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 TJP1 in samples. No significant cross-reactivity or interference between Human TJP1 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 TJP1. 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 TJP1 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 TJP1, 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 TJP1. The concentration of Human TJP1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	ZO1: a scaffolding protein of the MAGUK (membrane-associated guanylate kinase) family associated with tight junctions (TJ). May mediate dynamic changes in the composition of TJs. The N-terminal may be involved in transducing a signal required for tight junction assembly, while the C-terminal may have specific properties of tight junctions. The alpha domain might be involved in stabilizing junctions. Plays a role in the regulation of cell migration by targeting the protein kinase MRCKb to the leading edge of migrating cells. Interacts with BVES (via the C-terminus cytoplasmic tail). Interacts with HSPA4 and KIRREL1. Forms homodimers and heterodimers with ZO-2 or ZO-3. Interacts with occludin, CALM, claudins, CGN, CXADR, GJA12, GJD3 and UBN1. Interacts (via ZU5 domain) with MRCKb and MYZAP. Interacts (via PDZ domain) with Cx43. ZO-2 and ZO-3 shuttle between the TJ and the nucleus, where they may regulate gene expression. TJ, or zonula occludens, are the closely associated areas of two cells whose membranes are tightly joined together. They are composed of a branching network of strands, the efficiency of which increases exponentially with the number of strands. The strands associate with transmembrane proteins that bind to similar proteins on adjacent cells, and with submembranous proteins that are anchored to the actin component of the cytoskeleton. Thus, TJs join together the cytoskeletons of adjacent cells. They endow tissues with substantial form, shape and location, enforce cellular polarity, and form barriers to molecules and pathogens. Molecules forming the membranous part of TJs include occludin, claudins, tricellulin and junctional adhesion molecules. These molecules interact with the proteins ZO-1, ZO-2 and ZO-3. Tight junction proteins can be up- or downregulated in cancer and are involved in the epithelial-mesenchymal transitions (EMT) in tumors. Alternative splicing produces two isoforms. The long isoform is found in most epithelial cell junctions. The short isoform is found both in endothelial cells and the highly specialized epithelial junctions of renal glomeruli and Sertoli cells of the seminiferous tubules.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Adaptor/scaffold Chromosomal Location of Human Ortholog: 15q13 Cellular Component: apical junction complex; intercellular canaliculus; tight junction; apicolateral plasma membrane; basolateral plasma membrane; cytosol; cell-cell adherens junction; apical part of cell; cytoplasm; apical plasma membrane; plasma membrane; gap junction; nucleus; cell junction Molecular Function:protein C-terminus binding; calmodulin binding; protein domain specific binding; protein binding Biological Process: response to drug; intercellular junction assembly; response to ethanol; sensory perception of sound; apoptosis; negative regulation of vascular permeability; blastocyst formation; response to lipopolysaccharide; cell structure disassembly during apoptosis
NCBI Summary:	This gene encodes a protein located on a cytoplasmic membrane surface of intercellular tight junctions. The encoded protein may be involved in signal transduction at cell-cell junctions. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, Jul 2014]
UniProt Code:	Q07157
NCBI GenInfo Identifier:	85700443
NCBI Gene ID:	7082
NCBI Accession:	Q07157. 3
UniProt Secondary Accession:	Q07157,Q2NKP3, Q4ZGJ6, B4E3K1,
UniProt Related Accession:	Q07157
Molecular Weight:	1748
NCBI Full Name:	Tight junction protein ZO-1
NCBI Synonym Full Names:	tight junction protein 1
NCBI Official Symbol:	TJP1
NCBI Official Synonym Symbols:	ZO-1
NCBI Protein Information:	tight junction protein ZO-1; zona occludens 1; zonula occludens 1 protein
UniProt Protein Name:	Tight junction protein ZO-1
UniProt Synonym Protein Names:	Tight junction protein 1; Zona occludens protein 1; Zonula occludens protein 1
UniProt Gene Name:	TJP1
UniProt Entry Name:	ZO1_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
2000	53514 57928	55721	55693
1000	23965 23983	23974	23946
500	11066 10998	11032	11004
250	5046 5542	5294	5266
125	2781 2435	2608	2580
62.5	1333 1289	1311	1283
31.25	629 719	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TJP1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TJP1 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)	91.68	208.30	937.40	90.75	189.42	1028.95
Standard deviation	7.64	20.68	63.56	11.57	16.76	83.45
C V (%)	8.33	9.93	6.78	12.75	8.85	8.11

Recovery

The recovery of Human TJP1 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)	95-111	102
EDTA plasma (n=5)	92-104	99
Cell culture media (n=5)	92-103	97

Linearity

Samples were spiked with high concentrations of Human TJP1 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 (%)	95-112	103-119	93-104
1:2	Average (%)	103	110	98
1:4	Range (%)	99-112	92-105	96-112
1:4	Average (%)	104	100	103
1:8	Range (%)	93-110	102-115	104-121
1:8	Average (%)	100	109	110
1:16	Range (%)	95-111	86-99	99-112
1:16	Average (%)	103	91	105

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.