Mouse fecal IgA titers were measured using the Mouse IgA ELISA Quantitation Set (Bethyl Laboratories) following the manufacturer’s instructions. Mouse fecal albumin was measured using the Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories) following the manufacturer’s instructions. For both ELISAs, fecal samples were diluted 1/100 or 1/1,000, and concentration was determined based on a standard curve. For measurement of C. rodentium–specific antibodies in serum or fecal supernatants by ELISA, 10 µg/ml C. rodentium antigen was coated on 96-well plates, and sera were incubated in doubling dilutions. Antigen-specific IgG and antigen-specific IgA were detected using an anti-mouse IgG-HRP antibody (BD Biosciences) and an anti-mouse IgA-HRP (Bethyl Laboratories). For measurement of mouse IL-4 in cell culture, a capture anti-mouse IL-4 antibody (clone 11B11; donated by A. MacDonald, University of Manchester, Manchester, UK), recombinant mouse IL-4 (BioLegend) and a detection biotinylated anti-mouse IL-4 antibody (clone 24G2; BioLegend) were used. The mouse IL17A ELISA kit (Invitrogen) were used to detect cytokines in cells stimulated with C. rodentium antigen. Plates were developed with TMB peroxidase substrate (BD Biosciences), and optical densities were measured using a plate spectrophotometer.
Mouse iga elisa quantitation set
Manufactured by Fortis Life Sciences
Sourced in United States
The Mouse IgA ELISA Quantitation Set is a laboratory equipment used for the quantitative determination of mouse immunoglobulin A (IgA) levels in biological samples. It is designed to provide a sensitive and accurate method for measuring mouse IgA concentrations.
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Lab products found in correlation
Mouse igg elisa quantitation set, by Fortis Life Sciences (3 mentions)
Maxisorp nunc immuno plate, by Thermo Fisher Scientific (2 mentions)
Pierce coomassie plus bradford assay kit, by Thermo Fisher Scientific (2 mentions)
Tmb substrate, by BD (2 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (2 mentions)
Anti mouse iga hrp, by Fortis Life Sciences (1 mentions)
Recombinant mouse il 4, by BioLegend (1 mentions)
Tmb peroxidase substrate, by BD (1 mentions)
Mouse albumin elisa quantitation set, by Fortis Life Sciences (1 mentions)
Mouse il 17a elisa kit, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg hrp antibody, by BD (1 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (1 mentions)
Topo ta cloning kit for sequencing, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Merck Group (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
1 step ultra tmb elisa, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Fecal mucin assay kit, by Cosmo Bio (1 mentions)
20 protocols using mouse iga elisa quantitation set
1
Measuring Murine IgA, Albumin, and Cytokines
2
Quantification of Intestinal IgA
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The quantification of intestinal IgA was performed using Cosmo Bio Co., Ltd. (Tokyo, Japan). Intestinal IgA content was quantified through ELISA using a mouse IgA ELISA Quantitation Set (Bethyl Laboratories, Inc., Montgomery, TX, USA). Approximately 50 mg of feces was used for IgA analysis, and the values were expressed as values per feces weight.
3
IgA Sequencing and Quantification
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IgA sequencing was performed as previously described (Lindner et al, 2012 ). Briefly, PP were harvested and kept in RNAlater (Sigma‐Aldrich) until processing. PP were homogenized using a TissueLyser II (20 Hz, 2 min; Qiagen), and RNA was extracted using RNeasy Mini Kit (Qiagen). cDNA synthesis was performed with iScript Select cDNA Synthesis Kit (Bio‐Rad) using a mix of three primers to enrich in IgA transcripts (Lindner et al, 2012 ), and IgA heavy chain was amplified with Phusion High‐Fidelity DNA Polymerase (New England Biolabs). IgA amplicons were cloned using TOPO TA Cloning Kit for Sequencing (Invitrogen), and DNA sequencing was performed by Eurofins MWG Operon (Germany). Sequences were subjected to quality control (Wu et al, 2010 ) before submitting to IMGT/HighV‐QUEST (www.imgt.org/ ) for identification of IGHV genes.
For faecal IgA detection, fresh stool pellets were homogenized in PBS (100 μl/10 mg faeces) and spun at 400 × g for 5 min to remove large particles. The supernatant was collected and spun 10 min at 8,000 × g to separate bacteria‐coating IgA (pellet) from soluble IgA (faecal supernatant) (Kawamoto et al,2012 ). Free IgA levels were detected in the faecal supernatant after centrifugation at 21,000 × g using Mouse IgA ELISA Quantitation Set (Bethyl). Serum IgA and IgG levels were quantified using IgA and IgG ELISA Quantitation Sets (Bethyl), respectively.
For faecal IgA detection, fresh stool pellets were homogenized in PBS (100 μl/10 mg faeces) and spun at 400 × g for 5 min to remove large particles. The supernatant was collected and spun 10 min at 8,000 × g to separate bacteria‐coating IgA (pellet) from soluble IgA (faecal supernatant) (Kawamoto et al,
4
Oral Immunization with Recombinant Salmonella Expressing TT
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Recombinant Salmonella (rSalmonella)–ToxC (ΔaroA, ΔaroD) and TT were kindly provided by the BIKEN Foundation (Osaka, Japan). Sox8−/− or littermate control mice were orally immunized with 5 × 107 CFU of rSalmonella-ToxC. TT-specific IgA and IgG in feces and serum were measured by ELISA. Flat-bottomed, 96-well MaxiSorp Nunc-Immuno plates were coated overnight with 500 ng/well of TT. Plates were blocked with 2% BSA in PBS, and optically diluted fecal extracts and sera were added into the plate wells. The Mouse IgA ELISA Quantitation Set (Bethyl Laboratories) and Mouse IgG ELISA Quantitation Set (Bethyl Laboratories) were used for antibody detection. To produce HRP signals, a 1-Step Ultra TMB-ELISA was used.
5
Quantifying IgA Levels in Plasma Samples
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The plasma samples were subjected to IgA ELISA using a mouse IgA ELISA Quantitation Set (Bethyl Laboratories, Inc.; Montgomery, TX, USA), following the manufacturer’s instructions.
6
Quantitative IgA Measurement in Intestine
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IgA concentrations in small intestine fluid or cell culture supernatants were determined using a sandwich ELISA kit (Mouse IgA ELISA Quantitation Set; Bethyl Laboratories, Montgomery, TX, USA). The kit was used according to the manufacturer’s instructions. In brief, 96-well immunoplates were coated with antibodies. After washing and blocking the immunoplates, the samples and standards were added, followed by incubation. After washing, a horseradish peroxidase-labeled monoclonal antibody was added, and the plates were incubated at room temperature for 1 hr. The plates were washed again and incubated with the substrate 3,3′,5,5′-tetramethylbenzidine (Moss Inc., Elk Grove Village, IL, USA). A stop solution was added, and absorbance was measured at 450 nm.
7
Salmonella Vaccine Immunogenicity in Mice
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rSalmonella-ToxC (ΔaroA, ΔaroD) and TT were kindly provided by the BIKEN Foundation (Osaka, Japan). Opg−/− mice and co-housed WT mice were orally or intraperitoneally infected with 5 × 107 or 5 × 105 colony-forming units (c.f.u.) of rSalmonella-ToxC1 (link),25 (link). TT-specific IgA and IgG in feces and serum were measured by ELISA. Flat-bottomed, 96-well Maxisorp Nunc-immuno plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight with 500 ng/well of TT. Plates were blocked with 2% BSA in PBS, and optically diluted fecal extracts and sera were added into the plate wells. The Mouse IgA ELISA Quantitation Set (Bethyl Laboratories, Inc., Montgomery, TX, USA.) and Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, Inc.) were used for antibody detection. To produce horse radish peroxidase signals, a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific) was used.
8
Mouse Fecal IgA Quantification
9
Murine Stool IgA and IgG ELISA
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Murine stool (50 mg) was incubated in 500 μL of PBS at room temperature for 10 minutes, followed by vortexing for 5 minutes and bead-beating for 2 minutes. The sample supernatants were collected after centrifugation.
ELISA was performed using Mouse IgA ELISA Quantitation Set or Mouse IgG ELISA Quantitation Set (Bethyl Laboratories) according to the manufacturer’s protocol with minor modifications. MaxiSorp ELISA plates (Thermo Fisher Scientific) for total IgA measurement were pre-coated with 100 μL of diluted purified IgA antibody in 0.1M carbonate buffer (10 μl/mL). For OVA-specific IgA or IgG measurement, MaxiSorp ELISA plate were pre-coated with 5μg of OVA protein in 0.1M carbonate buffer. After overnight incubation at 4°C, plates were washed with PBS and incubated with PBS containing 1% BSA for 1 hour at room temperature. After washing, 100 μL of stool supernatant or serum were plated and incubated for 1 hour. Plates were washed again and 100 μL of anti-mouse IgA-HRP or anti-mouse IgG HRP (1:40 000 dilutions) was added before a 1-hour of incubation. TMB substrate (BD Pharmingen) was added to each well, followed by supplementation of 100 μL of 1M H2SO4 as a stop solution. Absorbance (450 nm) was measured using an ELISA reader.
ELISA was performed using Mouse IgA ELISA Quantitation Set or Mouse IgG ELISA Quantitation Set (Bethyl Laboratories) according to the manufacturer’s protocol with minor modifications. MaxiSorp ELISA plates (Thermo Fisher Scientific) for total IgA measurement were pre-coated with 100 μL of diluted purified IgA antibody in 0.1M carbonate buffer (10 μl/mL). For OVA-specific IgA or IgG measurement, MaxiSorp ELISA plate were pre-coated with 5μg of OVA protein in 0.1M carbonate buffer. After overnight incubation at 4°C, plates were washed with PBS and incubated with PBS containing 1% BSA for 1 hour at room temperature. After washing, 100 μL of stool supernatant or serum were plated and incubated for 1 hour. Plates were washed again and 100 μL of anti-mouse IgA-HRP or anti-mouse IgG HRP (1:40 000 dilutions) was added before a 1-hour of incubation. TMB substrate (BD Pharmingen) was added to each well, followed by supplementation of 100 μL of 1M H2SO4 as a stop solution. Absorbance (450 nm) was measured using an ELISA reader.
10
Quantifying Fecal Immunoglobulin A
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The concentration of fecal IgA in the aforementioned fecal supernatant was measured using a Mouse IgA ELISA Quantitation Set (Bethyl) according to the manufacturer’s instructions. The
absorbance of each well was read at 450 nm using a microplate reader (Bio-Rad).
absorbance of each well was read at 450 nm using a microplate reader (Bio-Rad).
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