BAFF soluble (mouse), detection set



APO-54N-013-KI01	4x96 wells	1'050.00 USD

Figure: left) Serum levels of BAFF (mouse) in wild-type (red) and lupus-prone mice (green) measured with the matched pair MAbs 5A8 and 1C9. right) Standard curve: coated with 5A8 (5µg/ml) and detected with 1C9 (Biotin) followed by SAP-HRP.

Standard Curve Method: A 96-well ELISA plate was coated O/N at 4°C with 100µl/well of a 5µg/ml solution of MAb to BAFF (mouse) (5A8) (Prod. No. ALX-804-158) diluted in 50mM sodium bicarbonate buffer (pH 9.6). After two washes, wells were blocked with 100µl of PBS containing 1% BSA at 37°C for two hours. To reduce interference from rheumatoid factor (RF) in the serum, Ig were cleared by treating serum dilutions with 0.05 volume of protein G-Sepharose (Amersham Biosciences) twice for one hour at 4°C before pelleting the beads and collecting supernatants (precleared sera). A total of 20µl/well serum or rmsBAFF (Prod. No. ALX-522-052) serial dilutions in ELISA buffer (PBS, 0.05% Tween, 1% BSA) were added to the plates. After incubation for one hour at 37°C, wells were washed three times. Then, 100µl/well of a 2µg/ml solution of MAb to BAFF (mouse) (1C9) (Biotin) (Prod. No. ALX-804-157B) in PBS containing 1% BSA was added. After incubation for one hour at 37°C, wells were washed three times and incubated with 100µl/well of HRP-labeled streptavidin (DAKO) for 30 min. The plates were washed four times before the enzymatic reaction was conducted using the tetramethylbenzidine substrate kit (BD PharMingen). The reaction was stopped by adding 2M H₂SO₄, and the OD was measured at 450nm in an ELISA reader.Washing buffer: PBS containing 0.1% Tween 20.

Figure: Standard curve: coated with MAb to BAFF (mouse) (5A8) (Prod. No. ALX-804-158) and detected with MAb to BAFF (mouse) (1C9) (Biotin) (Prod. No. ALX-804-157B) followed by SAP-HRP. Standard Curve Method: A 96-well ELISA plate was coated O/N at RT with 100µl/well of a 5µg/ml solution of MAb to BAFF (mouse) (5A8) (Prod. No. ALX-804-158) diluted in PBS, pH 7.4. After extensive washes, and a blocking step with 100µl of PBS containing 5% FCS for 1 hour at 37°C, the indicated concentrations of rmsBAFF (Prod. No. ALX-522-052) diluted in PBS containing 0.5% FCS were added. After incubation at 37°C for 1 hour, wells were extensively washed. Then 100µl/well of a 2µg/ml solution of MAb to BAFF (mouse) (1C9) (Biotin) (Prod. No. ALX-804-157B) diluted in PBS containing 0.5% FCS were added. After incubation for 1 hour at 37°C, wells were extensively washed. 100µl/well of SAP-HRP (1:500 dilution, S5512 SIGMA) were added for 30 min. to detect bound biotinylated 1C9. After extensive washes, OPD was used as a substrate, and absorbance was measured at 405nm in an ELISA reader.

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Product Specification

Alternative Name:	BLyS, TALL-1, THANK, zTNF4, TNFSF 13B/20, CD257

Sensitivity:	0.3ng/ml (range 0 to 20ng/ml)

Kit/Set Contains:	1 vial Standard (lyophilized) (STD).1 vial Coating Antibody (COAT).1 vial Detection Antibody (DET).

Application:	For the quantitative determination of soluble mouse BAFF from biological fluids (serum and cell culture supernatant).

Short Term Storage:	+4°C

Handling:	Avoid freeze/thaw cycles. For long term storage keep the standard (STD) at -20°C.

Miscellaneous/General:	BAFF (B cell activation factor of the TNF family, also known as BLyS or TALL1) is a cytokine expressed predominantly by cells of the immune system such as neutrophils, monocytes, macrophages, dendritic cells, follicular dendritic cells, activated T cells and some malignant B cells. BAFF binds three distinct receptors (BAFF-R, TACI and BCMA) expressed predominantly on B cells, although activated T cells also express BAFF-R. BAFF is a master regulator of peripheral B cell survival, and also acts in processes such as immunoglobulin isotype switch and B cell co-stimulation. Besides its major role in B cell biology, BAFF co-stimulates activated T cells. Deregulated expression of this membrane-bound protein, which can readily be released in a soluble form, leads to autoimmune disorders in mice. In humans, elevated levels of soluble BAFF have been detected in the serum of patients with various autoimmune diseases.

Background / Technical Information:	UniProt ID Q9WU72: BAFF (mouse)

Product Literature References

Development of autoantibody responses in NC/Nga mice: its prevention by pulverized konjac glucomannan feeding: N. Onishi, et al.; Arch. Dermatol. Res. 300, 95 (2008), Abstract;

A role for B cell-activating factor of the TNF family in chemically induced autoimmunity: Y. Zheng, et al.; J. Immunol. 175, 6163 (2005), Abstract;

BAFF Augments Certain Th1-Associated Inflammatory Responses: A.P. Sutherland, et al.; J. Immunol. 174, 5537 (2005), Abstract; Full Text

Regulation of B-cell survival by BAFF-dependent PKCdelta-mediated nuclear signalling: I. Mecklenbrauker, et al.; Nature 431, 456 (2004), Abstract;

Toll-like Receptor 9-Dependent and -Independent Dendritic Cell Activation by Chromatin-Immunoglobulin G Complexes: M.W. Boule, et al.; J. Exp. Med. 199, 1631 (2004), Abstract;

General Literature References

The BAFF/APRIL system: an important player in systemic rheumatic diseases: F. Mackay, et al.; Curr. Dir. Autoimmun. 8, 243 (2005), Abstract;

TNF deficiency fails to protect BAFF transgenic mice against autoimmunity and reveals a predisposition to B cell lymphoma: M. Batten, et al.; J. Immunol. 172, 812 (2004), Abstract;

The TNF family members BAFF and APRIL: the growing complexity: F. Mackay & C. Ambrose; Cytokine Growth Factor Rev. 14, 311 (2003), Abstract;