Rat ALAD(Aminolevulinate Delta Dehydratase)ELISA Kit (RTES01093)

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RTES01093
€649

Description

ELISA Kit Technical ManualMSDS

Rat ALAD(Aminolevulinate Delta Dehydratase)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 Rat ALAD . 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 Rat ALAD 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 ALAD, 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 Rat ALAD. The concentration of Rat ALAD in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection MethodColormetric
Detection Range0.78-50 ng/mL
Sensitivity0.47 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Rat ALAD in samples. No significant cross-reactivity or interference between Rat ALAD 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
502.381
2.401
2.3912.322
251.683
1.705
1.6941.625
12.50.914
0.874
0.8940.825
6.250.434
0.47
0.4520.383
3.130.266
0.258
0.2620.193
1.570.174
0.166
0.170.101
0.780.114
0.128
0.1210.052
00.06
0.078
0.069--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)2.307.0321.882.407.0719.81
Standard deviation0.120.371.120.150.360.71
C V (%)5.225.265.126.255.093.58

Recovery

The recovery of Rat ALAD 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)90-10597
EDTA plasma (n=5)88-10294
Cell culture media (n=5)94-106100

Linearity

Samples were spiked with high concentrations of Rat ALAD 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 (%)90-10088-10298-108
Average (%)9595103
1:4Range (%)93-10683-9589-103
Average (%)998994
1:8Range (%)87-10185-9781-94
Average (%)949087
1:16Range (%)89-10079-9289-104
Average (%)958595

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

Rat ALAD(Aminolevulinate Delta Dehydratase)ELISA Kit (RTES01093) 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.

Rat ALAD(Aminolevulinate Delta Dehydratase)ELISA Kit (RTES01093) Protein Information

UniProt Protein Function:ALAD: Catalyzes an early step in the biosynthesis of tetrapyrroles. Binds two molecules of 5-aminolevulinate per subunit, each at a distinct site, and catalyzes their condensation to form porphobilinogen. Defects in ALAD are the cause of acute hepatic porphyria (AHEPP). A form of porphyria. Porphyrias are inherited defects in the biosynthesis of heme, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors. They are classified as erythropoietic or hepatic, depending on whether the enzyme deficiency occurs in red blood cells or in the liver. AHP is characterized by attacks of gastrointestinal disturbances, abdominal colic, paralysis, and peripheral neuropathy. Most attacks are precipitated by drugs, alcohol, caloric deprivation, infections, or endocrine factors. Belongs to the ALADH family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cofactor and Vitamin Metabolism - porphyrin and chlorophyll; EC 4.2.1.24; Lyase

Cellular Component: cytosol; extracellular space; nucleus

Molecular Function:identical protein binding; lead ion binding; porphobilinogen synthase activity; zinc ion binding

Biological Process: heme biosynthetic process; protein homooligomerization; protoporphyrinogen IX biosynthetic process; response to activity; response to aluminum ion; response to amino acid stimulus; response to arsenic; response to cadmium ion; response to cobalt ion; response to drug; response to ethanol; response to glucocorticoid stimulus; response to herbicide; response to hormone stimulus; response to hypoxia; response to inorganic substance; response to ionizing radiation; response to iron ion; response to lead ion; response to lipopolysaccharide; response to mercury ion; response to metal ion; response to methylmercury; response to nutrient; response to nutrient levels; response to organic cyclic substance; response to organic substance; response to oxidative stress; response to selenium ion; response to toxin; response to vitamin; response to vitamin B1; response to vitamin E; response to zinc ion

NCBI Summary:catalyzes the conversion of 5-aminolevulinate to porphobilinogen in heme biosynthesis [RGD, Feb 2006]
UniProt Code:P06214
NCBI GenInfo Identifier:6978483
NCBI Gene ID:25374
NCBI Accession:NP_037031.1
UniProt Related Accession:P06214
Molecular Weight:36,032 Da
NCBI Full Name:delta-aminolevulinic acid dehydratase
NCBI Synonym Full Names:aminolevulinate dehydratase
NCBI Official Symbol:Alad  
NCBI Official Synonym Symbols:ALADR; aminolevulinatedelta-dehydratase  
NCBI Protein Information:delta-aminolevulinic acid dehydratase
UniProt Protein Name:Delta-aminolevulinic acid dehydratase
UniProt Synonym Protein Names:Porphobilinogen synthase
Protein Family:Delta-aminolevulinic acid dehydratase
UniProt Gene Name:Alad  
UniProt Entry Name:HEM2_RAT
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Additional Information

Reactivity:
Rat
ELISA Type:
Sandwich
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