ELISAs were performed as described (Bates et al., 2021b (link)). Plates were coated overnight at 4°C with 1 mg/mL recombinant SARS-CoV-2 spike receptor binding domain (RBD) protein (Bates et al., 2021c (link)) (BEI Resources NR-52309) or recombinant SARS-CoV-2 nucleocapsid (N) protein (BEI Resources NR-53797). Serum dilutions (6 x 3-fold for RBD, 6 x 4-fold for N) in duplicate were prepared in 5% milk powder, 0.05% Tween 20, in phosphate buffered saline (PBS), starting at 1:1600 (pan-Ig), 1:50 (IgA), 1:200 (IgG). The secondary antibodies used were pan-Ig (1:10,000 anti-human GOXHU IgG/A/M-HRP, A18847 Invitrogen), IgA (1:3,000 anti-human IgA-HRP, 411,002 Biolegend), and IgG (1:3,000 anti-human IgG-HRP 555788, BD Biosciences). Plates were developed with o-phenylenediamine (OPD) (ThermoScientific). Absorbance at 492nm was measured on a CLARIOstar plate reader and normalized by subtracting the average of negative control wells and dividing by the highest concentration from a positive control dilution series. ELISA EC50 values were calculated by fitting normalized A492 as described (Bates et al., 2021b (link)). The limit of detection (LOD) was defined by the lowest dilution tested for RBD and half of the lowest dilution for N. Values below the LOD were set to LOD – 1.
Anti human iga hrp
Manufactured by BioLegend
Anti-human IgA-HRP is a reagent that includes horseradish peroxidase (HRP) conjugated to anti-human IgA antibodies. It is designed for the detection and quantification of human IgA in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti human goxhu igg a m hrp, by Thermo Fisher Scientific (2 mentions)
Anti human igg hrp 555788, by BD (2 mentions)
O phenylenediamine opd, by Thermo Fisher Scientific (2 mentions)
Anti human igg hrp, by Merck Group (2 mentions)
Anti human igm hrp, by LGC (2 mentions)
Nr 52309, by BEI Resources (1 mentions)
Synergy ht plate reader, by Agilent Technologies (1 mentions)
Prism 8, by GraphPad (1 mentions)
4 protocols using anti human iga hrp
1
SARS-CoV-2 Antibody Detection by ELISA
2
DENV-Specific Antibody Detection ELISA
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DENV-reactive serum IgM/IgG/IgA levels were assessed using a modified 4G2 DENV capture ELISA protocol. 96 well NUNC MaxSorb flat-bottom plates were coated with 2 μg/ml flavivirus group-reactive mouse monoclonal antibody 4G2 (Envigo Bioproducts, Inc.) diluted in borate saline buffer. Plates were washed and blocked with 0.25% BSA +1% Normal Goat Serum in PBS after overnight incubation. DENV-1 (strain Nauru/West Pac/1974) was captured for 2 h in separate wells, followed by extensive washing. Serially diluted serum samples were plated in duplicate and incubated for 1 h at RT on the captured virus. DENV-specific IgM/IgG/IgA levels were quantified using anti-human IgM HRP (Seracare, 5220-0328), anti-human IgG HRP (Sigma-Aldrich, SAB3701362), and anti-human IgA HRP (Biolegend, 411,002). Secondary antibody binding was quantified using the TMB Microwell Peroxidase Substrate System (KPL, cat. #50-76-00) and Synergy HT plate reader. Single-dilution ELISA was performed using a 1/500 serum dilution for IgM and IgA assays, and a 1/20,000 serum dilution for IgG assays.
3
SARS-CoV-2 Antibody Quantification ELISA
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ELISAs were performed as described (Bates et al., 2021b (link)). Plates were coated overnight at 4°C with 1 mg/mL recombinant SARS-CoV-2 spike receptor binding domain (RBD) protein (Bates et al., 2021c ) (BEI Resources NR-52309) or recombinant SARS-CoV-2 nucleocapsid (N) protein (BEI Resources NR-53797). Serum dilutions (6 × 3-fold for RBD, 6 × 4-fold for N) in duplicate were prepared in 5% milk powder, 0.05% Tween-20, in phosphate buffered saline (PBS), starting at 1:1600 (pan-Ig), 1:50 (IgA), 1:200 (IgG). The secondary antibodies used were pan-Ig (1:10,000 anti-human GOXHU IgG/A/M-HRP, A18847 Invitrogen), IgA (1:3,000 anti-human IgA-HRP, 411002 Biolegend), and IgG (1:3,000 anti-human IgG-HRP 555788, BD Biosciences). Plates were developed with o-phenylenediamine (OPD) (ThermoScientific). Absorbance at 492nm was measured on a CLARIOstar plate reader and normalized by subtracting the average of negative control wells and dividing by the highest concentration from a positive control dilution series. ELISA EC50 values were calculated by fitting normalized A492 as described (Bates et al., 2021b (link)). The limit of detection (LOD) was defined by the lowest dilution tested for RBD and half of the lowest dilution for N. Values below the LOD were set to LOD – 1.
4
Quantifying DENV-Reactive Antibodies
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Monoclonal antibody and plasma DENV-reactivity was assessed using a 4G2 DENV capture ELISA protocol. In short, 96 well NUNC MaxSorb flat-bottom plates were coated with 2 μg/ml flavivirus group-reactive mouse monoclonal antibody 4G2 (Envigo Bioproducts, Inc.) diluted in borate saline buffer. Plates were washed and blocked with 0.25% BSA + 1% Normal Goat Serum in PBS after overnight incubation. DENV-3 (strain CH53489) diluted in blocking buffer was captured for 2 hr, followed by extensive washing with PBS + 0.1% Tween 20. Serially diluted monoclonal antibody or plasma samples were incubated for 1 hr at RT on the captured virus, and DENV-specific antibody binding quantified using anti-human IgG HRP (Sigma-Aldrich, SAB3701362), anti-human IgA HRP (Biolegend, 411,002), or anti-human IgM HRP (SeraCare, 5220-0328). Secondary antibody binding was quantified using the TMB Microwell Peroxidase Substrate System (KPL, cat. #50-76-00) and Synergy HT plate reader (BioTek, Winooski, VT). Monoclonal antibody binding data were analyzed by nonlinear regression (One site total binding) to determine EC50 titers in GraphPad Prism 8 (GraphPad Software, La Jolla, CA). End-point titers of DENV-reactive plasma samples were determined as the reciprocal of the final dilution at which the optical density (OD) was greater than 2× of a control flavivirus naïve serum.
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