DESCRIPTION

Complement factor H (CFH) is an abundant plasma glycoprotein that regulates the function of the alternative complement pathway in the fluid phase and on cellular surfaces. Factor H binds to C3b, accelerates the decay of the alternative pathway C3convertase (C3bBb), and acts as a cofactor for the factor I-mediated proteolytic inactivation of C3b. It plays a critical role in the homeostasis of the complement system in plasma and in the protection of bystander host cells and tissues from damage by complement activation. Abnormalities in factor H have been associated with renal disease.

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	3,145.5	69.0	2.2
2	8	794.7	13.6	1.7
3	8	403.0	5.5	1.4

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	3,124.7	59.7	1.9
2	16	778.3	20.8	2.7
3	16	396.1	8.3	2.1

RECOVERY

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

Sample Type		Average% of Expected	Range (%)
Human plasma	1:400,000	112	93-130
Cell culture supernatant	1:40	118	108-124
Saliva	1:40	104	93-117
	1:80	107	102-113

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 1:25,000. The cell culture supernatant was initially diluted 1:2.5. The saliva was initially diluted 1:5.)

		Human plasma	Cell culture supernatant	Saliva
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	100	93	107
	Range (%)	99-102	91-96	101-112
1:8	Average% of Expected	96	90	110
	Range (%)	94-99	89-92	106-115
1:16	Average% of Expected	95	84	103
	Range (%)	92-97	84-85	89-116

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human Complement factor H/CFH One-Step ELISA Kit produced at Sunlong Medical™.