DESCRIPTION

CD100 (also known as SEMA4D), a 150 kDa homodimer, belongs to the family of immune semaphorins, and CD100 is abundantly expressed on resting T cells, NK cells, and antigen-presenting cells. As a transmembrane glycoprotein, it is digested to its soluble form, sCD100, by specific matrix metalloproteinases. CD100 has been reported that the expression levels of CD100, and one of its receptors, plexin-B1 (PLXNB1), are increased in head and neck, prostate, colon, breast, and lung cancers, and the interaction between them provides oncogenic signaling essential for tumor growth and metastasis. Elevated levels of CD100 have been observed in inflammatory and infectious diseases, although its exact role in these conditions is still under investigation. CD100 and its signaling pathways are increasingly being linked to various immunological diseases, making it a potential diagnostic marker and therapeutic target.

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	1,657.7	57.5	3.5
2	8	463.8	9.9	2.1
3	8	247.2	4.5	1.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,644.2	66.8	4.1
2	16	458.2	12.8	2.8
3	16	246.3	6.6	2.7

RECOVERY

The recovery of human CD100 spiked to three different levels throughout the range of the assay in human plasma was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:16	118	113-125
	1:32	110	109-112

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 samples were initially diluted 1:2.)

		Human plasma
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	92
	Range (%)	92-93
1:8	Average% of Expected	86
	Range (%)	85-86
1:16	Average% of Expected	77
	Range (%)	76-77

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CD100 produced at Sunlong Medical™.