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:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human S100B in samples. No significant cross-reactivity or interference between Human S100B 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 S100B. 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 S100B 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 S100B, 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 S100B. The concentration of Human S100B in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	S100B: Weakly binds calcium but binds zinc very tightly- distinct binding sites with different affinities exist for both ions on each monomer. Physiological concentrations of potassium ion antagonize the binding of both divalent cations, especially affecting high-affinity calcium-binding sites. Binds to and initiates the activation of STK38 by releasing autoinhibitory intramolecular interactions within the kinase. Interaction with AGER after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53/TP53 signaling. Dimer of either two alpha chains, or two beta chains, or one alpha and one beta chain. The S100B dimer binds two molecules of STK38. Interacts with AGER. The S100B dimer interacts with two molecules of CAPZA1. Although predominant among the water-soluble brain proteins, S100 is also found in a variety of other tissues. Belongs to the S-101 family.
UniProt Protein Details:	Protein type:Calcium-binding Chromosomal Location of Human Ortholog: 21q22. 3 Cellular Component: ruffle; extracellular space; cell soma; intracellular membrane-bound organelle; perinuclear region of cytoplasm; cytoplasm; extracellular region; nucleus Molecular Function:identical protein binding; protein binding; RAGE receptor binding; protein homodimerization activity; zinc ion binding; calcium ion binding; calcium-dependent protein binding; tau protein binding Biological Process: central nervous system development; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of apoptosis; response to glucocorticoid stimulus; memory; cell proliferation; regulation of cell shape; learning and/or memory; astrocyte differentiation; axonogenesis; response to methylmercury; positive regulation of cell proliferation; innate immune response; regulation of neuronal synaptic plasticity
NCBI Summary:	The protein encoded by this gene is a member of the S100 family of proteins containing 2 EF-hand calcium-binding motifs. S100 proteins are localized in the cytoplasm and/or nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. S100 genes include at least 13 members which are located as a cluster on chromosome 1q21; however, this gene is located at 21q22. 3. This protein may function in Neurite extension, proliferation of melanoma cells, stimulation of Ca2+ fluxes, inhibition of PKC-mediated phosphorylation, astrocytosis and axonal proliferation, and inhibition of microtubule assembly. Chromosomal rearrangements and altered expression of this gene have been implicated in several neurological, neoplastic, and other types of diseases, including Alzheimer's disease, Down's syndrome, epilepsy, amyotrophic lateral sclerosis, melanoma, and type I diabetes. [provided by RefSeq, Jul 2008]
UniProt Code:	P04271
NCBI GenInfo Identifier:	134138
NCBI Gene ID:	6285
NCBI Accession:	P04271. 2
UniProt Secondary Accession:	P04271,D3DSN6,
UniProt Related Accession:	P04271
Molecular Weight:	10,713 Da
NCBI Full Name:	Protein S100-B
NCBI Synonym Full Names:	S100 calcium binding protein B
NCBI Official Symbol:	S100B
NCBI Official Synonym Symbols:	NEF; S100; S100-B; S100beta
NCBI Protein Information:	protein S100-B; S-100 protein subunit beta; S-100 calcium-binding protein, beta chain; S100 calcium-binding protein, beta (neural)
UniProt Protein Name:	Protein S100-B
UniProt Synonym Protein Names:	S-100 protein beta chain; S-100 protein subunit beta; S100 calcium-binding protein B
Protein Family:	Protein
UniProt Gene Name:	S100B
UniProt Entry Name:	S100B_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	50729 56621	53675	53650
500	21956 23022	22489	22464
250	10382 10040	10211	10186
125	4566 5236	4901	4876
62.5	2548 2358	2453	2428
31.25	1372 1190	1281	1256
15.63	698 718	708	683
0	24 26	25	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human S100B were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human S100B 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)	51.86	97.33	353.43	48.09	100.17	365.56
Standard deviation	4.45	10.45	31.70	3.97	8.45	22.19
C V (%)	8.58	10.74	8.97	8.26	8.44	6.07

Recovery

The recovery of Human S100B 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)	94-109	100
EDTA plasma (n=5)	88-102	95
Cell culture media (n=5)	91-106	99

Linearity

Samples were spiked with high concentrations of Human S100B 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 (%)	94-110	95-108	89-102
1:2	Average (%)	102	102	94
1:4	Range (%)	97-113	93-107	100-114
1:4	Average (%)	105	99	107
1:8	Range (%)	85-97	85-98	93-104
1:8	Average (%)	92	90	98
1:16	Range (%)	85-99	96-111	87-99
1:16	Average (%)	92	102	92

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.