Serum samples containing IgG red blood cell (RBC) antibodies were collected without reference to clinical information from 131 pregnant alloimmunized women. Anti-D and anti-K were present in sera from 75 and 20 patients respectively. Antibody titres were determined by indirect antiglobulin test (IAGT), anti-D levels were measured by AutoAnalyzer, RBC-binding IgG was quantified using an enzyme-linked immunosorbent assay (SOL-ELISA), and functional activities were measured using the monocyte chemiluminescence (CL) test, antibody-dependent monocyte-mediated and K cell-mediated cytotoxicity (ADCC) assays, and rosette formation with U937 cells. Details of clinical outcomes were obtained retrospectively from 104 pregnancies. Forty-one babies were 'antigen-negative', and of the remainder, four required top-up transfusions, 12 required exchange transfusions, three received intrauterine transfusions, and two died in utero. A comparison of test results with severity of haemolytic disease of the newborn (HDN) showed that, provided sera tested were collected within 8 weeks of the expected delivery date, the CL test and the monocyte-mediated ADCC assay differentiated those D-positive babies which required exchange transfusions from those unaffected or only mildly affected. The usefulness of results from the AutoAnalyzer and IAGT in predicting disease severity was compromised by the wide range of results from mothers of unaffected babies. This variability was less apparent in the SOL-ELISA which predicted severe HDN with greater precision. Results from the U937 rosette assay and the K cell-mediated ADCC assay failed to correlate with disease severity.
Thirty-four IgG anti-D human monoclonal antibodies (mAb) derived from 18 donor were assessed for their ability to mediate lysis of D+ red cells by lymphocytes in antibody-dependent cell-mediated cytotoxicity assays. Cell-bound antibody was quantified and the mAb were compared at similar levels of sensitization. The majority (23/31) of IgG1 and all (3/3) IgG3 mAb were ineffective; two donors produced both lytic and non-lytic anti-D mAb. Greater sensitivity was achieved using fluid-phase antibody (as culture supernatants) in the assay than was obtained with pre-sensitized red cells. Minimum levels of 2000 anti-D molecules per cell were required for lysis using pre-sensitized cells. Partial D red cells (DIVa, DVa and DVI) were lysed by three mAb that were lytic with normal D+ cells. There was no relationship between lytic ability and Gm allotype or D epitope specificity of the antibodies. Four mAb to other blood group specificities were tested: two (anti-E and anti-G) were lytic and two (anti-c and anti-Kell) were not lytic. Possible reasons for the heterogeneity of the lytic activity by the mAb are discussed.
The response of human monocytes to red cells sensitized with known levels of monoclonal antibody to the Rh antigen D (anti-D) was compared with that of polyclonal anti-D. Monocyte response was determined by measuring red cell adherence, erythrophagocytosis, monocyte-mediated red cell lysis and luminol-dependent chemiluminescence. By all criteria, monoclonal and polyclonal antibodies showed comparable activity, with IgG3 antibodies promoting a greater monocyte-red cell interaction than IgG1 antibodies. It is suggested that monoclonal anti-D may be effective in the prophylaxis of haemolytic disease of the newborn, providing such material is clinically acceptable.
It has been postulated that agalactosyl immunoglobulin G (IgG) self-associates to form pathological aggregates in the rheumatoid joint. To examine this hypothesis, IgG aggregates from synovial fluid (SF) of 22 patients with RA were prepared by precipitation with polyethylene glycol (PEG) 6000. The PEG precipitates and SFs were reduced with 2-mercaptoethanol (2ME) and bound to protein G. This procedure isolated the IgG in the PEG precipitates from other contaminating glycosylated proteins. The levels of galactose and N-acetylglucosamine (GlcNAc) residues present on the reduced IgG were quantified by their ability to bind the lectins Ricinus communis (RCA)120 and Bandeiraea simplicifolia (BS) II. Proportionally less galactose (expressed as a ratio of bound RCA120 to BS II) was present on the IgG from the PEG precipitates than on the IgG in the paired SF (P = 0.001). However, in many cases more RCA120 as well as BS II bound to IgG from PEG precipitates than from the corresponding SF. It is considered that agalactosyl IgG occurs preferentially in RA SF PEG precipitates and that this IgG may also exhibit increased Fab glycosylation.
SUMMARYWe have studied the ability of lymphocytes from the blood, thyroid and lymph nodcsof patients with autoimmune thyroid disease (AITD) to produce aiitoantibodies to thyroglobulin (Tg) and/or thyroid peroxidase (TPO) in SCID mice. Human IgG class Tg and/or TPO antibodies were delectable in plasma from SCiD mice 7 days after transfer of 15-25 x 10'' cells/moiise and ihc highest levels were recorded 2-3 weeks later. In contrast. Tg and/or TPO antibodies were undetcctable in recipients of lymphocytes from thyroid antibody negative controls. AITD thyroid lymphocytes produced the mosi antibody in recipient mice and lower levels were observed in recipients of AITD blood and lymph node lymphocytes. The amounls of Tg and/or TPO tmtibody detected were in accordance with the ability of Ihyroid and lymph node lymphocytes to secrete these autoanlibodies spontaneously in culture (indicating ihc presence of cells activated in the patient) and with the capacity of blood lymphocytes (probably B memory cells) to secrete Tgand/or TPO antibodies in ctilture in response to pokeweed mitogen. Tg antibodies in plasma from SCID recipients of thyroid lymphocytes were of subclasses IgGl. IgG2and !gG4and the proportions closely resembled those of the donor's serum Tg antibodies. Blood lymphocytes transferred to SCID recipients were also able to produce Tg antibodies of subclasses 1. 2 and 4 but the subclass distribution varied between mice and the reason for this is not clear at present. Since SCID mice provide an environment in which B lymphocytes from paiients with AITD can be activated without mitogen to secrete thyroid antibodies, this model will provide a powerful system for elucidating the mechanisms regulating the secretion of hutiian antibodies to Tg and TPO-
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