Human TACI / TNFRSF13B ELISA Kit

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SKU:
HUFI00265
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O14836
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich
Synonyms:
TNFRSF13B, TACI, CD267, CVID, CD267 antigen, CVID2, Transmembrane activator and CAML interactor, tumor necrosis factor receptor superfamily member 13B,
Reactivity:
Human

Description

system_update_altDatasheet - तकनीकी मैनुअलsystem_update_altMSDS - तकनीकी मैनुअल
Product Name:Human TACI / TNFRSF13B ELISA Kit
Product Code:HUFI00265
Size:96 Assays
Alias:TNFRSF13B, TACI, CD267, CVID, CD267 antigen, CVID2, Transmembrane activator and CAML interactor, tumor necrosis factor receptor superfamily member 13B
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human TNFRSF13B/TACI concentrations in serum plasma and other biological fluids.
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human TNFRSF13B/TACI and the recovery rates were calculated by comparing the measured value to the expected amount of Human TNFRSF13B/TACI in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)86-9893
EDTA plasma(n=5)85-10595
UFH plasma(n=5)89-10397
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TNFRSF13B/TACI and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)90-98%87-104%85-105%
EDTA plasma(n=5)85-100%86-100%85-97%
UFH plasma(n=5)82-100%80-97%82-99%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotO14836
UniProt Protein Function:TNFRSF13B: Receptor for TNFSF13/APRIL and TNFSF13B/TALL1/BAFF/BLYS that binds both ligands with similar high affinity. Mediates calcineurin-dependent activation of NF-AT, as well as activation of NF-kappa-B and AP-1. Involved in the stimulation of B- and T- cell function and the regulation of humoral immunity. Defects in TNFRSF13B are the cause of immunodeficiency common variable type 2 (CVID2). CVID2 is a primary immunodeficiency characterized by antibody deficiency, hypogammaglobulinemia, recurrent bacterial infections and an inability to mount an antibody response to antigen. The defect results from a failure of B-cell differentiation and impaired secretion of immunoglobulins; the numbers of circulating B-cells is usually in the normal range, but can be low. Defects in TNFRSF13B are a cause of immunoglobulin A deficiency 2 (IGAD2). Selective deficiency of immunoglobulin A (IGAD) is the most common form of primary immunodeficiency, with an incidence of approximately 1 in 600 individuals in the western world. Individuals with symptomatic IGAD often have deficiency of IgG subclasses or decreased antibody response to carbohydrate antigens such as pneumococcal polysaccharide vaccine. Individuals with IGAD also suffer from recurrent sinopulmonary and gastrointestinal infections and have an increased incidence of autoimmune disorders and of lymphoid and non-lymphoid malignancies. In vitro studies have suggested that some individuals with IGAD have impaired isotype class switching to IgA and others may have a post-switch defect. IGAD and CVID have been known to coexist in families. Some individuals initially present with IGAD1 and then develop CVID. These observations suggest that some cases of IGAD and CVID may have a common etiology. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:Cell cycle regulation; Membrane protein, integral; Receptor, misc.

Chromosomal Location of Human Ortholog: 17p11.2

Cellular Component: integral to plasma membrane; external side of plasma membrane

Molecular Function:protein binding; receptor activity

Biological Process: cell surface receptor linked signal transduction; B cell homeostasis; hemopoietic progenitor cell differentiation; negative regulation of B cell proliferation

Disease: Immunodeficiency, Common Variable, 2; Immunoglobulin A Deficiency 2

NCBI Summary:The protein encoded by this gene is a lymphocyte-specific member of the tumor necrosis factor (TNF) receptor superfamily. It interacts with calcium-modulator and cyclophilin ligand (CAML). The protein induces activation of the transcription factors NFAT, AP1, and NF-kappa-B and plays a crucial role in humoral immunity by interacting with a TNF ligand. This gene is located within the Smith-Magenis syndrome region on chromosome 17. [provided by RefSeq, Jul 2008]
UniProt Code:O14836
NCBI GenInfo Identifier:21264086
NCBI Gene ID:23495
NCBI Accession:O14836.1
UniProt Secondary Accession:O14836,Q32LX4, Q7Z6F5, B2R8B0, B7Z6V8,
UniProt Related Accession:O14836
Molecular Weight:19,865 Da
NCBI Full Name:Tumor necrosis factor receptor superfamily member 13B
NCBI Synonym Full Names:tumor necrosis factor receptor superfamily, member 13B
NCBI Official Symbol:TNFRSF13B  
NCBI Official Synonym Symbols:CVID; RYZN; TACI; CD267; CVID2; IGAD2; TNFRSF14B  
NCBI Protein Information:tumor necrosis factor receptor superfamily member 13B; tumor necrosis factor receptor 13B; transmembrane activator and CAML interactor
UniProt Protein Name:Tumor necrosis factor receptor superfamily member 13B
UniProt Synonym Protein Names:Transmembrane activator and CAML interactor; CD_antigen: CD267
UniProt Gene Name:TNFRSF13B  
UniProt Entry Name:TR13B_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol
SerumIf using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal FluidCollect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatantCollect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysatesSolubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenatesThe preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysatesRinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast MilkCollect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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