DESCRIPTION

The synaptotagmins are integral membrane proteins of synaptic vesicles thought to serve as Ca(2+) sensors in the process of vesicular trafficking and exocytosis. Synaptotagmin-1 acts as the primary calcium sensor for fast synchronous neurotransmitter release. It contains two C2 domains (C2A and C2B) that mediate Ca²⁺-dependent membrane interactions. Calcium binding to synaptotagmin-1 participates in triggering neurotransmitter release at the synapse. In addition to regulating exocytosis, synaptotagmin-1 has also been implicated in endocytosis and neurite outgrowth.

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	1,620.3	41.5	2.6
2	8	427.7	10.7	2.5
3	8	233.0	9.4	4.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,648.4	67.2	4.1
2	16	442.3	28.0	6.3
3	16	237.7	14.6	6.1

RECOVERY

The recovery of human/mouse/rat Synaptotagmin-1 spiked to three different levels throughout the range of the assay in tissue lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Tissue lysate	1:12,800	97	87-105
	1:25,600	90	76-100

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 tissue lysate samples were initially diluted 1:1,600.)

		Tissue lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	103
	Range (%)	89-117
1:8	Average% of Expected	105
	Range (%)	82-128
1:16	Average% of Expected	117
	Range (%)	108-125

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human/Mouse/Rat Synaptotagmin-1 One-Step ELISA Kit produced at Sunlong Medical™.