Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	62.50-4000 pg/mL
Sensitivity:	37.50 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human Bcl2L11 in samples. No significant cross-reactivity or interference between Human Bcl2L11 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 Bcl2L11. 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 Bcl2L11 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 Bcl2L11, 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 Bcl2L11. The concentration of Human Bcl2L11 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	BIM: a pro-apoptotic member of the BCL-2 protein family. Interacts with other members of the BCL-2 protein family, including BCL2, BCL2L1/BCL-X(L), and MCL1, and act as an apoptotic activator. Its expression can be induced by nerve growth factor (NGF), as well as by the forkhead transcription factor FKHR-L1, which suggests a role in neuronal and lymphocyte apoptosis. Transgenic studies in the mouse suggested that this protein functions as an essential initiator of the apoptosis in thymocyte-negative selection. Nineteen alternatively spliced transcript variants of this gene have been reported.
UniProt Protein Details:	Protein type:Apoptosis Chromosomal Location of Human Ortholog: 2q13 Cellular Component: microtubule; mitochondrial outer membrane; extrinsic to membrane; endomembrane system; cytosol Molecular Function:protein binding; microtubule binding Biological Process: nerve growth factor receptor signaling pathway; positive regulation of apoptosis; apoptosis; cell-matrix adhesion; pigmentation during development; myeloid cell homeostasis; ear development; positive regulation of caspase activity; B cell apoptosis; cellular process regulating host cell cycle in response to virus; positive regulation of apoptosis by virus; mammary gland development; B cell homeostasis; positive regulation of neuron apoptosis; T cell homeostasis; kidney development; post-embryonic organ morphogenesis; caspase activation; spleen development; thymus development; in utero embryonic development; positive regulation of protein homooligomerization; male gonad development; positive regulation of cell cycle; regulation of organ growth; lumen formation; odontogenesis of dentine-containing teeth; DNA damage response, signal transduction resulting in induction of apoptosis; regulation of pigmentation during development; spermatogenesis; brain development
NCBI Summary:	The protein encoded by this gene belongs to the BCL-2 protein family. BCL-2 family members form hetero- or homodimers and act as anti- or pro-apoptotic regulators that are involved in a wide variety of cellular activities. The protein encoded by this gene contains a Bcl-2 homology domain 3 (BH3). It has been shown to interact with other members of the BCL-2 protein family and to act as an apoptotic activator. The expression of this gene can be induced by nerve growth factor (NGF), as well as by the forkhead transcription factor FKHR-L1, which suggests a role of this gene in neuronal and lymphocyte apoptosis. Transgenic studies of the mouse counterpart suggested that this gene functions as an essential initiator of apoptosis in thymocyte-negative selection. Several alternatively spliced transcript variants of this gene have been identified. [provided by RefSeq, Jun 2013]
UniProt Code:	O43521
NCBI GenInfo Identifier:	20336315
NCBI Gene ID:	10018
NCBI Accession:	NP_619527. 1
UniProt Secondary Accession:	O43521,O43522, Q0MSE7, Q0MSE8, Q0MSE9, Q53R28, Q6JTU6 Q6T851, Q6TE14, Q6TE15, Q6TE16, A8K2W2,
UniProt Related Accession:	O43521
Molecular Weight:	22,171 Da
NCBI Full Name:	bcl-2-like protein 11 isoform 1
NCBI Synonym Full Names:	BCL2-like 11 (apoptosis facilitator)
NCBI Official Symbol:	BCL2L11
NCBI Official Synonym Symbols:	BAM; BIM; BOD
NCBI Protein Information:	bcl-2-like protein 11; bcl-2 interacting protein Bim; bcl-2-related ovarian death agonist; bcl-2 interacting mediator of cell death
UniProt Protein Name:	Bcl-2-like protein 11
UniProt Synonym Protein Names:	Bcl2-interacting mediator of cell death
Protein Family:	Bcl-2-like protein
UniProt Gene Name:	BCL2L11
UniProt Entry Name:	B2L11_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
4000	59061 59101	59081	59046
2000	26486 29232	27859	27824
1000	14016 12952	13484	13449
500	6372 6838	6605	6570
250	3459 3027	3243	3208
125	1702 1460	1581	1546
62.50	744 766	755	720
0	34 36	35	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Bcl2L11 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Bcl2L11 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)	221.90	415.89	1938.17	222.30	409.00	1846.81
Standard deviation	27.87	36.52	218.43	26.12	45.52	205.73
C V (%)	12.56	8.78	11.27	11.75	11.13	11.14

Recovery

The recovery of Human Bcl2L11 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)	93-107	100
EDTA plasma (n=5)	94-107	101
Cell culture media (n=5)	99-115	106

Linearity

Samples were spiked with high concentrations of Human Bcl2L11 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 (%)	94-105	90-103	92-107
1:2	Average (%)	100	98	98
1:4	Range (%)	84-96	89-106	86-100
1:4	Average (%)	91	97	92
1:8	Range (%)	92-106	87-101	93-108
1:8	Average (%)	98	94	99
1:16	Range (%)	85-96	88-100	85-99
1:16	Average (%)	90	93	91

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.