Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.16-10 ng/mL
Sensitivity:	0.10 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human TJP1 in samples. No significant cross-reactivity or interference between Human TJP1 and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA 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 ELISA 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 blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human TJP1. The concentration of Human TJP1 in samples can be calculated by comparing the OD 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 OD 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 (ng/mL)	O.D	Average	Corrected
10	2.56 2.578	2.569	2.503
5	1.734 1.78	1.757	1.691
2.5	1.031 1.027	1.029	0.963
1.25	0.484 0.49	0.487	0.421
0.63	0.292 0.282	0.287	0.221
0.32	0.182 0.17	0.176	0.11
0.16	0.121 0.123	0.122	0.056
0	0.059 0.073	0.066	--

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 (ng/mL)	0.50	1.29	4.40	0.55	1.17	4.57
Standard deviation	0.03	0.06	0.24	0.03	0.05	0.14
C V (%)	6.00	4.65	5.45	5.45	4.27	3.06

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)	88-100	93
EDTA plasma (n=5)	96-108	101
Cell culture media (n=5)	90-107	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 (%)	84-97	90-102	98-112
1:2	Average (%)	91	96	104
1:4	Range (%)	88-100	82-94	88-99
1:4	Average (%)	94	88	94
1:8	Range (%)	93-106	84-98	82-94
1:8	Average (%)	99	90	87
1:16	Range (%)	85-100	82-95	84-98
1:16	Average (%)	92	88	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 ELISA 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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.