DESCRIPTION

Gastrin-releasing peptide, also known as GRP, is normally formed by mucosal cells in the gastric antrum and by the D cells of the pancreatic islets, and its main function is to stimulate secretion of HCl by the gastric mucosa. HCl, in turn, inhibits gastrin formation. Its 148-amino acid preproprotein, following cleavage of a signal peptide, is further processed to produce either the 27-amino acid gastrin-releasing peptide or the 10-amino acid neuromedin C. These smaller peptides regulate numerous functions of the gastrointestinal and central nervous systems, including release of gastrointestinal hormones, smooth muscle cell contraction, and epithelial cell proliferation. These peptides are also likely to play a role in human cancers of the lung, colon, stomach, pancreas, breast, and prostate.

PRINCIPLE OF THE ASSAY

A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody also binds to the analyte. Horseradish peroxidase (HRP)-conjugated secondary antibody binds to the detection antibody. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns 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 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	20	13.3	0.90	6.9
2	20	4.6	0.20	4.6
3	20	1.1	0.06	5.3

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	24	13.8	0.70	4.9
2	24	3.4	0.30	7.8
3	24	0.6	0.05	8.8

RECOVERY

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

Sample Type		Average% of Expected	Range (%)
Human plasma	1:4	106	86-123
	1:8	108	83-128
Cell culture supernatant	1:2	83	77-88
	1:4	98	95-103

LINEARITY

To assess the linearity of the assay, three samples were spiked with high concentrations of human GRP in various matrices and 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(Sample Diluent PT 1-ec)	Cell culture supernatant (SampleDiluent PT 1-dg)
1:2	Average% of Expected	94	88
	Range (%)	85-103	81-94
1:4	Average% of Expected	111	118
	Range (%)	94-123	116-119
1:8	Average% of Expected	104	125
	Range (%)	99-109	120-130
1:16	Average% of Expected	123	108
	Range (%)	113-133	106-109

CALIBRATION

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