Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	7.81-500 pg/mL
Sensitivity:	4.69 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human MCP-4/CCL13 in samples. No significant cross-reactivity or interference between Human MCP-4/CCL13 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 MCP-4/CCL13. 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 MCP-4/CCL13 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 MCP-4/CCL13, 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 MCP-4/CCL13. The concentration of Human MCP-4/CCL13 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	CCL13: Chemotactic factor that attracts monocytes, lymphocytes, basophils and eosinophils, but not neutrophils. Signals through CCR2B and CCR3 receptors. Plays a role in the accumulation of leukocytes at both sides of allergic and non-allergic inflammation. May be involved in the recruitment of monocytes into the arterial wall during the disease process of atherosclerosis. May play a role in the monocyte attraction in tissues chronically exposed to exogenous pathogens. By IL1/interleukin-1 and TNF. Widely expressed. Found in small intestine, thymus, colon, lung, trachea, stomach and lymph node. Low levels seen in the pulmonary artery smooth muscle cells. Belongs to the intercrine beta (chemokine CC) family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Motility/polarity/chemotaxis; Chemokine; Secreted Chromosomal Location of Human Ortholog: 17q11. 2 Cellular Component: extracellular space Molecular Function:chemokine activity; receptor binding Biological Process: cellular calcium ion homeostasis; regulation of cell shape; cell-cell signaling; eosinophil chemotaxis; cytoskeleton organization and biogenesis; immune response; signal transduction; inflammatory response; chemotaxis
NCBI Summary:	This antimicrobial gene is one of several Cys-Cys (CC) cytokine genes clustered on the q-arm of chromosome 17. Cytokines are a family of secreted proteins involved in immunoregulatory and inflammatory processes. The CC cytokines are proteins characterized by two adjacent cysteines. The cytokine encoded by this gene displays chemotactic activity for monocytes, lymphocytes, basophils and eosinophils, but not neutrophils. This chemokine plays a role in accumulation of leukocytes during inflammation. It may also be involved in the recruitment of monocytes into the arterial wall during artherosclerosis. [provided by RefSeq, Sep 2014]
UniProt Code:	Q99616
NCBI GenInfo Identifier:	3024128
NCBI Gene ID:	6357
NCBI Accession:	Q99616. 1
UniProt Related Accession:	Q99616
Molecular Weight:
NCBI Full Name:	C-C motif chemokine 13
NCBI Synonym Full Names:	C-C motif chemokine ligand 13
NCBI Official Symbol:	CCL13
NCBI Official Synonym Symbols:	NCC1; CKb10; MCP-4; NCC-1; SCYL1; SCYA13
NCBI Protein Information:	C-C motif chemokine 13
UniProt Protein Name:	C-C motif chemokine 13
UniProt Synonym Protein Names:	CK-beta-10; Monocyte chemoattractant protein 4; Monocyte chemotactic protein 4; MCP-4; NCC-1; Small-inducible cytokine A13Cleaved into the following 3 chains:C-C motif chemokine 13, long chain; C-C motif chemokine 13, medium chain; C-C motif chemokine 13, short chain
Protein Family:	Meiotic coiled-coil protein
UniProt Gene Name:	CCL13
UniProt Entry Name:	CCL13_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
500	51246 51828	51537	51508
250	19040 22956	20998	20969
125	10117 8675	9396	9367
62.5	4113 4911	4512	4483
31.25	2392 2206	2299	2270
15.63	1264 1236	1250	1221
7.81	716 764	740	711
0	28 30	29	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MCP-4/CCL13 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human MCP-4/CCL13 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)	27.15	45.20	168.45	24.84	40.99	182.55
Standard deviation	3.23	3.93	11.62	2.92	2.87	12.40
C V (%)	11.90	8.69	6.90	11.76	7.00	6.79

Recovery

The recovery of Human MCP-4/CCL13 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)	86-99	91
EDTA plasma (n=5)	96-111	104
Cell culture media (n=5)	100-114	106

Linearity

Samples were spiked with high concentrations of Human MCP-4/CCL13 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 (%)	90-104	97-109	95-112
1:2	Average (%)	97	102	103
1:4	Range (%)	87-103	94-106	99-110
1:4	Average (%)	94	100	105
1:8	Range (%)	84-95	93-107	85-96
1:8	Average (%)	90	98	90
1:16	Range (%)	88-100	93-104	96-110
1:16	Average (%)	93	99	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.