96-well plates were coated overnight at 4°C with one of the following: NPDP19 at 50 ng per well, NANP5 at 125 ng per well or PfCSP 25 ng per well. Plates were washed 5 times with 0.05% Tween 20 in PBS, blocked with 100 µl of 3% BSA in PBS for 1 h at room temperature (RT), and washed again prior to incubation with 1:3 or 1:5 serially diluted mouse serum samples for 1 h at RT. Wells were washed and incubated with Alkaline Phosphatase AffiniPure Goat Anti-Mouse IgG (Jackson Immuno Research) at 1:1,000 in PBS with 0.5% BSA for 1h at RT. p-Nitrophenyl phosphate dissolved in ddH2O (50 µl/well, RT, 25 min) was used for detection. A chimeric version of the anti-PfCSP antibody 2A10 (Fisher et al., 2017 ; Hollingdale et al., 1984 (link)) with human Ig heavy and Ig kappa and the fully human mature anti-PfCSP CIS43 antibody were used as standard reference materials. ELISA curves were calculated and analyzed using GraphPad Prism 8.4.3 (GraphPad).
Alkaline phosphatase affinipure goat anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alkaline Phosphatase AffiniPure Goat Anti-Mouse IgG is a secondary antibody conjugated with alkaline phosphatase enzyme. It is designed for use in immunoassay applications that require detection of mouse immunoglobulin G (IgG).
Automatically generated - may contain errors
Lab products found in correlation
Prism 8, by GraphPad (2 mentions)
P nitrophenyl phosphate, by Merck Group (2 mentions)
Synergy neo2, by Agilent Technologies (2 mentions)
Prism 9, by GraphPad (1 mentions)
Immobilon p pvdf, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
4 protocols using alkaline phosphatase affinipure goat anti mouse igg
1
Comparative ELISA of anti-PfCSP Antibodies
2
Quantifying Antigen-Specific IgG Titers
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To assess the IgG titers of immunized mice, 200 ng per well of either H3ssF, H7ssF, or their respective epitope KO versions, were pre-coated in 96-well plates at 4°C overnight. After incubation with blocking buffer (3% BSA in PBS + 0.01% tween 20) for 2 h at RT, 3-fold serial diluted sera (7 dilutions) with starting dilution 1:100 from priming or boosting mice were incubated with pre-coated protein for 2 h at RT. Wells were washed and incubated with Alkaline Phosphatase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, #115–055-071) at 1:1,000 in PBS with 0.5% BSA for 1 h at RT. P-Nitrophenyl phosphate (Sigma, # N2770) dissolved in ddH2O (50 μL/well, RT, 25 min) was used for detection. Absorbance at 405 nm was determined with a plate reader (Synergy Neo2, BioTek). ELISA curves were calculated and analyzed using GraphPad Prism 9.5.1 (GraphPad).
3
Antigen-Specific Antibody Titration ELISA
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Antigen specific antibody titers were detected by ELISA, using anti-His Ab (2 μg/ml) to capture GT2, GT5 or -KO antigen (2 μg/ml) on 96-well. Plates were washed 5 times with 0.05% Tween 20 in PBS, blocked with 100 μL of 3% BSA in PBS for 1 h at room temperature (RT), and washed again prior to incubation with 1:2 or 1:5 serially diluted mouse serum samples for 1 h at RT. Wells were washed and incubated with Alkaline Phosphatase AffiniPure Goat Anti-Mouse IgG (Jackson Immuno Research,#115-055-071) at 1:1,000 in PBS with 0.5% BSA for 1 h at RT. p-Nitrophenyl phosphate (Sigma, # N2770) dissolved in ddH2O (50 μL/well, RT, 25 min) was used for detection. Absorbance at 405 nm was determined with a plate reader (Synergy Neo2, BioTek). ELISA curves were calculated and analyzed using GraphPad Prism 8.4.3 (GraphPad).
4
Western Blot Analysis of PGRN Protein
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Equal amounts of total protein were run on 8% polyacrylamide gels at 70–90 volts. Proteins were transferred to Immobilon-P PVDF membranes (Merck Millipore, Burlington, MA, USA) for 2 h at 1000 mA and blocked at RT for 1 h in 5% non-fat milk diluted in TBS 1× (25 mM Tris-HCl, 150 mM NaCl) with 0.1% Tween-20 (TBS-T), following primary antibody anti-PGRN (1:1000, Abcam ab208777, Cambridge, UK) incubation at 4 °C overnight. After 3 washes in TBS-T, the membranes were incubated with the appropriate secondary antibody (1:10,000) at RT for 2 h. The membranes were then washed 3 times in TBS-T and incubated with Vistra EFC (Enhanced Chemifluorescence) substrate (Merk, Darmstadt, Germany ) for 5 min at RT. The fluorescence signal was visualized using a ChemiDoc System (Bio-Rad, Hercules, CA, USA) and analysis was performed using the Image Lab Software (Bio-Rad, Hercules, CA, USA). Anti-GAPDH (1:5000, Merck Millipore MAB374, Burlington, MA, USA) was used as a loading control. The secondary antibodies employed were alkaline phosphatase affinipure goat anti-mouse IgG (Jackson Immunoresearch, #115-055-146, West Grove, PA, USA) and alkaline phosphatase affinipure mouse anti-rabbit IgG (Jackson Immunoresearch, #211-055-109, West Grove, PA, USA).
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