DESCRIPTION

FLT4, also known as Vascular Endothelial Growth Factor Receptor 3 (VEGFR-3), is a receptor tyrosine kinase primarily involved in lymphangiogenesis (the formation of lymphatic vessels) and vascular development. It binds to its ligands, VEGF-C and VEGF-D, activating signaling pathways that regulate lymphatic endothelial cell proliferation, migration, and survival. FLT4 plays a critical role in embryonic development, maintaining lymphatic vessel integrity, and pathological conditions such as lymphedema, tumor metastasis, and cardiovascular diseases. Mutations in FLT4 can lead to conditions like lymphedema.

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 (ng/mL)	SD	CV%
1	8	4.31	0.19	4.41
2	8	1.09	0.04	3.67
3	8	0.57	0.03	5.26

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	4.53	0.30	6.62
2	16	1.11	0.06	5.41
3	16	0.58	0.03	5.17

RECOVERY

The recovery of human FLT4/VEGFR3 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:80	106	92-118
	1:160	98	93-103
Cell lysate	1:4	84	81-90
	1:8	91	80-102

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 1:10.)

		Human plasma	Cell lysate
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	102	89
	Range (%)	99-106	86-92
1:8	Average% of Expected	90	99
	Range (%)	84-95	87-111
1:16	Average% of Expected	103	-
	Range (%)	96-110	-

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human FLT4/VEGFR3 One-Step ELISA Kit produced at Sunlong Medical™.