Inhibition of acute passive transfer experimental autoimmune myasthenia gravis with Fab antibody to complement C6

The role of the lytic complement C5b-9 membrane attack complex (MAC) in acute passive transfer experimental autoimmune myasthenia gravis (EAMG) produced in rats was investigated by in vivo inhibition of MAC formation with anti-C6 Fab. Anti-C6 Fab totally inhibited in vitro serum hemolytic activity, but did not consume or inhibit early complement pathways. Injection of rats with 0.12 mg/ml anti-C6 Fab reduced serum C6 to 8% and inhibited the muscle weakness, electrophysiologic abnormalities and loss of acetylcholine receptor (AChR) associated with acute EAMG. This level of C6 inhibition reduced the total serum complement hemolytic activity to 29% of normal but did not reduce the serum levels of complement components C3, C5, or C7. Treatment of rats with lower amounts of anti-C6 Fab (0.08 mg/ml) also inhibited clinical and electrophysiologic signs of EAMG, however, the lower amount of anti-C6 did not prevent the loss of muscle AChR. Both the higher and the lower amount of anti-C6 Fab inhibited the accumulation of macrophages at muscle motor end-plates. The inhibition by anti-C6 indicates that muscle weakness and electrophysiologic abnormalities associated with EAMG are dependent on the complement MAC, and that muscle weakness results from tissue injury in addition to loss of muscle membrane and AChR.     

Chronic experimental autoimmune myasthenia gravis induced by monoclonal antibody to acetylcholine receptor: biochemical and electrophysiologic criteria

To determine whether the chronic presence of antibody to acetylcholine receptor (AChR) can account for the neuromuscular abnormalities in myasthenia gravis (MG), rats injected repeatedly with monoclonal antibody (mAb) to AChR were compared with those injected with control mAb. In a previous report, those receiving anti-AChR mAb, studied ultrastructurally, had grossly simplified endplates when compared with normal controls. In this report, animals injected once or chronically for 9 to 12 wk had reduced content of muscle AChR. The chronically injected animals also had diminished miniature endplate potential amplitudes, but to a lesser extent than the reduction in AChR content. These studies establish the pathogenetic role of antibody to AChR in the induction of the ultrastructural, biochemical, and electrophysiologic hallmarks of MG.     

Prevention of passively transferred experimental autoimmune myasthenia gravis by an in vitro selected RNA aptamer

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are mainly caused by autoantibodies directed against acetylcholine receptors (AChR) located in the postsynaptic muscle membrane. Previously, we isolated an RNA aptamer with 2'-fluoropyrimidines using in vitro selection techniques that acted as an effective decoy against both a rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the AChR, and a significant fraction of patient autoantibodies with MG. To investigate the therapeutic potential of the RNA, we tested the ability of the RNA aptamer to protect the receptors in vivo from mAb198. Clinical symptoms of EAMG in rats engendered by passive transfer of mAb198 were efficiently inhibited by a truncated RNA aptamer that was modified with polyethylene glycol, but not by control scrambled RNA. Moreover, the loss of AChR in the animals induced by the antibody was also significantly blocked with the modified RNA aptamer. These results suggested that RNA aptamers could be applied for antigen-specific treatment for autoimmune diseases including MG.     

C5-blocking antibody reduces fluid requirements and improves responsiveness to fluid infusion in hemorrhagic shock managed with hypotensive resuscitation.

Hypotensive resuscitation strategies and inhibition of complement may both be of benefit in hemorrhagic shock. We asked if C5-blocking antibody (anti-C5) could diminish the amount of fluid required and improve responsiveness to resuscitation from hemorrhage. Awake, male Sprague-Dawley rats underwent controlled hemorrhage followed by prolonged (3 h) hypotensive resuscitation with lactated Ringer's or Hextend, with or without anti-C5. Anti-C5 treatment led to an estimated 62.3 and 58.5% reduction in the volume of Hextend and lactated Ringer's, respectively. In the subgroup of animals with a positive mean arterial pressure (MAP) response to fluid infusion following prolonged hypotension, anti-C5 treatment led to an estimated 4.7- and 4.1-fold increase in mean arterial pressure response per unit Hextend and lactated Ringer's infused, respectively. We observed no significant postresuscitation metabolic differences between the anti-C5 groups and controls. Whether anti-C5 could serve as a volume-sparing adjunct that improves responsiveness to fluid administration in humans deserves further study.     

Role of the main immunogenic region of acetylcholine receptor in myasthenia gravis. An Fab monoclonal antibody protects against antigenic modulation by human sera

Antigenic modulation of acetylcholine receptor (AChR), i.e., acceleration of its internalization and degradation rate by antibody-cross-linking, is considered to be one of the two main causes of AChR loss in myasthenia gravis (MG). The majority of the antibodies to AChR are directed to the main immunogenic region (MIR) on the alpha-subunit of the receptor. We here examine the relative contribution of the anti-MIR antibody fraction (as well as of another fraction) to the antigenic modulation caused by MG patients' sera. Fab fragments of an anti-MIR monoclonal antibody (mAb) or a mAb to the beta-subunit (neither of which causes antigenic modulation) were allowed to shield their corresponding regions on the AChR on the mouse muscle cell line BC3H1. The 27 MG sera subsequently added thus bound to all other regions except to the protected one, and the resulting antigenic modulation was measured. The anti-MIR mAb protected the AChR by 68 +/- 16%. This is interpreted as the contribution to antigenic modulation of the anti-MIR antibody fraction in the human sera. This percentage correlated very well with the occurrence of the anti-MIR antibodies in the same sera. The anti-beta mAb gave only small protection of the AChR. No significant pattern differences were observed between sexes, early and recent onset of the disease, or high and low antibody titers. It is concluded that as far as it concerns the one of the pathogenic mechanisms in MG, i.e., the antigenic modulation, the MIR seems to be the main pathogenic region. The observation that a single mAb can efficiently protect the AChR in this system may prove to be of therapeutic interest.     

Breakdown of tolerance to a self-peptide of acetylcholine receptor alpha-subunit induces experimental myasthenia gravis in rats

Experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia (MG), is routinely induced in susceptible rat strains by a single immunization with Torpedo acetylcholine receptor (TAChR). TAChR immunization induces anti-AChR Abs that cross-react with self AChR, activate the complement cascade, and promote degradation of the postsynaptic membrane of the neuromuscular junction. In parallel, TAChR-specific T cells are induced, and their specific immunodominant epitope has been mapped to the sequence 97-116 of the AChR alpha subunit. A proliferative T cell response against the corresponding rat sequence (R97-116) was also found in TAChR-immunized rats. To test whether the rat (self) sequence can be pathogenic, we immunized Lewis rats with R97-116 or T97-116 peptides and evaluated clinical, neurophysiological, and immunological parameters. Clinical signs of the disease were noted only in R97-116-immunized animals and were confirmed by electrophysiological signs of impaired neuromuscular transmission. All animals produced Abs against the immunizing peptide, but anti-rat AChR Abs were observed only in animals immunized with the rat peptide. These findings suggested that EAMG in rats can be induced by a single peptide of the self AChR, that this sequence is recognized by T cells and Abs, and that breakdown of tolerance to a self epitope might be an initiating event in the pathogenesis of rat EAMG and MG.     

Antigenic modulation of junctional acetylcholine receptor is not sufficient to account for the development of myasthenia gravis in receptor immunized mice

Myasthenia gravis (MG) is a disease thought to result from an autoimmune response against the nicotinic acetylcholine receptor of the neuromuscular junction. Although there is little doubt that the muscular weakness characteristic of MG can be attributed to an antibody-mediated reduction in the density of AChR, the mechanism responsible for this reduction remains uncertain. In the present studies we have used a mouse model of MG, termed experimental myasthenia gravis (EMG), to test the possibility that antigenic modulation of AChR may be the principle mechanism whereby this reduction in AChR density is achieved. We found that immunization of mice with AChR, on average, leads to a twofold increase in the rate of junctional AChR degradation. Because this effect occurred to the same extent in mice that developed severe paralysis and in those that gave no indication of muscular weakness, the role of antigenic modulation as a major pathologic mechanism in MG is questioned.     

Suppression of development of experimental autoimmune myasthenia gravis with isogeneic monoclonal anti-idiotopic antibody

We have made use of isogeneic anti-idiotopic (anti-Id) monoclonal antibodies (mAb to modify experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. High-avidity anti-Id mAb HC-4A (Kd = 0.1 nM) and HC-29 (Kd = 0.1 nM) were produced against an anti-acetylcholine receptor (anti-AChR) Lewis-rat mAb 132A (Kd = 0.34 nM) that is capable of inducing passive-transfer EAMG. mAb HC-4A and HC-29 define separate framework Id cross-reactive with anti-AChR mAb recognizing different AChR epitopes. Animals were preinjected i.p. with either anti-Id mAb or with control mAb and then were actively immunized 2 wk later with purified AChR. All animals had elevated total serum anti-AChR antibody titers, despite the absence of weakness or decremental electromyographic findings. Animals preinjected with control mAb developed serum anti-AChR titers of 1.34 +/- 0.29 microM (mean +/- SEM) and reduced muscle AChR content to 30 percent of normal. Animals injected with 0.5 mg/kg of either anti-Id had significantly lower serum anti-AChR titers, 0.55 +/- 0.1, p less than 0.05, and normal muscle AChR content. Both the 132A Id and the anti-Id complementary to 132A were detected in the serum of all of the animals preinjected with this dose of either anti-Id HC-29 or HC-4A, whereas both were detected in a much smaller percentage of the animals receiving control mAb. These results show that pretreatment with anti-Id not only perturbs this Id-anti-Id network, but also suppresses the overall polyclonal anti-AChR response with resultant protection of actively immunized animals from EAMG.     

Absence of IL-4 facilitates the development of chronic autoimmune myasthenia gravis in C57BL/6 mice

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. Ab against muscle acetylcholine receptor (AChR) cause the muscular weakness that characterizes MG and its animal model, experimental MG (EMG). EMG is induced in C57BL6 (B6) mice by three injections of Torpedo AChR (TAChR) in adjuvant. B6 mice develop anti-TAChR Ab that cross-react with mouse muscle AChR, but their CD4+ T cells do not cross-react with mouse AChR sequences. Moreover, murine EMG is not self-maintaining as is human MG, and it has limited duration. Several studies suggest that IL-4 has a protecting function in EMG. Here we show that B6 mice genetically deficient in IL-4 (IL-4-/-) develop long-lasting muscle weakness after a single immunization with TAChR. They develop chronic self-reactive Ab, and their CD4+ T cells respond not only to the TAChR and TAChR subunit peptides, but also to several mouse AChR subunit peptides. These results suggest that in B6 mice, regulatory mechanisms that involve IL-4 contribute to preventing the development of a chronic Ab-mediated autoimmune response to the AChR.     

Immunotherapy for myasthenia gravis: a murine model

In vivo therapy with monoclonal antibody (mAb) GK1.5, which recognizes a glycoprotein antigen designated L3T4 on murine helper T lymphocytes, either prevented or suppressed the development of murine lupus, autoimmune encephalomyelitis, and collagen arthritis. The L3T4 antigen in the mouse is analogous to the human Leu-3/T4 antigen expressed on helper T lymphocytes, because they both participate in the T cell response to class II major histocompatibility complex (MHC) antigens. Class II MHC genes and I-A antigens mediate murine experimental autoimmune myasthenia gravis (EAMG) induced by acetylcholine receptor (AChR) autoimmunity. We studied the efficacy of mAb GK1.5 as an immunotherapeutic agent for murine EAMG. Therapy with mAb GK1.5 not only suppressed established autoimmunity to AChR but also prevented loss of muscle AChR in mice with EAMG. Moreover, permanent remission of clinical muscle weakness was induced if mAb GK1.5 therapy was initiated after the onset of clinical disease. Because the function of the Leu-3/T4 determinant on human helper T lymphocytes is analogous to the murine L3T4 determinant, use of antibody to the Leu-3/T4 determinant as an immunotherapeutic agent may provide a way to control the progression of human MG.     

Split tolerance in a novel transgenic model of autoimmune myasthenia gravis

Because it is one of the few autoimmune disorders in which the target autoantigen has been definitively identified, myasthenia gravis (MG) provides a unique opportunity for testing basic concepts of immune tolerance. In most MG patients, Abs against the acetylcholine receptors (AChR) at the neuromuscular junction can be readily identified and have been directly shown to cause muscle weakness. T cells have also been implicated and appear to play a role in regulating the pathogenic B cells. A murine MG model, generated by immunizing mice with heterologous AChR from the electric fish Torpedo californica, has been used extensively. In these animals, Abs cross-react with murine AChR; however, the T cells do not. Thus, to study tolerance to AChR, a transgenic mouse model was generated in which the immunodominant Torpedo AChR (T-AChR) alpha subunit is expressed in appropriate tissues. Upon immunization, these mice showed greatly reduced T cell responses to T-AChR and the immunodominant alpha-chain peptide. Limiting dilution assays suggest the likely mechanism of tolerance is deletion or anergy. Despite this tolerance, immunization with intact T-AChR induced anti-AChR Abs, including Abs against the alpha subunit, and the incidence of MG-like symptoms was similar to that of wild-type animals. Furthermore, evidence suggests that this B cell response to the alpha-chain receives help from T cells directed against the other AChR polypeptides (beta, gamma, or delta). This model offers a novel opportunity to elucidate mechanisms of tolerance regulation to muscle AChR and to clarify the role of T cells in MG.     

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.     

A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.     

Improvement of RNA aptamer activity against myasthenic autoantibodies by extended sequence selection.

Myasthenia gravis (MG) is mainly engendered by autoantibodies directed against acetylcholine receptors (AChRs) located in the postsynaptic muscle cell membrane. Previously, we isolated an RNA aptamer with 2'-amino pyrimidines using in vitro selection techniques that acted as a decoy against both a rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the AChR, and patient autoantibodies with MG (1). However, low affinity of this RNA to mAb198 relative to that of AChR might limit potential of the RNA as an inhibitor of the autoantibodies. To improve decoy activity of the RNA aptamer against autoantibodies, here we employed in vitro selection methods with RNA libraries containing extra random nucleotides extended to the 3' end of previously selected RNA sequences. RNAs isolated in this study showed significant increases in the binding affinities to mAb198 as well as bioactivities protecting AChRs on human cells from both mAb198 and patient autoantibodies, compared with the previous RNA aptamers. These results have important implications for the development of antigen-specific modulation of autoimmune diseases including MG.     

Specificity of the T cell immune response to acetylcholine receptor in experimental autoimmune myasthenia gravis. Response to subunits and synthetic peptides

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are T cell-dependent diseases mediated by antibodies against acetylcholine receptor (AChR) on skeletal muscle. Most of the antibodies are directed toward conformation-dependent epitopes on the AChR, whereas T cells recognize denatured AChR. In search of T cell epitopes in EAMG, we tested 24 synthetic peptides covering 62% of the alpha-subunit sequence of Torpedo californica electric organ AChR in the T cell proliferation assay with lymph node cells from rats immunized with AChR. In Lewis rats, 2 of these peptides, [Tyr 100]alpha 100-116 and [Gly 89, Tyr 90]alpha 73-90, strongly stimulated T cells and, of these, [Tyr 100]alpha 100-116 was much more potent; 4 other peptides were weakly mitogenic and 18 were ineffective. None of the 24 synthetic peptides alone stimulated anti-AChR production and, when added to cultures along with AChR, [Tyr 100]alpha 100-116 and [Gly 89, Tyr 90]alpha 73-90 suppressed antibody production. Of twelve cloned T cell lines specific to AChR, 4 responded to [Tyr 100]alpha 100-116, indicating the importance of the epitope in alpha 101-116 in Lewis rats. In three other strains of rats whose responses to AChR and its subunits were similar to those in the Lewis rat, neither [Tyr 100]alpha 100-116 nor [Gly 89, Tyr 90]alpha 73-90 was stimulatory. Instead, completely different sets of peptides stimulated their T cells. When peptides were used as immunogens, each strain (except Lewis rats) responded only to the peptides that stimulated AChR-immune T cells from the same strain. Genetically restricted T cell recognition of AChR peptides in rats suggests that T cells from MG patients with different major histocompatibility haplotypes may recognize different AChR peptides.     

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.     

Amelioration of experimental autoimmune myasthenia gravis in rats by neonatal FcR blockade

The neonatal FcR (FcRn) plays a critical role in IgG homeostasis by protecting it from a lysosomal degradation pathway. It has been shown that IgG has an abnormally short half-life in FcRn-deficient mice and that FcRn blockade significantly increases the catabolism of serum IgG in mice. Therefore, reduction of serum IgG half-life may have therapeutic benefits in Ab-mediated autoimmune diseases. We have studied the therapeutic effects of an anti-rat FcRn mAb, 1G3, in two rat models of myasthenia gravis, a prototypical Ab-mediated autoimmune disease. Passive experimental autoimmune myasthenia gravis was induced by administration of an anti-acetylcholine receptor (AChR) mAb, and it was shown that treatment with 1G3 resulted in dose-dependent amelioration of the disease symptoms. In addition, the concentration of pathogenic Ab in the serum was reduced significantly. The effect of 1G3 was also studied in an active model of experimental autoimmune myasthenia gravis in which rats were immunized with AChR. Treatment with 1G3 significantly reduced the severity of the disease symptoms as well as the levels of total IgG and anti-AChR IgG relative to untreated animals. These data suggest that FcRn blockade may be an effective way to treat Ab-mediated autoimmune diseases.     

Direct Proof of the In Vivo Pathogenic Role of the AChR Autoantibodies from Myasthenia Gravis Patients

Several studies have suggested that the autoantibodies (autoAbs) against muscle acetylcholine receptor (AChR) of myasthenia gravis (MG) patients are the main pathogenic factor in MG; however, this belief has not yet been confirmed with direct observations. Although animals immunized with AChR or injected with anti-AChR monoclonal Abs, or with crude human MG Ig fractions exhibit MG symptoms, the pathogenic role of isolated anti-AChR autoAbs, and, more importantly, the absence of pathogenic factor(s) in the autoAb-depleted MG sera has not yet been shown by in vivo studies. Using recombinant extracellular domains of the human AChR α and β subunits, we have isolated autoAbs from the sera of four MG patients. The ability of these isolated anti-subunit Abs and of the Ab-depleted sera to passively transfer experimental autoimmune MG in Lewis rats was investigated. We found that the isolated anti-subunit Abs were at least as efficient as the corresponding whole sera or whole Ig in causing experimental MG. Abs to both α- and β-subunit were pathogenic although the anti-α-subunit were much more efficient than the anti-β-subunit ones. Interestingly, the autoAb-depleted sera were free of pathogenic activity. The later suggests that the myasthenogenic potency of the studied anti-AChR MG sera is totally due to their anti-AChR autoAbs, and therefore selective elimination of the anti-AChR autoAbs from MG patients may be an efficient therapy for MG.     

A Novel Antibody against Human Properdin Inhibits the Alternative Complement System and Specifically Detects Properdin from Blood Samples

The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1–10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H₂O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80–125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13–30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.