Sandwich ELISA to human AT3 AEE0018

Description

Antithrombin III/AT3

All names and symbols: Antithrombin III; Antithrombin-III; serpin family C member 1; Serpin C1; SerpinC1; AT3; AT3D; ATIII; THPH7; ATIII-R2; ATIII-T1; ATIII-T2

Confirmed species reactivity	Human
Sensitivity	12.6 ng/ml
Linear calculation range	31.25 – 1000 ng/ml
Precision intra-assay / inter-assay	<10% / <12%
Aeonian Rating®	90
RRID

Matrix	Recovery Range (%)	Average (%)
Serum (n=5)	80-102	91
EDTA plasma (n=5)	81-100	90
Heparin plasma (n=5)	80-89	84

Matrix	1:2	1:4	1:8	1:16
Serum (n=5)	87-91%	87-107%	74-101%	92-97%
EDTA plasma (n=5)	90-105%	84-101%	90-101%	79-108%
Heparin plasma (n=5)	84-95%	92-105%	82-105%	89-91%

Calibratrion curve:

AEE0018 to AT3 was successfully used to determine the concentration of AT3 in serum, plasma and buffer. Recommended serum dilution 1:16, and EDTA plasma dilution 1:8.

Selectivity of the ELISA kit:

AEE0018 to AT3 was successfully confirmed selective as there were no signals found in the presence of other closely related proteins at high and low concentrations.

ELISA Test:

The microtiter plate provided in this kit has been pre-coated with an antibody specific to AT3. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to AT3. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain AT3, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of AT3 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

ELISA Procedure:

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 2 hours at 37℃;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37℃;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 1 hour at 37℃;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37℃;
8. Add 50µL Stop Solution. Read at 450nm immediately.

Stability:

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage conditions.
Note:
To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.