TY - JOUR
T1 - The use of Poly-L-Lysine as a capture agent to enhance the detection of antinuclear antibodies by ELISA
AU - Stearns, Nancy A.
AU - Zhou, Shuxia
AU - Petri, Michelle
AU - Binder, Steven R.
AU - Pisetsky, David S.
N1 - Funding Information:
This study received funding from VA Merit Review, Alliance for Lupus Research, and National Institutes of Health AR43727. Co-authors Shuxia Zhou and Steven R. Binder received salary from Bio-Rad, a commercial company, during this study. The contributions of Bio-Rad relate to the performance of the comparative study, establishment of cut-off values and review of the manuscript. Bio-Rad did not have any additional role in the study design, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.
Publisher Copyright:
© This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
PY - 2016/9
Y1 - 2016/9
N2 - 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 BioPlex1 2200 system indicated good agreement in results for a panel of lupus sera. Together, these studies indicate that a precoat 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 chargecharge interactions.
AB - 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 BioPlex1 2200 system indicated good agreement in results for a panel of lupus sera. Together, these studies indicate that a precoat 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 chargecharge interactions.
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U2 - 10.1371/journal.pone.0161818
DO - 10.1371/journal.pone.0161818
M3 - Article
C2 - 27611194
AN - SCOPUS:84991228070
SN - 1932-6203
VL - 11
JO - PloS one
JF - PloS one
IS - 9
M1 - e0161818
ER -