The recombinant human ASM (2 µg ml−1, Genscript) in ELISA coating buffer (Abcam, ab210899) was coated onto MaxiSorp ELISA plates (Thermo Fisher Scientific) overnight at 4 °C. Wells were incubated with blocking buffer (Abcam, ab210904) for 2 h at RT, washed with wash buffer (Abcam, ab206977). ASM antibody (23A12C23) or diluted plasma in blocking buffer were incubated for 2 h at RT. Following washing with wash buffer, Anti-mouse IgG/Biotin (Sigma-Aldrich, B7264) in blocking buffer was added and incubated for 1 h at RT. After standard washing, wells were further incubated with streptavidin HRP (Abcam, ab210901) in blocking buffer for 1 h at RT and results were developed with TMB substrate (Abcam, ab210902). Absorbance was measured at 450 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The KD was analyzed using the SigmaPlot 13.0 software. Each experiment was performed in triplicate. The ASM titers were defined as the dilution factor referring to 50 % of the maximal optical density (ODmax/2).
Anti mouse igg biotin
Manufactured by Merck Group
Sourced in United States
Anti-mouse IgG-biotin is a laboratory reagent used for the detection and quantification of mouse immunoglobulin G (IgG) in various immunoassay and research applications. It is a conjugate of anti-mouse IgG antibodies and the small molecule biotin. The biotin label allows for signal amplification and sensitive detection when used in conjunction with streptavidin-based detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Immunosorbent plates, by Thermo Fisher Scientific (2 mentions)
Mouse albumin elisa kit, by Fortis Life Sciences (2 mentions)
Thymic dna, by Merck Group (2 mentions)
Streptavidin hrp, by BD (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Ab210901, by Abcam (1 mentions)
B7264, by Merck Group (1 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Varioskan lux multimode microplate reader, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Welgene (1 mentions)
3 protocols using anti mouse igg biotin
1
ASM Antibody Quantification by ELISA
2
Anti-dsDNA IgG Antibody Quantification
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Titers of anti-dsDNA IgG Ab were measured by ELISA, as described previously [35 (link)]. In brief, sera were diluted 1/10 000 to 1/50 000 in PBS and applied to immunosorbent plates (Nunc) precoated with 5 μg/ml poly-L-lysine and 5 μg/ml thymic DNA (Sigma Aldrich). The serum containing the highest titer of anti-dsDNA IgG was serially diluted and used as standard. The plates were incubated with anti-mouse IgG-biotin (Sigma Aldrich) and streptavidin-HRP (BD Biosciences). The concentration of albumin in urine was measured by quantitative ELISA using a mouse albumin ELISA kit (Bethyl), according to the manufacturer’s instructions. The concentration of serum B-cell activating factor of the TNF family (BAFF) was quantitated using a BAFF/BLys/TNFSF13B ELISA Kit (R&D Systems).
3
Serological Biomarkers in Murine Lupus
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Sera were collected from NZB/W mice at 2-to 4-week intervals to measure the levels of Abs by ELISA, as described previously (16). In brief, to measure the levels of anti-dsDNA IgG, sera were diluted 1:5,000 in PBS (WelGene) and applied to immunosorbent plates (Nunc, Rochester, NY, USA) precoated with 5 µg/ml poly-Llysine and 5 µg/ml thymic DNA (Sigma-Aldrich, St. Louis, MO, USA). A serum containing the highest titer of anti-dsDNA IgG was serially diluted and used as standard. The plates were incubated with anti-mouse IgG-biotin (Sigma-Aldrich) and streptavidin-HRP (BD Biosciences, San Jose, CA, USA). To measure the levels of anti-SSA/ Ro and anti-SSB/La Abs, sera were diluted 1:1,000 and assayed according to the manufacturer's instructions (Alpha Diagnostic, San Antonio, TX, USA).
To evaluate proteinuria, the concentration of albumin in urine was measured by quantitative ELISA using a mouse albumin ELISA kit (Bethyl, Montgomery, TX, USA), according to the manufacturer's instructions. Mice were scored as nephritic if the concentration of urinary albumin exceeded 100 mg/dl (5) .
To evaluate proteinuria, the concentration of albumin in urine was measured by quantitative ELISA using a mouse albumin ELISA kit (Bethyl, Montgomery, TX, USA), according to the manufacturer's instructions. Mice were scored as nephritic if the concentration of urinary albumin exceeded 100 mg/dl (5) .
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