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Quantitative determination of the Complement activation via SC5b-9 or C3a - in vitro ELISA-Assay

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This in vitro method is used to determine the influence of a test material on complement activation in human whole blood.

The complement system is a part of the body’s innate nonspecific humoral immune system. It is activated through the surface of pathogens or antibody presented antigens and complements the activity of phagocytic cells and the adaptive immune system as well as promoting inflammation and attacking the cell membranes of bacterial pathogens.1 It consists of over 30 plasma and surface-expressed proteins with the 9 major component factors C1-C9.2 There are three complement initiation pathways: the classical pathway (antibody mediated response), the mannose-binding lectin (MBL) pathway (bacterial carbohydrate motifs e.g. mannose) and the alternative pathway (spontaneous/continuous activation at low rate or by pathogen surface).1

The complement factors are activated via protease-mediated cleavage into a small fragment termed as a (e.g. C3a) and a lager fragment designated as b (e.g. C5b). These fragments in turn cleave their substrates resulting in the complement cascade. Cleavage of C3 is the pivotal step for the complement activation through all three initiation pathways, leading to a feedback loop via C3b interaction with fB to form more C3 convertases.2

The small fragments C3a and C5a, also called anaphylatoxins, are recruiters of leucocytes (white blood cells), such as neutrophils, granulocytes and macrophages. Anaphylatoxins are one of the main mediators in inflammation reactions and also involved in sepsis and allergic responses.3,4

One consequence of C5b formation is the creation of Membrane Attack Complexes (MACs) together with other complement factors at the cell membrane, leading to the destruction of target cells inducing cell lysis.2 However, a large part of the C5b formed stays in the fluid phase (i.e. the plasma) by forming complexes with Protein S. One of them is the SC5b-9 complex, consisting of Protein S, C5b and C9. This free SC5b-9-Komplex can only form upon the terminal steps of the complement activation pathway in vivo and can therefore be used as a blood plasma marker for its activation.5

C3a on the other hand is generated early in the complement cascade and its concentration therefore gives insight into the initiating complement response.6

The complement activation tests are performed in accordance with the ISO guidelines 10993-11, 10993-42 and 10993-128 at Eurofins BioPharma Product Testing Munich GmbH.

Procedure

Human whole blood samples are incubated with the test item. After Incubation the Plasma is gained from the blood samples and used to determine complement component concentration using an ELISA based method. Zymosan is used as a positive control, as it is a repetitive glucose carbohydrate, which triggers the MBL pathway complement activation.

 

Historical Data

LCL:           Lower control limit (95%, mean-2SD)

UCL:          Upper control limit (95%, mean+2SD)

n:               number of assays

SD:             Standard Deviation

Historical data were generated from 2018 to 2022.

 

References

  1. Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. The complement system and innate immunity.
  2. Mathern DR, Heeger PS. Molecules Great and Small: The Complement System. Clin J Am Soc Nephrol. 2015 Sep 4;10(9):1636-50. doi: 10.2215/CJN.06230614.
  3. Giang J, Seelen MAJ, van Doorn MBA, Rissmann R, Prens EP, Damman J. Complement Activation in Inflammatory Skin Diseases. Front Immunol. 2018 Apr 16;9:639. doi: 10.3389/fimmu.2018.00639.
  4. Hotchkiss RS, Moldawer LL, Opal SM, Reinhart K, Turnbull IR, Vincent JL. Sepsis and septic shock. Nat Rev Dis Primers. 2016 Jun 30;2:16045. doi: 10.1038/nrdp.2016.45.
  5. Mollnes TE. Complement and biocompatibility. Vox Sang. 1998;74 Suppl 2:303-7. doi: 10.1111/j.1423-0410.1998.tb05435.x.
  6. Bajic G, Yatime L, Klos A, Andersen GR. Human C3a and C3a desArg anaphylatoxins have conserved structures, in contrast to C5a and C5a desArg. Protein Sci. 2013 Feb;22(2):204-12. doi: 10.1002/pro.2200. Epub 2012 Dec 16.
  7. ISO 10993-1: “Evaluation and testing within a risk management process”
  8. ISO 10993-4: “Selection of tests for interactions with blood”
  9. ISO 10993-12: “Sample preparation and reference materials”