Serum collected from mice was aliquoted before storage at -70°C to ensure only one freeze-thaw cycle for all samples. The night before our ELISAs were performed, Immulon Two ELISA plates were coated with 5μg/ml of NP-BSA at either a 2:1 or 28:1 NP:BSA ratio, hereafter referred to as NP(2)-BSA or NP(28)-BSA (purchased from LGC Biosearch Technologies). To detect low-affinity antibodies, NP(28)-BSA was used, and to detect high-affinity antibodies, NP(2)-BSA was used. The next day, plates were washed and blocked with 0.5% BSA in PBS for 1 hr at RT. Plates were washed again and serum was added for 1 hr at RT. After another washing, an anti-mouse IgG-HRP antibody (Southern Biotech, cat. no. 1030-05) was added at 1:5,000 for 45 min at RT. Final washes were performed, One-Step Ultra TMB Substrate was added to each well for 2 minutes before adding H2SO4 and measuring A450. Endpoint titers were determined as previously described38 (link). Control serum was generated from Tcra−/− mice that had received LLO cell transfers but were immunized with the unconjugated protein (LLOLT-N) instead of NP-LLOLT-N.
Anti mouse igg hrp antibody
Manufactured by Southern Biotech
The Anti-mouse IgG-HRP antibody is a laboratory reagent used in immunoassays, such as ELISA, to detect and quantify mouse immunoglobulin G (IgG) in biological samples. The antibody is conjugated with horseradish peroxidase (HRP), an enzyme that catalyzes a colorimetric reaction, allowing for the visualization and measurement of target mouse IgG.
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2 protocols using anti mouse igg hrp antibody
1
NP-BSA ELISA Quantification of Antibody Affinity
2
NP-BSA ELISA Quantification of Antibody Affinity
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Serum collected from mice was aliquoted before storage at -70°C to ensure only one freeze-thaw cycle for all samples. The night before our ELISAs were performed, Immulon Two ELISA plates were coated with 5μg/ml of NP-BSA at either a 2:1 or 28:1 NP:BSA ratio, hereafter referred to as NP(2)-BSA or NP(28)-BSA (purchased from LGC Biosearch Technologies). To detect low-affinity antibodies, NP(28)-BSA was used, and to detect high-affinity antibodies, NP(2)-BSA was used. The next day, plates were washed and blocked with 0.5% BSA in PBS for 1 hr at RT. Plates were washed again and serum was added for 1 hr at RT. After another washing, an anti-mouse IgG-HRP antibody (Southern Biotech, cat. no. 1030-05) was added at 1:5,000 for 45 min at RT. Final washes were performed, One-Step Ultra TMB Substrate was added to each well for 2 minutes before adding H2SO4 and measuring A450. Endpoint titers were determined as previously described38 (link). Control serum was generated from Tcra−/− mice that had received LLO cell transfers but were immunized with the unconjugated protein (LLOLT-N) instead of NP-LLOLT-N.
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