DESCRIPTION

FAP, also known as fibroblast activation protein-alpha (FAP-alpha) is a cell surface glycoprotein serine protease, which can participate in extracellular matrix degradation and is involved in many cellular processes including tissue remodeling, fibrosis, wound healing, inflammation and tumor growth. FAP is a single transmembrane protein that locates the cytoplasm and plasma membrane. Immunohistochemical studies have shown that FAP is transiently expressed in certain normal fetal mesenchymal tissues but that normal adult tissues as well as malignant epithelial, neural, and hematopoietic cells are generally FAP-negative. However, most of the common types of epithelial cancers contain abundant FAP-reactive stromal fibroblasts.

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	1,234.8	56.1	4.5
2	8	332.5	12.6	3.8
3	8	99.4	2.0	2.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,263.7	66.9	5.3
2	16	333.6	13.2	4.0
3	16	99.9	2.6	2.6

RECOVERY

The recovery of human FAP spiked to three different levels throughout the range of the assay in human plasma was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:1,600	105	100-108
	1:3,200	107	103-111

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 1:200.)

		Human plasma
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	100
	Range (%)	99-100
1:8	Average% of Expected	102
	Range (%)	99-104
1:16	Average% of Expected	92
	Range (%)	90-94

CALIBRATION

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