DESCRIPTION

ErbB3/HER3 is a member of the ErbB family of receptor tyrosine kinases, which play crucial roles in cell proliferation, survival, and differentiation. ErbB3/HER3 is a type I transmembrane glycoprotein and is unique within the ErbB family because it lacks intrinsic kinase activity. Instead, it relies on forming heterodimers with other ErbB receptors, particularly HER2, to mediate signaling. HER3 has emerged as a key player in resistance to other HER family-targeted therapies, such as tyrosine kinase inhibitors and monoclonal antibodies against EGFR or HER2.

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	2,185.8	131.7	6.0
2	8	534.1	7.5	1.4
3	8	268.0	10.4	3.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,166.8	93.9	4.3
2	16	539.5	19.7	3.7
3	16	274.7	19.9	7.2

RECOVERY

The recovery of human ErbB3/HER3 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:32	107	103-111
	1:64	105	95-113
Cell culture supernatant	1:2	108	107-111
	1:4	113	109-119
Urine	1:4	112	109-117
	1:8	113	110-120
Saliva	1:8	93	87-98
	1:16	100	97-104
Human milk	1:16	94	90-101
	1:32	96	95-97
Cell lysate	1:8	81	77-86
	1:16	87	85-91

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 1:4. The saliva and human milk was initially diluted 1:2.)

		Humanplasma	Cell culturesupernatant	Urine	Saliva	Human milk	Cell lysate
1:2	Average% ofExpected	100	100	100	100	100	100
	Range (%)	-	-	-	-	-	-
1:4	Average% ofExpected	98	94	114	112	99	110
	Range (%)	93-103	93-96	111-116	106-118	99-100	100-120
1:8	Average% ofExpected	106	94	123	122	115	118
	Range (%)	99-113	93-95	120-125	111-132	114-115	114-121
1:16	Average% ofExpected	108	92	118	125	113	118
	Range (%)	102-115	91-93	106-129	119-131	110-115	113-123

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human ErbB3/HER3 One-Step ELISA Kit produced at Sunlong Medical™.