Titin Protein N-Fragment

Solid phase sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of Titin N-Fragment in human and rat urine. For research use only, not for use in diagnostic procedures. Titin protein (connectin) consists of 34,350 amino-acids and specifically expresses in a cross-striated muscle. The molecular weight of human titin is 3,816 kDa and it is known to be the largest protein in a living body. It is a sarcomere structured protein that has minimum units of myofibrillary proteins and it has a role as an elastic protein for recovering the length of shortened sarcomeres by its contraction. It has been known that titin is cleaved by protein metabolic enzymes such as calpain and matrix metalloprotease if muscles are damaged. In fact, it has been confirmed that titin is dramatically cleaved in skeletal muscles of muscular dystrophy patients and various titin fragments are confirmed in blood and urine. It has been reported that the N fragment (26kDa) and the C fragment (12kDa) are especially increased in urine of muscular dystrophy patients and it is also suggested that it can be a candidate as a noninvasive biomarker for monitoring status of muscles. The Titin Protein N-Fragment ELISA kit can measure titin protein N fragment in human and rat urine.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01707
Species Human
Design Solid phase sandwich enzyme-linked immunosorbent assay (ELISA).
Standards 7 standards, serially diluted from 1 prepared lyophilized standard.
Controls None provided.
Sample Types Human and rat urine.
Sample Volume 100 μL of properly diluted unknown / determination.
Assay Desc. 1 hour incubation (37°C) + 30 min. (37°C) + 30 min. (RT) = 2 hours total incubation time.
Standard Range 46.88 - 3000.0 pmol/L
Sensitivity 27.91 pmol/L
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 Titin Protein N-Fragment 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 Titin Protein N-Fragment. Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Titin Protein N-Fragment 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 Titin Protein N-Fragment, 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 Titin Protein N-Fragment. You can calculate the concentration of Titin Protein N-Fragment 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 Titin Protein N-Fragment. And the sample of Titin Protein N-Fragment were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Titin Protein N-Fragment. And the sample of Titin Protein N-Fragment were tested on 3 different plates, 30 replicates in each plate.

Recovery

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

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 94-104 95
EDTA plasma (n=5) 90-97 95
heparin plasma (n=5) 92-96 95

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Titin Protein N-Fragment 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 (%) 87-103 92-103 87-102
Average (%) 93 95 95
1:4 Range (%) 94-102 93-99 88-108
Average (%) 95 95 95
1:8 Range (%) 92-107 88-102 93-106
Average (%) 94 94 95
1:16 Range (%) 87-102 93-107 89-102
Average (%) 94 95 94

Stability

The stability of Titin Protein N-Fragment is determined by the loss rate of activity. The loss rate of Titin Protein N-Fragment 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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