DESCRIPTION

Complement factor D, also known as adipsin, is a highly specific serine protease. Complement factor D is the rate-limiting enzyme in the alternative pathway of complement. It activates C3 convertase by cleaving factor B, which is a key step in the alternative pathway. Complement factor D catalyzes the cleavage of factor B to generate Ba (non-catalytic chain) and Bb (active catalytic subunit), which together with C3(H2O) (or C3b) constitute the active C3 convertase C3(H2O)Bb (or C3bBb).

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	268.6	4.6	1.7
2	8	68.3	2.9	4.2
3	8	32.7	1.6	4.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	275.1	9.0	3.3
2	16	68.0	2.8	4.1
3	16	32.2	1.5	4.7

RECOVERY

The recovery of human Complement factor D spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:64,000	89	84-94
Cell culture supernatant	1:256	97	85-112
Urine	1:16	101	88-115

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The human plasma was initially diluted 1:4,000. The cell culture supernatant was initially diluted 1:16. The urine was initially diluted 1:4.)

		Human plasma	Cell culturesupernatant	Urine
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	98	108	104
	Range (%)	97-99	92-123	98-110
1:8	Average% of Expected	109	90	104
	Range (%)	101-118	85-94	98-111
1:16	Average% of Expected	91	90	89
	Range (%)	80-102	87-93	83-96

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Complement factor D produced at Sunlong Medical™.