Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Mouse |
| Detection method: | Chemiluminescence |
| Detection range: | 1.56-100 ng/mL |
| Sensitivity: | 0.94 ng/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Mouse TTR in samples. No significant cross-reactivity or interference between Mouse TTR and analogues was observed. |
This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Mouse TTR. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse TTR and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse TTR, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Mouse TTR. The concentration of Mouse TTR in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | TTR: Thyroid hormone-binding protein. Probably transports thyroxine from the bloodstream to the brain. Defects in TTR are the cause of amyloidosis transthyretin-related (AMYL-TTR). A hereditary generalized amyloidosis due to transthyretin amyloid deposition. Protein fibrils can form in different tissues leading to amyloid polyneuropathies, amyloidotic cardiomyopathy, carpal tunnel syndrome, systemic senile amyloidosis. The disease includes leptomeningeal amyloidosis that is characterized by primary involvement of the central nervous system. Neuropathologic examination shows amyloid in the walls of leptomeningeal vessels, in pia arachnoid, and subpial deposits. Some patients also develop vitreous amyloid deposition that leads to visual impairment (oculoleptomeningeal amyloidosis). Clinical features include seizures, stroke-like episodes, dementia, psychomotor deterioration, variable amyloid deposition in the vitreous humor. Defects in TTR are a cause of hyperthyroxinemia dystransthyretinemic euthyroidal (HTDE). It is a condition characterized by elevation of total and free thyroxine in healthy, euthyroid persons without detectable binding protein abnormalities. Defects in TTR are a cause of carpal tunnel syndrome type 1 (CTS1). It is a condition characterized by entrapment of the median nerve within the carpal tunnel. Symptoms include burning pain and paresthesias involving the ventral surface of the hand and fingers which may radiate proximally. Impairment of sensation in the distribution of the median nerve and thenar muscle atrophy may occur. This condition may be associated with repetitive occupational trauma, wrist injuries, amyloid neuropathies, rheumatoid arthritis. Belongs to the transthyretin family. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Cellular Component: extracellular region; extracellular space; protein complex Molecular Function:hormone activity; hormone binding; identical protein binding; protein binding; protein heterodimerization activity Biological Process: retinol metabolic process; transport |
| NCBI Summary: | This gene encodes a carrier protein responsible for the transport of thyroid hormones and retinol. The protein consists of a tetramer of identical subunits. Due to increased stability of the tetramer form of this encoded protein in mouse, compared to the human protein, this gene product has a reduced tendency to form amyloid fibrils. In humans, this protein binds beta-amyloid preventing its aggregation and providing a neuroprotective role in Alzheimer's disease. [provided by RefSeq, Mar 2010] |
| UniProt Code: | P07309 |
| NCBI GenInfo Identifier: | 7305599 |
| NCBI Gene ID: | 22139 |
| NCBI Accession: | NP_038725. 1 |
| UniProt Related Accession: | P07309 |
| Molecular Weight: | 15. 6 kDa |
| NCBI Full Name: | transthyretin |
| NCBI Synonym Full Names: | transthyretin |
| NCBI Official Symbol: | Ttr |
| NCBI Official Synonym Symbols: | D17860; AA408768; AI787086; prealbumin |
| NCBI Protein Information: | transthyretin |
| UniProt Protein Name: | Transthyretin |
| UniProt Synonym Protein Names: | Prealbumin |
| Protein Family: | Transthyretin |
| UniProt Gene Name: | Ttr |
| UniProt Entry Name: | TTHY_MOUSE |
As the RLU 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.
| Concentration (ng/mL) | RLU | Average | Corrected |
| 100 | 35683 39425 | 37554 | 37521 |
| 50 | 15877 16605 | 16241 | 16208 |
| 25 | 7985 7099 | 7542 | 7509 |
| 12.5 | 3582 3782 | 3682 | 3649 |
| 6.25 | 1948 1802 | 1875 | 1842 |
| 3.13 | 1066 936 | 1001 | 968 |
| 1.56 | 542 602 | 572 | 539 |
| 0 | 33 33 | 33 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse TTR were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse TTR were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 4.81 | 8.74 | 35.15 | 4.42 | 8.49 | 37.81 |
| Standard deviation | 0.52 | 1.04 | 2.58 | 0.43 | 0.89 | 3.55 |
| C V (%) | 10.81 | 11.90 | 7.34 | 9.73 | 10.48 | 9.39 |
Recovery
The recovery of Mouse TTR spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 86-101 | 92 |
| EDTA plasma (n=5) | 99-112 | 104 |
| Cell culture media (n=5) | 98-112 | 105 |
Linearity
Samples were spiked with high concentrations of Mouse TTR and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 100-116 | 92-104 | 101-118 |
| Average (%) | 108 | 97 | 107 | |
| 1:4 | Range (%) | 95-112 | 88-99 | 89-102 |
| Average (%) | 102 | 93 | 95 | |
| 1:8 | Range (%) | 90-104 | 90-100 | 91-105 |
| Average (%) | 96 | 95 | 96 | |
| 1:16 | Range (%) | 100-113 | 88-100 | 91-106 |
| Average (%) | 105 | 94 | 98 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro CLIA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 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 A | 1 vial, 5 mL | 4°C (shading light) |
| Substrate Reagent B | 1 vial, 5 mL | 4°C (shading light) |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
- Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37 °C.
- Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 100 µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37 °C. Protect the plate from light.
- Determine the RLU value of each well immediately.
