Two-site ELISA for quantification of the terminal C5b-9 complement complex in plasma

Accardo-Palumbo, Antonina; Triolo, G; Casiglia, D; Sallì, L; Giardina, E

doi:10.1016/0022-1759(93)90119-r

Cited by 4 publications

(1 citation statement)

References 10 publications

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“…Purification of Fluid Phase Pre-assembled C5b-9-Fluid phase C5b-9 was isolated from pooled normal sera as described previously (29,30). In brief, 200 mg of zymosan A was added to 20 ml of serum, mixed, and incubated at 37°C for 1 h. The C5b-9 was then precipitated by adding 7% polyethylene glycol 6000 weight by volume, and the mixture was incubated overnight at 4°C.…”

Section: Purification Of Ics From Sle Patients-thementioning

confidence: 99%

T Cell Activation by Terminal Complex of Complement and Immune Complexes

Chauhan

Moore

2011

Journal of Biological Chemistry

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T cell hyperactivation and complement consumption are prominent features of the immunopathology of systemic lupus erythematosus. Although complement activation is secondary to autoantibodies that form immune complexes (ICs), the trigger for alterations in human peripheral blood T cells is poorly understood. To study the impact (on T cells) of several types of preformed ICs and terminal complement complex, also referred to as C5b-9, we incubated these immune reactants with peripheral blood naive CD4؉ T cells as well as Jurkat cells and analyzed their effects on cellular behavior. We first assembled the C5b-9 in situ on the membrane and observed its assembly primarily on a single site where it promoted aggregation of membrane rafts and recruitment of the CD3 signaling complex. However, C5b-9 alone did not initiate proliferation or commencement of downstream signaling events associated with T cell activation. When T cells were treated with ICs together with nonlytic C5b-9, changes associated with T cell activation by possible antigen engagement then occurred. T cell antigen receptor signaling proteins, including -chain, ZAP-70, Syk, Src, and Lck, were phosphorylated and organized in a synapse-like structure. The cytoskeleton formed F-actin spindles and a distal pole complex, resulting in a bipolar distribution of phosphorylated ezrin-radixin-moesin and F-actin. Furthermore, ICs and nonlytic C5b-9 induced T cell proliferation and IFN-␥ production. These results raise the possibility that ICs and the nonlytic C5b-9 modulate T cell-mediated responses in systemic lupus erythematosus and other related chronic inflammatory disorders.

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Section: Purification Of Ics From Sle Patients-thementioning

confidence: 99%

T Cell Activation by Terminal Complex of Complement and Immune Complexes

Chauhan

Moore

2011

Journal of Biological Chemistry

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

Moskovich¹,

Fishelson²

2013

Methods in Molecular Biology

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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 death, whereas at a low copy number, it will transmit various signals into cells. Quantification of C5b-9 deposition is useful for assessments of the capacity of cells and antibodies to activate complement. By using an antibody that identifies a novel antigen of the C5b-9 complex, the amount of C5b-9 complexes on cells can be quantified by flow cytometry. The detailed protocol is described in this chapter.

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The Clinical Laboratory: Testing the Complement System

Kirschfink¹

1998

The Complement System

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Two-site ELISA for quantification of the terminal C5b-9 complement complex in plasma

Cited by 4 publications

References 10 publications

T Cell Activation by Terminal Complex of Complement and Immune Complexes

T Cell Activation by Terminal Complex of Complement and Immune Complexes

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

The Clinical Laboratory: Testing the Complement System

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