A MAXISORP immunoplate (442404; NUNC) was coated with mouse anti-E protein, 4G2 (a fusion loop murine Ab which crossreacts to ZIKV). Plates were blocked with 3% BSA for one hour followed by incubation with viral supernatant. After one hour, 10 μg/ml anti-DENV humAbs or serially diluted plasma was added. The reaction was visualized by ALP-conjugated anti-human IgG antibody at a 1:10,000 dilution (A9544; Sigma) and PNPP substrate. Reactions were stopped with NaOH and the absorbance was measured at 405nm. Endpoint titers (EPTs) were defined as reciprocal plasma dilutions that corresponded to 2 times the average OD values obtained with mock antigen.
A9544
Manufactured by Merck Group
A9544 is a laboratory equipment product. It serves as a core function for specific laboratory tasks. The detailed description of the product's features and intended use is not available.
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Lab products found in correlation
Maxisorp immunoplate, by Thermo Fisher Scientific (3 mentions)
Anti human igg ap, by Merck Group (1 mentions)
A9316, by Merck Group (1 mentions)
Ma1 21315, by Thermo Fisher Scientific (1 mentions)
A3438, by Merck Group (1 mentions)
Microtiter plate, by Thermo Fisher Scientific (1 mentions)
Opsys mr plate reader, by Dynex (1 mentions)
Recombinant human il 12, by Thermo Fisher Scientific (1 mentions)
Elisa plate, by Corning (1 mentions)
Alkaline phosphatase substrate, by Merck Group (1 mentions)
7 protocols using a9544
1
ZIKV E Protein Antibody Binding Assay
2
SARS-CoV-2 Spike Protein Binding Assay
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MAXISORP immunoplates were coated with 5 μg/mL of Wuhan S. After blocking with 2% BSA, serial diluted ACE2-mouse Fc, starting from 80 μg/mL with fourfold dilution, were added into each plate. After 1 h incubation at 37 °C, plates were washed with PBST, and 5 μg/mL of SD1 mAbs or mAb222 were added into the plates and incubated for 1 h at 37 °C, followed by washing with PBST. To detect the binding of antibodies with S, anti-human IgG-AP (1:10,000 dilution, A9544, Sigma-Aldrich) was added after wash, incubated for 1 h at 37 °C, and developed by PNPP. To detect the binding of ACE2-mouse Fc on S, the same assay was performed, except that anti-mouse IgG-AP (1:10,000 dilution, A9316, Sigma-Aldrich) was added instead of anti-human IgG-AP after adding SD1 mAb or mAb222, and plates were further developed by PNPP.
3
SARS-CoV-2 Spike Protein Western Blot
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Culture samples were centrifuged at 200× g for 10 min and supernatants collected and stored at −80 °C until further analysis. Western blot analysis was performed as reported elsewhere [29 (link)]. For S identification, the human monoclonal antibody SARS-CoV-2 spike protein (MA5-35948, Thermo Scientific, Breda, The Netherlands) was used at dilution of 1:3000. For 6his-tag recognition on S proteins identification, a mouse monoclonal antibody anti-6His tag (MA1-21315, Thermo Scientific, Breda, The Netherlands) was used at a dilution of 1:1000. As secondary antibody, an anti-mouse IgG (A3438, Sigma, Amsterdam, The Netherlands) and an anti-human IgG antibody (A9544, Sigma, Amsterdam, The Netherlands) conjugated with alkaline phosphatase were used at a dilution of 1:5000.
4
ZIKV E Protein Antibody Binding Assay
5
IgG Immune Response Quantification by ELISA
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The immunoglobulin G (IgG) immune response against the synthetic P27A and overlapping 20-mers was measured using an enzyme-linked immunosorbent assay (ELISA) (5 (link), 15 (link)). Microtiter plates (Nunc™ Maxisorp, Denmark) were coated at a concentration of 2 and 5 μg/mL, respectively, at RT for 1 h then blocked with PBS-3% milk for 1 h. Samples and controls were serially diluted in PBS-1.5% milk-0-05% Tween and added at 50 μL per well. Controls were present on each ELISA plate. Anti-hu IgG secondary antibodies conjugated to alkaline phosphatase (A-9544, Sigma, St. Louis, MO), diluted 1:1000 in PBS-1.5% milk-0-05% Tween, were added to each well. p-Nitrophenyl phosphate substrate tablets (Sigmafast™, Sigma, St. Louis, MO) were dissolved and added to each well. The optical density (OD) of each well was measured at 405 nm using an OpsysMR plate reader (Dynex Technologies). Results were expressed as AU/mL based on the standard curve obtained from the response of the positive control pool of sera to P27A.
6
DENV Serotype Quantification Using 4G2 Antibody
7
ELISA Protocol for Antibody Quantification
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96-well ELISA plates (Costar) were coated with recombinant human IL-12 (0.5 μg/mL; PeproTech), or goat polyclonal anti-human Fc specific antibody (produced in-house) or anti-human IgG (#I1886, 1:3000, Sigma-Aldrich) diluted to 1 μg/mL in PBS. The plates were blocked with PBS/S for 1 hour at RT followed by washing 4 times with PBS/T. Samples collected from HERA experiments were added directly to wells and titrated 4 times by diluting 1:1 in PBS/S/T and incubated for 2 hours before washing as described above. Captured antibodies were detected with a polyclonal alkaline phosphatase-conjugated anti-human Fc antibody produced in goat (#A9544, 1:4000, Sigma-Aldrich). Binding was visualised by adding of 100 μL alkaline phosphatase substrate (#S0942, Sigma-Aldrich) solved in diethanolamine buffer (made in-house, pH 9.8). Absorbance was measured at 405 nm using a TECAN spectrophotometer (Sunrise).
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