Recombinant proteins solubilized in the respective refolding buffers were further diluted to a concentration of 5 μg/ml using bicarbonate coating buffer (pH 9.6) of which 100 μl per well were used to coat Maxisorb ELISA plates (NUNC, Thermofisher, Braunschweig, Germany) overnight at 4 °C. After washing, wells were blocked by 5% skim milk-TBST) for 2 h at room temperature and then washed four times with TBST [44 (link)]. Individual sera were diluted 1:200 (for the NP iELISA) or 1:500 (other antigens) in appropriate sample dilution buffer. Sera were incubated at room temperature for 1 h. Wells were washed again four times with TBST before 100 μl of appropriately diluted goat-anti-chicken or -turkey IgY POD conjugate (Dianova, Hamburg, Germany) was added for 1 h at room temperature. Antibody was removed and after a final washing cycle with TBST, 50 μl of chromogenic TMB substrate was added. OD450 values were measured after 10 min of incubation and addition of 50 μl of 1 N H2SO4 to each well. Results were calculated and expressed in S/P units:
Maxisorb elisa plate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Maxisorb ELISA plates are a type of microplate used in enzyme-linked immunosorbent assay (ELISA) experiments. The plates are designed with a high-binding surface to facilitate the immobilization of target analytes or capture antibodies. The Maxisorb plates are suitable for a wide range of ELISA applications.
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Lab products found in correlation
Skim milk, by Merck Group (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Elx808 plate reader, by Agilent Technologies (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (2 mentions)
Anti mouse igg hrp, by Agilent Technologies (2 mentions)
5 tetramethylbenzidine, by Merck Group (1 mentions)
Hrp conjugated anti mouse iga, by Fortis Life Sciences (1 mentions)
Hrp conjugated anti mouse igg, by Fortis Life Sciences (1 mentions)
Ovalbumin (ova), by Seikagaku (1 mentions)
Sigmafast opd peroxidase substrate, by Merck Group (1 mentions)
Fast opd, by Merck Group (1 mentions)
Skimmed milk, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg antibody, by Merck Group (1 mentions)
Anti ha, by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Anti human igg, by Jackson ImmunoResearch (1 mentions)
Step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions)
1 step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions)
Anti mouse iga hrp, by Thermo Fisher Scientific (1 mentions)
Gentamicin sulfate, by Biowest (1 mentions)
23 protocols using maxisorb elisa plate
1
ELISA-Based Antigen Detection Assay
2
ELISA Assay for Trimeric Env Glycoprotein
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Titer of IgG antibodies binding to trimeric Env glycoprotein was determined by ELISA using a D7324-tagged version of the recently described soluble HIV-1 Env trimer BG505 SOSIP.664 gp140 and following the methods described by Sanders et al [22 (link)]. Briefly, Maxisorb Elisa plates (Nunc) were coating with D7324 antibody (10 μg/mL) and blocked using TBS+10%FBS for two hours at room temperature. After washing, the BG505 SOSIP.664 protein was added at 1ng/ml in blocking buffer and incubated overnight at 4°C. Then, plates were washed and blocked again using TBS/2% skimmed milk. After washing, plasma samples diluted 1/1000 in TBS/5%FBS/2%skimmed milk were added and incubated for two hours at room temperature. HRP-Goat anti-human IgG (Fc specific) (Jackson-Immunoresearch) was used as secondary antibody. The reaction was revealed using 3, 3′,5, 5′-Tetramethylbenzidine (Sigma-Aldrich) and stopped using 2M of H2SO4. 2G12 antibody was used as standard and the results are showed as arbitrary units. The IgGb12 antibody, which does not bind to the BG505 SOSIP.664, was used as negative control. Plasma samples were assayed in parallel in D7324 antibody coated and antigen free wells to evaluate background. The signal obtained in these wells was subtracted to the signal obtained with antigen.
3
Measuring Autoantibody Levels in Sera
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We measured the serum natural autoantibody IgM levels against Hsp60, Hsp70, and CS using indirect ELISA [12 (link),40 (link)]. Briefly, we coated Nunc Maxisorb ELISA plates with 5 μg of recombinant Hsp60, -Hsp70, or citrate-synthase in carbonate coating buffer overnight at 4 °C. After the coating, plates were washed 4 times and blocked for 1 h at RT followed by washing 4 times again. The sera were added at a 1:100 dilution and incubated for 2 h at 37 °C. Next, we washed the plates 4 times, before adding the secondary PO-conjugated anti-mouse IgM at a 1:1000 dilution in PBS and incubated for 1 h at RT. The color reaction was developed with the addition of OPD substrate solution, which was stopped with sulfuric acid after 20 min. Optical density values were read at 492 nm with the iEMS reader (MF Thermo Labsystems, Philadelphia, PA, USA).
ANA antibodies were measured using the QANTA Lite ELISA Kit (Inova Diagnostics, San Diego, CA, USA) according to the manufacturer’s instructions, with slight modifications. The reaction was developed using peroxidase-conjugated anti-mouse-IgG1 (BD Bioscience, San Jose, CA, USA) as secondary antibodies [38 (link)].
ANA antibodies were measured using the QANTA Lite ELISA Kit (Inova Diagnostics, San Diego, CA, USA) according to the manufacturer’s instructions, with slight modifications. The reaction was developed using peroxidase-conjugated anti-mouse-IgG1 (BD Bioscience, San Jose, CA, USA) as secondary antibodies [38 (link)].
4
Measuring Cytokines and Antibodies in Cell Supernatant
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The concentrations of cytokine and antibodies in cell culture supernatant, plasma and ICs were measured using a commercially available enzyme-linked immunosorbent assay kit: OptEIATM mouse IL-12 (p40) ELISA was purchased from BD Pharmingen. Mouse IgG quantitation kit and mouse IgA quantitation kit were purchased from Bethyl Laboratories (Montgomery, TX, USA). Anti-OVA IgG in plasma and anti-OVA IgA in the ICs were measured as previously reported, with some modification [28 (link)]. Briefly, Maxisorb ELISA plates (Nunc) were coated with 20 μg/mL OVA (Seikagaku Corp., Tokyo, Japan) at room temperature, overnight. After washing and blocking, collected plasma or the ICs were added and incubated at room temperature for 2 h. HRP-conjugated anti-mouse IgG (Bethyl Laboratories) or HRP-conjugated anti-mouse IgA (Bethyl Laboratories) was used for detection. TMB was used as the substrate (e-Bioscience), and the reaction was stopped by the addition of 1M phosphoric acid. Absorbance was measured at 405 nm.
5
Canine Respiratory Virus ELISA Protocol
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Antigen was adsorbed overnight at 4 °C onto 96 well Nunc Maxisorb™ ELISA plates, and blocked with 5% milk in 0.05% PBS Tween. Serum samples were diluted 1:100 in blocking buffer and 50 μl dispensed in duplicate onto positive and control antigens and incubated at 37 °C for 1 h. Following three washes with 0.05% PBST, the anti-dog IgG peroxidase conjugate (Sigma-Aldrich, Poole) secondary antibody was diluted 1:2500 (CRCoV, CnPnV) or 1:5000 (M. cynos) in blocking buffer, and 50 μl dispensed into each well and incubated. The plates were washed as before and bound antibody was detected using Sigma-Fast™ OPD peroxidase substrate (Sigma Aldrich, UK) according to the manufacturer’s instructions, and stopped with 2 M H2SO4. Optical densities (OD) were measured at 490 nm (Optimax automatic plate reader Molecular Devices, Wokingham). Positive and negative control sera were included in duplicate on each plate. For each serum sample, the average OD value for the negative antigen was subtracted from that of the positive antigen to give the average corrected OD value. The positive cut-off was defined as the average corrected OD value for a panel of reference serum (IgG negative for the antigen used) plus three times the standard deviation. Samples were considered positive if the ‘average corrected OD value’ was greater than or equal to the positive cut-off value for the ELISA.
6
Quantifying Antibody Levels in Biological Samples
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Maxisorb ELISA plates (Nunc, USA) were coated overnight at 4° C with vCCI (35 K) capture antibody (R&D systems) at 1 μg/ml in PBS. Plates were washed 5 × with dH2O then non-specific binding was blocked with blocking buffer (PBS/0.25% BSA/1 mM EDTA/0.05% Tween 20) for 2 hours at room temperature. Lavage samples or standards were directly incubated on the plate, or diluted in blocking buffer to within the standard curve as required and plasma samples were diluted at least 1:2 in RIPA buffer (Tris pH7.3 50 mM, NaCl 150 mM, SDS 0.1%, NP-40 1%). Samples were incubated in the ELISA plate for 2 hours, washed as above, then incubated with anti-human Fc HRP antibody (1:5000 in blocking buffer, Jackson ImmunoResearch). The plate was incubated with substrate (Sigma Fast OPD) then the reaction stopped with 3 M H2SO4.
7
ELISA Protocol for Nanobody Screening
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The ELISA procedure was based on a previously described method (23 (link), 24 (link)) with minor modifications. Briefly, Maxisorb ELISA plates (Nunc) were coated at 4°C for 16 h (ON) with 100 μl/well of FMDV 3ABC protein at a concentration of 60 ng/ml suspended in PBS. Following incubation plates were washed three times with washing buffer [PBS containing 0.05 % Tween 20 (PBS-T)] and then blocked for 1 h at RT with 5 % skimmed milk (Sigma). The same washing procedure was performed after each incubation step. Next, serial dilutions of tested Nbs in concentrations from 4 μg/ml to 60 ng/ml were added at a volume of 100 μl/well and incubated for 1 h at RT. Following incubation plates were washed and 100 μl/well of 1 μg/ml anti-HA (Sigma) was added and incubated for 1 h at RT. Afterward, plates were washed and 100 μl/well of 1 μg/ml HRP-conjugated anti-mouse IgG antibody (Sigma) was added and incubated for an additional 1 h at RT. Finally, plates were washed four times and 90 μl/well of TMB One solution (SouthernBiotech) was added to develop the color reaction. The color reaction was stopped after 15 min with 1 M Sulfuric acid and readouts were obtained with absorbance read at 470 nm using a standard luminometer (Thermolabsystems-Luminoskan Ascent).
8
ASFV Antibody Detection by ELISA
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ASFV antibody detection was performed using an in-house ELISA, as previously described [21 (link)]. Briefly, ELISA antigen was prepared from ASFV-infected Vero cells. Maxisorb ELISA plates (Nunc, St Louis, MO, USA) were coated with 1 µg per well of infected or uninfected cell extract. The plates were blocked with phosphate-buffered saline containing 10% skim milk (Merck, Kenilworth, NJ, USA) and 5% normal goat serum (Sigma, Saint Louis, MO, USA). Each swine serum was tested at multiple dilutions against both infected and uninfected cell antigens. ASFV-specific antibodies in the swine sera were detected using an anti-swine IgG-horseradish peroxidase conjugate (KPL, Gaithersburg, MD, USA) and SureBlue Reserve peroxidase substrate (KPL, Milford, MA, USA). Plates were read at OD630 nm in an ELx808 plate reader (BioTek, Shoreline, WA, USA). Sera titers were expressed as the log10 of the highest dilution, where the OD630 reading of the tested sera at least duplicated the reading of the mock-infected sera.
9
ELISA-based Quantification of Anti-ASFV Antibodies
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Anti-ASFV antibodies in the sera of infected animals were quantified using an in-house developed ELISA as previously described [9 (link)]. Briefly, ASFV was amplified in Vero cells [16 (link)], infected cells were then treated with Tween 80 (G-Biosciences, Saint Louis, MO, USA) and sodium deoxycholate (Sigma, Saint Louis, MO, USA) to a final concentration of 1% (v/v) and cell antigens were stored at ≤−70 °C. Maxisorb ELISA plates (Nunc, Saint Louis, MO, USA) were coated with 1 µg per well of either infected cell or uninfected cell antigen and blocked with phosphate buffered saline containing 10% skim milk (Merck, Kenilworth, NJ, USA) and 5% normal goat serum (Sigma). Swine sera were tested at multiple dilutions against both infected and uninfected cell antigen, with reactivity detected by an anti-swine IgG–horseradish peroxidase conjugate (KPL, Gaithersburg, MD, USA) and SureBlue Reserve peroxidase substrate (KPL). Plates were read at OD630 in an ELx808 plate reader (BioTek, Shoreline, WA, USA). Swine sera were considered positive for ASFV-specific antibodies if the OD630 ratio of the reaction against infected cell antigen to uninfected cell antigen was higher than 2.2.
10
Quantification of Pathogen-Specific Antibodies
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Maxisorb ELISA-plates (Nunc) were coated with 0.5–2 µg/ml pathogen antigens, derived from human vaccines (Influvac 2012/2013 (0.5 µg/ml), Abbott Biologicals), Act-HIB (Sanofi pasteur MSD (0.5 µg/ml)), DTP (1/200, Nederland vaccine instituut), or 25 µl inactivated RSV A2 or rhinovirus (kindly provided by Prof. S Johnston, Imperial College London, UK). Plates were blocked with 0.5% gelatine/PBS, washed (0.05% tween-20/PBS), and samples were added and titrated. Plates were washed four times and 1/2000 HRP-conjugated sheep anti bovine IgG1 (Abd Serotec, Kidlington, UK) or 1/6000 anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA) were used as detector antibody. Plates were washed extensively and developed with TMB and read at 450 nm. For inhibition ELISAs purified IgG from human plasma (Intravenous Immunoglobulin, pooled from >1000 donors, or IVIg) (Sanquin blood supply, The Netherlands) or bIgG equivalent to 167 µg/ml was pre-incubated at room temperature with twice the amount used for coating of hRSV or vaccine. Binding to F protein of bRSV was analysed by a commercial ELISA, according to the manufacturer’s descriptions (Bio-X Diagnostics, Jemelle, Belgium).
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