DESCRIPTION

Used for the quantitative determination of Anti-Semaglutide Neutralizing Antibody concentration in serum and plasma.

PRINCIPLE OF THE ASSAY

This assay employs the quantitative competitive enzyme immunoassay technique. Anti-GLP1 antibody has been pre-coated onto a microplate. Standards or samples are premixed with biotin-labeled Semaglutide and then pipetted into the wells. Anti-Semaglutide Neutralizing Antibody in the sample competitively binds to biotin-labeled Semaglutide with the pre-coated anti-GLP1 antibody. After washing away any unbound substances, Streptavidin-HRP is added to the wells. Following a wash to remove any unbound enzyme reagent, a substrate solution is added to the wells and color develops in inversely proportion to the amount of Anti-Semaglutide Neutralizing Antibody bound in the initial step. The color development is stopped and the intensity of the color is measured.

PRECISION

Intra-Assay Precision (Precision within an assay): <20% Three samples of known concentration were tested sixteen times on one plate to assess intra-assay precision. Inter-Assay Precision (Precision between assays): <20% Three samples of known concentration were tested in twenty four separate assays to assess inter-assay precision.

Sample		1	2	3	1	2	3
n		16	16	16	24	24	24
Mean (ng/mL)		8068.8	1316.8	246.8	7292.5	1333.2	271.1
Standard deviation		1207.5	145.5	35.0	1183.3	169.9	52.1
CV \(%\)		15.0	11.1	14.2	16.2	12.7	19.2

RECOVERY

The recovery of Anti-Semaglutide Neutralizing Antibody spiked to three different levels in human serum samples throughout the range of the assay was evaluated.

Sample	Dilution factor	Average (%)	Range (%)
Human serum (n=3)	1:10	95	80-111
	1:20	90	80-112

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of Anti-Semaglutide Neutralizing Antibody and diluted with the Standard Diluent to produce samples with values within the dynamic range of the assay.

Sample	Dilution factor	Average (%)	Range (%)
Human serum (n=3)	1:2	106	95-114
	1:4	106	94-115
	1:8	92	82-100
	1:16	98	84-119