Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Rat
Detection method:	Chemiluminescence
Detection range:	3.13-200 ng/mL
Sensitivity:	1.88 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Rat DRD1 in samples. No significant cross-reactivity or interference between Rat DRD1 and analogues was observed.

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 DRD1. 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 DRD1 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 DRD1, 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 DRD1. The concentration of Rat DRD1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	DRD1: a G-protein coupled receptor. One of the five types (D1 to D5) of receptors for dopamine. The most abundant dopamine receptor in the central nervous system. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase. Interacts with calcyon.
UniProt Protein Details:	Protein type:Receptor, GPCR; Membrane protein, multi-pass; Membrane protein, integral; GPCR, family 1 Cellular Component: axon; caveola; cell soma; cytosol; dendrite; dendritic shaft; dendritic spine; endomembrane system; endoplasmic reticulum; endoplasmic reticulum membrane; integral to membrane; integral to plasma membrane; membrane; nerve terminal; nonmotile primary cilium; nucleus; plasma membrane Molecular Function:angiotensin receptor binding; ATPase binding; D3 dopamine receptor binding; dopamine binding; dopamine D1 receptor-like receptor activity; dopamine receptor activity; drug binding; G-protein alpha-subunit binding; G-protein coupled receptor activity; protein binding; protein complex binding; protein heterodimerization activity; protein phosphatase binding; receptor binding Biological Process: adenylate cyclase activation; adult walking behavior; associative learning; astrocyte development; behavioral fear response; behavioral response to cocaine; calcium-mediated signaling; cellular response to insulin stimulus; cerebral cortex GABAergic interneuron migration; conditioned taste aversion; dentate gyrus development; dopamine receptor signaling pathway; dopamine receptor, adenylate cyclase activating pathway; dopamine receptor, phospholipase C activating pathway; dopamine transport; elevation of cytosolic calcium ion concentration during G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); feeding behavior; G-protein coupled receptor protein signaling pathway; G-protein signaling, adenylate cyclase activating pathway; G-protein signaling, coupled to cyclic nucleotide second messenger; generation of action potential; glucose import; grooming behavior; habituation; hippocampus development; intracellular protein transport; learning; locomotory behavior; maternal behavior; mating behavior; memory; muscle contraction; negative regulation of cell migration; negative regulation of circadian sleep/wake cycle, sleep; negative regulation of protein kinase activity; operant conditioning; orbitofrontal cortex development; peristalsis; phosphatidylinositol catabolic process; phosphatidylinositol metabolic process; positive regulation of adenylate cyclase activity; positive regulation of cAMP biosynthetic process; positive regulation of cell migration; positive regulation of membrane potential; positive regulation of release of sequestered calcium ion into cytosol; positive regulation of synaptic transmission, glutamatergic; prepulse inhibition; protein import into nucleus; regulation of dopamine metabolic process; regulation of ion transport; regulation of long-term neuronal synaptic plasticity; regulation of vasoconstriction; response to activity; response to amino acid stimulus; response to amphetamine; response to cocaine; response to drug; response to estradiol stimulus; response to ethanol; response to food; response to morphine; response to nicotine; response to organic cyclic substance; response to organic nitrogen; response to retinoic acid; response to steroid hormone stimulus; sensitization; social behavior; startle response; striatum development; synaptic transmission, dopaminergic; synaptogenesis; thermoregulation; transmission of nerve impulse; vasodilation; visual learning
NCBI Summary:	induces dopamine sensitive adenylate cyclase activation; plays a role in regulating food intake [RGD, Feb 2006]
UniProt Code:	P18901
NCBI GenInfo Identifier:	118229
NCBI Gene ID:	24316
NCBI Accession:	P18901. 2
UniProt Secondary Accession:	P18901,P21669,
UniProt Related Accession:	P18901
Molecular Weight:	49,428 Da
NCBI Full Name:	D(1A) dopamine receptor
NCBI Synonym Full Names:	dopamine receptor D1
NCBI Official Symbol:	Drd1
NCBI Official Synonym Symbols:	D1a; Drd-1; Drd1a
NCBI Protein Information:	D(1A) dopamine receptor
UniProt Protein Name:	D(1A) dopamine receptor
UniProt Synonym Protein Names:	Dopamine D1 receptor
Protein Family:	D(1A) dopamine receptor
UniProt Gene Name:	Drd1
UniProt Entry Name:	DRD1_RAT

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 (ng/mL)	RLU	Average	Corrected
200	43392 44754	44073	44035
100	17220 19202	18211	18173
50	8372 8040	8206	8168
25	3922 3948	3935	3897
12.5	2114 1850	1982	1944
6.25	1105 999	1052	1014
3.13	561 635	598	560
0	38 38	38	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat DRD1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat DRD1 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	10.82	30.90	84.42	10.62	30.48	90.49
Standard deviation	1.17	2.70	6.42	1.18	2.15	6.42
C V (%)	10.81	8.74	7.60	11.11	7.05	7.09

Recovery

The recovery of Rat DRD1 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)	88-99	94
EDTA plasma (n=5)	91-103	96
Cell culture media (n=5)	97-109	103

Linearity

Samples were spiked with high concentrations of Rat DRD1 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)
1:2	Range (%)	89-102	91-106	90-105
1:2	Average (%)	94	97	98
1:4	Range (%)	96-110	103-119	104-120
1:4	Average (%)	102	110	110
1:8	Range (%)	98-112	93-108	99-111
1:8	Average (%)	106	99	105
1:16	Range (%)	88-100	96-111	84-95
1:16	Average (%)	93	103	90

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 ×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 A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
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 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.
Determine the RLU value of each well immediately.