DESCRIPTION

LAG-3, also known as CD223, is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. LAG-3 is expressed on activated T cells, NK cells, regulatory T cells, and plasmacytoid dendritic cells. It is a CD4-related molecule that binds MHC class II. Additionally, LAG-3 plays an important role in modulating T cell expansion and function, and blockade of LAG3 with monoclonal antibodies can augment T cell function.

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	2,802.4	79.7	2.8
2	8	666.9	22.5	3.4
3	8	331.5	9.6	2.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,727.9	104.4	3.8
2	16	659.1	22.7	3.4
3	16	325.6	11.8	3.6

RECOVERY

The recovery of human LAG-3/CD223 spiked to three different levels throughout the range of the assay in human plasma was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:2	74	73-75

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.

		Human plasma
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	120
	Range (%)	115-124
1:8	Average% of Expected	118
	Range (%)	111-125
1:16	Average% of Expected	96
	Range (%)	86-106

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human LAG-3/CD223 One-Step ELISA Kit produced at Sunlong Medical™.