DESCRIPTION

Mesothelin (MSLN) is a glycosylphosphatidylinositol-linked membrane glycoprotein which is highly expressed in a variety of tumors and is also expressed in mesothelial cells of healthy individuals, but at low levels. Therefore, it can be considered as a promising target protein for tumor-targeted therapy. In the tumor environment, MSLN plays an important role in survival, proliferation, and migration/invasion of cancer cells as well as in drug resistance. Over the years, evidence has accumulated with regards to the importance of MSLN as a tumor-associated antigen (TAA) overexpressed in almost one-third of human cancers. (PMID: 35795680)

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	1,397.1	26.8	1.9
2	8	313.4	4.6	1.5
3	8	148.7	1.9	1.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,360.2	50.2	3.7
2	16	306.8	9.0	2.9
3	16	147.5	2.9	2.0

RECOVERY

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

Sample Type		Average% of Expected	Range (%)
Human plasma	1:160	98	96-100
	1:320	95	86-101
Urine	1:4	107	106-108
	1:8	108	106-111
Saliva	1:16	87	85-89

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 1:20. The urine was initially diluted 1:2.)

		Human plasma(Sample Diluent PT 4B1)	Urine(Sample Diluent PT 4B1)	Saliva(Sample Diluent PT 1)
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	103	113	101
	Range (%)	100-105	112-114	98-105
1:8	Average% of Expected	105	117	98
	Range (%)	100-110	116-118	97-99
1:16	Average% of Expected	106	114	91
	Range (%)	102-110	113-116	90-91

CALIBRATION

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