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Elisa plate

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ELISA plates are a type of laboratory equipment used in enzyme-linked immunosorbent assay (ELISA) techniques. They are multi-well plates, typically made of polystyrene, that are designed to facilitate the detection and quantification of various biomolecules, such as proteins, antibodies, or antigens, within a sample.

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Lab products found in correlation

Microplate reader, by Molecular Devices (7 mentions) 3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (7 mentions) Tmb substrate, by Thermo Fisher Scientific (6 mentions) Microplate reader, by Agilent Technologies (5 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (4 mentions) Bovine serum albumin (bsa), by Merck Group (4 mentions) Sunrise spectrophotometer, by Tecan (3 mentions) 1 step ultra tmb elisa substrate, by Thermo Fisher Scientific (3 mentions) Infinite 200, by Tecan (3 mentions) Sodium bicarbonate, by Merck Group (3 mentions) Sodium carbonate, by Merck Group (3 mentions) Phosphate buffered saline (pbs), by Merck Group (3 mentions) O phenylenediamine dihydrochloride, by Merck Group (3 mentions) Imark microplate reader, by Bio-Rad (3 mentions) A80229ap, by Fortis Life Sciences (2 mentions) Based mbp affinity chromatography, by GE Healthcare (2 mentions) Sigmaplot v12, by Grafiti LLC (2 mentions) Spectramax m2e, by Molecular Devices (2 mentions) Human vegf165, by Merck Group (2 mentions) Peroxidase substrate, by Thermo Fisher Scientific (2 mentions)

223 protocols using elisa plate

1

ELISA Binding Kinetics Characterization

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ELISAs were performed as previously described (Hastie et al., 2017 (link), 2019 (link)). Briefly, ELISA plates (Corning, Kennebunk, ME) were coated with 2.5 μg/mL streptavidin in PBS for 3 h at RT, and blocked with 3% BSA in PBS for 2 h at RT. LASV pfGP-TD (1 μg/mL) either alone or in complex with mAb (10 μg/mL) was diluted in PBS for 1hr at RT before coating on ELISA plates (Corning, Kennebunk, ME). After blocking, a dilution series of LAMP1-RbFc starting at 10 μg/mL was bound to coated protein for 2 h at RT in 50 mM citrate pH 5.0 with 150 mM NaCl buffer. Wells were washed extensively and bound LAMP1-RbFc was detected using goat anti-rabbit-IgG(H + L), mouse/human ads-HRP antibody (Southern Biotech, 1:5,500) for 1 h at RT.
Enriquez A.S., Buck T.K., Li H., Norris M.J., Moon-Walker A., Zandonatti M.A., Harkins S.S., Robinson J.E., Branco L.M., Garry R.F., Saphire E.O, & Hastie K.M. (2022). Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies. Cell reports, 39(8), 110841.
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2

ELISA Binding Kinetics Characterization

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ELISAs were performed as previously described (Hastie et al., 2017 (link), 2019 (link)). Briefly, ELISA plates (Corning, Kennebunk, ME) were coated with 2.5 μg/mL streptavidin in PBS for 3 h at RT, and blocked with 3% BSA in PBS for 2 h at RT. LASV pfGP-TD (1 μg/mL) either alone or in complex with mAb (10 μg/mL) was diluted in PBS for 1hr at RT before coating on ELISA plates (Corning, Kennebunk, ME). After blocking, a dilution series of LAMP1-RbFc starting at 10 μg/mL was bound to coated protein for 2 h at RT in 50 mM citrate pH 5.0 with 150 mM NaCl buffer. Wells were washed extensively and bound LAMP1-RbFc was detected using goat anti-rabbit-IgG(H + L), mouse/human ads-HRP antibody (Southern Biotech, 1:5,500) for 1 h at RT.
Enriquez A.S., Buck T.K., Li H., Norris M.J., Moon-Walker A., Zandonatti M.A., Harkins S.S., Robinson J.E., Branco L.M., Garry R.F., Saphire E.O, & Hastie K.M. (2022). Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies. Cell reports, 39(8), 110841.
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3

ELISA Protocol for IFN-gamma Detection

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IFN-γ capture antibody (2 μg/ml, BioLegend) and IFN-γ detection antibody (1 μg/ml, BioLegend) were used to detect cytokine. The ELISA plate (Corning) was coated overnight with a capture antibody at 4°C and washed three times with PBS containing 0.05% Tween 20 (PBST), then blocked with 3% BSA at 37°C for 1 h. Then, supernatants were added to the ELISA plate (50 μl/well) and incubated at 37°C for 1 h. After washing with PBST for five times, the detection antibody was added and incubated at 37°C for 30 min. The plate was incubated with streptavidin-ALP (1:1,000) at 37°C for 30 min after washing with PBST five times. Next, the plate was washed with PBST six times, followed by incubation with 50 μl pNPP solution (Mabtech, Stockholm, Sweden) at 37°C for 40 min. Finally, the plate was analyzed via the Varioskan LUX Multimode Microplate Reader at 405 nm.
Li L., Chen Q., Han X., Shen M., Hu C., Chen S., Zhang J., Wang Y., Li T., Huang J., Li S., Hao Y, & Jin A. (2021). T Cell Immunity Evaluation and Immunodominant Epitope T Cell Receptor Identification of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Glycoprotein in COVID-19 Convalescent Patients. Frontiers in Cell and Developmental Biology, 9, 696662.
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4

FcRn-mediated HSA binding ELISA

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Ninety-six-well ELISA plates (Costar) were coated with 8 μg/ml of hIgG1-YTE/KF diluted in PBS, and incubated ON at 4 °C. Plates were blocked with PBS/S for 1 h at RT, and then washed four times with PBS/T. His-tagged hFcRn (10 μg/ml) in pH 5.5 or pH 7.4 PBS/S/T was added and incubated for 1 h at RT. The plates were washed four times with either pH 5.5 or pH 7.4 PBS/T, before serial dilutions (15,000–7.3 ng/ml) of WT HSA or HSA-EQTMVAKP in pH 5.5 or pH 7.4 PBS/S/T were added, and incubated for 1 h at RT. The plates were washed four times as above, before alkaline phosphatase-conjugated polyclonal anti-HSA antibody from goat (#A80229AP; Bethyl Laboratories, Inc., 1:5000), diluted (1:3000) in pH 5.5 or pH 7.4 PBS/S/T was added for 1 h at RT. The plates were washed four times and developed as above.
Grevys A., Nilsen J., Sand K.M., Daba M.B., Øynebråten I., Bern M., McAdam M.B., Foss S., Schlothauer T., Michaelsen T.E., Christianson G.J., Roopenian D.C., Blumberg R.S., Sandlie I, & Andersen J.T. (2018). A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nature Communications, 9, 621.
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5

Quantification of Serum Protein Levels

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Ninety-six-well ELISA plates (Costar) were coated with 1.0 µg/ml of polyclonal goat anti-HSA (#A1151, Sigma-Aldrich) or polyclonal anti-MSA (#ab19194, Abcam) antibodies, 1.0 µg/ml diluted in PBS, and incubated ON at 4 °C. Plates were blocked with PBS/S ON at 4 °C and washed four times with PBS/T before serial dilutions of MSA or HSA (250.0–0.1 ng/ml) in PBS/S/T were applied in parallel with samples from HERA followed by 2 h incubation at RT. Bound MSA and HSA were detected using horseradish peroxidase-conjugated polyclonal anti-MSA antibody from goat (#ab19195; Abcam, 1:4000) or alkaline phosphatase-conjugated polyclonal anti-HSA antibody from goat (#A80229AP; Bethyl Laboratories, Inc., 1:5000). ELISAs were developed by adding 100 µl of 3,3´,5,5´-tetramethylbenzinide solution (Calbiochem) and the reaction was stopped by adding 100 µl of 1 M HCl for MSA, while for HSA 100 µl of the p-nitropenylphospate substrate (Sigma-Aldrich) diluted to 10 µg/ml in diethanolamine buffer was added. The absorbance was measured at 450 or 405 nm using a Sunrise spectrophotometer (TECAN).
Grevys A., Nilsen J., Sand K.M., Daba M.B., Øynebråten I., Bern M., McAdam M.B., Foss S., Schlothauer T., Michaelsen T.E., Christianson G.J., Roopenian D.C., Blumberg R.S., Sandlie I, & Andersen J.T. (2018). A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nature Communications, 9, 621.
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6

Quantifying Alternative Pathway Complement

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ELISA plates (Costar) were sensitized overnight with five micrograms of SGE in 50 µl of coating buffer (35 mM Na2CO3, 15 mM NaHCO3, pH 9.6) at 4°C. Wells incubated with 1% BSA were used as negative controls. The wells were blocked for one hour with 200 µl of blocking buffer (PBS + 1% BSA) under agitation and then incubated with 0.2 µg of each purified complement component from the alternative pathway (C3, factor B, factor D and properdin) (CompTech USA) in 50 µl of PBS for 30 min at room temperature. After two washes with 200 µl of washing buffer (PBS + 0.05% Tween 20) for two minutes, the wells were incubated for 30 min with 50 µl of blocking buffer containing a primary antibody specific to each tested component that was diluted 1:5000. After two more washes, the wells were incubated with a secondary antibody diluted 1:3000 in blocking buffer over 30 min. The plate was washed two more times and then developed as described for the deposition assays, and the absorbance was read in a microplate reader at 450 nm after 5 min of incubation at 37 °C. The assays were conducted in duplicate, and the mean absorbance for each complement component was compared to its respective negative control (BSA).
Mendes-Sousa A.F., Vale V.F., Queiroz D.C., Pereira-Filho A.A., da Silva N.C., Koerich L.B., Moreira L.A., Pereira M.H., Sant’Anna M.R., Araújo R.N., Andersen J., Valenzuela J.G, & Gontijo N.F. (2017). Inhibition of the complement system by saliva of Anopheles (Nyssorhynchus) aquasalis. Insect biochemistry and molecular biology, 92, 12-20.
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7

ELISA Assay for Complement C3 Activation

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ELISA plates (Costar) were coated with 50 μl of coating buffer (35 mM
Na2CO3, 15 mM NaHCO3, pH 9.6) containing 100
μg/ml of mannan from Saccharomyces cerevisiae (Sigma) or
1% BSA (negative control) and incubated overnight at 4°C. After a one hour
blockage of the wells with blocking buffer (1% BSA in PBS), 1% NHS diluted
in GVB2+ was added together with different concentrations of
An. albimanus SGH or recombinant protein dissolved in PBS (final volume
of 100 μl/well) and incubated for 30 minutes at 37°C. Wells incubated with
serum and no inhibitor were used as positive controls. The wells were washed with PBS-Tw
(0.05% Tween-20 in PBS) and incubated for 60 minutes at room temperature with
blocking buffer containing anti-C3 antibody diluted 1:1000. After two washes, 50
μl of blocking buffer containing conjugated antibody (Sigma) diluted 1:1500 were
added to the wells. After two more washes, the wells were filled with 200 μL of
developing buffer (50 mM Na3C6H5O7, 50 mM
Na2HPO4, 1 mg/ml o-phenylenediamine (Sigma) and 0.075 %
H2O2, pH 5.0) and read at 450 nm and 37°C for 10 min in
the kinetic mode. The assays were done in duplicate and the means of the negative control
were subtracted of the other means. The results were transformed in percentage of C3
activation, considering the positive control as 100% of activation and then
analyzed using ANOVA and the Tukey test.
Mendes-Sousa A.F., Queiroz D.C., Vale V.F., Ribeiro J.M., Valenzuela J.G., Gontijo N.F, & Andersen J.F. (2016). An inhibitor of the alternative pathway of complement in saliva of new-world anopheline mosquitoes. Journal of immunology (Baltimore, Md. : 1950), 197(2), 599-610.
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8

Evaluating Vaccine-Specific Antibody Responses

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Vaccine-specific IgG and IgA levels were measured from serum and specific secretory IgA levels were measured from the feces of immunized and control mice from each model of vaccination by using an antibody enzyme-linked immunosorbent assay (ELISA). ELISA plates (Corning-Costar) coated at 4°C overnight with 100 µl of 0.5-µg/ml vaccine supernatant were washed three times with PBS–0.05% Tween 20 (PBS/T) prior to blocking with 100 µl PBS–2% bovine serum albumin (BSA)–0.05% Tween 20 (PBS/BSA/T) for 1 h at room temperature. Dilutions of mouse serum in PBS–0.5% BSA were added to the plates (100 µl) and incubated at 37°C for 2 h prior to washing three times with PBS/T. To detect total IgG or IgA, 100 µl of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or IgA (1:1,000; Santa Cruz Biotechnology) was added, and the plates were incubated for 1 h at 37°C. After three washes in PBS/T, the reaction mixture was developed with 100 µl of 3,3′,5,5′-tetramethylbenzidine substrate solution (BD Biosciences) for 20 min and stopped with 50 µl of 3 M H2SO4. The OD450 was measured.
Ferreira R.B., Valdez Y., Coombes B.K., Sad S., Gouw J.W., Brown E.M., Li Y., Grassl G.A., Antunes L.C., Gill N., Truong M., Scholz R., Reynolds L.A., Krishnan L., Zafer A.A., Sal-Man N., Lowden M.J., Auweter S.D., Foster L.J, & Finlay B.B. (2015). A Highly Effective Component Vaccine against Nontyphoidal Salmonella enterica Infections. mBio, 6(5), e01421-15.
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9

Antiprothrombin Antibody Characterization

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Purified human prothrombin was from Synapse Research Institute (Maastricht, The Netherlands) and rivaroxaban (Xarelto) was from Bayer Schering Pharma AG. Anti‐FcγRIIA monoclonal antibody (mAb) IV.3 was purified in the laboratory from a hybridoma.12 (link) The antiprothrombin mAb 28F4 is a mouse IgG antibody, as described previously.13 (link), 14 (link) Antiprothrombin fragment 1+2 mAbs 3B1, 6A3, 11H2, and 8H11 were developed and produced according to standard procedures.15 (link) LAC was measured by activated partial thromboplastin time (Diagnostica Stago), dilute Russell Viper Venom Time (Diagnostica Stago), and Ecarin Clotting Time (ECT; Diagnostic Reagents). The Pierce Classic IP Kit from Thermo Fisher Scientific was used for immunoprecipitation. High‐binding enzyme‐linked immunosorbent assay (ELISA) plates from Costar were used for the ELISAs. Sheep anti‐human prothrombin IgG horseradish peroxidase–labeled antibody was from Affinity Biologicals, Inc., prothrombin fragment 1+2 was from Haematologic Technologies, Inc. All other reagents are previously described or are from Sigma‐Aldrich.16 (link)
Chayoua W., Nicolson P.L., Meijers J.C., Kardeby C., Garcia‐Quintanilla L., Devreese K.M., de Laat B., Watson S.P, & de Groot P.G. (2021). Antiprothrombin antibodies induce platelet activation: A possible explanation for anti‐FXa therapy failure in patients with antiphospholipid syndrome?. Journal of Thrombosis and Haemostasis, 19(7), 1776-1782.
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10

Antibody Profiling of Chagas Disease

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For mice, ELISA plates were coated with 1 μg/well of rASP-2, rTS or TRASP, reacted with sera diluted 1:200, followed by incubation with secondary antibodies anti-total IgG, anti-IgG1, or anti-IgG2c conjugated with streptavidin-HRP (Cat 1030-05, 1070-05, 1079-05, Southern Biotech), all diluted 1:5000. For dogs, we used ELISA plates (Costar) coated with 100 ng/well of T. cruzi recombinant antigens, sera diluted 1:100 and anti-total IgG, IgG1, IgG2, and IgM conjugated with streptavidin-HRP (Cat A40-123P, A40-120P, A40121P, BEYA40-116P, Bethyl Laboratories, INC), all diluted 1:100,000. The reactions were developed with the substrate 3,3’ 5,5’-tetrametylbenzidine (TMB) from SIGMA and read at 450 nm19 (link),73 (link).
Castro J.T., Brito R., Hojo-Souza N.S., Azevedo B., Salazar N., Ferreira C.P., Junqueira C., Fernandes A.P., Vasconcellos R., Cardoso J.M., Aguiar-Soares R.D., Vieira P.M., Carneiro C.M., Valiate B., Toledo C., Salazar A.M., Caballero O., Lannes-Vieira J., Teixeira S.R., Reis A.B, & Gazzinelli R.T. (2023). ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease. NPJ Vaccines, 8, 81.
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