Human TNFRSF1A (Tumor Necrosis Factor Receptor Superfamily, Member 1A) ELISA Kit (HUES01474)

SKU:
HUES01474
€299

Description

ELISA Kit Technical ManualMSDS

Human TNFRSF1A (Tumor Necrosis Factor Receptor Superfamily, Member 1A) 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 TNFRSF1A . 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 TNFRSF1A 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 TNFRSF1A, 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 TNFRSF1A. The concentration of Human TNFRSF1A in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range7.81-500 pg/mL
Sensitivity4.69 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human TNFRSF1A in samples. No significant cross-reactivity or interference between Human TNFRSF1A 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
5002.325
2.351
2.3382.28
2501.572
1.572
1.5721.514
1250.967
0.965
0.9660.908
62.50.438
0.46
0.4490.391
31.250.236
0.226
0.2310.173
15.630.16
0.158
0.1590.101
7.810.104
0.116
0.110.052
00.05
0.066
0.058--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TNFRSF1A were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TNFRSF1A were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)23.2174.91187.9020.9167.85199.93
Standard deviation1.273.037.271.404.0510.96
C V (%)5.474.043.876.705.975.48

Recovery

The recovery of Human TNFRSF1A 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)88-10395
EDTA plasma (n=5)94-108101
Cell culture media (n=5)92-10397

Linearity

Samples were spiked with high concentrations of Human TNFRSF1A 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)
1:2Range (%)97-11293-10691-106
Average (%)1029997
1:4Range (%)90-10583-9485-100
Average (%)988992
1:8Range (%)91-10585-9687-97
Average (%)969092
1:16Range (%)91-10583-9483-95
Average (%)998888

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 Standard2 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 Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human TNFRSF1A (Tumor Necrosis Factor Receptor Superfamily, Member 1A) ELISA Kit (HUES01474) 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 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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 TNFRSF1A (Tumor Necrosis Factor Receptor Superfamily, Member 1A) ELISA Kit (HUES01474) Protein Information

UniProt Protein Function:TNF-R1: Receptor for TNFSF2/TNF-alpha and homotrimeric TNFSF1/lymphotoxin-alpha. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death-inducing signaling complex (DISC) performs caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspartate- specific cysteine proteases) mediating apoptosis. Contributes to the induction of non-cytocidal TNF effects including anti-viral state and activation of the acid sphingomyelinase. Binding of TNF to the extracellular domain leads to homotrimerization. The aggregated death domains provide a novel molecular interface that interacts specifically with the death domain of TRADD. Various TRADD-interacting proteins such as TRAFS, RIPK1 and possibly FADD, are recruited to the complex by their association with TRADD. This complex activates at least two distinct signaling cascades, apoptosis and NF-kappa-B signaling. Interacts with BAG4, BRE, FEM1B, GRB2, SQSTM1 and TRPC4AP. Interacts with HCV core protein. Interacts with human cytomegalovirus/HHV-5 protein UL138. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Receptor, cytokine; Membrane protein, integral

Chromosomal Location of Human Ortholog: 12p13.2

Cellular Component: Golgi membrane; extracellular space; cell surface; mitochondrion; integral to plasma membrane; axon; extracellular region; plasma membrane; synapse; nucleus; cytosol; receptor complex; lipid raft

Molecular Function:protein binding; tumor necrosis factor receptor activity; protease binding; protein complex binding; tumor necrosis factor binding

Biological Process: response to alkaloid; viral reproduction; protein heterooligomerization; response to lipopolysaccharide; tumor necrosis factor-mediated signaling pathway; positive regulation of neuron apoptosis; negative regulation of interleukin-6 production; response to wounding; tetrapyrrole metabolic process; inflammatory response; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of protein import into nucleus, translocation; cytokine and chemokine mediated signaling pathway; positive regulation of tumor necrosis factor production; response to amino acid stimulus; prostaglandin metabolic process; positive regulation of tyrosine phosphorylation of Stat1 protein; positive regulation of angiogenesis; response to ethanol; induction of apoptosis via death domain receptors; DNA damage response, signal transduction resulting in induction of apoptosis; negative regulation of inflammatory response; defense response to bacterium; response to hypoxia; positive regulation of transcription from RNA polymerase II promoter; positive regulation of inflammatory response; negative regulation of apoptosis

Disease: Multiple Sclerosis, Susceptibility To, 5; Periodic Fever, Familial, Autosomal Dominant

NCBI Summary:The protein encoded by this gene is a member of the TNF-receptor superfamily. This protein is one of the major receptors for the tumor necrosis factor-alpha. This receptor can activate NF-kappaB, mediate apoptosis, and function as a regulator of inflammation. Antiapoptotic protein BCL2-associated athanogene 4 (BAG4/SODD) and adaptor proteins TRADD and TRAF2 have been shown to interact with this receptor, and thus play regulatory roles in the signal transduction mediated by the receptor. Germline mutations of the extracellular domains of this receptor were found to be associated with the autosomal dominant periodic fever syndrome. The impaired receptor clearance is thought to be a mechanism of the disease. [provided by RefSeq, Jul 2008]
UniProt Code:P19438
NCBI GenInfo Identifier:135959
NCBI Gene ID:7132
NCBI Accession:P19438.1
UniProt Secondary Accession:P19438,Q9UCA4, A8K4X3, B2RDE4, B3KPQ1, B4DQB7, B4E309 B5M0B5, D3DUR1,
UniProt Related Accession:P19438
Molecular Weight:24,194 Da
NCBI Full Name:Tumor necrosis factor receptor superfamily member 1A
NCBI Synonym Full Names:tumor necrosis factor receptor superfamily, member 1A
NCBI Official Symbol:TNFRSF1A
NCBI Official Synonym Symbols:FPF; MS5; p55; p60; TBP1; TNF-R; TNFAR; TNFR1; p55-R; CD120a; TNFR55; TNFR60; TNF-R-I; TNF-R55; TNFR1-d2
NCBI Protein Information:tumor necrosis factor receptor superfamily member 1A; TNF-R1; TNF-RI; TNFR-I; tumor necrosis factor-alpha receptor; tumor necrosis factor receptor type 1; tumor necrosis factor binding protein 1; tumor necrosis factor receptor 1A isoform beta
UniProt Protein Name:Tumor necrosis factor receptor superfamily member 1A
UniProt Synonym Protein Names:Tumor necrosis factor receptor 1; TNF-R1; Tumor necrosis factor receptor type I; TNF-RI; TNFR-I; p55; p60; CD_antigen: CD120aCleaved into the following 2 chains:Tumor necrosis factor receptor superfamily member 1A, membrane form; Tumor necrosis factor-binding protein 1; TBPI
UniProt Gene Name:TNFRSF1A
UniProt Entry Name:TNR1A_HUMAN
View AllClose

Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
View AllClose