Unexpected effects of donor gender on the storage of liquid plasma.

BACKGROUND: Swedish regulations in effect since 2006 allow the storage of plasma for transfusion up to 14 days at 2-6 degrees C and for 3 years at < or = -30 degrees C. In this study, the quality of currently used plasma components was investigated. MATERIALS AND METHODS: Plasma components, prepared from whole blood or by apheresis, either leucocyte depleted or not leucocyte depleted, were stored at 2-6 degrees C as liquid plasma or as thawed fresh-frozen plasma; 31% were from female donors. Concentration, function and activation markers of the plasma coagulation systems were investigated during storage for up to 42 days. RESULTS: Cold-induced contact activation was the dominant storage lesion, occurring earlier and at higher frequency in plasma from females. Increased kallikrein-like activity led to changes in activated partial thromboplastin time, prothrombin time, protein C and C1 inhibitor (C1INH). C1INH function dropped to 53% on Day 14 in cold-activated plasma components. CONCLUSION: Contact activation may be triggered before Day 14, especially in plasma from females, and may progress as a result of the consumption of C1INH. The data suggest that lack of cold-induced contact activation may be an important quality criterion. To achieve this, plasma from male donors could be selected for transfusion and the storage time limited to 7 days.

Immune complexes from SLE sera induce IL10 production from normal peripheral blood mononuclear cells by an FcgammaRII dependent mechanism: implications for a possible vicious cycle maintaining B cell hyperactivity in SLE.

BACKGROUND: Raised interleukin (IL)6 and IL10 levels are thought to contribute to the pathogenesis of systemic lupus erythematosus (SLE) by enhancing autoantibody production and immune complex (IC) formation. These immune complexes can then stimulate cellular reactions through Fc and complement receptors. OBJECTIVE: To investigate whether circulating SLE ICs stimulate type 2 cytokine production. METHODS: Twenty serum samples from patients with active SLE were compared with sera from 18 healthy controls. Sera and polyethylene glycol (PEG) precipitates from sera were added to peripheral blood mononuclear cell (PBMC) cultures, and the production of IL10 and IL6 was investigated by enzyme linked immunospot assay (ELISPOT) and enzyme linked immunosorbent assay (ELISA). Fc gamma receptor (FcgammaR) antibodies were used in blocking experiments, and flow cytometry was used to assess the correlation between monocyte FcgammaR expression and IC-induced cytokine production. RESULTS: Ten per cent dilutions of the SLE sera induced a significantly increased number of IL10-producing cells in comparison with control sera (median, 11.75 v 1.25 spot forming cells/50 000 PBMC; p<0.0001). PEG precipitates from SLE sera also induced significantly increased levels of IL10 (p=0.016) and IL6 (p=0.042) in comparison with control PEG precipitates. IL10 production induced by SLE PEG precipitates or by artificial ICs could be blocked by anti-FcgammaRII antibodies, and the FcgammaRII expression on CD14+ monocytes correlated with the IC-induced production of IL10 and IL6. CONCLUSIONS: SLE sera stimulate IL10 and IL6 production from PBMC, and this effect is at least partly explained by precipitable ICs acting through FcgammaRII. This effect provides a possible mechanism for the enhanced production of IL10 in SLE, whereby B cell activation, antibody production, IC stimulated monocytes/macrophages, and type 2 cytokines create a vicious cycle that may help to maintain B cell hyperactivity in SLE.

Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation.

BACKGROUND: Intraportal transplantation of pancreatic islets offers improved glycaemic control and insulin independence in type 1 diabetes mellitus, but intraportal thrombosis remains a possible complication. The thrombotic reaction may explain why graft loss occurs and islets from more than one donor are needed, since contact between human islets and ABO-compatible blood in vitro triggers a thrombotic reaction that damages the islets. We investigated the possible mechanism and treatment of such thrombotic reactions. METHODS: Coagulation activation and islet damage were monitored in four patients undergoing clinical islet transplantation according to a modified Edmonton protocol. Expression of tissue factor (TF) in the islet preparations was investigated by immunohistochemistry, immunoprecipitation, electron microscopy, and RT-PCR. To assess TF activity in purified islets, human islets were mixed with non-anticoagulated ABO-compatible blood in tubing loops coated with heparin. FINDINGS: Coagulation activation and subsequent release of insulin were found consistently after clinical islet transplantation, even in the absence of signs of intraportal thrombosis. The endocrine, but not the exocrine, cells of the pancreas were found to synthesise and secrete active TF. The clotting reaction triggered by pancreatic islets in vitro could be abrogated by blocking the active site of TF with specific antibodies or site-inactivated factor VIIa, a candidate drug for inhibition of TF activity in vivo. INTERPRETATION: Blockade of TF represents a new therapeutic approach that might increase the success of islet transplantation in patients with type 1 diabetes, in terms of both the risk of intraportal thrombosis and the need for islets from more than one donor.

Opsonization of yeast cells with equine iC3b, C3b, and IgG.

The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells (Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS-PAGE and Western blot analyses. Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3-6-fold, indicating spatial competition between C3 and IgG at binding. Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With > or =6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents. It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.

Phosphorylation of C3 by a casein kinase released from activated human platelets increases opsonization of immune complexes and binding to complement receptor type 1.

We have previously demonstrated that complement component C3 is phosphorylated both in vitro and in vivo by a casein kinase released from activated human platelets. In vitro, the studies have shown that cleavage of C3b by factor I is decreased, and binding to various target surfaces is enhanced by affecting the thiol ester. In the present study we have examined the effect of phosphorylation on the binding of C3b to complement receptor 1 (CR1, CD35). Upon phosphorylation by platelet casein kinase, C3b covalently bound to activated thiol Sepharose bound higher amounts of soluble recombinant CR1. Similar effects were demonstrated with two ELISA systems in which microtiter plates were coated with phosphorylated or unphosphorylated purified C3b or with C3 activated by the alternative pathway convertase. Phosphorylated C3b was also four times more efficient than unphosphorylated C3b in inhibiting the binding of complement-opsonized human aggregated gammaglobulin to erythrocytes. A similar increase in binding was found at low serum concentrations when the C3 activation occurred in C3-deficient serum reconstituted with phosphorylated or unphosphorylated C3. In this serum system, using a monoclonal antibody specific for iC3b, we also demonstrated that the phosphorylated C3b was protected against cleavage to iC3b. Corresponding experiments using factor H showed a decrease in binding of both fluid-phase and bound C3b to factor H. We postulate that phosphorylation of C3 by activated platelets amplifies the complement-mediated binding of immune complexes to CR1 by three different mechanisms: decreased cleavage of C3b to iC3b, increased deposition of C3b to immune complexes, and increased binding of C3b to CR1.

Phosphorylation of plasma proteins with emphasis on complement component C3.

Phosphorylation of complement component C3 by different protein kinases in vitro has been demonstrated to alter the functional properties of the protein. Extracellular phosphorylation mediated by activated platelets is a newly described mechanism by which the function of plasma proteins can be regulated. Upon activation of platelets a casein kinase is released concomitant with large amounts of ATP and Ca2+. These components are sufficient to phosphorylate proteins e.g., C3 extracellularly. In vivo, in patients with SLE, the phosphate content in plasma proteins, including C3 has been demonstrated to increase during exacerbation. The changes were linked to platelet activation by a covariation with the levels of beta-thromboglobulin. The purpose of this review is to summarise the findings in this field.

A new in vitro model to study interaction between whole blood and biomaterials. Studies of platelet and coagulation activation and the effect of aspirin.

We have developed a versatile in vitro chamber model with a double purpose: first, to be able to study mechanisms of bio-incompatibility, and, second, to test biomaterials at all levels of interactions, in whole blood. The use of biomaterials in the form of microscope slides as walls in the chamber makes it possible to analyse both the biomaterial surface with regard to protein and cell binding, as well as the molecular events taking place in the fluid. Incubation of blood in the chamber, for 60 min at 37 degrees C resulted in the rapid binding of complement and coagulation proteins and of leukocytes and platelets to polyvinylchloride (PVC) slides. The cells formed a layer which more or less covered the underlying surface. Unlike complement activation, as reflected by soluble C3a and C5b-9, the thrombin-antithrombin formation was completely nullified in cell-depleted plasma. Despite the fact that thrombin-antithrombin generation was also negligible in platelet-rich plasma, inhibition of platelet aggregation on the material surface with aspirin resulted in suppressed generation of thrombin antithrombin complexes. Taken together, the coagulation activation in the chamber was dependent on the presence of blood cells which suggests that bound/aggregated platelets initiate a sequence of events involving leukocytes that results in coagulation activation.

Defective elimination of C3b/iC3b-coated autologous erythrocytes in patients with primary biliary cirrhosis, alcoholic cirrhosis, and ulcerative colitis.

Delayed in vivo elimination of autologous erythrocytes coated with immunoglobulin G has been reported in autoimmune and inflammatory gastrointestinal diseases. Our aim was to elucidate whether impairment of the macrophages was restricted to the Fc receptors of the reticuloendothelial system or whether complement receptors were also affected. We studied elimination by complement receptors of autologous erythrocytes coated with fragments of C3 and C4 in patients with primary biliary cirrhosis, ulcerative colitis, and alcoholic cirrhosis. Impaired function was seen in all patient groups as compared with function in normal subjects, both concerning the mean half-life of the injected cells and the total number of eliminated erythrocytes. Neither of these parameters correlated with the levels of C3 fragments bound to the injected autologous erythrocytes. This is the first report of defective complement receptor function in ulcerative colitis and alcoholic cirrhosis. Immunoglobulin G-dependent elimination of erythrocytes was confirmed to be lowered in all patient groups. The results suggest severe macrophage functional aberrations involving both complement receptors and Fc receptors as the basis of phagocytic defects in autoimmune/inflammatory conditions. In contrast, a general loss of macrophages might cause the functional loss in alcoholic cirrhosis.

Correlation between anti-C1q and immune conglutinin levels, but not between levels of antibodies to the structurally related autoantigens C1q and type II collagen in SLE or RA.

The simultaneous appearance of autoantibodies with either a functional or structural relationship to anti-C1q antibodies (anti-C1q) was investigated in 39 systemic lupus erythematosus (SLE) patients and in 28 rheumatoid arthritis (RA) patients, in both cross-sectional and longitudinal design. Levels of anti-C1q showed an isotype-specific correlation to levels of immune con-glutinin (IK) in SLE patients, whereas no correlation was evident to levels of antibodies to the structurally related antigen type II collagen (anti-CII) in SLE or RA patients. IgG anti-C1q levels correlated with serum levels of the terminal complement complex (sC5b-9) in SLE patients. In two longi-tudinally followed patients, the IK response preceded the anti-C1q response. Possibilities for regulation of the humoral anti-complement response are discussed.

Phosphorylation of complement component C3 after synthesis in U937 cells by a putative protein kinase, casein kinase 2, which is regulated by CD11b: evidence that membrane-bound proteases preferentially cleave phosphorylated C3.

It was our aim in this study to investigate the possibility that the third component of complement (C3) is phosphorylated during synthesis and secretion in U937 cells. Labelling of U937 cells with [32P]Pi, followed by immunoprecipitation of C3 from cell lysates and culture supernatants at different time points, showed that C3 was phosphorylated intracellularly immediately before release into the medium, which initiated cleavage of the protein into an iC3b-like fragment. Stimulation of CD11b/CD18 increased phosphorylation 7-fold, from a basal level of 2%. The phosphorylation sites in C3 did not resemble those described previously for casein kinase (CK) 1, cAMP-dependent protein kinase A or calcium- and phospholipid-dependent protein kinase C. Instead, protein kinase CK2 was suggested inasmuch as: (1) CK2 was detected both on the cell surface and on shed microparticles; (2) phosphorylation of purified C3 by microparticles was abolished by a monoclonal antibody, anti-CK2; (3) the [32P]Pi tag of both phosphorylated C3 (secreted from U937 cells) and of microparticle-phosphorylated C3, which was cleaved either by membrane proteases or by leucocyte elastase, was found in a 40 and a 70 kDa polypeptide; (4) both secreted C3 and C3 phosphorylated in vitro were much more susceptible to cleavage by proteases. Generation of C3 fragments provides a means by which U937 cells can stimulate nearby cells which are expressing complement receptors. The present study demonstrates that the cleavage of C3 is controlled by an intracellular phosphorylation event regulated by CD11b/CD18.

Evidence for iC3 generation during cardiopulmonary bypass as the result of blood-gas interaction.

Earlier we have shown that iC3 is generated at the blood-gas interface in vitro and that the generation of this molecule is independent of complement activation and the composition of the gas. In order to investigate whether iC3 is also generated during cardiopulmonary bypass where blood comes into contact with oxygen bubbles, two bubble oxygenators were incubated at 37 degrees C with human heparinized blood. A continuous increase in the level of iC3 was shown in the oxygen-perfused bubble oxygenator (up to 100 nmol/l after 180 min) in contrast to the unbubbled control. Similarly, in plasma drawn from patients undergoing cardiopulmonary bypass using either bubble or membrane oxygenators, the levels of iC3 were shown to increase continuously during the operation. Furthermore, this form of C3 was found to be susceptible to cleavage by factor I. The formation of iC3 at the blood-gas interface in vivo could be a mechanism by which gas bubbles induce clinical manifestations associated with complement activation, e.g. during cardiopulmonary bypass, adult respiratory distress syndrome and decompression sickness.

Modification of the complement binding properties of polystyrene: effects of end-point heparin attachment.

In recent years, conjugation of heparin to biomaterials has been shown to improve its biocompatibility. The purpose of the present work was to compare complement activation and binding of C3 to unmodified and heparin-treated polystyrene surfaces of microtitre plates. When polystyrene was incubated with human serum, C3 was deposited on the surface by both adsorption and binding dependent on activation of the classical (CPW) and alternative (APW) pathways. After end-point attachment of heparin, significant C3 deposition, although at reduced levels, occurred only by CPW-mediated mechanisms, while adsorption and APW-mediated binding were strongly reduced. Generally, the modified surface bound lower amounts of protein, e.g. serum albumin and IgG, than the unmodified. By contrast, it had increased affinity for C1q which leads to binding of C1 and activation of complement via the CPW. Nevertheless, the net effect of the surface modification on the complement reaction was an overall reduction of C3 binding due to obliteration of APW. This can be related to an enhanced factor H/I-dependent down-regulation of C3b and to the lowered protein-adsorbing property of the surface, both of which have inhibitory effects on APW and on the C3 shunt-dependent activation of the complement system.

Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression.

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.

RF-plasma-modified polystyrene surfaces for studying complement activation.

Five different plasma modified surfaces were made for studying different aspects of biocompatibility. These surfaces were: 1,2-diaminocyclohexane (DACH), acrylic acid (AA), Hydroxyethylmethacrylate (HEMA), methane and hexamethylene-disiloxane (HMDSO). In addition a polyethylene-glycol (PEG) was made by grafting aldehyde functional PEG to the DACH surface. PEG and HMDSO which are the most hydrophilic and the most hydrophobic surface shows the lowest amount of adsorbed protein of the three proteins studied here (albumin, IgG and C3). Methane, HMDSO and HEMA was found to activate via the classical (complement activation) pathway while the others activated via the alternative pathway.

Generation of iC3 at the interface between blood and gas.

Earlier studies have shown that C3 can be denatured when blood comes in contact with a polystyrene surface. This study was undertaken to see if similar denaturation of C3 occurs at the gas-plasma interface which is found in all kinds of oxygenator used during cardio-pulmonary operations. An in vitro system consisting of gas bubbling through human blood, serum or plasma was used. The generation of C3a, as an indicator of complement activation, and iC3 and iC3 fragments were monitored. Both C3a and iC3/iC3 fragments levels were increased during bubbling. In contrast to the C3a level, no reduction in iC3/iC3 fragments formation was seen in the presence of EDTA, indicating that it was independent of complement activation. The rate of iC3/iC3 fragments generation was unaffected by the composition of the gas (pure oxygen, pure nitrogen or air), suggesting that the denaturation of C3 indeed occurred at the serum-gas interface. C3 and iC3/iC3 fragments were isolated from bubbled EDTA-chelated serum by PEG precipitation and chromatography on FPLC, using a Mono S column and detected by two ELISAs, specific for native C3 and iC3/iC3 fragments. After 240 min approximately 20% of the total amount of C3 consisted of intact iC3 and it was confirmed that this population bound to human erythrocytes.

An improved method to study complement receptor-mediated function of the fixed macrophage system in vivo.

A method to coat unsensitized erythrocytes with fragments of C3 and C4 using autologous serum, in order to study complement receptor-dependent function of the fixed macrophage system, is presented. After incubation with serum under optimal conditions, at least 90% of the cells had C3b/iC3b deposited on the surface, with an average of 20 x 10(3) molecules per cell. Elimination of the coated cells by the fixed macrophage system was studied in 12 normal subjects. With a dose of 4.5 x 10(8) red cells injected, 75% of the cells were eliminated with a half-life of approximately 2.4 +/- 0.3 min (n = 7). In subjects receiving ten times more cells, there was a rapid decrease in the amount of C3-coated cells, reaching a nadir with 85% remaining for 4-6 min, after which there was a gradual release of cells for another 20 min (n = 5). In absolute numbers, 3 x 10(8) of labeled cells were eliminated regardless of the dose injected. The coating procedure presented here is simple, does not introduce heterologous blood components and makes it possible to control the amount and the degree of fragmentation of the C3 and C4 deposited on the erythrocyte surface.

C3d-mediated negative and positive signals on the proliferation of human B cells separated from blood.

Soluble C3d applied to human blood-derived B lymphocytes inhibited anti-mu, T cell-produced growth factor, and EBV-induced DNA synthesis in serum-free culture. C3d added to the B cell cultures 1 and 2 days after the stimulus, still exerted inhibition, though with gradually diminishing efficiency. C3d, fixed on zymosan or attached to the culture wells, induced [3H]thymidine incorporation of the B cells in serum-free medium. The concentration of C3d used to coat the wells was critical, with optimal stimulatory effect of 8.3 micrograms/ml. These C3d molecules were shown to be denatured. Our results are in line with earlier data on B cells derived from mouse spleen and human tonsils showing that depending on the way of presentation and its amounts, the natural ligand of CR2 can exert negative or positive signals. Moreover, we demonstrate that C3d can inhibit even the proliferative stimulus of EBV.

Enhanced local production of complement components in the small intestines of patients with Crohn's disease.

There is evidence that complement components may be formed locally in inflammatory lesions containing monocytes and macrophages. To investigate the role of complement in Crohn's disease we measured jejunal-fluid concentrations of the complement components C4, C3, and factor B by perfusion of a closed segment of the jejunum in 22 patients with Crohn's disease thought to be limited to the terminal ileum. The mean (+/- SEM) jejunal-fluid C4 concentration was 2.0 +/- 0.3 mg per liter, significantly higher than the mean level in 35 healthy controls (0.7 +/- 0.1 mg per liter; P less than 0.001). The mean C3 concentration was 1.0 +/- 0.1 mg per liter in the patients and 0.7 +/- 0.1 mg per liter in the controls (P less than 0.05). The factor B levels were similar in the two groups. Calculated rates of intestinal secretion of these components showed differences of the same magnitude. Leakage of protein from plasma was not increased. The jejunal-fluid:serum ratios of these complement proteins indicated that their appearance in the lumen of the jejunum was due to at least in part to local mucosal synthesis. The increased jejunal secretion of C4, but not C3 or factor B, paralleled the clinical activity of Crohn's disease. Values were normal in first-degree relatives of the patients (n = 13), patients with celiac disease (n = 8), and patients with ulcerative colitis (n = 4). We conclude that increased secretion of complement by clinically unaffected jejunal tissue in patients with Crohn's disease reflects the systemic nature of this disorder and may be due to the stimulated synthesis of complement by activated intestinal monocytes and macrophages.

Neoantigens in complement component C3 as detected by monoclonal antibodies. Mapping of the recognized epitopes by synthetic peptides.

The different fragments of the third complement component, C3, generated upon complement activation/inactivation have the ability to bind to several other complement components and receptors as well as to proteins of foreign origin. These multiple reactivities of C3 fragments are associated with a series of conformational changes occurring in the C3 molecule during its degradation. The conformations acquired by the different C3 fragments are also associated with the exposure of neoantigenic epitopes that are specific for (a) particular fragment(s). In order to study these epitopes and thus the conformational changes occurring in C3, monoclonal antibodies (mAbs) recognizing such epitopes were produced in Balb/c mice after immunization with denatured human C3. Two of the three antibodies (7D84.1 and 7D264.6) presented in this study recognized predominantly surface-bound iC3b, and one mAb (7D323.1) recognized both surface-bound and fluid-phase iC3b. Although none of the mAbs recognized any other fluid-phase C3 fragment, all three antibodies detected micro-titre-plate-fixed C3b and iC3b, but not C3c or C3d. In addition to the reaction with human C3, mAb 7D323.1 also bound to micro-titre-plate-fixed rabbit C3. The epitopes recognized by the three mAbs were further localized by using synthetic peptides that were designed on the basis of the differential binding of the mAbs to the C3 fragments. All three antibodies reacted with C3-(924-965)-peptide, which represents the region of C3 between the kallikrein-cleavage site (923-924) and the elastase-cleavage site (965-966). On the basis of the binding of the mAbs to five different overlapping peptides spanning the region between residues 924 and 965 of the human C3 sequence, and the sequence similarity between human C3 and rabbit C3 within this area, the epitopes recognized by these antibodies are mapped. The contribution of the individual amino acid residues in the formation of the epitopes is discussed.

Development of an immunoassay for the detection of minute amounts of IgG-coated erythrocytes in whole blood and its application for the assessment of Fc-mediated clearance of anti-D-coated erythrocytes in vivo.

A rapid and sensitive immunoassay for the detection of minute quantities of IgG-coated erythrocytes in whole blood was developed. Washed red blood cells were incubated in two steps with anti-human IgG antiserum followed by 125I-labelled protein A. The assay was able to detect amounts of sensitized erythrocytes as small as 0.5 ml of packed erythrocytes in a total blood volume of 5 liters and hematocrit 40%. A linear relation between increasing amounts of IgG-coated red cells in whole blood and the binding of 125I-labelled protein A was obtained. We applied the technique on the assessment of the removal of IgG anti-D-coated erythrocytes from the circulation of test individuals. T1/2 for the elimination of approximately 4 ml packed red cells sensitized with 62 micrograms of anti-D in 14 normal subjects was 20 +/- 5 min (mean +/- SEM). A splenectomized person did not clear the injected cells from the circulation during the test period of 70 min. If a standard curve was constructed the total blood volume in the test subjects could be calculated. This value correlated well (r = 0.99) with the blood volume calculated from the height and weight of the test individuals.