Complement has a complex role in immune mediated red blood cell (RBC) destruction and usually induces extravascular hemolysis of C3bcoated RBCs by erythrophagocytosis and by acting synergistically with cell-bound immunoglobulins. A sensitive two-stage enzyme-linked direct antiglobulin test (ELDAT) was developed and used to measure RBC-bound C3b and C3d in 120 healthy adult individuals and in 60 patients suffering from a variety of conditions, including warm-and cold-type autoimmune hemolytic anemia, neoplasia, and collagen diseases. The results were compared with those of standard agglutination tests employing polyclonal and monoclonal antiglobulin reagents. Small amounts of C3b and C3d were detected on RBCs of the healthy individuals only by the ELDAT and probably reflected the continuing low-grade activation of complement necessary for the maintenance of homeostasis of a variety of physiological systems. The quantity did not vary with age or gender. In the patients, increased amounts of RBCbound C3b and C3d were relatively common and probably resulted from autoantibody activity, immune-complexes, and nonspecific adsorption. There was no association between positive ELDAT results and the presence of active hemolysis. The ELDAT was far more sensitive than the agglutination tests for detecting RBC-bound C3b and also for C3d if the monoclonal reagent was employed.
An 80-year-old female presented with melena and anemia due to bleeding from a benign gastric ulcer. Her blood group was O, D+. The serum contained anti-B and a weak anti-A (titer 2 at 18°C). She was inadvertently transfused with approximately 3.5 units of group A red blood cells with no initial ill effects. One week later, the antiA titer increased to 8 and the direct antiglobulin test (DAT) was weakly positive (IgG and C3d). The next day, intravascular hemolysis became evident. The DAT was still weakly positive and the serum contained a weak cold autoagglutinin, which did not correlate with the severity of the hemolysis. A Donath-Landsteiner test was performed and found to be strongly positive. The antibody showed P specificity, confirming a diagnosis of paroxysmal cold hemoglobinuria (PCH). Exchange transfusion was followed by rapid recovery even though the Donath-Landsteiner test remained positive for at least a month. The patient was well when last seen 11 months after presentation. It was thought that the original low titer of anti-A reflected compromised immune homeostasis in an elderly patient and that stimulation by incompatible blood in those circumstances resulted in a delayed hemolytic transfusion reaction that triggered, exacerbated, or was accompanied by an autoimmune response manifesting as PCH.
The monoclonal autoantibody (mAb) V-88 was derived from a (NZB x NZW)Fl (BWF1) female mouse with lupus [l]. The mAb V88 binds ssDNA preferentially but also binds dsDNA and other nuclear material. Studies made by epitope scanning, using synthetic peptides, revealed the locations of idiotopes in both the CDR and framework regions [2]. A dominant idiotope corresponding to the peptide sequence EWVATISG was identified in the FR-H2/CDR-H2 region.The gene sequence encoding the EWVATISG peptide was found to be homologous to the human 16/6 Id family. The 16/6 Id was first described on a human IgM anti-DNA monoclonal antibody 13-41 and is thought to be involved with the pathology of lupus. Increased levels of 16/6 Id have been detected in serum of lupus patients with disease activity [5].Cross reactive idiotypes (CRI) are expressed on sets of related antibodies which are often associated with similar germline encoded antibodies. Private idiotypes probably occur as a result of extensive somatic mutation in the CDR regions of a given antibody and are likely to be unique to a highly modified antibody. The Ids of DNA antibodies are primarily of the CRI type which implies that the CRI+ antibodies could be encoded by relatively few unmutated germline genes.The aim of this study was to determine whether the EWVATISG encoding gene segment was a cross reactive idiotope shared by antibodies that have the same or related VH germline gene sequence, or whether they are private idiotopes, a result of antigendriven somatic mutation.DNA was isolated from livers of BWF1, NZW, NZB, Balblc and MRUMp-lpr/lpr (MRL) mice and from the mAb V88 hybridoma which was used as reference material. A PCR method was used to amplify the IgVH gene segments, using either the gene sequences of EWVATISG itself or SSYVMS (CDR-H1) as the forward primer with the gene sequence of EDTALYY from the conserved FR-H3 region as the reverse primer. The PCR products were subcloned in pCRT3111 and sequencedl61.
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