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Human ALAD ELISA Kit

SKU:
HUFI01758
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P13716
Sensitivity:
9.375pg/ml
Range:
15.625-1000pg/ml
ELISA Type:
Sandwich
Synonyms:
ALAD, ALADH, Porphobilinogen synthase
Reactivity:
Human
Research Area:
Metabolism
€599
Frequently bought together:

Description

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Human ALAD ELISA Kit

The Human ALAD (Aminolevulinate Dehydratase) ELISA Kit is specifically designed for the accurate quantification of ALAD levels in human serum, plasma, and cell culture supernatants. With outstanding sensitivity and specificity, this kit ensures reliable and reproducible results, making it an excellent tool for a variety of research applications.ALAD is a crucial enzyme involved in heme biosynthesis, playing a key role in the production of hemoglobin and other heme-containing proteins. Dysregulation of ALAD has been linked to various diseases such as lead poisoning, porphyrias, and certain types of cancer, making it a valuable biomarker for studying these conditions and evaluating potential therapeutic interventions.

With the Human ALAD ELISA Kit, researchers can accurately measure ALAD levels in biological samples, facilitating the advancement of scientific knowledge and the development of novel diagnostic and treatment strategies. Order your kit today and unlock new insights into the role of ALAD in health and disease.

Product Name:Human ALAD ELISA Kit
Product Code:HUFI01758
Size:96 Assays
Alias:ALAD, ALADH, Porphobilinogen synthase
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human ALAD concentrations in serum plasma and other biological fluids.
Sensitivity:9.375pg/ml
Range:15.625-1000pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human ALAD and the recovery rates were calculated by comparing the measured value to the expected amount of Human ALAD in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)90-10398
EDTA plasma(n=5)85-10596
UFH plasma(n=5)88-10596
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ALAD and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)92-104%88-105%87-103%
EDTA plasma(n=5)94-98%92-98%84-101%
UFH plasma(n=5)86-99%80-94%87-97%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotP13716
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

Chromosomal Location of Human Ortholog: 9q32

Cellular Component: cytosol; extracellular region; nucleus

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

Biological Process: heme biosynthetic process; neutrophil degranulation; protein homooligomerization

Disease: Porphyria, Acute Hepatic

NCBI Summary:The ALAD enzyme is composed of 8 identical subunits and catalyzes the condensation of 2 molecules of delta-aminolevulinate to form porphobilinogen (a precursor of heme, cytochromes and other hemoproteins). ALAD catalyzes the second step in the porphyrin and heme biosynthetic pathway; zinc is essential for enzymatic activity. ALAD enzymatic activity is inhibited by lead and a defect in the ALAD structural gene can cause increased sensitivity to lead poisoning and acute hepatic porphyria. Alternative splicing of this gene results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Dec 2015]
UniProt Code:P13716
NCBI GenInfo Identifier:122833
NCBI Gene ID:210
NCBI Accession:P13716.1
UniProt Secondary Accession:P13716,Q16870, Q16871, Q9BVQ9, A8K375, B2R6F2,
UniProt Related Accession:P13716
Molecular Weight:36kDa
NCBI Full Name:Delta-aminolevulinic acid dehydratase
NCBI Synonym Full Names:aminolevulinate dehydratase
NCBI Official Symbol:ALAD
NCBI Official Synonym Symbols:PBGS; ALADH
NCBI Protein Information:delta-aminolevulinic acid dehydratase
UniProt Protein Name:Delta-aminolevulinic acid dehydratase
UniProt Synonym Protein Names:Porphobilinogen synthase
Protein Family:Aladin
UniProt Gene Name:ALAD

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol
SerumIf using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal FluidCollect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatantCollect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysatesSolubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenatesThe preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysatesRinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast MilkCollect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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