The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA

Abstract

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.

Discovery of IgG4 Anti‐Gliadin Autoantibody as a Potential Biomarker of Psoriasis Using an Autoantigen Array

“…Enzyme‐linked immunosorbent assay (ELISA) microplates (Nunc MaxiSorp flat‐bottom) were precoated with poly‐ l ‐lysine at 2 µg mL −1 in 4 °C overnight prior to the experiment . Recombinant wheat gliadin protein (Abcam) was then coated onto the plate at 2 µg m L −1 , 100 µL per well at 37 °C and incubated on a shaker (New Brunswick Scientific) for 30 min.…”

SLE, An Overlooked Disease: Possibilities for Early Rescue by Early Diagnosis

“…ELISA is one of the most renowned techniques that enjoy a high versatility that allowed its use in a variety of biomedical applications including the detection of ANAs and anti-DNA antibodies in patients with SLE and other autoimmune diseases [102][103][104][105][106][107]. ELISA is based on coating a solid surface with the antigens of interest such as DNA substrates or an array of nuclear antigens so that they are tightly bound and can withstand subsequent washing steps.…”

Evaluation of a standardised Vi poly-l-lysine ELISA for serology of Vi capsular polysaccharide antibodies

“…The latter observation agrees with previous studies that reported poor binding of bacterial polysaccharides to the micro-plate surface resulting in inconsistent ELISA results, which can be mitigated by pre- or co-coating the polysaccharide with a protein or by chemical modification of the polysaccharide [ 20 , 22 , 23 ] . Poly- l -lysine is a representative of nucleic acid binding polymers and has been used to bind DNA and RNA avidly on the basis of their charge and thus increase their adherence to the solid phase [ 24 ]. Like these polymers, polysaccharides have a negative charge and a PLL coating will capture these molecules efficiently.…”