Circular dichroism of immune complexes, IgG and Fab gamma with unique antigenic determinants from rheumatoid serum.

IgG-IgG and IgG-IgM complexes were isolated from one rheumatoid arthritis (RA) serum by affinity chromatography to immobilized F(ag')2 gamma of specific antibodies against unique determinants in the complexes. Both IgG nd IgM, when isolated from these complexes, contained the unique determinants. The circular dichroism (CD) spectrum of IgG differed from that of normal IgG at neutral pH. At pH 3 both IgG and IgM displayed normal CD spectra, and only one third of the molecules now had affinity for the immobilized ligand. The molecules with affinity at pH 3 exhibited an abnormal CD spectrum at pH 3, and a normal CD spectrum was obtained only of those components that lacked affinity. One-third of the Fab gamma isolated from the IgG and IgG complexes with the unique determinants contained the unique determinants that were lacking in the rest of the same principal the Fc gamma fragments. The CD of the two Fab gamma preparations showed the same principal differences as the CD of the molecules with and without affinity to the ligand at acidic pH.

Circular dichroism of immune complexes with rheumatoid factor activity.

The circular dichroism (CD) of IgG complexes with RF activity and that of monomeric IgG without this activity, and isolated from the same individual, were compared. The IgG complexes had a significantly deviant CD spectrum in the near u.v. region, both before and after dissociation, whereas the monomeric IgG had a normal spectrum. Immunoglobulins were isolated from the same serum with the use of specific antiserum against unique determinants in some IgG complexes with RF activity. Both before and after dissociation the CD spectrum in the far u.v. region of these immunoglobulins differed significantly from that of the above-mentioned preparations. The results confirmed that the structure of IgG in the RF-active complexes differed from that of normal IgG. The immunoglobulins with the unique determinants had, in turn, a structure that was not found in the pool of the RF-active IgG molecules or in normal IgG.

Quantitative precipitin analysis of free and hidden RF IgM.

Quantitative precipitin analysis using rabbit IgG anti-human Fc mu was performed with 16 RF IgM, six macroglobulinaemic IgM and four normal IgM. Abnormal precipitin curves were obtained for all RF IgM, even when the latter were not readily demonstrated with conventional serological tests for RF, and for macroglobulinaemic IgM with sedimentation rates greater than 19S. These IgM formed significantly more precipitates with IgG anti-Fcmu in the antigen excess zone than did normal IgM, but the precipitin curves for the other zones were similar for all IgM. The underlying mechanisms of some of the reactions were studied and discussed. Because the divergence in the precipitin reaction for normal IgM and RF IgM was so pronounced, a model precipitin curve was constructed. This could be used to detect RF IgM, even when not readily demonstrable with conventional serological tests for RF, by direct analysis of serum. The results obtained for RF IgM suggested that the method might be applied to RF IgG and intermediate complexes comprised of IgG. The mechanisms demonstrated here might be used to develop immunological methods for routine use.

Isolation and characterization of intermediate complexes and other components with common antigenic determinants.

Fractions with affinity for heat-denatured human IgG (HDG) were isolated from four sera that contained intermediate complexes (IC). These IC fractions contained part of the 75 IgG, all IC, and part of the rapidly sedimenting complexes (RC) found in the sera. The IC consisted of IgG1 or IgG3 and the RC of IgG and IgM with kappa and lambda light chains. The IgG in the IC fractions contained an abnormally large amount of neuraminic acid. No correlation between IgG subclass or content of neuraminic aid and complex formation was found. There were indications that the formation of IC was not only the result of self-association of IgG molecules with anit-gamma-globulin activity. Specific rabbit antisera were prepared against two of the IC fractions. Affinity chromatography wtih immobilized IgG and F(ab')2gamma from these antisera confirmed the presence of common antigenic determinants in the sera. These determinants occurred mainly in 7S components in one individual, in IC in one and in RC in another. Only a minor part of the serum components with affinity for HDG contained the determinants. RF activity was found in the components that lacked and in those that contained the common antigenic determinants.