Enzyme immunometric assay (ELISA) for the determination of Myeloperoxidase (MPO) in sputum and nasal lavage supernatants, lithium heparin plasma, culture supernates, urine, and other standard buffers. For research use only, not for use in diagnostic procedures.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01532
Species Human
Design Enzyme immunometric assay (ELISA) technique.
Standards 8 standards, serially diluted from 1 concentrated standard.
Controls None provided.
Sample Types Sputum and nasal lavage supernatants, lithium heparin plasma, culture supernates, urine, and other standard buffers.
Sample Volume 100 μL of properly prepared unknown / determination.
Assay Desc. 1 hour incubation (RT) + 1 hour (RT) + 30 min. (RT) + 30 min. (RT) = 3 hours total incubation time.
Standard Range 0 / 0.195 - 12.5 ng/mL
Sensitivity 0.028 ng/mL in Assay Buffer 13, and 0.019 ng/mL in Assay Buffer 31.
Radioactivity
Other Names
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 Myeloperoxidase (MPO) 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 Myeloperoxidase (MPO). Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Myeloperoxidase (MPO) 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 Myeloperoxidase (MPO), 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 Myeloperoxidase (MPO). You can calculate the concentration of Myeloperoxidase (MPO) 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.

Precision

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

Recovery

Matrices listed below were spiked with certain level of Myeloperoxidase (MPO) and the recovery rates were calculated by comparing the measured value to the expected amount of Myeloperoxidase (MPO) in samples.

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 89-102 95
EDTA plasma (n=5) 88-100 96
heparin plasma (n=5) 89-101 94

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Myeloperoxidase (MPO) 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 (%) 90-102 90-100 92-106
Average (%) 95 95 93
1:4 Range (%) 92-102 94-99 94-100
Average (%) 93 94 94
1:8 Range (%) 87-102 91-107 92-107
Average (%) 93 93 94
1:16 Range (%) 87-106 88-104 88-103
Average (%) 95 93 93

Stability

The stability of Myeloperoxidase (MPO) is determined by the loss rate of activity. The loss rate of Myeloperoxidase (MPO) 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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