To detect IgG against surface proteins of non-infected RBCs, each well of a 96-well, flat-bottomed, polystyrene microplate (Corning Incorporation, Corning, NY, USA) was coated with approximately one million RBCs (obtained from a healthy non-exposed individual whose blood type is O+) diluted in PBS containing 1 % (w/v) BSA (PBS/BSA), following an overnight incubation at 4 °C. After five washes with PBS the plate was blocked with 5 % BSA for 2 h at 37 °C. Plates were washed again and were incubated with sera samples in duplicate diluted 1:100 in PBS/BSA for 2 h at 37 °C. Wells were washed again and then incubated with HRP-conjugated anti-human IgG antibody (Sigma-Aldrich) diluted 1:500 in PBS/BSA for 90 min at 37 °C. The binding was revealed with OPD substrate as described in the section ‘Detection of total IgG’. The levels of specific IgG were expressed as reactivity index (RI), which was calculated as the ratio between the mean optical density (OD) by each sample duplicate and the mean OD plus three standard deviations of samples from 11 malaria-naïve volunteers never exposed to malaria.
Hrp conjugated anti human igg antibody
Manufactured by Merck Group
Sourced in United States
The HRP-conjugated anti-human IgG antibody is a laboratory reagent used to detect and quantify the presence of human immunoglobulin G (IgG) in various samples. It consists of an anti-human IgG antibody coupled with the enzyme horseradish peroxidase (HRP), which can catalyze a colorimetric reaction for visualization and measurement purposes.
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Lab products found in correlation
Flat bottomed polystyrene microplate, by Corning (1 mentions)
Tmb substrate kpl, by LGC (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Streptavidin peroxidase polymer, by Merck Group (1 mentions)
Pgex 4t 1 vector, by GE Healthcare (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit antibody, by Merck Group (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Rna extraction kit, by GeneAll (1 mentions)
Ampicillin, by Duchefa Biochemie (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Duchefa Biochemie (1 mentions)
6 protocols using hrp conjugated anti human igg antibody
1
Anti-RBC Surface IgG Detection
2
SARS-CoV-2 Spike Antibody ELISA
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Recombinant Spike (Sp) and receptor binding domain (RBD)-specific antibodies were determined by ELISA, as previously described (11) with minor modification. Briefly, 1 µg/ml Sp or 0.5 µg/ml RBD antigen (both prepared in-house) were used to coat 96-well ELISA plates (100 µl/well). After blocking plates, 100 µl/well of 1:1000-diluted serum samples were added to wells. After 2 h incubated at RT, plates were washed and wells received 100 µl/well HRP-conjugated anti-human IgG antibody (Sigma-Aldrich) then incubated 1 h at RT. After washing, TMB substrate (KPL, SeraCare, Gaithersburg, MD, USA) was added and incubated for 10 min at RT before the reaction was stopped with 100 µl 1 M Phosphoric Acid. The optical density was measured at 450 nm (OD450nm) using a VersaMax plate reader and analyzed using SoftMax Pro Software. Average OD450nm values obtained from negative control wells were subtracted.
3
SARS-CoV-2 Antibody Binding Assay
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ELISA plates were coated with SARS-CoV-2 RBD protein, S1 protein, S2 protein, S protein trimer, and mutant S1 protein (Jiangsu East-Mab Biomedical Technology, China) at 4 °C overnight. Following washing with PBST, serial dilutions of testing antibodies start at 1 μg/mL or serial dilutions of plasma start at 1:100 were added to each well and incubated at 37 °C for 30 min. After washing with PBST, horseradish peroxidase (HRP)-conjugated anti-human IgG antibody (Sigma, USA) was added at the dilution of 1:20,000 and incubated at 37 °C for 30 min. The absorbance was detected at 450 nm. The data were analyzed using GraphPad Prism 8.0.
4
SDS-PAGE, BN-PAGE, and Western Blot Analysis
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blue-native PAGE (BN-PAGE), and western blot analyses were performed as described elsewhere (55 (link)). For western blot analysis, samples were separated through 3%–12% NuPAGE Gels and transferred onto polyvinylidene difluoride membranes (Invitrogen). The membranes were blocked in a solution of Tris-buffered saline containing 5% non-fat dry milk and 0.05% Tween 20 and subsequently probed with anti-Env antibodies (2G12 and VRC01) or biotinylated-goat anti-mouse C3d polyclonal antibody. Proteins were visualized with HRP-conjugated anti-human IgG antibody or streptavidin-peroxidase polymer according to the manufacturer’s instructions (Sigma).
5
Serological detection of Leptospirosis
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Recombinant proteins (500 ng/well) were immobilized into ELISA plates, which were washed with PBS-T and blocked with PBS-T/milk for 2 h at 37°C. Leptospirosis patient’s serum samples at the onset (MAT-, n = 20) or convalescent phase (MAT+, n = 20) were diluted (1:100) in PBS-T/milk and then incubated for 1 h at room temperature. Specificity was evaluated by employing serum samples of patients diagnosed with dengue (n = 10), Chagas disease (n = 10) malaria (n = 10) and HIV (n = 10). Reactivity was assessed using HRP-conjugated anti-human IgG antibody (1:5,000, Sigma-Aldrich). Reaction was detected by adding 1 mg/mL o-phenylenediamine dihydrochloride (OPD, P8287, Sigma-Aldrich) in citrate phosphate buffer (pH 5.0) plus 1 μL/mL H2O2. The reaction carried out for 10 min and was stopped by the addition of 50 μL 2 M H2SO4. The serum samples used in this experiment were previously treated with 10% E. coli whole cell lysate in PBS-T/milk solution in order to avoid reactivity with anti-E. coli antibodies, as described previously [34 ]. The cut-off was calculated based on the mean absorbance obtained with commercial normal human serum (NHS, A3912, Sigma-Aldrich) plus three times the standard deviation (SD) between the same samples [40 ]. The samples with values above the cut-off were considered positive for this experiment.
6
Recombinant Protein Expression Protocol
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Horseradish peroxidase (HRP)-conjugated anti-rabbit antibody and HRP-conjugated anti-human IgG antibody were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Rabbit monoclonal GST antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). The pGEX-4T-1 vector and BL21 (DE3) pLysS E. coli were from GE Healthcare (Little Chalfont, UK). RNA extraction kit and plasmid prep kit were from Gene All (Seoul, Korea). The cDNA synthesis kit was from Clontech Laboratories (Mountain View, California, USA). DH5α E. coli, PCR synthesis kit, T4 ligation kit and restriction enzyme digestion kit were from Enzynomics (Seoul, Korea). Primers were synthesized in Bioneer Corporation (Daejeon, Korea). Isopropyl β-D-1-thiogalactopyranoside (IPTG) and ampicillin were from Duchefa Biochemie (Haarlem, The Netherlands).
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