Human CPT1A (Carnitine Palmitoyltransferase 1A, Liver) ELISA Kit (HUES02915)

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HUES02915
€539

Description

ELISA Kit Technical ManualMSDS

Human CPT1A (Carnitine Palmitoyltransferase 1A, Liver) 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 CPT1A . 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 CPT1A 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 CPT1A, 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 CPT1A. The concentration of Human CPT1A 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 Range0.63-40 ng/mL
Sensitivity0.38 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human CPT1A in samples. No significant cross-reactivity or interference between Human CPT1A 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
402.382
2.416
2.3992.309
201.638
1.686
1.6621.572
101.018
0.978
0.9980.908
50.46
0.472
0.4660.376
2.50.311
0.281
0.2960.206
1.250.196
0.184
0.190.1
0.630.14
0.144
0.1420.052
00.086
0.094
0.09--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CPT1A were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CPT1A were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)1.974.6716.912.084.6918.17
Standard deviation0.110.230.720.140.190.69
C V (%)5.584.934.266.734.053.80

Recovery

The recovery of Human CPT1A 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)90-10697
EDTA plasma (n=5)84-9690
Cell culture media (n=5)91-10697

Linearity

Samples were spiked with high concentrations of Human CPT1A 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 (%)85-9697-11597-112
Average (%)91105103
1:4Range (%)92-10678-9183-98
Average (%)988589
1:8Range (%)93-10880-9286-99
Average (%)988692
1:16Range (%)95-10787-10182-96
Average (%)1009288

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 CPT1A (Carnitine Palmitoyltransferase 1A, Liver) ELISA Kit (HUES02915) 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 CPT1A (Carnitine Palmitoyltransferase 1A, Liver) ELISA Kit (HUES02915) Protein Information

UniProt Protein Function:CPT1A: Belongs to the carnitine/choline acetyltransferase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Lipid Metabolism - fatty acid; Membrane protein, integral; EC 2.3.1.21; Mitochondrial; Membrane protein, multi-pass; Transferase

Chromosomal Location of Human Ortholog: 11q13.2

Cellular Component: mitochondrial outer membrane; membrane; mitochondrion; mitochondrial inner membrane; integral to mitochondrial outer membrane

Molecular Function:identical protein binding; carnitine O-palmitoyltransferase activity

Biological Process: circadian rhythm; response to drug; eating behavior; glucose metabolic process; long-chain fatty acid metabolic process; carnitine shuttle; cellular lipid metabolic process; response to organic cyclic substance; positive regulation of fatty acid beta-oxidation; fatty acid beta-oxidation; triacylglycerol metabolic process; epithelial cell differentiation; carnitine metabolic process; regulation of insulin secretion; protein homooligomerization

Disease: Carnitine Palmitoyltransferase I Deficiency

NCBI Summary:The mitochondrial oxidation of long-chain fatty acids is initiated by the sequential action of carnitine palmitoyltransferase I (which is located in the outer membrane and is detergent-labile) and carnitine palmitoyltransferase II (which is located in the inner membrane and is detergent-stable), together with a carnitine-acylcarnitine translocase. CPT I is the key enzyme in the carnitine-dependent transport across the mitochondrial inner membrane and its deficiency results in a decreased rate of fatty acid beta-oxidation. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:P50416
NCBI GenInfo Identifier:56405343
NCBI Gene ID:1374
NCBI Accession:P50416.2
UniProt Secondary Accession:P50416,Q8TCU0, Q9BWK0,
UniProt Related Accession:P50416
Molecular Weight:773
NCBI Full Name:Carnitine O-palmitoyltransferase 1, liver isoform
NCBI Synonym Full Names:carnitine palmitoyltransferase 1A (liver)
NCBI Official Symbol:CPT1A  
NCBI Official Synonym Symbols:CPT1; CPT1-L; L-CPT1  
NCBI Protein Information:carnitine O-palmitoyltransferase 1, liver isoform; CPT I; CPTI-L; carnitine palmitoyltransferase I, liver; carnitine O-palmitoyltransferase I, liver isoform
UniProt Protein Name:Carnitine O-palmitoyltransferase 1, liver isoform
UniProt Synonym Protein Names:Carnitine O-palmitoyltransferase I, liver isoform; CPT I; CPTI-L; Carnitine palmitoyltransferase 1A
Protein Family:Carnitine O-palmitoyltransferase
UniProt Gene Name:CPT1A  
UniProt Entry Name:CPT1A_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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