MASP2, FNC1, and C4BPB levels were determined using an in-house ELISA setup. Briefly, polyclonal antibody anti-MASP2, anti-FCN1, or anti-C4BPB (LifeSpan Biosciences) was used to coat overnight at 4 °C 96-well MaxiSorp Nunc plates (Thermo Fisher Scientific, Waltham, MA, USA). The plates were then blocked with 3% BSA in PBS. Exosome fractions were solubilized in 10 µl of mild detergent solution (1% Nonidet P-40, 0.5% Tween-20 in PBS), supplemented with 90 µl 3% BSA in PBST and incubated at 4 °C overnight. After three washes in PBST, the plates were incubated for 4h with the corresponding monoclonal antibody (anti-MASP2, anti-FCN1, or anti-C4BPB, all from LifeSpan Biosciences, Seattle, WA, USA) diluted 1:1000 with 1% BSA in PBST. After three washes with PBST, the plates were incubated with HRP-conjugated anti-mouse IgG diluted 1:5000 in 1% BSA in PBST for 1h. After washing, the peroxidase substrate (TMB, Bio-Rad, Hercules, CA, USA) was added. The reaction was stopped with an H2SO4 solution. The absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad, Hercules, CA, USA). To standardize the response of the antibodies, we used a pool of strongly positive controls. The optical density results were expressed as relative units per milliliter (RU/ml).
Maxisorp nunc plates
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark
The MaxiSorp Nunc plates are high-quality polystyrene microplates designed for use in enzyme-linked immunosorbent assay (ELISA) and other immunoassay applications. These plates feature a MaxiSorp surface that provides enhanced protein binding capacity, allowing for efficient capture of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Tristar lb 941 luminometer, by Berthold Technologies (2 mentions)
Western lightning ecl reagent, by PerkinElmer (2 mentions)
Anti masp2, by LifeSpan BioSciences (1 mentions)
Anti fcn1, by LifeSpan BioSciences (1 mentions)
Anti c4bpb, by LifeSpan BioSciences (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
Goat anti rabbit igg conjugated to hrp, by Merck Group (1 mentions)
P nitrophenyl phosphate substrate, by Merck Group (1 mentions)
Microplate autoreader, by Agilent Technologies (1 mentions)
Hb 123, by American Type Culture Collection (1 mentions)
Goat anti mouse igg conjugated to alkaline phosphatase, by Southern Biotech (1 mentions)
Anti human igg fab specific, by Merck Group (1 mentions)
Tmb solution, by Merck Group (1 mentions)
Goat anti aqp4 antibody, by Santa Cruz Biotechnology (1 mentions)
Sa202, by Merck Group (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
10 protocols using maxisorp nunc plates
1
Quantifying Exosomal Biomarkers via ELISA
2
ELISA Assay for Malaria Antibodies
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The standard operating procedure developed by the African Malaria Network Trust was used to assess total IgG concentrations by ELISA to CSP and GLURP R2, as described previously [20 (link)]. Briefly, recombinant proteins (0.1 µg/well) diluted in PBS were coated on MaxiSorp Nunc plates (Thermo Fisher Scientific, Denmark) and blocked with 3% powdered-milk + 0.1% of PBS-Tween 20. Sera samples were diluted at 1:200 for all recombinant proteins. Polyclonal goat anti-human IgG (Gamma) (Caltag) conjugated to HRPO diluted 1:3000 (Skybio, France) was used for revealing the reaction with 3,3′,5,5′-tetramethylbenzidine TMB as substrate and 0.2 M H2SO4 to stop the reaction. Standard curves were established using human IgG purified proteins (Binding Site, France) to determine the concentration of specific antibodies. Concentrations of the standard curve are as follows: 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 151 and 3.9 µg/mL. Each curve is run in duplicate on each plate. The ADAMSEL FLP b039 software [21 (link)] is used to analyse absorbance at 450 nm and interpolate the standard curve (µg/ml). Discordant duplicates (with a variation coefficient > 15%) are dropped.
3
Quantifying SARS-CoV-2 RBD-ACE2 Binding and Complement Activation
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Ninety-six well flat-bottom MaxiSorp Nunc plates (ThermoFischer Scientific) were coated with 5 μg/ml Avidin in PBS overnight, blocked, and then incubated with either two-fold dilution of biotinylated RBD (69 (link)) or 2.5 μg/ml in 0.1% casein for 1 hour at RT. The ACE2-Fc proteins were then added over the indicated concentration range. In experiments to measure C5b-C9 fixation, the plates were incubated with 10% fresh human serum for 30 minutes at RT followed by 1/2000 dilution of rabbit anti-C5b-C9 (Millipore) for 1 hour at RT, washed and then incubated with goat anti-rabbit IgG conjugated to HRP (Millipore) at 1/2000 dilution for 1 hour at RT, followed by TMB substrate for 15-20 minutes at RT (70 (link)). Reactivity was stopped using 1 M sulfuric acid and absorbance was measured at 450 nm. Test samples and reagents were prepared in PBS 0.1% (w/v) casein and plates washed thrice between each step using PBS, 0.05% (v/v) Tween 20. Samples were tested in duplicate and corrected for background reactivity using negative control wells from which ACE2-Fc proteins were omitted. The mean and SEM from independent experiments are shown.
4
Quantifying Anti-Insulin Antibodies
5
Bovine Coronavirus Antibody ELISA
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IgG1 Ab titers to BCoV were measured using a double sandwich ELISA. Briefly, 96-well Maxisorp NUNC plates (Thermo Scientific®, USA) were coated with 100 μl of guinea pig hyperimmune serum to BCoV (1:5000 dilution in carbonate/bicarbonate buffer, pH 9.6) and incubated overnight (ON) at 4 °C. Plates were then washed and blocked with 10% non-fat milk solution diluted in PBS-0.05% Tween 20. After incubation during 1 hour at 37 °C, BCoV Mebus strain-infected (107 FFU/ml) and mock-infected HRT-18 supernatants were added as positive and negative antigens, respectively. Serial 4-fold dilutions of the serum samples were tested in duplicate followed by incubation with an HRP-conjugated commercial Ab to bovine IgG1 (Bethyl Laboratories, Inc., USA). The reaction was developed using hydrogen peroxide and ABTS as substrate/chromogen system (Sigma Aldrich, USA) and read at a wavelength of 405 nm (Multiskan Ex, Labsystems Inc.). The titer of each sample was expressed as the reciprocal of the highest serum dilution with a corrected optical density (OD405C; OD405 values in the BCoV-coated wells minus OD405 values in the HRT-18-coated wells) greater than the cut-off value of the assay. The cut-off value was established as the average of OD405C values of four blank wells (PBS-0.05% Tween 20) plus three standard deviations (SD).
6
Standardized Malaria Antibody ELISA
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Enzyme-Linked ImmunoSorbent Assay (ELISA) [15 (link),16 (link)] was performed to assess malaria antibody concentrations; Afro Immuno Assay (AIA) protocols were followed that are developed to standardize methods for evaluating malaria vaccines and sponsored by the African Malaria Network Trust (AMANET [www.amanet-trust.org ]). Briefly, standard curves were established using purified human IgG (Binding Site, France) to determine the concentration of specific antibodies. Each point was tested in duplicate.
Specific and total IgG were measured using recombinant proteins diluted in phosphate-buffered saline (PBS) for specific IgG or an anti-human IgG (Fab-specific, Sigma Aldrich, France) diluted in carbonate buffer for total IgG, both were coated at 0.1-μg/well on MaxiSorp Nunc plates (Thermo Fisher Scientific, Denmark) and blocked with 3% powdered milk, 0.1% Tween, 20 PBS. Maternal and cord blood samples were diluted at 1:200 for all IgG-specific, recombinant proteins except for AMA1 (1:2,000) and total IgG (1:1,000,000). An anti-human IgG coupled with peroxidase (1:3,000, Caltag, UK) was used to reveal the reaction with TMB One (3, 3′, 5, 5′-tetramethylbenzidine, Mast Diagnostic, France). Plates were read at 450 nm.
Specific and total IgG were measured using recombinant proteins diluted in phosphate-buffered saline (PBS) for specific IgG or an anti-human IgG (Fab-specific, Sigma Aldrich, France) diluted in carbonate buffer for total IgG, both were coated at 0.1-μg/well on MaxiSorp Nunc plates (Thermo Fisher Scientific, Denmark) and blocked with 3% powdered milk, 0.1% Tween, 20 PBS. Maternal and cord blood samples were diluted at 1:200 for all IgG-specific, recombinant proteins except for AMA1 (1:2,000) and total IgG (1:1,000,000). An anti-human IgG coupled with peroxidase (1:3,000, Caltag, UK) was used to reveal the reaction with TMB One (3, 3′, 5, 5′-tetramethylbenzidine, Mast Diagnostic, France). Plates were read at 450 nm.
7
Evaluating gp120 Protein Interactions
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The capacity of recombinant gp120 proteins to interact with CD4, CoRBS and cluster A Abs was tested by ELISA, as previously described (67 (link)). BSA and the recombinant gp120 proteins (ΔV1V2V3V5 WT, ΔV1V2V3V5 D368R, and ID2) were prepared in PBS (0.1 μg/ml) and adsorbed to MaxiSorp Nunc plates (Thermo Fisher Scientific) overnight at 4°C. BSA was used as a negative control. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween 20 and 2% [wt/vol] BSA) for 90 min at room temperature. The wells were then washed four times with washing buffer (TBS containing 0.1% Tween 20). Abs (17b, N5i5, and CD4-Ig) were diluted in blocking buffer, followed by incubation for 120 min at room temperature. The wells were washed four times with washing buffer, followed by the incubation of HRP-conjugated antibody specific for the Fc region of human IgG (Pierce) for 90 min at room temperature. The wells were then washed four times with washing buffer. The HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning ECL reagents (Perkin-Elmer). Light emission was measured with an LB 941 TriStar luminometer (Berthold Technologies, Bad Wildbad, Germany).
8
ELISA for Cytophilic IgG1 and IgG3
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The standard operating procedures developed by the African Malaria Network Trust was used to assess cytophilic IgG1 and IgG3 concentrations by enzyme linked immunosorbent assay (ELISA) to a panel of recombinant proteins, as described previously [13 (link)]. Briefly, recombinant proteins (0.1 µg/well) diluted in phosphate buffered saline (PBS) were coated on MaxiSorp Nunc plates (Thermo Fisher Scientific, Denmark) and blocked with 3% powdered-milk 0.1% PBS-Tween 20. Plasma samples were diluted 1:50 for all recombinant proteins. Peroxidase conjugated anti-human IgG1 (NL16 clone) diluted 1:2000 and anti-human IgG3 (ZG4 clone) diluted 1:5000 (Skybio, France) were used for revealing the reaction with 3,3′,5,5′-tetramethylbenzidine (TMB) as substrate. Standard curves were established using human IgG1 and IgG3 purified proteins (Binding Site, France) to determine the concentration of specific antibodies. Each point was tested in duplicate.
9
Enzyme-linked immunosorbent assay for gp120 antibodies
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Bovine serum albumin (BSA), gp120core and stabilized gp120 inner domain (ID2 [33 (link)]) were prepared in PBS (0.1 µg/mL) and adsorbed to MaxiSorp; Nunc plates (Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 °C. BSA was used as a negative control. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline (TBS) containing 0.1% Tween 20 and 2% [wt/vol] BSA) for 90 min at room temperature. Wells were then washed 4 times with washing buffer (Tris-buffered saline [TBS] containing 0.1% Tween 20). Sera from the final bleed of the immunized guinea pigs (1:10,000 dilution) were diluted in blocking buffer and incubated for 120 min at room temperature. Wells were then washed 4 times with washing buffer. This was followed by incubation of horseradish peroxidase (HRP)-conjugated antibody specific for the Fc region of guinea pig IgG (0.4 µg/mL; Thermo Fisher Scientific, Waltham, MA, USA) for 90 min at room temperature. Wells were then washed 4 times with washing buffer. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning ECL reagents (Perkin Elmer Life Sciences, Waltham, MA, USA). Light emission was measured with an LB 941 TriStar luminometer (Berthold Technologies, Bad Wildbad, Germany).
10
AQP4 ELISA Assay Protocol
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The ELISA was performed as previously described by Pisani et al., 35 . Briefly, Maxisorp NUNC Plates (Thermo) were coated with 0.2 μg of commercial goat anti‐AQP4 antibody (Santa Cruz Biotechnology, sc‐9888) overnight at 4°C or for 2 hrs at 37°C. After coating, wells were washed and coated with approximately 35 ng of AQP4. Negative control wells were only coated with the buffer. After incubation for 1 hr, sera diluted from 1:1000 to 1:8000 and goat anti‐AQP4 were incubated in the wells for 1 hr under shaking. Wells were then washed, incubated with anti‐human biotinylated secondary antibody (Millipore AP112B), washed again and incubated with streptavidin‐HRP (Millipore SA202, 1:1000 in A). After 1 hr, 100 μl of TMB solution was added (Millipore) for 20 min., and the reaction was stopped by adding 100 μl of 0.3M sulphuric acid solution. Finally, absorbance was read at 450 nm. Normalized absorbance was calculated as follows: absorbance of AQP4‐coated well minus absorbance of negative control well. Absorbance was read using a Flex Station 3 (Molecular Devices, Sunnyvale, California, USA).
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