Fig 1.
The effect of nucleic acid binding polymers on anti-DNA assays.
ELISA plate wells were pre-coated with 2 μg/ml of PAMAM, HDMBr, PLL, or protamine sulfate, and then used to capture native calf thymus (CT) DNA at concentrations from 10 ng/ml to 5,000 ng/ml. Control was incubated with buffer alone. The same concentrations of CT DNA were coated onto plate wells without polymer pre-coat. Binding by antibodies in SLE plasmas 1, 2 and 3 at 1/1,600 dilution was assayed in a standard ELISA assay. Data points represent the average OD450 value of two wells except for the data point for 2,000 ng CT DNA coated directly on plate detected with no plasma. That data point represents the OD450 value of only one well. Squares show data for plasma 1, diamonds for plasma 2, triangles for plasma 3, and circles for buffer alone. The following are shown: (A) DNA coated directly on plate; (B) DNA with a PAMAM pre-coat; (C) DNA with an HDMBr pre-coat; (D) DNA with a PLL pre-coat; and (E) DNA with a protamine sulfate pre-coat.
Fig 2.
The effect of PLL pre-coat on assays of anti-nucleosome antibodies.
ELISA plate wells were pre-coated with 2 μg/ml PLL, and then used to capture nucleosomes at various concentrations (determined from OD260 readings) from 10 ng/ml to 5,000 ng/ml, or buffer alone as control. The same concentrations of nucleosomes were directly coated onto plate wells. Binding by antibodies in three SLE plasmas was determined by ELISA. Each point is the average OD450 of two wells. Squares show data for nucleosomes directly coated on plate; diamonds show data for nucleosomes captured by polymer. (A) Binding by SLE plasma 1 at 1/800 dilution. (B) Binding by SLE plasma 2 at 1/800 dilution. (C) Binding by SLE plasma 3 at 1/400 dilution.
Fig 3.
The effect of PLL on ANA assays using supernatant of apoptotic cells as antigen.
ELISA plate wells were pre-coated with 2 μg/ml PLL, and then used to capture STS-supernatant at concentrations from 1 ng/ml to 2,000 ng/ml of DNA or buffer alone as control; the concentration refers to that of DNA. The same concentrations of STS-supernatant were coated to plate wells without polymer pre-coat. Binding by antibodies in three SLE plasmas was assayed in a standard ELISA assay. Points shown are the average OD450 of two wells. Squares show data for STS-supernatant directly coated on plate; circles show data for STS-supernatant captured by polymer. (A) Binding by SLE plasma 1 at 1/1,600 dilution; (B) Binding by SLE plasma 2 at 1/1,600 dilution; and (C) Binding by SLE plasma 3 at 1/800 dilution.
Fig 4.
Index plasma binding to STS-supernatant captured with PLL or coated directly onto ELISA plate wells.
ELISA plate wells were pre-coated with 500 ng/ml PLL, and then used to capture STS-supernatant at concentrations from 250 ng/ml to 2,000 ng/ml of DNA (left panel) or from 1 ng/ml to 1,000 ng/ml (right panel), or with buffer alone as control. Index plasmas were used at 1/800 dilution in the ELISA. The data points for the anti-dsDNA and anti-SSA index plasmas represent a single well. The data points for anti-RNP, anti-histone, anti-SSB and anti-Sm index plasmas are the average of two wells. Squares show data for STS-supernatant directly coated on the plate; circles show data for STS-supernatant captured by polymer.
Table 1.
The effect of DNase digestion on the antigenic activity of the supernatant.
Fig 5.
The effect of RNase A treatment of apoptotic supernatant on binding in the ELISA.
STS-supernatant was prepared as described and treated with a range of RNase A concentrations from 1.5 Kunitz units/ml to 0.015 Kunitz units/ml or with buffer alone. Samples that were digested and controls were assayed in both a direct coat ELISA and a PLL capture ELISA. SLE plasma 1 was used at 1/800 dilution. Squares show data for PLL capture; circles show data for direct coat ELISA. Data represent 1 well per condition.
Fig 6.
Titration of plasmas against STS-supernatant captured with PLL or coated directly in ELISA plate wells.
Index plasmas and normal plasmas were titrated against 1 μg/ml STS-supernatant coated directly in ELISA plate wells or captured onto wells pre-coated with 500 ng/ml PLL. Plasma dilutions ranged from 1/800 to 1/25,600; buffer only was used as control. Squares show data for STS-supernatant directly coated on plate; circles show data for STS-supernatant captured by polymer. Index plasmas used contained the specificities indicated on the charts in the figure.
Fig 7.
Effect of PLL on assay of antibodies to tetanus toxoid.
ELISA plate wells were pre-coated with 500 ng/ml PLL, and then used to capture tetanus toxoid at various concentrations from 25 pg/ml to 500 ng/ml, or buffer alone as control. The same concentrations of tetanus toxoid were coated onto plate wells without the PLL pre-coat. Binding by antibodies in three SLE plasmas was assayed in a standard ELISA assay. Squares show data for direct coat; circles show data for PLL pre-coated plate. All plasmas were tested at 1/800 dilution. (A) Binding by SLE plasma 1. (B) Binding by SLE plasma 2. (C) Binding by SLE plasma 3.
Table 2.
Comparison of prototype ANA capture assay with BioPlex® 2200 ANA assay data.