Detection of native human complement components C3 and C5 and their primary activation peptides C3a and C5a (anaphylatoxic peptides) by ELISAs with monoclonal antibodies

doi:10.1016/0022-1759(88)90133-0

Journal of Immunological Methods

Volume 111, Issue 2, 22 July 1988, Pages 241-252

https://doi.org/10.1016/0022-1759(88)90133-0 Get rights and content

Abstract

Monoclonal antibodies (mAbs) were raised against human C3a, C3b, C5a, and C5b after immunization of BALB/c mice with the native components C3 and C5. Using different combinations of these mAbs we have developed four sensitive sandwich-enzyme-linked immunosorbent assays (ELISAs) for the detection of native C3 or C5 in samples with low concentrations of these proteins, e.g., in cell culture supernatants or synovial fluids and cerebrospinal fluids (CSF) and for the detection of the anaphylatoxic peptides (AT-peptides) C3 or C5a in human EDTA-plasma. The C3- and C5-ELISAs were found to be specific for the uncleaved complement proteins. Two different anti-C3a or anti-C5a mAbs were combined for the C3a- and C5a-ELISA. Before assaying a sample in the C3a- or C5a-ELISA a precipitation step to eliminate uncleaved C3 and C5 was necessary. The sensitivity and specificity of the four ELISAs were tested with purified antigens and EDTA-plasma or Cobra venom factor-activated EGTA-plasma samples as a source of C3a and C5a. The detection limits were 1 ng/ml for C3, 1 ng/ml for C3a, 2 ng/ml for C5, and 100 pg/ml for C5a. Plasma samples from patients undergoing cardiopulmonary bypass (CPB) surgery were used as a source of pathological material.

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The microsphere-induced complement activation is studied by measuring the anaphylatoxins C3a and C5a (Fig. 7D). The levels of C3a and C5a have an insignificant or modest increase after the incubation, indicating different degrees of activation of the complement system [49]. Complete blood count.
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Hyperkalemia (serum potassium >5 mmol/L) is a common complication in chronic renal failure patients. The limitations of existing treatments prompt a shift to wearable artificial kidney technology for clinical convenience and efficacy. Existing treatments have limitations, and we turn to adsorbent-based miniaturized blood purification devices in the prospect of wearable artificial kidney technology. There exists a lack of ion-specific adsorbents applied in extracorporeal circuits to redress electrolyte imbalances like hyperkalemia. Inspired by cells, we aim at the favorable permeation and enrichment of K⁺ by microspheres. The microspheres have a microphase-isolated core-shell structure, whose nano-spaced groups form cation-affinitive clusters. Selective K⁺ removal and blood compatibility are achieved. We expect this strategy to enlighten alternative hyperkalemia therapy for these high-risk patients.
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Presence of C3a provides indirect evidence of C5a generation as C3 cleavage results in the formation of the C5 convertase, which in turn cleaves C5 into C5a and C5b (Müller-Eberhard, 1986). Indeed, under conditions that favor high levels of complement activation in the absence of cells, C5a can be detected in plasma and shows a similar kinetic when compared with C3a generation (Klos et al., 1988). Regression analyses with complement factors as dependent and albumin and hormones as independent variables identified albumin, cortisol (with a time lag of 3 h) and epinephrine as best predictors for levels of C3 and C4 (Table 1).
The sleep-wake cycle is characterized by complex interactions among the central nervous, the endocrine and the immune systems. Continuous 24-h wakefulness prevents sleep-associated hormone regulation resulting in impaired pro-inflammatory cytokine production. Importantly, cytokines and hormones also modulate the complement system, which in turn regulates several adaptive immune responses. However, it is unknown whether sleep affects the activation and the immunoregulatory properties of the complement system. Here, we determined whether the 24-h sleep-wake cycle has an impact on: (i) the levels of circulating complement factors; and (ii) TLR4-mediated IL-12 production from human IFN-γ primed monocytes in the presence or absence of C5a receptor signaling. For this purpose, we analyzed the blood and blood-derived monocytes of 13 healthy donors during a regular sleep-wake cycle in comparison to 24 h of continuous wakefulness. We found decreased plasma levels of C3 and C4 during nighttime hours that were not affected by sleep. In contrast, sleep was associated with increased complement activation as reflected by elevated C3a plasma levels during nighttime sleep. Sleep deprivation prevented such activation. At the cellular level, C5a negatively regulated TLR4-mediated IL-12p40 and p70 production from human monocytes. Importantly, this regulatory effect of C5a on IL-12p70 production was effective only during daytime hours. Thus, similar to hormones, some complement factors and immunoregulatory properties of C5a are influenced by sleep and the circadian rhythm. Our findings that continuous wakefulness has a negative impact on complement activation may provide a rationale for the immunosupportive functions of sleep.
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Fig. 15A shows that the convertases formed with humanized CVF and purified human complement proteins do not cleave purified human C5 in vitro (Fritzinger et al., 2009). Using either a hemolytic assay that is based on fluid-phase C5 cleavage (Vogel and Müller-Eberhard, 1984) or an ELISA to measure C5a production (Klos et al., 1988), undetectable or only very low levels of C5 activation were observed in guinea pig or monkey serum after incubation with humanized CVF in vitro (Fig. 15B and C) (Fritzinger et al., 2008c, 2009). Similar results were obtained in vivo after decomplementation with the humanized CVF protein HC3-1496, both in mice (Gorsuch et al., 2009) and monkeys (see Fig. 20, below) (Fritzinger et al., 2008c).
Cobra venom factor (CVF) is the complement-activating protein in cobra venom. This manuscript reviews the structure and function of CVF, how it interacts with the complement system, the structural and functional homology to complement component C3, and the use of CVF as an experimental tool to decomplement laboratory animals to study the functions of complement in host defense and immune response as well as in the pathogenesis of diseases. This manuscript also reviews the recent progress in using the homology between CVF and C3 to study C3 structure and function, and to develop human C3 derivatives with the complement-depleting function of CVF. These human C3 derivatives represent humanized CVF, and are a conceptually different concept for pharmacological intervention of the complement system, therapeutic complement depletion. The use of humanized CVF for therapeutic complement depletion in several pre-clinical models of human diseases is also reviewed.
C5a mutants are potent antagonists of the C5a receptor (CD88) and of C5L2. Position 69 is the locus that determines agonism or antagonism
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The next day, the periplasmic fraction was prepared by freezing and thawing as described (26). CNBr-activated Sepharose (Amersham Biosciences) was coated with C5a-specific mAb 561 (36) according to the manufacturer's instructions. A polyethylene-column (Biognosis) was loaded with 100 μl of gel matrix.
The anaphylatoxin C5a exerts a plethora of biologic activities critical in the pathogenesis of systemic inflammatory diseases. Recently, we reported on a C5a mutant, jun/fos-A8, as a potent antagonist for the human and mouse C5a receptor (CD88). Addressing the molecular mechanism accounting for CD88 receptor antagonism by site-directed mutagenesis, we found that a positively charged amino acid at position 69 is crucial. Replacements by either hydrophobic or negatively charged amino acids switched the CD88 antagonist jun/fos-A8 to a CD88 agonist. In addition to CD88, the seven-transmembrane receptor C5L2 has recently been found to provide high affinity binding sites for C5a and its desarginated form, C5adesArg⁷⁴. A jun/fos-A8 mutant in which the jun/ fos moieties and amino acids at positions 71–73 were deleted, A8^Δ71–73, blocked C5a and C5adesArg⁷⁴ binding to CD88 and C5L2. In contrast, the cyclic C5a C-terminal analog peptide AcF-[OP-d-ChaWR] inhibited binding of the two anaphylatoxins to CD88 but not to C5L2, suggesting that the C5a core segment is important for high affinity binding to C5L2. Both receptors are coexpressed on human monocytes and the human mast cell line HMC-1; however, C5L2 expression on monocytes is weaker as compared with HMC-1 cells and highly variable. In contrast, no C5L2 expression was found on human neutrophils. A8^Δ71–73 is the first antagonist that blocks C5a and C5adesArg⁷⁴ binding to both C5a receptors, CD88 and C5L2, making it a valuable tool for studying C5L2 functions and for blocking the biological activities of C5a and C5adesArg⁷⁴ in mice and humans.
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We constructed combinatorial immunoglobulin libraries from the whole rabbit antibody repertoire of bone marrow, spleen and peripheral blood of a rabbit immunized with guinea pig complement protein C3. By means of the phage display technology we selected guinea pig C3 specific single chain Fv (scFv) antibodies from each of the libraries. None of the scFv antibodies cross reacted with guinea pig C3a, human C3 or rat C3. The frequency of bone marrow derived C3 positive clones was much higher as compared to blood or spleen derived clones. Additionally bone marrow and spleen derived clones show higher diversity than clones, obtained from blood, as determined by fingerprint analysis with the restriction enzyme AluI. Dissociation rate constants for all scFvs were similar, indicating that the source of the scFvs had no influence on affinities. The antibody fragments were used to analyze complement activation during xenotransplantation. Several blood or bone marrow derived scFvs bound to C3 located on rat liver endothelium after hyperacute rejection of a heterotopically transplanted rat liver into guinea pig. These data demonstrate that monoclonal rabbit scFvs can be easily generated from recombinant phage display libraries, constructed from spleen, blood or bone marrow. The selected guinea pig C3 specific scFvs appear to be useful to detect complement activation during xenotranplantation in guinea pigs.
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The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35–40 was randomly mutated and the resulting C5a[35–40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A₃₅E₃₆R₃₇A₃₈S₃₉R₄₀], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q₃₅E₃₆R₃₇I₃₈L₃₉N₄₀] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.

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^☆: Part of this work was done in fulfillment of the requirements of the Medical Faculty Johannes Gutenberg University of Mainz for the MD-Thesis of A. Klos and V. Ihrig.

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Detection of native human complement components C3 and C5 and their primary activation peptides C3a and C5a (anaphylatoxic peptides) by ELISAs with monoclonal antibodies☆

Abstract

Immunochemistry

Anal. Biochem.

J. Immunol. Methods

Lancet

Methods Enzymol.

J. Immunol. Methods

Anal. Biochem.

J. Immunol. Methods

Bypass activation of the complement system starting with C3

Immunology

Identification of functionally relevant determinants on the complement component C3 with monoclonal antibodies

J. Immunol.

Complement activation during cardiopulmonary bypass

New Engl. J. Med.

The complement system

Hepatocellular carcinoma after thorotrast exposure: establishment of a new cell line (Mz-Hep-1)

Hepatology

Complement activation in cystic fibrosis respiratory fluids: in vivo and in vitro generation of C5a and chemotactic activity

Pediatr. Res.