes / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry*
D000974:Antibodies, Antinuclear; D009707:Nucleosomes; D011107:Polylysine; D004247:DNA; D003850:Deoxyribonuclease I