DESCRIPTION

The complement system is an important effector that bridges the innate and adaptive immune systems. The fifth component of complement, C5, is part of the complement cascade and plays an important role in inflammation and cell killing. It consists of disulfide-linked alpha and beta polypeptide chains but can be cleaved into two active peptides (C5a and C5b) by C5 convertases. Mutations and defects in C5 are associated with severe recurrent infections.

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	1,131.1	86.8	7.7
2	8	327.1	11.5	3.5
3	8	188.0	16.2	8.6

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,131.3	59.8	5.3
2	16	319.9	12.5	3.9
3	16	182.0	15.4	8.5

RECOVERY

The recovery of mouse C5a spiked to three different levels throughout the range of the assay in mouse serum was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:400	93	90-98
	1:800	93	90-97

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 mouse serum was initially diluted 1:50.)

		Mouse serum
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	102
	Range (%)	100-104
1:8	Average% of Expected	107
	Range (%)	104-109
1:16	Average% of Expected	111
	Range (%)	109-114

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse C5a produced at Sunlong Medical™.