Induction of Anti-DNA IgG and IgE Antibodies in BALB/c Mice (1)
Varying amounts of reaginic IgE antibodies specific for native DNA, denatured DNA (dnDNA), RNA and tRNA were present in the sera of SLE patients, along with IgG antibodies. DNA fragments present in DNA/anti-DNA immune complexes isolated from SLE patients were found to be 45-300 base pairs in length.
Male BALB/c mice (8- to 10-weeks-old) were bled before immunization. Each animal received calf thymus dnDNA (100 ug) complexed with MBSA (100 ug) and 5 mg of aluminium hydroxide in a volume of 300 ul of normal saline. A booster of the same antigen was given on day 21.
Although anti-DNA antibodies of IgG and IgE classes were detectable in the sera of these animals 2 weeks after immunization, the titers reached optimum levels 2 weeks after secondary immunization (day 35). Anti-DNA antibodies were not detectable in the sera of animals injected with dnDNA and adjuvant or adjuvant (aluminium hydroxide) alone.
NOTE: mBSA must be used for inducing anti-DNA antibodies.
Anti-DNA IgG antibodies present in the sera were found to be predominantly of IgG1 subclass. Negligible titers of IgG3 and IgG2b antibodies specific for dnDNA were present in the immune sera while antibodies of IgG2a subclass were not detectable.
Approximately 1pg/ml of the antigen (aDNA, dnDNA or dsDNA) was required to give 50% inhibition of the binding of the IgGl antibodies. Similarly, about 1.5 pg/ml of aDNA, dnDNA or dsDNA were required to cause 50% inhibition of the binding of IgE antibodies.
Considerable inhibition was obtained with cibacron blue (88 and 49%) and chondroitin sulphate (37 and 38%), respectively, for IgGl and IgE.
NOTE: Cibacron Blue has been used for affinity purification of anti-DNA antibodies
The PCA reaction was abrogated when the serum was incubated at 56°C for 2 h prior to sensitization of the rats, thus confirming that the DNA-specific antibodies responsible for the PCA reaction were of the IgE class.
The anti-DNA antibodies present in the sera of SLE prone mice were found to be predominantly of IgG2 subclass. IgG eluted for B x SB male kidney is dominated by IgG2b antibody, with smaller amounts of IgG3, IgG2a and IgG1. Similarly, IgG eluted from MRL/l kidneys is predominantly of IgG2a and IgG2b subclasses, whereas in NZB and NZB x W, IgG2a exceeds the smaller amounts of remaining subclasses.
NOTE: Only IgG2a and IgG2b are responsible for causing inflammation in the kidney?
Anti-DNA antibodies show extensive cross-reactivity with phospholipids, proteins, proteoglycans and some dyes.