Human Complement Factor H DuoSet ELISA Summary

Product Summary

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Complement Factor H. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet ELISA.

Preparation and Storage

Background: Complement Factor H

Complement Factor H is a 155 kDa glycoprotein that provides critical negative regulation to the alternative pathway of complement cascade. It is secreted by Kupffer cells, hepatocytes, vascular endothelial cells, and platelets, and circulates in the serum at high concentration. Complement Factor H is composed of 20 SCRs (short consensus repeats), each of which consists of approximately 60 amino acids with four invariant Cys residues. Alternate splicing generates a Complement Factor H isoform that is truncated following SCR7.

Complement Factor H interacts with cell surface polyanions including heparin and sialoglycoproteins, and immobilized Complement Factor H supports the CD11b/CD18 integrin-dependent adhesion of neutrophils. It prevents local complement activation by sequestering complement component C3b, accelerating the decay of C3 and C5 convertases, and functions as a cofactor for the C3b inactivator, Factor I.

Short consensus repeats 15-20 (SCR15-20) encompass the primary binding sites for heparin and C3b, as well as for the peptide hormone adrenomedullin. Within SCR15-20, human Complement Factor H shares 60% and 63% amino acid sequence identity with mouse and rat Complement Factor H, respectively. Dozens of mutations clustered in SCR15-20 are associated with atypical hemolytic uremic syndrome, a disorder characterized by anemia, thrombocytopenia, and renal failure. Binding of Complement Factor H to tumor cell-associated dentin matrix protein 1, bone sialoprotein, or osteopontin results in the protection of that cell from complement-mediated lysis. A variety of pathogenic microbes also express Complement Factor H binding molecules that interfere with immune clearance of the infection.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1、Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2、Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3、Add 50 μL of Assay Diluent to each well.

4、Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5、Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6、Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7、Aspirate and wash 5 times.

8、Add 100 μL Substrate Solution to each well.

9、Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.