Human TTR (Transthyretin) ELISA Kit (HUES03217)

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HUES03217
€539

Description

ELISA Kit Technical ManualMSDS

Human TTR (Transthyretin) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human TTR . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human 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 Human TTR, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding 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 Human TTR. The concentration of Human TTR in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range15.63-1000 ng/mL
Sensitivity9.38 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human TTR in samples. No significant cross-reactivity or interference between Human TTR and analogues was observed.

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.

Concentration
(ng/mL)
O.D Average Corrected
10002.48
2.498
2.4892.411
5001.659
1.661
1.661.582
2500.97
0.936
0.9530.875
1250.498
0.506
0.5020.424
62.50.295
0.283
0.2890.211
31.250.192
0.166
0.1790.101
15.630.125
0.135
0.130.052
00.068
0.088
0.078--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TTR were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TTR were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)47.91143.38417.4649.48133.33397.96
Standard deviation3.267.9617.532.786.6115.96
C V (%)6.805.554.205.624.964.01

Recovery

The recovery of Human 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)85-9792
EDTA plasma (n=5)87-9993
Cell culture media (n=5)87-9992

Linearity

Samples were spiked with high concentrations of Human 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)
1:2Range (%)88-10190-10391-106
Average (%)939898
1:4Range (%)88-10184-9882-98
Average (%)959189
1:8Range (%)93-10687-10285-101
Average (%)989392
1:16Range (%)88-9979-9181-93
Average (%)938687

Kit Components & Storage

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 ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human TTR (Transthyretin) ELISA Kit (HUES03217) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL 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.
    7. Aspirate or decant the solution from the plate and add 350µL 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human TTR (Transthyretin) ELISA Kit (HUES03217) Protein Information

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; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 18q12.1

Cellular Component: extracellular space; protein complex; cytoplasm; extracellular region

Molecular Function:identical protein binding; protein binding; protein heterodimerization activity; hormone activity

Biological Process: phototransduction, visible light; extracellular matrix organization and biogenesis; retinol metabolic process; transport; retinoid metabolic process

Disease: Hyperthyroxinemia, Dystransthyretinemic; Carpal Tunnel Syndrome; Amyloidosis, Hereditary, Transthyretin-related

NCBI Summary:This gene encodes transthyretin, one of the three prealbumins including alpha-1-antitrypsin, transthyretin and orosomucoid. Transthyretin is a carrier protein; it transports thyroid hormones in the plasma and cerebrospinal fluid, and also transports retinol (vitamin A) in the plasma. The protein consists of a tetramer of identical subunits. More than 80 different mutations in this gene have been reported; most mutations are related to amyloid deposition, affecting predominantly peripheral nerve and/or the heart, and a small portion of the gene mutations is non-amyloidogenic. The diseases caused by mutations include amyloidotic polyneuropathy, euthyroid hyperthyroxinaemia, amyloidotic vitreous opacities, cardiomyopathy, oculoleptomeningeal amyloidosis, meningocerebrovascular amyloidosis, carpal tunnel syndrome, etc. [provided by RefSeq, Jan 2009]
UniProt Code:P02766
NCBI GenInfo Identifier:136464
NCBI Gene ID:7276
NCBI Accession:P02766.1
UniProt Secondary Accession:P02766,Q549C7, Q6IB96, Q9UBZ6, Q9UCM9,
UniProt Related Accession:P02766
Molecular Weight:55,000
NCBI Full Name:Transthyretin
NCBI Synonym Full Names:transthyretin
NCBI Official Symbol:TTR  
NCBI Official Synonym Symbols:CTS; CTS1; PALB; TBPA; HEL111; HsT2651  
NCBI Protein Information:transthyretin; ATTR; carpal tunnel syndrome 1; thyroxine-binding prealbumin; epididymis luminal protein 111; prealbumin, amyloidosis type I
UniProt Protein Name:Transthyretin
UniProt Synonym Protein Names:ATTR; Prealbumin; TBPA
Protein Family:Transthyretin
UniProt Gene Name:TTR  
UniProt Entry Name:TTHY_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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