Antigen-specific IgG concentrations were analyzed as described previously.24 (link) Briefly, ELISA plates coated with recombinant PhtD or PcpA were incubated with duplicate serial dilutions of test sera or human AB serum standard (Sigma, St. Louis, MO, USA). Bound antibody was detected using horseradish peroxidase-conjugated goat-anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA) followed by tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO, USA). Antibody concentrations were determined by comparison with the human AB serum standard curve. The lower limit of quantitation was 0.123 ELISA units (EU)/ml for PhtD and 1.11 EU/ml for PcpA.
Horseradish peroxidase conjugated goat anti human igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase-conjugated goat anti-human IgG is a secondary antibody that binds to human immunoglobulin G (IgG) antibodies. The horseradish peroxidase enzyme conjugated to the antibody can be used in various immunoassays and detection methods to visualize the presence and distribution of human IgG.
Automatically generated - may contain errors
Lab products found in correlation
Tetramethylbenzidine substrate, by Merck Group (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Maxi binding, by SPL Life Sciences (1 mentions)
Odyssey fc, by LI COR (1 mentions)
Image studio version 5, by LI COR (1 mentions)
Bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Ab49132, by Abcam (1 mentions)
Iblot 2 gel transfer device, by Thermo Fisher Scientific (1 mentions)
Spectramax plus 384, by Molecular Devices (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Softmax pro 7, by Molecular Devices (1 mentions)
Super aquablue elisa substrate, by Thermo Fisher Scientific (1 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
8 protocols using horseradish peroxidase conjugated goat anti human igg
1
Quantification of Antigen-specific IgG Levels
2
ELISA for Anti-rK39 Antibodies
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Microplates (Maxi binding, SPL Life Sciences, Eumhyeon, South Korea) were coated with 100 µl/well of purified Li-rK39 at a concentration of 0.5 μg/ml in 0.1 M carbonate/bicarbonate buffer (pH 9.6) overnight at 4 °C. Blocking was performed for 1 h at ambient temperature with 400 μl of PBS-Blotto (0.01 M phosphate, 0.14 M NaCl, pH 7.4). Diluted serum (100 μl of 1:100 dilution in PBS-Blotto) was added to each well and incubated for 30 min at ambient temperature. After three times washing with 350 µl/well of PBS-Tween (0.01 M, pH 7.4), 100 μl of horseradish peroxidase-conjugated goat anti-human IgG (Cat No. 109-035-003, Jackson ImmunoResearch, West Grove, PA, USA), diluted 1:40,000 in PBS-Tween (0.01 M, pH 7.4), were applied to the wells and incubated for 30 min at ambient temperature. After five times washing with 350 µl/well of PBS-Tween (0.01 M, pH 7.4), 100 µl/well of TMB substrate solution (Cat No. 34029, Thermo Scientific, Rockford, IL, USA) were added. The reaction was stopped with 100 µl/well of 1 N H2SO4. The absorbance (optical density, OD) at 450 nm and 630 nm was measured with a microplate reader (Epoch, Biotek Instruments, Winooski, VT, USA). The cut-off value for positive was defined as the OD corresponding with the highest value of the Youden index (J = sensitivity + specificity − 1).
3
Exosome Characterization by Western Blot
4
Eculizumab Quantification in Serum by ELISA
5
Western Blot Analysis of JEV Antibodies
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Protein samples were analyzed using the NuPAGE Bis-Tris Gel Electrophoresis system (Life Technologies). For western blotting, proteins that had been separated by gel electrophoresis were transferred onto a PVDF membrane using an iBlot 2 Gel Transfer Device (Life Technologies). The blots were blocked for 1 h with TBS-Tween (TBST; USB Corporation, Cleveland, OH, USA) containing 5% skim milk and then reacted for 1 h with a 1:100 dilution of JEV-positive human sera in the same buffer. The JEV-positive sera were pooled from five patients, which were diagnosed by using a commercial ELISA (InBios, Seattle, WA, USA) and an in-house IgG IFA [23 (link)] at the Division of Arboviruses in the KNIH. The blot was washed three times in TBST (10 min per wash) and incubated for 1 h in a 1:2,500 dilution of horseradish peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch, Westgrove, PA, USA). After three further washes with TBST, the blot was developed using Pierce enhanced chemiluminescence detection reagents (Thermo Scientific, Waltham, MA, USA).
6
Quantifying Anti-RTN3 Antibody Titers in MS
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To validate the high antibody titers to RTN1–300, an independent enzyme-linked immunosorbent assay was performed. As antigen, a recombinant version of RTN1–300 omitting the ABD tag was used. MaxiSorp plates were coated overnight with 100 μl of RTN31–300 or BSA (5 μg/ml; no. SH30574.02, HyClone), washed five times, and blocked with 1% BSA for 1 hour. Plates were washed five times before sera from patients with MS (n = 11) and HCs (n = 11), diluted 1:30 in PBS containing 0.2% BSA and 0.05% Tween 20, and were added and incubated for 2 hours at room temperature. Plates were again washed five times before developing using horseradish peroxidase–conjugated goat anti-human IgG (catalog no. 109-035-003, Jackson ImmunoResearch) for 1 hour and secondary 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich) for 10 min before stopping the reaction with 100 μl of 0.5 M H2SO4. Plates were read at 450 nm using a SpectraMax Plus 384 (Molecular Devices, USA) using the SoftMax Pro 7.03 software (Molecular Devices). All tests were performed in duplicate. Each individual’s optical density in the BSA wells was subtracted from the RTN1–300 wells before statistical analysis.
7
ELISA for Antibody Binding Quantification
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Recombinant protein antigen was coated at a concentration of 2 µg/ml overnight at 4°C in carbonate-binding buffer. Eight 1:4 serial dilutions of antibody were prepared from a starting concentration of 10 µg/ml. Intermediate washes were performed with PBS/0.05% Tween. Plates were blocked for 1 h at 37°C with 20% FBS in PBS, and then incubated with horseradish peroxidase-conjugated goat anti–human IgG (Jackson ImmunoResearch Laboratories) for 1 h at 37°C to detect binding. Development was performed using Super AquaBlue ELISA Substrate (eBioscience) at an absorbance of OD405.
8
Quantifying Antibody Binding to Bacteria
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Bacteria from an overnight culture were washed in ice-cold PBS (VWR International) and adjusted to an OD600 nm ~ 0.1 in ice-cold PBS. ELISA plates (Nunc MaxiSorp, Nalge Nunc International, Rochester, NY) were coated with 100 µL of bacteria and incubated overnight at 4 °C. Plates with S. aureus were then blocked with 400 µg · mL−1 rabbit IgG (Sigma-Aldrich, St. Louis, MO), and plates with other bacterial species were blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) in PBS for 2 h at 4 °C and then washed 3× with 0.1% Tween-20 (vol/vol) in PBS (PBST). Serial dilutions of 100 µL of SAC55 or isotype control R347 were then applied to the plates and incubated for 2 h at 4 °C. The plates were washed 3 times with PBST, and bound antibody was detected with 100 µL of 1 µg · mL−1 horseradish peroxidase-conjugated goat-anti-human IgG (Jackson Immunoresearch Laboratory, West Grove, PA) and 3,3′,5,5′-tetramethylbenzidine substrate (KPL, SeraCare, Milford, MA). The reaction was stopped after 10 min with 100 μL 0.2 mol · L−1 H2SO4, and the OD450 was measured in a spectrophotometer (Molecular Devices, Sunnyvale, CA).
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