Figure 3.
Binding of affinity-purified human IgG molecules to dsDNA. The affinity-purified human IgG antibodies were tested by ELISA for their binding to dsDNA. These experiments were carried out on two separate occasions and representative data are shown. Diluted serum from a patient with systemic lupus erythematosus was run on every plate as a positive control. The standard deviation (SD) of the standard serum optical density (OD) value between plates was ± 0.07 at a concentration of 5 IU/ml. ODs in the control wells containing no antigen were always lower than 0.068. (a) The dsDNA binding of the affinity-purified antibodies when diluted in sample-enzyme-conjugate (SEC) buffer but not treated with DNaseI. Only B3VH/B33VL binds to dsDNA under these conditions. The SDs were as follows: <0.121 OD units for all points on the curve B3VH/B33VL and <0.002 OD units for all points on curves B3VH/B3VL, B3VH/B3(R27aS)VL, and B3VH/BUVL. (b) The dsDNA binding of the affinity-purified antibodies when diluted in SEC buffer and treated with DNaseI before testing in the ELISA. The DNaseI treatment appears to have abolished the binding of the affinity-purified B3VH/B33VL to dsDNA. The SD for all points on all four curves was less than 0.002 OD units. (c) The dsDNA binding of the affinity-purified antibodies diluted in supernatant derived from COS-7 cells and treated with DNaseI before testing in the ELISA. The addition of the COS-7 supernatant appears to have reinstated the binding of B3VH/B33VL to dsDNA and also allows the binding of B3VH/B3VL and B3VH/B3(R27aS)VL to dsDNA. B3VH/BUVL does not bind to dsDNA. The SDs were as follows: <0.176 OD units for all points on the curve B3VH/B33VL, <0.185 OD for all points on curve B3VH/B3VL, <0.185 OD for all points on curve B3VH/B3(R27aS)VL, and <0.01 OD for all points on curve B3VH/BUVL.