Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human NF-kBp65 in samples. No significant cross-reactivity or interference between Human NF-kBp65 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 NF-kBp65. 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 NF-kBp65 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 NF-kBp65, 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 NF-kBp65. The concentration of Human NF-kBp65 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	NFkB-p65: a subunit of NF-kappa-B transcription complex, which plays a crucial role in inflammatory and immune responses. The inhibitory effect of I-kappa-B upon NF-kappa-B in the cytoplasm is exerted primarily through the interaction with p65. P65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. There are five NFkB proteins in mammals (RelA/NFkB-p65, RelB, c-Rel, NF-_B1/NFkB-p105, and NF-_B2/NFkB-p100). They form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes. Three splice-variant isoforms have been identified.
UniProt Protein Details:	Protein type:DNA-binding; Nuclear receptor co-regulator; Transcription factor Chromosomal Location of Human Ortholog: 11q13. 1 Cellular Component: cytoplasm; cytosol; nuclear chromatin; nucleoplasm; nucleus; transcription factor complex Molecular Function:actinin binding; chromatin binding; chromatin DNA binding; DNA binding; histone deacetylase binding; identical protein binding; NF-kappaB binding; phosphate binding; protein binding; protein heterodimerization activity; protein homodimerization activity; protein kinase binding; protein N-terminus binding; RNA polymerase II transcription factor activity, enhancer binding; transcription activator binding; transcription factor activity; transcription factor binding; ubiquitin protein ligase binding Biological Process: activation of NF-kappaB transcription factor; cytokine and chemokine mediated signaling pathway; inflammatory response; membrane protein intracellular domain proteolysis; negative regulation of apoptosis; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of cell proliferation; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of interferon type I production; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of inflammatory response; response to organic substance; response to UV-B; stimulatory C-type lectin receptor signaling pathway; T cell receptor signaling pathway
NCBI Summary:	NF-kappa-B is a ubiquitous transcription factor involved in several biological processes. It is held in the cytoplasm in an inactive state by specific inhibitors. Upon degradation of the inhibitor, NF-kappa-B moves to the nucleus and activates transcription of specific genes. NF-kappa-B is composed of NFKB1 or NFKB2 bound to either REL, RELA, or RELB. The most abundant form of NF-kappa-B is NFKB1 complexed with the product of this gene, RELA. Four transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011]
UniProt Code:	Q04206
NCBI GenInfo Identifier:	62906901
NCBI Gene ID:	5970
NCBI Accession:	Q04206. 2
UniProt Secondary Accession:	Q04206,Q6GTV1, Q6SLK1,
UniProt Related Accession:	Q04206
Molecular Weight:	59,910 Da
NCBI Full Name:	Transcription factor p65
NCBI Synonym Full Names:	RELA proto-oncogene, NF-kB subunit
NCBI Official Symbol:	RELA
NCBI Official Synonym Symbols:	p65; NFKB3
NCBI Protein Information:	transcription factor p65
UniProt Protein Name:	Transcription factor p65
UniProt Synonym Protein Names:	Nuclear factor NF-kappa-B p65 subunit; Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3
Protein Family:	Proline-rich P65 protein
UniProt Gene Name:	RELA

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
2000	54233 57209	55721	55693
1000	22595 25353	23974	23946
500	11611 10453	11032	11004
250	4827 5761	5294	5266
125	2644 2572	2608	2580
62.5	1369 1253	1311	1283
31.25	667 681	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NF-kBp65 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NF-kBp65 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)	107.99	203.70	965.39	100.87	215.26	983.84
Standard deviation	11.57	23.34	81.09	11.84	20.56	96.22
C V (%)	10.71	11.46	8.40	11.74	9.55	9.78

Recovery

The recovery of Human NF-kBp65 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)	97-110	104
EDTA plasma (n=5)	99-113	105
Cell culture media (n=5)	89-103	96

Linearity

Samples were spiked with high concentrations of Human NF-kBp65 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 (%)	93-109	92-104	87-99
1:2	Average (%)	100	98	92
1:4	Range (%)	101-115	96-111	100-113
1:4	Average (%)	107	103	107
1:8	Range (%)	101-118	96-108	98-116
1:8	Average (%)	108	102	106
1:16	Range (%)	88-98	85-98	95-109
1:16	Average (%)	93	90	100

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.