Complement Factor D Functional Test

Overview of Complement Factor B Functional Test

Complement factor D(CFD), also acknowledged as adipsin, assumes the guise of a circulating serine protease within the human bloodstream. In its capacity as a catalytic protease, CFD occupies a pivotal niche in orchestrating the initiation of the alternate route within the complement cascade. Notably, complement factor B stands as the solitary verified natural substrate of CFD in the realm of the complement system. CFD identifies and enzymatically cleaves complement factor B, birthing the fragments Ba and Bb, concomitant with the spatial coordinates Arg234-Lys235. This cleavage ensues under the proviso that Complement Factor B has attained activation and adherence to C3b (or C3•H₂O).

The catalytic prowess showcased by CFD wields a constructive and unparalleled influence upon the activation of the complement system, holding the mantle of the rate-determining step within the alternate pathway cascade. Thus, any functional aberration befalling CFD casts a shadow over the initiation of the complement alternate pathway, consequently unfurling a cascade of maladies.

Fig. 1 Complement's alternative pathway: unveiling the CFD position.¹

Introduction of Complement Factor D Functional Test

Why test CFD?

The complement system has traditionally held the primary role of a guardian against microbial intruders. However, a fresh perspective has recently emerged, with a plethora of inquiries shedding light on the intricate involvement of complement in orchestrating pathogenic processes across immune responses, inflammation, neurodegeneration, ischemia, and age-related ailments. A consensus has been reached that the pervasiveness and activation of complement assume a pivotal position in the causation of a wide spectrum of diseases.

CFD, as the rate-determining element within the alternate pathway, stands at the crux of this system. In patients afflicted with heart failure, the levels of CFD have exhibited correlations with markers of systemic inflammation, neurohormonal degradation, and cardiac functionality.

What is the test object?

As a forefront global biologics enterprise, boasting over a decade of dedicated inquiry, Creative Biolabs presents an exhaustive array of functional assays for CFD through expeditious enzyme-linked immunosorbent assay (ELISA) and hemolysis evaluation techniques.

Which methods are recommended to be used?

Semi-quantitative enzyme-linked immunosorbent assay (ELISA);

Hemolysis test.

Workflow of Complement Factor D Functional Test

ELISA

Creative Biolabs recommended approach is to employ the enzyme-linked immunosorbent assay (ELISA) to achieve a concise functional delineation of CFD within serum or tissue specimens, as the role played by CFD is of paramount significance and singular distinction, as it orchestrates the cleavage of activated complement factor B into fragments Bb and Ba. Through this method, the operational dynamics of CFD can be effectively deduced by analyzing the resultant ELISA curve.

Fig.2 Complement CFD ELISA Workflow.

Hemolysis test

Creative Biolabs provides a tailored hemolysis assay involving the deployment of CFD-depleted serum (CFD-Dpl serum), which stands as a suitable method to functionally authenticate mutants of CFD.

The conventional hemolysis assay, employing rabbit erythrocytes, traditionally serves to unveil the impact of mutations on CFD. In this context, equivalent quantities of both CFD mutants and unaltered CFD are separately introduced into the CFD-Dpl serum, alongside rabbit erythrocytes. By gauging the extent of hemolysis, the ramifications of a mutation on CFD can be discerned. A comparative assessment can also be undertaken, appraising the quantum of CFD or its mutants requisite to restore 50% or 100% hemolysis within partially depleted CFD serum, thereby permitting the characterization of the mutational influence.

Fig.3 Workflow of hemolysis test of CFD.

Published Data of CFD Functional Tests

Discoveries spotlighted in articles delineating functional testing of CFD:

ELISA analysis

Fig. 4 Enhanced complement activation velocity through reconstitution of factor D-deficient serum with purified CFD.^{2, 5}.

Fig. 5 Lufaxin impact on CFD activity and direct complement component binding.^{3, 5}.

Hemolysis test

Fig. 6 A result of serum and patients with hemolytic assays.^{4, 5}.

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Creative Biolabs stands as a pioneering research and development service provider, furnishing a robust platform for complement assays that bolsters comprehensive complement research endeavors. This encompassing platform encompasses tailored functional tests for factor D, thereby amplifying the efficacy of drug discovery within this domain, welcome to contact with us for more details.

References

1. From Wikipedia: By Rantes, oringinally at pl.wikipedia, CC BY-SA 3.0 https://commons.wikimedia.org/wiki/File:Droga_klasyczna.png.
2. Langereis, J. D.; et al. Complement factor D haplodeficiency is associated with a reduced complement activation speed and diminished bacterial killing. Clin Transl Immunology. 2021.10(4): e1256.
3. Mendes-Sousa, A. F.; et al. The Sand Fly Salivary Protein Lufaxin Inhibits the Early Steps of the Alternative Pathway of Complement by Direct Binding to the Proconvertase C3b-B. Front Immunol. 2017. 8: 1065.
4. Aradottir, Sigridur Sunna, et al. "Factor D inhibition blocks complement activation induced by mutant factor B associated with atypical hemolytic uremic syndrome and membranoproliferative glomerulonephritis." Frontiers in Immunology 12 (2021): 690821.
5. under Open Access license CC BY 4.0, without modification

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