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ELISAGenie/Human TNFRSF5(Tumor Necrosis Factor Receptor Superfamily, Member 5)ELISA Kit (HUES03330)/Tumor Necrosis Factor Receptor Superfamily, Member 5)ELISA Kit (HUES03330/HUES03330

Description
Additional Information

Description

Human TNFRSF5(Tumor Necrosis Factor Receptor Superfamily, Member 5)ELISA Kit

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 TNFRSF5 . 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 TNFRSF5 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 TNFRSF5, 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 TNFRSF5. The concentration of Human TNFRSF5 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay type	Sandwich
Format	96T
Assay time	4.5h
Reactivity	Human
Detection Method	Colormetric
Detection Range	31.25-2000 pg/mL
Sensitivity	18.75 pg/mL
Sample Volume Required Per Well	100uL
Sample Type	Serum, plasma and other biological fluids

Specificity

This kit recognizes Human TNFRSF5 in samples. No significant cross-reactivity or interference between Human TNFRSF5 and analogues was observed.

Typical Data

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(pg/mL)	O.D	Average	Corrected
2000	2.5292.567	2.548	2.47
1000	1.5871.587	1.587	1.509
500	0.9520.916	0.934	0.856
250	0.4430.473	0.458	0.38
125	0.2650.241	0.253	0.175
62.5	0.190.17	0.18	0.102
31.25	0.1220.138	0.13	0.052
0	0.0760.08	0.078	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TNFRSF5 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TNFRSF5 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)	96.99	311.47	706.69	102.82	303.17	661.19
Standard deviation	6.19	17.69	32.44	5.43	17.52	26.38
C V (%)	6.38	5.68	4.59	5.28	5.78	3.99

Recovery

The recovery of Human TNFRSF5 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)	89-102	96
EDTA plasma (n=5)	90-101	96
Cell culture media (n=5)	87-97	92

Linearity

Samples were spiked with high concentrations of Human TNFRSF5 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 (%)	87-102	91-102	95-111
1:2	Average (%)	93	97	101
1:4	Range (%)	91-105	83-96	82-93
1:4	Average (%)	97	90	87
1:8	Range (%)	89-104	87-101	87-103
1:8	Average (%)	96	93	94
1:16	Range (%)	91-105	82-92	84-96
1:16	Average (%)	96	87	89

Kit Components & Storage

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 X12 strips	-20"C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100X)	1 vial, 120 uL
Concentrated HRP Conjugate (100X)	1 vial, 120 uL	-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 (25X)	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

Human TNFRSF5(Tumor Necrosis Factor Receptor Superfamily, Member 5)ELISA Kit (HUES03330) Assay procedure

1. 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.

2. Aliquot 100µl of standard solutions into the standard wells.

3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.

4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.

5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.

6. 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.

7. 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.

8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.

9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.

10. 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.

11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.

12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human TNFRSF5(Tumor Necrosis Factor Receptor Superfamily, Member 5)ELISA Kit (HUES03330) Protein Information

UniProt Protein Function:	CD40: a member of the TNF-receptor superfamily. This receptor for CD40L mediates a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation.Defects in CD40 are the cause of hyper-IgM immunodeficiency type 3 (HIGM3). HIGM3 is an autosomal recessive disorder which includes an inability of B cells to undergo isotype switching, one of the final differentiation steps in the humoral immune system, an inability to mount an antibody-specific immune response, and a lack of germinal center formation. Two alternatively spliced isoforms have been reported. Isoform I is a type I membrane protein; isoform II is secreted.
UniProt Protein Details:	Protein type:Membrane protein, integral; Receptor, cytokine Chromosomal Location of Human Ortholog: 20q12-q13.2 Cellular Component: cell surface; integral to plasma membrane; plasma membrane Molecular Function:enzyme binding; protein binding; receptor activity; signal transducer activity; tumor necrosis factor receptor activity; ubiquitin protein ligase binding Biological Process: activation of NF-kappaB transcription factor; cellular calcium ion homeostasis; immune response; multicellular organismal development; positive regulation of GTPase activity; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of MAP kinase activity; positive regulation of protein amino acid phosphorylation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of tyrosine phosphorylation of Stat1 protein; protein complex assembly; regulation of cell proliferation; regulation of immune response; response to lipopolysaccharide; tumor necrosis factor-mediated signaling pathway Disease: Immunodeficiency With Hyper-igm, Type 3
NCBI Summary:	This gene is a member of the TNF-receptor superfamily. The encoded protein is a receptor on antigen-presenting cells of the immune system and is essential for mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. AT-hook transcription factor AKNA is reported to coordinately regulate the expression of this receptor and its ligand, which may be important for homotypic cell interactions. Adaptor protein TNFR2 interacts with this receptor and serves as a mediator of the signal transduction. The interaction of this receptor and its ligand is found to be necessary for amyloid-beta-induced microglial activation, and thus is thought to be an early event in Alzheimer disease pathogenesis. Mutations affecting this gene are the cause of autosomal recessive hyper-IgM immunodeficiency type 3 (HIGM3). Multiple alternatively spliced transcript variants of this gene encoding distinct isoforms have been reported. [provided by RefSeq, Nov 2014]
UniProt Code:	P25942
NCBI GenInfo Identifier:	116000
NCBI Gene ID:	958
NCBI Accession:	P25942.1
UniProt Secondary Accession:	P25942,Q53GN5, Q5JY15, Q5U007, Q7M4Q8, Q86YK5, Q9BYU0 E1P5S9,
UniProt Related Accession:	P25942
Molecular Weight:	22,259 Da
NCBI Full Name:	Tumor necrosis factor receptor superfamily member 5
NCBI Synonym Full Names:	CD40 molecule
NCBI Official Symbol:	CD40
NCBI Official Synonym Symbols:	p50; Bp50; CDW40; TNFRSF5
NCBI Protein Information:	tumor necrosis factor receptor superfamily member 5
UniProt Protein Name:	Tumor necrosis factor receptor superfamily member 5
UniProt Synonym Protein Names:	B-cell surface antigen CD40; Bp50; CD40L receptor; CDw40; CD_antigen: CD40
Protein Family:	CD40 ligand
UniProt Gene Name:	CD40
UniProt Entry Name:	TNR5_HUMAN

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Additional Information

Reactivity:	Human
ELISA Type:	Sandwich