Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains.

Abstract

Three human monoclonal rheumatoid factor (mRF) crossidiotype (XId) groups, WA, PO, and BLA, were defined using polyclonal antisera (1, 2) . Both the WA and the BLA XIds appeared to be associated with the antigen-combining site (2), and serologic studies (3, 4) indicated that the antisera defining the WA XId appeared to recognize a determinant requiring both H and L chains . Structural characterization of the Ids was not done. Extensive studies (5-7) have recently been performed, however, to determine the structural basis of the WA XId. Investigators using antisera to primary structures on the H and L chains postulated that the WA XId resides on the L chain . Our study, in which we used the original antisera that defined the WA and BLA XIds and additional polyclonal antisera, presents evidence that the WA and BLA XIds are conformational antigens requiring both L and H chains, and that with denaturation, the antigens that define the XId and the antigen-binding activity are lost in parallel . Volume 164 November 1986 1809-1814 Materials and Methods BriefDefinitive ReportPreparation of Reagents and Antisera . IgM mRFs were obtained and isolated as previously described (2). Antisera used to type the WA XId (anti-McD) and BLA XId (antiBla) were previously described (2). Anti-PSL2, a synthetic peptide corresponding to the second complementarity-determining region (CDR) and adjacent residues on the L chain of the IgM RF Sie (8), was provided by Drs. P. P. Chen and D. A. Carson (Scripps Clinic, La Jolla, CA).Preparation of H and L Chains. H and L chains of mRFs were prepared by routine partial reduction with 10 mM DTT, alkylated and isolated in 1 M propionic acid on a *100 Sephadex column . Purity of H and L chains was established, and recombination of H and L chains was performed by mixing H and L chain pools in a 1 :2 M ratio, respectively, and by dialyzing them for 72 h at 4°C against a 0 .5 M Tris, 0 .15 M NaCl buffer (pH 7 .2) . Recombined H and L chains were isolated with G200 Sephadex column chromatography . The recombinant pools were tested for free K or w chains by immunodiffusion in agarose gel using nonspecific antisera to x and ju chains .Immunodotting and Blotting. 100 A1 of each mRF diluted to 10 ug/ml in PBS was "dotted" onto 0 .45-gm nitrocellulose paper using a hybridot filtration manifold (Schleicher & Schuell, Inc ., Keene, NH) . Parallel sample sets were stained with Coomassie blue or blocked with 1 % BSA/PBS and probed with WA or BLA XId-typing reagents . The filter This work was supported by National Institutes of Health (Bethesda, MD) grant ROI AM-35487 and Veterans Administration merit review grant. J. Exp. MED.

Complete protein sequences of the variable regions of the cloned heavy and light chains of a human anti-cytomegalovirus antibody reveal a striking similarity to human monoclonal rheumatoid factors of the Wa idiotypic family.

“…5) and most likely account for the difference in the antigen binding characteristics, -as well as the observed differences in the expression of idiotype. This difference lends further support to the importance ofthe D-J4 region (22,25,58) ofthe RFs, both for expression of the Wa idiotype and for antigen binding. Until other related anti-CMV antibodies with the same specificity are described, no firm conclusions about the importance of parts of the variable regions to anti-CMV activity can be made.…”

“…The Wa idiotypic family, representing -60% of monoclonal RFs, has been found to be highly restricted in the use of light and heavy chain variable region gene segments (20)(21)(22)(23)(24)(25)(26)(27)(28). This is analogous to the restricted use of immunoglobulin variable region gene segments in murine antigen specific systems, many of which bear cross-reactive idiotypes.…”

Idiotypes and the structural diversity of human rheumatoid factors

“…Amino acid sequence showed that two CRI-positive RFs have homologous light chains, but not heavy chains [4]. Recently, the expression of the "Wa" CRI has been reported to be conformation dependent [2]. Kunkel and co-workers also described another CRI on 20 % of the RFs, termed the minor "Po" CRI [28].…”

Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor

“…The peak of HCV present in the void volume was contaminated with small amounts of immunoglobulins that were removed by using immobilized rProtein A (Repligen). Immoblotting (dot blots) to detect small amounts of protein was performed as described (7). Sensitivity of the assay was 100 pg for IgG and IgM and 200 pg for apolipoproteins B and E. Lipoproteins were quantitated by Lowry This paper was submitted directly (Track II) to the PNAS office.…”