Human BAFFR (B Cell Activating Factor Receptor) CLIA Kit (HUES00377)

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HUES00377
€599

Description

ELISA Kit Technical ManualMSDS

Human BAFFR (B Cell Activating Factor Receptor) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human BAFFR . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human BAFFR 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 BAFFR, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human BAFFR. The concentration of Human BAFFR in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

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

Typical Data

As the RLU 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)
RLU Average Corrected
200050410
61032
5572155693
100021886
26062
2397423946
50012017
10047
1103211004
2504959
5629
52945266
1252680
2536
26082580
62.51379
1243
13111283
31.25636
712
674646
027
29
28--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)97.02308.33944.42105.70315.38886.97
Standard deviation7.9632.3773.4812.2832.3979.47
C V (%)8.2010.507.7811.6210.278.96

Recovery

The recovery of Human BAFFR 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)99-113105
EDTA plasma (n=5)88-10394
Cell culture media (n=5)89-10495

Linearity

Samples were spiked with high concentrations of Human BAFFR 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 (%)94-11192-10686-98
Average (%)1019791
1:4Range (%)87-10190-105100-117
Average (%)9496109
1:8Range (%)97-11391-10591-105
Average (%)1039796
1:16Range (%)91-10685-97100-115
Average (%)9892108

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 CLIA 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 Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human BAFFR (B Cell Activating Factor Receptor) CLIA Kit (HUES00377) 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Human BAFFR (B Cell Activating Factor Receptor) CLIA Kit (HUES00377) Protein Information

UniProt Protein Function:BAFF-R: B-cell receptor specific for TNFSF13B/TALL1/BAFF/BLyS. Promotes the survival of mature B-cells and the B-cell response. Defects in TNFRSF13C are the cause of immunodeficiency common variable type 4 (CVID4); also called antibody deficiency due to BAFFR defect. CVID4 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. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Cell cycle regulation; Receptor, misc.; Cell surface

Chromosomal Location of Human Ortholog: 22q13.1-q13.31

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

Biological Process: B cell costimulation; B cell homeostasis; T cell costimulation; positive regulation of B cell proliferation; positive regulation of germinal center formation; positive regulation of T cell proliferation; positive regulation of interferon-gamma biosynthetic process

Disease: Immunodeficiency, Common Variable, 2; Immunodeficiency, Common Variable, 4

NCBI Summary:B cell-activating factor (BAFF) enhances B-cell survival in vitro and is a regulator of the peripheral B-cell population. Overexpression of Baff in mice results in mature B-cell hyperplasia and symptoms of systemic lupus erythematosus (SLE). Also, some SLE patients have increased levels of BAFF in serum. Therefore, it has been proposed that abnormally high levels of BAFF may contribute to the pathogenesis of autoimmune diseases by enhancing the survival of autoreactive B cells. The protein encoded by this gene is a receptor for BAFF and is a type III transmembrane protein containing a single extracellular cysteine-rich domain. It is thought that this receptor is the principal receptor required for BAFF-mediated mature B-cell survival. [provided by RefSeq, Jul 2008]
UniProt Code:Q96RJ3
NCBI GenInfo Identifier:21264093
NCBI Gene ID:115650
NCBI Accession:Q96RJ3.1
UniProt Related Accession:Q96RJ3
Molecular Weight:18,935 Da
NCBI Full Name:Tumor necrosis factor receptor superfamily member 13C
NCBI Synonym Full Names:tumor necrosis factor receptor superfamily, member 13C
NCBI Official Symbol:TNFRSF13C  
NCBI Official Synonym Symbols:BAFFR; CD268; CVID4; BAFF-R; BROMIX; prolixin  
NCBI Protein Information:tumor necrosis factor receptor superfamily member 13C; BAFF receptor; BLyS receptor 3; B cell-activating factor receptor; B-cell-activating factor receptor
UniProt Protein Name:Tumor necrosis factor receptor superfamily member 13C
UniProt Synonym Protein Names:B-cell-activating factor receptor; BAFF receptor; BAFF-R; BLyS receptor 3; CD_antigen: CD268
UniProt Gene Name:TNFRSF13C  
UniProt Entry Name:TR13C_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
CLIA
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