Rat DLD (Dihydrolipoyl Dehydrogenase) CLIA Kit (RTES00180)

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SKU:
RTES00180
€649

Description

ELISA Kit Technical ManualMSDS

Rat DLD (Dihydrolipoyl Dehydrogenase) CLIA Kit

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 Rat DLD . 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 Rat DLD 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 Rat DLD, 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 Rat DLD. The concentration of Rat DLD in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection methodChemiluminescence
Detection range15.63-1000 pg/mL
Sensitivity9.38 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

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

Typical Data

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
(pg/mL)
RLU Average Corrected
100053501
53849
5367553650
50021054
23924
2248922464
25011015
9407
1021110186
1254816
4986
49014876
62.52585
2321
24532428
31.251300
1262
12811256
15.63668
748
708683
025
25
25--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)53.26159.10368.2654.01172.76362.72
Standard deviation4.9616.6933.666.9813.9437.61
C V (%)9.3110.499.1412.928.0710.37

Recovery

The recovery of Rat DLD 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)96-111104
EDTA plasma (n=5)88-10495
Cell culture media (n=5)99-113104

Linearity

Samples were spiked with high concentrations of Rat DLD 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 (%)89-10390-10488-104
Average (%)969895
1:4Range (%)96-11090-10683-95
Average (%)1049890
1:8Range (%)86-10093-10994-107
Average (%)9299102
1:16Range (%)92-10786-99103-117
Average (%)9993110

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 CLIA 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 Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Rat DLD (Dihydrolipoyl Dehydrogenase) CLIA Kit (RTES00180) 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 100µL 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.
    11. Determine the RLU value of each well immediately.

Rat DLD (Dihydrolipoyl Dehydrogenase) CLIA Kit (RTES00180) Protein Information

UniProt Protein Function:DLD: a multi-functional mitochondrial enzyme. An enzymatic component of the mitochondrial glycine cleavage system, the pyruvate dehydrogenase complex (PDHC), the alpha-ketoglutarate dehydrogenase complex, and the branched-chain alpha-keto acide dehydrogenase complex. Is the E3 component of the PDHC that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO2. The E3 component has dihydrolipoamide dehydrogenase activity. The PDHC contains 20-30 copies of pyruvate decarboxylase tetramers (2 alpha:2 beta)(E1), 60 copies of dihydrolipoamide acetyltransferase (E2), six homodimers of dihydrolipoamide dehydrogenase (E3), plus E3 binding proteins. Defects in DLD are a cause of maple syrup urine disease (MSUD), characterized by mental and physical retardation, feeding problems and a maple syrup odor to the urine. The keto acids of the branched-chain amino acids are present in the urine, resulting from a block in oxidative decarboxylation. Differentially expressed in the Wernicke's Area from patients with schizophrenia. Inhibited by 5-methoxyindole-2-carboxylic acid (MICA).
UniProt Protein Details:

Protein type:Carbohydrate Metabolism - pyruvate; Amino Acid Metabolism - valine, leucine and isoleucine degradation; Carbohydrate Metabolism - glycolysis and gluconeogenesis; Carbohydrate Metabolism - citrate (TCA) cycle; EC 1.8.1.4; Oxidoreductase; Amino Acid Metabolism - glycine, serine and threonine; Mitochondrial

Cellular Component: acrosomal matrix; cilium; mitochondrial matrix; mitochondrion; myelin sheath; nucleoplasm; oxoglutarate dehydrogenase complex; pyruvate dehydrogenase complex

Molecular Function:dihydrolipoyl dehydrogenase activity; FAD binding; lipoamide binding; NAD binding

Biological Process: 2-oxoglutarate metabolic process; acetyl-CoA biosynthetic process from pyruvate; aging; cell redox homeostasis; dihydrolipoamide metabolic process; gastrulation; lipoate metabolic process; mitochondrial electron transport, NADH to ubiquinone; proteolysis; regulation of membrane potential; sperm capacitation

NCBI Summary:This gene encodes a member of the class-I pyridine nucleotide-disulfide oxidoreductase family. A similar protein in human, mouse, and pig has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. In homodimeric form, the protein functions as a dehydrogenase and is found in several multi-enzyme complexes that regulate energy metabolism. However, as a monomer, the protein can function as a protease. [provided by RefSeq, Jan 2014]
UniProt Code:Q6P6R2
NCBI GenInfo Identifier:40786469
NCBI Gene ID:298942
NCBI Accession:NP_955417.1
UniProt Related Accession:Q6P6R2
Molecular Weight:
NCBI Full Name:dihydrolipoyl dehydrogenase, mitochondrial
NCBI Synonym Full Names:dihydrolipoamide dehydrogenase
NCBI Official Symbol:Dld  
NCBI Protein Information:dihydrolipoyl dehydrogenase, mitochondrial
UniProt Protein Name:Dihydrolipoyl dehydrogenase, mitochondrial
UniProt Synonym Protein Names:Dihydrolipoamide dehydrogenase
Protein Family:Delta-like protein
UniProt Gene Name:Dld  
UniProt Entry Name:DLDH_RAT
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
CLIA
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