MOPC315.BM myeloma load was determined by measuring myeloma specific anti-DNP IgA in serum by ELISA. Briefly: 96 well plates were coated with 10 µg/ml DNP-BSA/PBS (1 h/RT). After washing the plates three times with PBS unspecific binding was blocked (0,5% BSA/PBS; 1 h/RT). Subsequently, sera were incubated in serial dilutions for 60 min. Then, detection antibody biotinylated goat anti-mouse IgA was added (1 µg/ml) (SouthernBiotech, Birmingham, USA), and later a 3000-fold dilution of streptavidin coupled alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany) and finally ALP (Roche Diagnostics GmbH, Mannheim, Germany). In-between incubation steps, plates were washed three to five times. Readout was performed on a FLUOstar Omega Microplate Reader (BMG LABTECH GmbH, Ortenberg, Germany); ODs were analyzed by a (5-PL) Non-Linear Regression Curve-Fitting Model.
Streptavidin coupled alkaline phosphatase
Manufactured by Roche
Sourced in United States, United Kingdom
Streptavidin-coupled alkaline phosphatase is a conjugate of the protein streptavidin and the enzyme alkaline phosphatase. Streptavidin has a high affinity for the biotin molecule, allowing this conjugate to be used as a detection reagent in various biotechnological applications.
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3 protocols using streptavidin coupled alkaline phosphatase
1
Myeloma Load Quantification by Anti-DNP IgA ELISA
2
Collagen VII-specific Autoantibody Induction
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Epidermolysis bullosa acquisita was induced by means of subcutaneous immunization and scored, as previously described. 42 Autoreactive plasma cells were identified by means of flow cytometry, as previously described. 42 For more information, see the Methods section in this article's Online Repository at www.jacionline.org. Collagen VII (amino acids 757-967) was produced, as recently described. 42 Collagen-specific serum antibodies were quantified by means of ELISA. Plates were coated with 500 ng of collagen VII. After blocking, wells were incubated with a 150-fold dilution of the serum samples for 60 minutes. Detection was performed with biotinylated goat anti-mouse IgG antibody (SouthernBiotech, Birmingham, Ala), followed by streptavidin-coupled alkaline phosphatase (Roche Diagnostics, Mannheim, Germany) and ALP (Roche Diagnostics GmbH). Polyclonal plasmacytosis was induced by means of intraperitoneal injection with 200 mL of goat-anti mouse IgD. Some groups received anti-IL-10 receptor (clone IBI.3, a generous gift from DNAX, Palo Alto, Calif).
3
Quantifying Myeloma-Specific IgA Antibodies
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As previously described, 43 murine myeloma was induced by means of intravenous injections of MOPC315.BM cells (5 3 10 5 cells) stably transfected with eGFP. MOPC315.BM myeloma-specific anti-DNP IgA antibodies were quantified by means of ELISA. Briefly, plates were coated with 10 mg/mL DNP-BSA/PBS (1 hour at room temperature). Nonspecific binding was blocked with 1 mg/mL BSA/PBS. Subsequently, sera were incubated for 1 hour at room temperature. Detection was done with biotinylated goat anti mouse IgA (SouthernBiotech), followed by streptavidin-coupled alkaline phosphatase (Roche Diagnostics) and ALP (Roche Diagnostics).
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