L. A. Aarden

C. luciliae are hemoflagellates nonpathogenic for man and easy to culture. They have a giant mitochondrion, in which the mitochondrial DNA is concentrated in a single large network, the kinetoplast. When used as a substrate for the indirect immunofluorescence technique, studying sera from patients with SLE, we could demonstrate a very good correlation between this test and the Farr assay for the demonstration of antibodies to double-stranded DNA. Although the sensitivity of both techniques is on the same order of magnitude, the IF technique has the following advantages over the Farr assay. It is easy to perform in laboratories equipped for autoimmune serology. It possesses an intrinsic check on the immunoglobulin character of the DNA-binding activity. It allows one to determine the Ig classes and subclasses of antibodies to DNA. It permits study of complement fixation to antibodies without interference of Clq fixation to DNA or anticomplementarity of the serum. There is an absence of interference with antibodies to single-stranded DNA.

To study the active site(s) of IL-6 we combined mutagenesis of IL-6 with epitope mapping of IL-6 specific mAb. In addition to amino-terminal deletion mutants we described previously, carboxyl-terminal deletion mutants were prepared. Functional analysis showed that deletion of only five carboxyl-terminal amino acids already reduced the bioactivity 1000-fold. A panel of mAb to IL-6 was subsequently analyzed by antibody competition experiments and binding to the amino- and carboxyl-terminal deletion mutants. On the basis of the competition experiments the six neutralizing mAb were divided in two groups (I and II). The binding pattern with the deletion mutants suggested that the region recognized by the four mAb in group I is composed of residues of amino- and carboxyl-terminus: binding of two mAb was abolished after deletion of amino acid Ala I-Ile26, of the third mAb after deletion of the four carboxyl-terminal amino acids whereas the fourth mAb did not bind to either mutant. Group II mAb retained binding to these mutants. Taken together these data suggest that in the native IL-6 molecule amino acid residues of amino and carboxyl terminus are in close proximity and that together they constitute an active site. Furthermore our data suggest that the part of the molecule recognized by group II antibodies is a second site involved in biologic activity.

A high-avidity human antibody (Ab) to double-stranded DNA (dsDNA) could be virtually completely dissociated from Crithidia luciliae kinetoplast dsDNA at pH 12 and a low-avidity Ab at pH 11. At low pH (pH 2), no dissociation occurs of either Ab. Low-avidity Ab could be dissociated at neutral pH with 1 M NaCl, but even with 5 M NaCl high avidity Ab could not be dissociated. Prolonged exposure to pH 12 did not affect DNA-binding by Ab after dialysis against PBS. A decrease in surface tension of the liquid medium in no case prompted dissociation. Contact angle measurements on DNA showed it to be very hydrophilic. It is concluded that concomitant with the strong negative charge of the antigen and the positive charge of the Ab, both antigenic determinant and antibody-active site are exceptionally hydrophilic, which causes their van der Waals attraction in aqueous media to be negligibly small. This particular antigen-antibody bond thus is mainly electrostatic and can be completely dissociated by abolishing the positive charge of the antibody-active site through a drastic increase in pH.

Recent results on the conditions of ionic strength needed to prevent the association of dsDNA with high avidity human anti-dsDNA, were compared with the insufficiency of even the highest practicable ionic strengths to effect the dissociation of such antigen-antibody complexes, once formed (1). Further analysis of these results make us conclude that such high avidity dsDNA-anti-dsDNA complexes, which in the initial stages of their formation are mainly of the Coulombic variety, subsequently evolve, at least in part, into hydrogen bonds.