In order to validate human IgG binding to different M1 protein regions ELISA was used. The recombinant, full-length M1 protein and the synthetic peptides from ProteoGenix (see above; all 10 μg ml−1) were immobilized on MaxiSorp 96-well ELISA plates (Thermo Fisher Scientific) overnight at 4 °C. The plates were washed three times with 1 × PBST, and blocked with 2% BSA in 1 × PBST for 1 h at 37 °C. The plates were washed again three times with 1 × PBST, and IVIG (4 mg ml−1) was added as a two-fold dilution series. The plates were incubated 1 h at 37 °C, washed three times with 1 × PBST and affinity-purified protein G HRP conjugate (Bio-Rad, catalog number 170-6425) diluted 1:3000 in 1 × PBST was added to the wells. The plates were incubated 1 h at 37 °C, washed three times with 1 × PBST and color developed with 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS; Sigma) for 5 min at RT in the dark, prior to determining the absorbance at 415 nm. The GFP-based peptide was used as a negative control in the assays, and its absorbance values were subtracted from the experimental data prior to analysis in Prism version 8.0.2 (GraphPad). Data analysis used two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Statistical significance levels were set at P < 0.0332, P < 0.0021, P < 0.0002 and P < 0.0001.
Maxisorp 96 well elisa plate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Maxisorp 96-well ELISA plates are a laboratory product designed for enzyme-linked immunosorbent assay (ELISA) experiments. The plates feature a high-binding polystyrene surface optimized for the immobilization of proteins, antibodies, or other biomolecules. The 96-well format allows for the simultaneous processing of multiple samples or standards.
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Lab products found in correlation
Hrp goat anti mouse igg fcγ fragment specific secondary antibody, by Jackson ImmunoResearch (3 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (2 mentions)
Filtermax f5 multi mode microplate reader, by Molecular Devices (2 mentions)
Spark 10m spectrophotometer, by Tecan (2 mentions)
Prism version 8, by GraphPad (1 mentions)
Protein g hrp conjugate, by Bio-Rad (1 mentions)
Carbonate buffer, by Fortis Life Sciences (1 mentions)
Opd substrate, by Merck Group (1 mentions)
Opd peroxidase substrate, by Merck Group (1 mentions)
Anti human igg peroxidase conjugate, by Merck Group (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti human igg fc antibody, by Jackson ImmunoResearch (1 mentions)
Carbonate bicarbonate, by Merck Group (1 mentions)
Tmb stop solution, by LGC (1 mentions)
Hrp rabbit anti human igg secondary antibody, by Agilent Technologies (1 mentions)
Tetramethylbenzidine dihydrochloride, by Merck Group (1 mentions)
Hrp conjugated donkey anti goat, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (1 mentions)
Superblock, by Thermo Fisher Scientific (1 mentions)
19 protocols using maxisorp 96 well elisa plate
1
IgG Binding to M1 Protein Regions
2
Quantifying Antibody Levels in Mouse Serum
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Goat anti-human IgG-Fc fragment antibodies (1 μg) or goat anti-mouse IgG-Fc fragment antibodies (0.2 μg) were diluted in carbonate buffer (pH 9.6) (Bethyl Laboratories, Montgomery, TX) and used to coat MaxiSorp 96-well ELISA plates (Nunc, ThermoFischer scientific, Waltham, MA) overnight at 4 °C. After blocking with PBS with 3% BSA for 1 h at room temperature, plates were washed three times with PBST and incubated with serially diluted wt or KI mouse serum (or human and mouse reference serum as quantification standards), at room temperature for 3 h. After washing, HRP-conjugated anti-human IgG (diluted 1:20,000; Bethyl Laboratories Inc.) or anti-mouse IgG (diluted 1:10,000; Bethyl Laboratories Inc.) were added, and plates were washed with PBST. Absorbance at 492 vs 620 nm was measured after development with the OPD substrate (Sigma Aldrich, Saint-Louis, MO).
3
Quantifying IgG Fab Titers Against M Proteins
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To investigate the plasma IgG titers against the different M proteins in 10 healthy donors (donors 1 to 10), an ELISA was performed. MaxiSorp 96-well ELISA plates (Thermo Fisher Scientific) were coated with the different M proteins (2.5 μg/mL) and left overnight at 4°C. Plasma from the 10 donors was pretreated with the specific IgG-cleaving enzyme IdeS (20 μg/mL) for 30 min at room temperature to generate Fab- and Fc-fragments. SDS-PAGE of IdeS-treated plasma was performed to confirm that IgG was cleaved by the treatment. The ELISA plate was washed three times with PBST and incubated with the IdeS-treated plasma (diluted 1:10) from the 10 donors for 1 h at 37°C, as previously described (26 (link)). The plate was washed, and bound IgG Fab-fragments were detected with horseradish peroxidase (HRP)-labeled anti-Fab (1:1,000) (Abcam; polyclonal). The plate was washed and developed with an ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] substrate (Sigma-Aldrich) before the absorbance at 415 nm was determined.
4
ELISA Screening of Recombinant Proteins
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Microtiter plates (Nunc Maxisorp 96-well ELISA plates) were coated with 100 μl of denatured E.coli-expressed ZNS1 (500 ng/well) for hybridoma screening, or with 100 μl of the indicated concentration of denatured E.coli-expressed NS1 proteins, or native or denatured HEK293-expressed ZNS1 in coating buffer (0.1 M Na2HPO4 buffer pH 9.5) for 18 h at 4°C. Following incubation in blocking buffer (5% skimmed milk in TBS) for 1 h at 37°C, the plates were further incubated with hybridoma supernatants for screening, or with 0.5 μg/ml of the indicated purified mAb for 1 h at RT in blocking buffer. Following four washing steps in TBS Tween-20 0.05%, plates were further incubated for 1 h at RT with HRP goat anti-mouse IgG Fcγ fragment specific secondary antibody (Jackson Immunoresearch) at a 1:6000 dilution. Finally, plates were washed four times in TBS Tween-20 0.05% and after incubation with the substrate [0.3% H2O2, 0.1% 3,3’,5,5’-tetramethylbemzidine (TMB) in 0.1 M citric acid pH 5] for 10 to 30 minutes at RT, the reaction was stopped with 0.2 M H2SO4. The absorbance at 450 nm was measured with a FilterMax F5 Multi-Mode microplate reader (Molecular Devices).
5
Serological Profiling of Recombinant cEDIII Immunogens
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Specific humoral responses in sera of mice immunized with bacterially expressed cEDIII alone or plant and CHO‐derived cEDIII‐PIGS were tested by ELISA. 10 μg/mL of recombinant cEDIII protein was coated on Nunc Maxisorp 96‐well ELISA plates and incubated overnight at 4 °C. After blocking, immunized sera were added in fivefold serial dilutions and incubated for 2 h at 37 °C. The wells were washed with PBS‐T and peroxidase‐conjugated detection antisera [anti‐mouse IgG, anti‐mouse IgG1 (The Binding Site), or anti‐mouse IgG2a‐peroxidase (Bio‐Rad)] were added at 1:1000, and incubated as before. Finally, the ELISA plates were developed by addition of the OPD peroxidase substrate (Sigma). In a further modification of the ELISA protocol, immune sera were preincubated with bacterially expressed cEDIII (10 μg/mL) and were then used to test reactivity in cEDIII‐ or cEDIII‐coated plates.
6
ELISA Validation of Microarray Findings
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To further validate the microarray findings, the peptide selected was then custom synthesized by the company Biomatik (Biomatik Corporation, Kitchener, ON, Canada) for ELISA assay. For this assay, a subset of the sera samples from the microarrays was used (Negative samples n = 11; WT-YFV samples n = 9; 17DD-YFV samples n = 13). To coat the plates, 8 µg/mL of the peptide diluted in coating buffer were added to Nunc MaxiSorp 96-well ELISA plates and incubated at 4 °C overnight. Next, the wells were washed three times using a washing buffer and a blocking buffer (2.5% w/v BSA, 2.5% w/v nonfat dry milk, 1X phosphate-buffered saline with 0.05% Tween20) were added followed by incubation at 37 °C for 1 h. After washing as described serum from each group, diluted in Standard Buffer for ELISA, (1:100) was added, followed by an incubation at 37 °C for 2 h. Anti-human IgG peroxidase conjugate (Sigma-Aldrich Corporation, Saint Louis, MO, USA), diluted in standard buffer for ELISA (1:10000) was added. After 1 h of incubation at 37 °C, the wells were washed and TMB Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well following by the addition of the Stop Solution. Absorbance was read on SpectraMax (Molecular Devices, San José, CA, USA) using a 450 nm wavelength.
7
ELISA Assay for EpCAM Protein Detection
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MaxiSorp™ 96-well ELISA plates (Nunc, Rochester, NY, USA) were coated with 100 μl per well of carbonate-bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) buffer containing mouse anti-EpCAM mAb (anti-GA733 mAb) (R&D Systems, Minneapolis, MN, USA) (50 ng) and were incubated overnight at 4 °C. The plates were washed four times with 1× PBS-T [1× PBS plus 0.5% (v/v) Tween 20]. After washing, serially diluted GA733-Fc and GA733-FcK protein samples (12.5–0.196 ng) were added to each well, and then the plates were incubated for 2 h at 37 °C. The wells were washed four times with 1× PBS-T. One hundred microliters of HRP-conjugated goat anti-human IgG Fc antibody (Jackson, West Grove, PA, USA; diluted 1:5,000) was added to each well as a secondary antibody. After incubating for 2 h at room temperature, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Seracare, Milford, MA, USA) was added to each well, and the color was allowed to develop for 3 min. TMB Stop Solution (Seracare, Milford, MA, USA) was used to stop the reaction. The absorbance of each well was read at 450 nm on a Gen5 microplate reader with version 2.01 software (Biotek, Winooski, VT, USA).
8
Quantification of Antibody Titers by ELISA
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Maxisorp 96‐well ELISA plates (Nunc) were coated overnight with purified eIL‐31‐C‐His or purified CuMVTT (5 mg/L) and continued as described in Fettelschoss‐Gabriel et al.19 Absorbance was measured at 450 nm by Tecan Spark 10M spectrophotometer (Tecan). The antibody titers as OD50 were calculated (serum dilution on a logarithmic scale where OD450 was half maximal). All antibody titers were calculated with naïve serum subtracted on logarithmic scales and presented as delta OD50 (ΔOD50). Titers ≤ 10 were considered background.
9
ELISA-based Detection of Antibodies
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Microtiter plates (Nunc Maxisorp 96-well ELISA plates) were coated with 100 μl of rZNS1-His (400 ng/well) in coating buffer (0.1 M Na2HPO4 buffer pH 9.5) for 18 h at 4 °C. Following incubation in blocking buffer (5 % skimmed milk in TBS) for 1 h at 37 °C, the plates were further incubated with human sera at a 1:100 dilution, or with mouse polyclonal sera at the indicated dilutions, for 1 h at RT in blocking buffer. Following four washing steps in TBS Tween-20 0.05 %, plates were further incubated in HRP rabbit anti-human IgG secondary antibody (Dako) at a 1:4000 dilution, or with HRP goat anti-mouse IgG Fcγ fragment specific secondary antibody (Jackson Immunoresearch) at a 1:2000 dilution, for 1 h at RT in blocking buffer. Finally, plates were washed four times in TBS Tween-20 0.05 % and after incubation with the substrate [0.3 % H2O2, 0.1 % 3,3′,5,5′-tetramethylbenzidine (TMB) in 0.1 M citric acid pH 5] for 15–20 min at RT, the reaction was stopped with 0.2 M H2SO4. The absorbance at 450 nm was measured with a FilterMax F5 Multi-Mode microplate reader (Molecular Devices).
10
Quantification of Complement Proteins
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MaxiSorp 96-well ELISA plates (Nalge Nunc International, Penfield, NY) were coated with 100 µl of diluted serum (1∶32,000 for C3; 1∶16,000 for C3a; 1∶6000 for C4), or with dilutions of purified C3, C3a or C4 human standards (all from Complement Technology). Plates were incubated at 4°C overnight, washed three times with PBS/Tween (0.2%), and wells were blocked with 200 µl of PBS containing 5% milk for 1 hr at 37°C. 100 µl of diluted primary goat anti-human C3 (1∶2000), rabbit anti-human C3a (1∶5000) or goat anti-human C4 (1∶2000) (all from Calbiochem) was added to each well and incubated for 1 h at room temperature (RT). Wells were washed five times with PBS/Tween before incubation at RT for 1 h with 100 µl per well of HRP-conjugated donkey anti-goat (1∶20,000 for C3 and C4) or goat anti-rabbit IgG (1∶50,000 for C3a) (Jackson ImmunoResearch Laboratories, PA) followed by development with substrate TMB (tetramethylbenzidine dihydrochloride, Sigma). The absorbance was determined at 450 nm on a ELx800 plate Absorbance Microplate Reader (Bio Tek Instruments, Inc., Vermont).
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