Enzyme-linked immunosorbent assay (ELISA) for the determination of α1-antitrypsin in stool. For research use only, not for use in diagnostic procedures.
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Protocol PDF Click for Protocol
Catalog Number TK01069
Species Human
Design Enzyme-linked immunosorbent assay (ELISA) technique.
Standards 5 standards, lyophilized.
Controls 2 controls, lyophilized.
Sample Types Stool.
Sample Volume 100 µL of properly prepared unknown / determination.
Assay Desc. 1 hour incubation (RT) + 1 hour (RT) + 10 min. (RT) = 2 hours, 10 min. total incubation time.
Standard Range 0 / 3.3 - 90.0 μg/L
Sensitivity 1.8 mg/dL
Other Names Alpha-1-Antitrypsin.
Storage 2 - 8°C
Postscript For research use only, not for use in diagnostic procedures.

Intended use

This ELISA kit applies to the in vitro quantitative determination of α1-Antitrypsin concentrations in serum, plasma and other biological fluids.

Test principle

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to α1-Antitrypsin. Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for α1-Antitrypsin and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain α1-Antitrypsin, biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of α1-Antitrypsin. You can calculate the concentration of α1-Antitrypsin in the samples by comparing the OD of the samples to the standard curve.

Typical data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.


Intra-assay Precision (Precision within an assay):
3 samples with low, middle,high concentration of α1-Antitrypsin. And the sample of α1-Antitrypsin were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of α1-Antitrypsin. And the sample of α1-Antitrypsin were tested on 3 different plates, 30 replicates in each plate.


Matrices listed below were spiked with certain level of α1-Antitrypsin and the recovery rates were calculated by comparing the measured value to the expected amount of α1-Antitrypsin in samples.

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 92-101 96
EDTA plasma (n=5) 91-100 96
heparin plasma (n=5) 89-96 95


The linearity of the kit was assayed by testing samples spiked with appropriate concentration of α1-Antitrypsin and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

    Serum (n=5) EDTA plasma (n=5) heparin plasma (n=5)
1:2 Range (%) 94-102 92-101 93-108
Average (%) 95 93 93
1:4 Range (%) 90-101 90-108 92-107
Average (%) 94 93 95
1:8 Range (%) 87-101 88-100 94-103
Average (%) 94 95 95
1:16 Range (%) 91-105 87-103 91-102
Average (%) 93 95 93


The stability of α1-Antitrypsin is determined by the loss rate of activity. The loss rate of α1-Antitrypsin is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Item Specifications Storage
Micro ELISA Plate(Dismountable) 8 wells ×12 strips -20℃, 6 months
Reference Standard 2 vials
Concentrated Biotinylated Detection Ab (100×) 1 vial, 120 μL
Concentrated HRP Conjugate (100×) 1 vial, 120 μL -20℃(shading light), 6 months
Reference Standard & Sample Diluent 1 vial, 20 mL 4℃, 6 months
Biotinylated Detection Ab Diluent 1 vial, 14 mL
HRP Conjugate Diluent 1 vial, 14 mL
Concentrated Wash Buffer (25×) 1 vial, 30 mL
Substrate Reagent 1 vial, 10 mL 4℃(shading light)
Stop Solution 1 vial, 10 mL 4℃
Plate Sealer 5 pieces
Product Description 1 copy
Certificate of Analysis 1 copy

Other supplies required

Microplate reader with 450 nm wavelength filter
High-precision transfer pipette, EP tubes and disposable pipette tips
Incubator capable of maintaining 37℃
Deionized or distilled water
Absorbent paper
Loading slot for Wash Buffer

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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