Human GAD1 (Glutamate Decarboxylase 1) ELISA Kit (HUES03264)
- Product Type:
- ELISA Kit
- 96 Assays
- ELISA Type:
- Tested Sample Types:
- Serum, plasma and other biological fluids
|Detection Range:||1.56-100 ng/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Human GAD1 in samples. No significant cross-reactivity or interference between Human GAD1 and analogues was observed.|
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 GAD1. 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 GAD1 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 GAD1, 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 GAD1. The concentration of Human GAD1 in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||GAD67: Catalyzes the production of GABA. Homodimer. Isoform 3 is expressed in pancreatic islets, testis, adrenal cortex, and perhaps other endocrine tissues, but not in brain. Belongs to the group II decarboxylase family. 3 isoforms of the human protein are produced by alternative splicing.|
|UniProt Protein Details:|
Protein type:EC 4. 1. 1. 15; Other Amino Acids Metabolism - beta-alanine; Other Amino Acids Metabolism - taurine and hypotaurine; Carbohydrate Metabolism - butanoate; Amino Acid Metabolism - alanine, aspartate and glutamate; Mitochondrial; Lyase
Chromosomal Location of Human Ortholog: 2q31
Cellular Component: plasma membrane; intracellular; vesicle membrane
Molecular Function:protein binding; glutamate binding; glutamate decarboxylase activity; protein heterodimerization activity; protein N-terminus binding; pyridoxal phosphate binding
Biological Process: response to drug; glutamate catabolic process; synaptic transmission; glutamate decarboxylation to succinate; neurotransmitter secretion; gamma-aminobutyric acid biosynthetic process; protein-pyridoxal-5-phosphate linkage; neurotransmitter biosynthetic process
Disease: Cerebral Palsy, Spastic Quadriplegic, 1
|NCBI Summary:||This gene encodes one of several forms of glutamic acid decarboxylase, identified as a major autoantigen in insulin-dependent diabetes. The enzyme encoded is responsible for catalyzing the production of gamma-aminobutyric acid from L-glutamic acid. A pathogenic role for this enzyme has been identified in the human pancreas since it has been identified as an autoantigen and an autoreactive T cell target in insulin-dependent diabetes. This gene may also play a role in the stiff man syndrome. Deficiency in this enzyme has been shown to lead to pyridoxine dependency with seizures. Alternative splicing of this gene results in two products, the predominant 67-kD form and a less-frequent 25-kD form. [provided by RefSeq, Jul 2008]|
|NCBI GenInfo Identifier:||1352213|
|NCBI Gene ID:||2571|
|NCBI Accession:||Q99259. 1|
|UniProt Secondary Accession:||Q99259,Q49AK1, Q53TQ7, Q9BU91, Q9UHH4,|
|UniProt Related Accession:||Q99259|
|Molecular Weight:||66,897 Da|
|NCBI Full Name:||Glutamate decarboxylase 1|
|NCBI Synonym Full Names:||glutamate decarboxylase 1 (brain, 67kDa)|
|NCBI Official Symbol:||GAD1|
|NCBI Official Synonym Symbols:||GAD; SCP; CPSQ1|
|NCBI Protein Information:||glutamate decarboxylase 1; GAD-67; 67 kDa glutamic acid decarboxylase; glutamate decarboxylase 67 kDa isoform|
|UniProt Protein Name:||Glutamate decarboxylase 1|
|UniProt Synonym Protein Names:||67 kDa glutamic acid decarboxylase; GAD-67; Glutamate decarboxylase 67 kDa isoform|
|Protein Family:||Glutamate decarboxylase|
|UniProt Gene Name:||GAD1|
|UniProt Entry Name:||DCE1_HUMAN|
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human GAD1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human GAD1 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.33||5.20||5.14||5.41||4.65||3.78|
The recovery of Human GAD1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||93-106||99|
|Cell culture media (n=5)||95-110||101|
Samples were spiked with high concentrations of Human GAD1 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)|
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.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20°C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 µL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 µL||-20°C(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4°C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25×)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4°C(shading light)|
|Stop Solution||1 vial, 10 mL||4°C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.