Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human HSP gp96 in samples. No significant cross-reactivity or interference between Human HSP gp96 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 HSPgp96. 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 HSPgp96 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 HSPgp96, 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 HSPgp96. The concentration of Human HSPgp96 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	GRP94: Molecular chaperone that functions in the processing and transport of secreted proteins. When associated with CNPY3, required for proper folding of Toll-like receptors. Functions in endoplasmic reticulum associated degradation (ERAD). Has ATPase activity. Homodimer; disulfide-linked. Component of an EIF2 complex at least composed of CELF1/CUGBP1, CALR, CALR3, EIF2S1, EIF2S2, HSP90B1 and HSPA5. Part a large chaperone multiprotein complex comprising DNAJB11, HSP90B1, HSPA5, HYOU, PDIA2, PDIA4, PDIA6, PPIB, SDF2L1, UGT1A1 and very small amounts of ERP29, but not, or at very low levels, CALR nor CANX. Interacts with AIMP1; regulates its retention in the endoplasmic reticulum. Interacts with OS9. Interacts with CNPY3. This interaction is disrupted in the presence of ATP. Interacts with TLR4 and TLR9, but not with TLR3. Belongs to the heat shock protein 90 family.
UniProt Protein Details:	Protein type:Apoptosis; Heat shock protein; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 12q24. 2-q24. 3 Cellular Component: endoplasmic reticulum membrane; focal adhesion; endoplasmic reticulum; endoplasmic reticulum lumen; extracellular region; cytosol; extracellular matrix; membrane; perinuclear region of cytoplasm; plasma membrane; melanosome; midbody; nucleus Molecular Function:protein binding; low-density lipoprotein receptor binding; RNA binding; unfolded protein binding; virion binding; calcium ion binding; protein phosphatase binding; ATP binding Biological Process: actin rod formation; receptor-mediated endocytosis; ER-associated protein catabolic process; protein transport; unfolded protein response, activation of signaling protein activity; cellular protein metabolic process; unfolded protein response; sequestering of calcium ion; response to hypoxia; toll-like receptor signaling pathway; innate immune response; regulation of phosphoprotein phosphatase activity; negative regulation of apoptosis
NCBI Summary:	This gene encodes a member of a family of adenosine triphosphate(ATP)-metabolizing molecular chaperones with roles in stabilizing and folding other proteins. The encoded protein is localized to melanosomes and the endoplasmic reticulum. Expression of this protein is associated with a variety of pathogenic states, including tumor formation. There is a microRNA gene located within the 5' exon of this gene. There are pseudogenes for this gene on chromosomes 1 and 15. [provided by RefSeq, Aug 2012]
UniProt Code:	P14625
NCBI GenInfo Identifier:	119360
NCBI Gene ID:	7184
NCBI Accession:	P14625. 1
UniProt Secondary Accession:	P14625,Q96A97,
UniProt Related Accession:	P14625
Molecular Weight:	803
NCBI Full Name:	Endoplasmin
NCBI Synonym Full Names:	heat shock protein 90kDa beta (Grp94), member 1
NCBI Official Symbol:	HSP90B1
NCBI Official Synonym Symbols:	ECGP; GP96; TRA1; GRP94; HEL35; HEL-S-125m
NCBI Protein Information:	endoplasmin; tumor rejection antigen 1; epididymis luminal protein 35; 94 kDa glucose-regulated protein; tumor rejection antigen (gp96) 1; endothelial cell (HBMEC) glycoprotein; heat shock protein 90 kDa beta member 1; stress-inducible tumor rejection antigen gp96; epididymis secretory sperm binding protein Li 125m
UniProt Protein Name:	Endoplasmin
UniProt Synonym Protein Names:	94 kDa glucose-regulated protein; GRP-94; Heat shock protein 90 kDa beta member 1; Tumor rejection antigen 1; gp96 homolog
Protein Family:	Endoplasmin
UniProt Gene Name:	HSP90B1
UniProt Entry Name:	ENPL_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
20	2.437 2.455	2.446	2.377
10	1.598 1.622	1.61	1.541
5	0.956 0.936	0.946	0.877
2.5	0.471 0.495	0.483	0.414
1.25	0.269 0.241	0.255	0.186
0.63	0.174 0.17	0.172	0.103
0.31	0.111 0.131	0.121	0.052
0	0.061 0.077	0.069	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human HSP gp96 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human HSP gp96 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)	0.90	2.90	9.60	0.90	3.10	9.70
Standard deviation	0.05	0.15	0.46	0.05	0.13	0.34
C V (%)	5.56	5.17	4.79	5.56	4.19	3.51

Recovery

The recovery of Human HSP gp96 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)	92-108	100
EDTA plasma (n=5)	88-105	96
Cell culture media (n=5)	90-101	95

Linearity

Samples were spiked with high concentrations of Human HSP gp96 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 (%)	95-108	89-102	93-109
1:2	Average (%)	100	96	101
1:4	Range (%)	99-112	85-98	94-109
1:4	Average (%)	105	90	100
1:8	Range (%)	97-109	82-93	91-102
1:8	Average (%)	103	87	97
1:16	Range (%)	98-112	86-100	94-108
1:16	Average (%)	104	93	101

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.