Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample volume:	100µL
Sample type:	Tissue homogenates
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human ACTG1 in samples. No significant cross-reactivity or interference between Human ACTG1 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 Human ACTG1. 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 Human ACTG1 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 ACTG1, 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 Human ACTG1. The concentration of Human ACTG1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	ACTG1: Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells. Polymerization of globular actin (G-actin) leads to a structural filament (F-actin) in the form of a two-stranded helix. Each actin can bind to 4 others. Belongs to the actin family.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Cytoskeletal Chromosomal Location of Human Ortholog: 17q25 Cellular Component: filamentous actin; extracellular space; cytoskeleton; myofibril; membrane; plasma membrane; nucleus; cytosol Molecular Function:identical protein binding; protein binding; structural constituent of cytoskeleton; ubiquitin protein ligase binding; ATP binding Biological Process: axon guidance; retinal homeostasis; intercellular junction assembly and maintenance; ephrin receptor signaling pathway; innate immune response; sarcomere organization; cell motility; vascular endothelial growth factor receptor signaling pathway Disease: Baraitser-winter Syndrome 2; Deafness, Autosomal Dominant 20
NCBI Summary:	Actins are highly conserved proteins that are involved in various types of cell motility, and maintenance of the cytoskeleton. In vertebrates, three main groups of actin isoforms, alpha, beta and gamma have been identified. The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus. The beta and gamma actins co-exist in most cell types as components of the cytoskeleton, and as mediators of internal cell motility. Actin, gamma 1, encoded by this gene, is a cytoplasmic actin found in non-muscle cells. Mutations in this gene are associated with DFNA20/26, a subtype of autosomal dominant non-syndromic sensorineural progressive hearing loss. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2011]
UniProt Code:	P63261
NCBI GenInfo Identifier:	54036678
NCBI Gene ID:	71
NCBI Accession:	P63261. 1
UniProt Secondary Accession:	P63261,P02571, P14104, P99022, Q5U032, Q96E67, A8K7C2
UniProt Related Accession:	P63261
Molecular Weight:	375
NCBI Full Name:	Actin, cytoplasmic 2
NCBI Synonym Full Names:	actin, gamma 1
NCBI Official Symbol:	ACTG1
NCBI Official Synonym Symbols:	ACT; ACTG; BRWS2; DFNA20; DFNA26; HEL-176
NCBI Protein Information:	actin, cytoplasmic 2; cytoskeletal gamma-actin; epididymis luminal protein 176
UniProt Protein Name:	Actin, cytoplasmic 2
UniProt Synonym Protein Names:	Gamma-actinActin, cytoplasmic 2, N-terminally processed
Protein Family:	Actin
UniProt Gene Name:	ACTG1
UniProt Entry Name:	ACTG_HUMAN

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
1000	52006 55344	53675	53650
500	20363 24615	22489	22464
250	10554 9868	10211	10186
125	4890 4912	4901	4876
62.5	2619 2287	2453	2428
31.25	1323 1239	1281	1256
15.63	675 741	708	683
0	25 25	25	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ACTG1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ACTG1 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 (pg/mL)	50.87	133.44	374.96	49.33	134.02	386.93
Standard deviation	4.94	15.68	42.03	5.34	11.24	31.65
C V (%)	9.71	11.75	11.21	10.83	8.39	8.18

Recovery

The recovery of Human ACTG1 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)	89-103	96
EDTA plasma (n=5)	95-110	103
Cell culture media (n=5)	89-101	95

Linearity

Samples were spiked with high concentrations of Human ACTG1 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 (%)	93-110	101-119	93-107
1:2	Average (%)	100	108	98
1:4	Range (%)	90-102	89-99	94-108
1:4	Average (%)	96	94	101
1:8	Range (%)	90-104	94-109	100-113
1:8	Average (%)	95	100	106
1:16	Range (%)	99-113	92-108	97-113
1:16	Average (%)	105	100	103

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.