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Chapter 14 : Analysis of Activity of Mannan-Binding Lectin, an Initiator of the Lectin Pathway of the Complement System

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Analysis of Activity of Mannan-Binding Lectin, an Initiator of the Lectin Pathway of the Complement System, Page 1 of 2

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Abstract:

The innate immune system has traditionally been described as the first line of the body's defense against invasive pathogens. Such a response then leads to an inflammatory response which may also include coagulation. Activation of the innate immune response is mediated by pattern recognition molecules, which may be membrane-bound (e.g., cell-associated Toll-like receptors, NOD-like receptors, and RIG-I-like receptors) or soluble proteins. Recognition of foreign or altered structures in the body by some of the soluble pattern recognition receptors may lead to activation of the complement system and thus trigger one of the innate antimicrobial defense mechanisms. Such complement-activating soluble pattern recognition molecules include the collectins (lectins, i.e., carbohydrate-binding proteins, that use a collagen helix for stabilization of the molecule), i.e., mannan-binding lectin (MBL, also known as mannose-binding lectin), collectin K1 (CL-K1), and collectin L1 (CL-L1), and the ficolins (proteins that contain a fibrinogen-like domain and use a collagen helix for stabilization of the molecule), i.e., H-ficolin, L-ficolin, and M-ficolin (1, 2).

Citation: Thiel S. 2016. Analysis of Activity of Mannan-Binding Lectin, an Initiator of the Lectin Pathway of the Complement System, p 133-137. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch14
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Figures

Image of FIGURE 1
FIGURE 1

Complement activation pathways. The figure shows the events leading to the formation of enzyme complexes that cleave C3. The three pathways thus converge on C3. Thereafter, formation of the terminal membrane attack complex, opsonic factors (C3b), and chemotactic factors (e.g., C5a) may proceed. The classical pathway is activated by C1q in complex with C1s and C1r upon binding to antibody-antigen complexes. The lectin pathway is activated when MBL in complex with MASPs recognizes foreign carbohydrate patterns. As indicated in the figure, ficolins may activate complement via this pathway, but the assay described in this chapter is not influenced by these molecules, as they bind to a different set of ligands than those presented on the surface and used in the assay. The alternative pathway is constitutively activated by the formation of C3(HO)-factor B complexes and may be propagated on foreign surfaces by the other factors indicated. The assay described in this chapter measures the cleavage of C4 and the following deposition of C4b by MASP-2 bound to MBL.

Citation: Thiel S. 2016. Analysis of Activity of Mannan-Binding Lectin, an Initiator of the Lectin Pathway of the Complement System, p 133-137. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch14
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Image of FIGURE 2
FIGURE 2

Assay of MBL pathway activity. Step 1, coating of the microtiter plate with mannan; step 2, MBL–MASP-2 complexes bind to immobilized mannan; step 3, incubation of C4, which is then cleaved, and deposition of C4b in the wells; step 4, anti-C4 antibody is bound to the deposited C4 fragment; step 5, binding of europium-labeled streptavidin to the biotinylated anti-C4 antibody; step 6, addition of enhancement solution to the wells, which is followed by reading of the plate by time-resolved fluorometry.

Citation: Thiel S. 2016. Analysis of Activity of Mannan-Binding Lectin, an Initiator of the Lectin Pathway of the Complement System, p 133-137. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch14
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Image of FIGURE 3
FIGURE 3

Appearance of a typical standard curve for the assay. The serum concentration is given on the axis (log scale), and the signal is given on the axis (log scale). In this figure, the highest serum concentration used is 1/20, followed by 3.5-fold dilutions. Other dilution schemes can also be used (e.g., 1/10 dilution followed by 3-fold dilutions).

Citation: Thiel S. 2016. Analysis of Activity of Mannan-Binding Lectin, an Initiator of the Lectin Pathway of the Complement System, p 133-137. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch14
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References

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1. Thiel S. 2007. Complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins. Mol Immunol 44:38753888.[CrossRef].[PubMed]
2. Holmskov U, Thiel S, Jensenius JC. 2003. Collectins and ficolins: humoral lectins of the innate immune defense. Annu Rev Immunol 21:547578.[CrossRef].[PubMed]
3. Ricklin D, Hajishengallis G, Yang K. 2010. Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11:785797.[CrossRef].[PubMed]
4. Thiel S, Frederiksen PD, Jensenius JC. 2006. Clinical manifestations of mannan-binding lectin deficiency. Mol Immunol 43:8696.[CrossRef].[PubMed]
5. Heitzeneder S, Seidel M, Förster-Waldl E, Heitger A. 2012. Mannan-binding lectin deficiency—good news, bad news, doesn't matter? Clin Immunol 143:2238.[CrossRef].[PubMed]
6. Møller-Kristensen M, Thiel S, Jensenius JC,. 2009. MBL polymorphisms and infectious diseases, p 303332. In Vasta GR, Ahmed H (ed), Animal Lectins: A Functional View. CRC Press, Taylor and Francis Group, Boca Raton, FL.
7. Stengaard-Pedersen K, Thiel S, Gadjeva M, Møller-Kristensen M, Sørensen R, Jensen LT, Sjøholm AG, Fugger L, Jensenius JC. 2003. Inherited deficiency of mannan-binding lectin-associated serine protease 2. N Engl J Med 349:554560.[CrossRef].[PubMed]
8. Degn SE, Jensenius JC, Thiel S. 2011. Disease-causing mutations in genes of the complement system. Am J Hum Genet 88:689705.[CrossRef].[PubMed]
9. Lovgren T, Hemmilä I, Halonen P,. 1985. Time-resolved fluorometry in immunoassay, p 203217. In Collins WP (ed), Alternative Immunoassays. Wiley, New York, NY.
10. Petersen SV, Thiel S, Jensen L, Steffensen R, Jensenius JC. 2001. An assay for the mannan-binding lectin pathway of complement activation. J Immunol Methods 257:107116.[PubMed].[CrossRef]
11. Dodds AW. 1993. Small-scale preparation of complement components C3 and C4. Methods Enzymol 223:4661.[PubMed].[CrossRef]

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