Human BAFF/CD257 (B-Cell Activating Factor) ELISA Kit (HUES01296)

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HUES01296
€539

Description

ELISA Kit Technical ManualMSDS

Human BAFF/CD257 (B-Cell Activating Factor) 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 BAFF/CD257 . 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 BAFF/CD257 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 BAFF/CD257, 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 BAFF/CD257. The concentration of Human BAFF/CD257 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 Range62.50-4000 pg/mL
Sensitivity37.50 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human BAFF/CD257 in samples. No significant cross-reactivity or interference between Human BAFF/CD257 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
40002.368
2.412
2.392.333
20001.55
1.586
1.5681.511
10000.866
0.86
0.8630.806
5000.482
0.484
0.4830.426
2500.256
0.24
0.2480.191
1250.155
0.149
0.1520.095
62.500.103
0.109
0.1060.049
00.048
0.066
0.057--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human BAFF/CD257 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human BAFF/CD257 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)185.79600.161343.28173.82543.691425.24
Standard deviation11.6535.4754.1311.9428.7145.04
C V (%)6.275.914.036.875.283.16

Recovery

The recovery of Human BAFF/CD257 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)87-10194
EDTA plasma (n=5)86-10091
Cell culture media (n=5)90-10596

Linearity

Samples were spiked with high concentrations of Human BAFF/CD257 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 (%)92-10595-10891-103
Average (%)10010197
1:4Range (%)91-10886-9987-102
Average (%)999394
1:8Range (%)86-9985-9785-100
Average (%)939091
1:16Range (%)91-10686-10085-96
Average (%)989290

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 BAFF/CD257 (B-Cell Activating Factor) ELISA Kit (HUES01296) 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 BAFF/CD257 (B-Cell Activating Factor) ELISA Kit (HUES01296) Protein Information

UniProt Protein Function:BAFF: Cytokine that binds to TNFRSF13B/TACI and TNFRSF17/BCMA. TNFSF13/APRIL binds to the same 2 receptors. Together, they form a 2 ligands -2 receptors pathway involved in the stimulation of B- and T-cell function and the regulation of humoral immunity. A third B-cell specific BAFF-receptor (BAFFR/BR3) promotes the survival of mature B-cells and the B-cell response. Belongs to the tumor necrosis factor family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Apoptosis; Membrane protein, integral; Cytokine; Cell cycle regulation

Chromosomal Location of Human Ortholog: 13q32-q34

Cellular Component: extracellular space; perinuclear region of cytoplasm; cytoplasm; plasma membrane; integral to membrane

Molecular Function:protein binding; cytokine activity; tumor necrosis factor receptor binding; receptor binding

Biological Process: cell proliferation; B cell costimulation; B cell homeostasis; immunoglobulin secretion; T cell costimulation; positive regulation of cell proliferation; positive regulation of germinal center formation; positive regulation of B cell proliferation; immune response; positive regulation of T cell proliferation; signal transduction

NCBI Summary:The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This cytokine is a ligand for receptors TNFRSF13B/TACI, TNFRSF17/BCMA, and TNFRSF13C/BAFFR. This cytokine is expressed in B cell lineage cells, and acts as a potent B cell activator. It has been also shown to play an important role in the proliferation and differentiation of B cells. Alternatively spliced transcript variants encoding distinct isoforms have been identified. [provided by RefSeq, Mar 2011]
UniProt Code:Q9Y275
NCBI GenInfo Identifier:13124573
NCBI Gene ID:10673
NCBI Accession:Q9Y275.1
UniProt Secondary Accession:Q9Y275,Q6FHD6, Q7Z5J2, E0ADT7,
UniProt Related Accession:Q9Y275
Molecular Weight:17,578 Da
NCBI Full Name:Tumor necrosis factor ligand superfamily member 13B
NCBI Synonym Full Names:tumor necrosis factor (ligand) superfamily, member 13b
NCBI Official Symbol:TNFSF13B  
NCBI Official Synonym Symbols:DTL; BAFF; BLYS; CD257; TALL1; THANK; ZTNF4; TALL-1; TNFSF20  
NCBI Protein Information:tumor necrosis factor ligand superfamily member 13B; delta BAFF; Delta4 BAFF; B-lymphocyte stimulator; B-cell-activating factor; ApoL related ligand TALL-1; TNF homolog that activates apoptosis; dendritic cell-derived TNF-like molecule; tumor necrosis factor-like protein ZTNF4; TNF and ApoL-related leukocyte expressed ligand 1; tumor necrosis factor (ligand) superfamily, member 20
UniProt Protein Name:Tumor necrosis factor ligand superfamily member 13B
UniProt Synonym Protein Names:B lymphocyte stimulator; BLyS; B-cell-activating factor; BAFF; Dendritic cell-derived TNF-like molecule; TNF- and APOL-related leukocyte expressed ligand 1; TALL-1; CD_antigen: CD257Cleaved into the following 2 chains:Tumor necrosis factor ligand superfamily member 13b, membrane form; Tumor necrosis factor ligand superfamily member 13b, soluble form
UniProt Gene Name:TNFSF13B  
UniProt Entry Name:TN13B_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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