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Quantification of Complement C5b-9 Binding to Cells by Flow Cytometry

Interaction of the complement system, directly or indirectly (e.g., via antibodies), with cells activates the early and late complement components and culminates in the deposition of a membrane-spanning C5b-9 complex on the cell surface. At a high copy number, this C5b-9 will activate cell de ...

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Purification of Human Complement Protein C5

Complement C5 is cleaved by proteolysis in the terminal phase of complement activation generating the pro-inflammatory C5a and membrane attack complex nucleator C5b. Whereas purification of its paralogues C3 and C4 from plasma is relatively straightforward, C5 purification is mo ...

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Purification and Characterization of Human and Mouse Complement C3

Complement component C3 is the most abundant complement protein in plasma, central to all three complement activation pathways and essential to complement amplification. Thus, it is one of the most extensively studied complement proteins. This chapter describes the purification of ...

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Expression and Purification Methods for the Production of Recombinant Human Complement Component C2

Human complement component C2 is a critical factor of the classical complement pathway. Here we provide a method for the production of recombinant human C2 (rhC2) protein for research purposes. The human complement component C2 (hC2) is cloned from a human cDNA library by polymerase chain reac ...

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Classical Complement Pathway Components C1r and C1s: Purification from Human Serum and in Recombinant Form and Functional Characterization

C1r and C1s are the proteases responsible for the activation and proteolytic activity of the C1 complex of the classical complement pathway, respectively. They are assembled into a Ca2+-dependent C1s–C1r–C1r–C1s tetramer which in turn associates with the recognition protein C1q. The C1 c ...

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Classical Complement Pathway Component C1q: Purification of Human C1q, Isolation of C1q Collagen-Like and Globular Head Fragments and Production of Re

The classical complement pathway (CCP) activation is a multimolecular complex, composed of three subcomponents namely C1q, C1r, and C1s. C1q is the recognition subunit of this complex and its binding to the specific targets leads to the formation of active C1, which in turn activates the CCP in an im ...

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Opsonophagocytic Assay

The opsonophagocytic killing (OPK) assay is used as a correlate for protection to measure the functional capacities of vaccine-candidate-raised antibodies. This in vitro assay aids selecting promising vaccines by demonstrating whether the vaccine-induced antibodies drive ...

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Analysis of the Complement Activation in Mice

Complement activation is an important immune mechanism in mice like in humans. Murine complement is also activated via three pathways, the classical, lectin, and alternative. Complement proteins are serine proteases; thus activation of complement is proteolytic cleavage of compl ...

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Detection of Soluble CR3 (CD11b/CD18) by Time-Resolved Immunofluorometry

In the cell membrane complement receptor 3 (CR3) consists of one alpha chain (CD11b) and one beta chain (CD18). CR3 participates in many immunological processes, especially those involving cell migration, adhesion, and phagocytosis of complement-opsonized microbes. Recent findi ...

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Evaluation of Complement Function by ELISA

Evaluation of total complement function in human serum is an essential component of laboratory diagnostics of the human complement system. During recent years, established hemolytic assays for classical pathway and alternative pathway function, CH50 and AP50 assays, respectiv ...

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Functional Analysis of CD59 Using Complement-Dependent Cytotoxicity Assay

CD59 overexpression has been shown to confer the resistance of tumors to complement lysis. Complement lysis is one of the two major killing mechanisms of therapeutic anticancer antibodies. This chapter provides a method that allows studying the extent of complement protection of tumors ...

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Detection of CD59 Using Flow Cytometry

CD59 is the single regulator of the terminal complement pathway. It has been implicated in disease such as Paroxysmal nocturnal hemoglobinuria (PMH) and cancer. Expression of CD59 protects normal and malignant cells from the cytotoxic potential of the complement system. Here we describe a ...

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Detection of Cell Membrane-Bound CD46 Using Flow Cytometry

CD46 is an important regulator of the complement system by preventing unwanted deposition of the complement activation products and opsonins C3b/C4b onto self-tissue. Recently, intracellular signals mediated by CD46 activation on several distinct human cell types have demonst ...

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Purification of C1q Receptors and Functional Analysis

The recognition subunit of C1, C1q, has emerged as an important player in various pathophysiologic conditions largely in part due to its ability to interact with pathogen-associated or cell surface expressed ligands and receptors. Identification and purification of these molecules ...

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Quantification of Complement Receptor 2 Calcium Signaling Enhancement Using Flow Cytometry

The complement receptor 2 (Cr2) gene is exclusively expressed in B cells and follicular dendritic cells (FDC) in mice and in humans. CR2 is a receptor for the complement component 3 (C3) cleavage fragments C3d(g) and iC3b. On B cells CR2 acts as the B cell co-receptor in which ligand binding of CR2 effectively ...

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Detection of Complement Receptors 1 and 2 on Mouse Splenic B Cells Using Flow Cytometry

The complement receptor 2 (Cr2) gene is exclusively expressed in B cells and follicular dendritic cells (FDC) in mice and in humans. However, mice also express an alternative splice variant, CR1, of the Cr2 gene. CR2 and CR1 are receptors for the complement component 3 (C3) cleavage fragments C3d(g) and ...

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Functional Analysis of C5a Effector Responses In Vitro and In Vivo

C5a is a powerful proinflammatory and immunomodulatory mediator as evidenced by its involvement in infectious, allergic, and autoimmune diseases as well as in cancer. C5a exerts most of its biologic functions through binding and activation of the G protein-coupled C5a receptor (C5aR). C ...

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Purification of Surfactant Protein D (SP-D) from Pooled Amniotic Fluid and Bronchoalveolar Lavage

Surfactant protein SP-D is a multimeric collagenous lectin, called collectin. SP-D is a multifunctional, pattern recognition innate immune molecule, which binds in a calcium dependent manner to an array of carbohydrates and lipids, thus offering resistance to invading pathogens, al ...

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Purification of Native Surfactant Protein SP-A from Pooled Amniotic Fluid and Bronchoalveolar Lavage

Surfactant protein SP-A is a hydrophilic glycoprotein, similar to SP-D, which plays an important role in pulmonary surfactant homeostasis and innate immunity. SP-A is actively expressed in the alveolar type II cells and Clara cells. Their basic structure consists of triple-helical coll ...

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Anti-Factor H Autoantibodies Assay

Non-Shiga-toxin-associated hemolytic uremic syndrome (atypical HUS) is a rare form of thrombotic microangiopathy which associates hemolytic anemia, thrombocytopenia, and acute renal failure. In 10 % of cases the disease is linked to presence of autoantibodies directed against ...

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