The frequency of HIV-specific ASC in total PBMC were determined by ELISpot similar to as previously described.65 (link),66 (link) Briefly, sterile 96-well PVDF membrane plates (MilliporeSigma, USA) were coated overnight at 4°C with 50 μL of 5 μg/ml HIV1 Env MN.B D11 gp120 or A244.AE D11 gp120 (NIH AIDS Reagent Program), 6 μg/ml FluZone (2006–2007) or 1 μg/ml anti-human IgG (Jackson Immunore-search, West Grove, PA) in PBS. Plates were blocked with RPMI1640 (Corning, VA, USA) media with 10% fetal bovine serum (Atlanta Biologicals, GA, USA) for 2 h at 37°C. Then 100,000 and 500,000 PBMCs (for Ag specific wells) or 10,000 and 20,000 PBMCs (for total IgG-specific wells) in a final volume of 200 μL were added per well in triplicate. The plates were incubated for ~40 hours at 37°C in 5% CO2 and then washed with PBS containing 0.1% Tween 20. Bound antibodies were detected with 50 μL of 1 μg/ml alkaline phosphatase-conjugated anti-human IgG (diluted in PBS containing 0.1% Tween 20 and 1% BSA) antibody (Jackson Immunore-search, West Grove, PA) for 2 hours at 37°C in 5% CO2 and then developed with VECTOR Blue, Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA). The spots per well were counted using the CTL immunospot reader (Cellular Technologies Ltd., Shaker Heights, OH, USA).
Alkaline phosphatase conjugated anti human igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alkaline phosphatase-conjugated anti-human IgG is a laboratory reagent used to detect and quantify human immunoglobulin G (IgG) in samples. The product consists of anti-human IgG antibodies conjugated to the enzyme alkaline phosphatase, which can be used to catalyze a colorimetric or chemiluminescent reaction for the identification and measurement of human IgG.
Automatically generated - may contain errors
Lab products found in correlation
Vector blue alkaline phosphatase substrate kit 3, by Vector Laboratories (2 mentions)
Anti human igg, by Jackson ImmunoResearch (2 mentions)
Pvdf membrane plates, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Rpmi 1640, by Corning (2 mentions)
Anti igg, by Jackson ImmunoResearch (1 mentions)
Elispot plate, by Merck Group (1 mentions)
96 well half area plate, by Corning (1 mentions)
O phenylenediamine opd, by Merck Group (1 mentions)
96 well assay plate, by Corning (1 mentions)
Gam broth, by Nissui Pharmaceutical (1 mentions)
7 protocols using alkaline phosphatase conjugated anti human igg
1
Quantifying HIV-specific Antibody-secreting Cells by ELISpot
2
Quantifying Antibody Responses to Vaccines
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Response to vaccination was determined by standard ELISAs for IgG levels against tetanus toxoid (TT), diphtheria toxoid (DT), Hepatitis B virus (Hep B), and Hib (using its polyribitol phosphate (PRP) antigen) [34 (link)–36 (link)]. Briefly, ELISA plates were coated with 1 µg/ml of TT (Massachusetts Biolabs, Cambridge, MA); 0·5 LF/ml (flocculation unit/ml) of DT (diphtheria Antitoxin Human Serum NIBSC code: 00/496); 1µg/ml of Hep B surface antigen (adw subtype, Fitzgerald Industries, Concord, MA, USA) or 1 µg/ml H. influenza type b oligosaccharide-human serum albumin conjugate (ATCC: NR-12268) and incubated overnight at 4ºC. After blocking and washing, diluted plasma samples were added to the plates and incubated for 1 h at room temperature. The plates were washed and alkaline phosphatase-conjugated anti-human IgG (Jackson ImmunoResearch, Malvern, PA) was added at 1:1000 for 1 h at 37°C. Substrate orthophenoylenediamine (o-p-NN) was added after the final wash. The reaction was stopped by adding 5% EDTA, and absorbance was read at 405 nm with an ELISA reader. For all assays, standard sera obtained from NIBSC (National Institute of Biological Standards and Control) were used to create standard curves for determination of the subjects’ anti-antigen IgG concentrations.
3
ELISPOT Assay for Quantifying Secreted Antibodies
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Serial diluted B cells were transferred directly to anti-IgG (Jackson ImmunoResearch) coated ELISPOT plates (Millipore) for 6 hr. Bound Ab was detected with alkaline phosphatase-conjugated anti-human IgG (Jackson ImmunoResearch) followed by development with alkaline phosphatase substrate (Moss, Inc). ELISPOTs were visualized using a CTL ELISPOT reader. The number of spots detected per well (following correction for non-specific background) was calculated.
4
SARS-CoV-2 RBD Antibody Binding Assay
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96-well half-area plates (Corning) were coated with 25 µl of 5 µg/ml of recombinant His-tagged WT or BA.1 RBD diluted in PBS overnight at 4 °C. The next day, wells were blocked with 50 µl of 3% bovine serum albumin (BSA) in 1X PBS for 1 h at room temperature, and subsequently, washed two times using wash buffer (1X PBS, 0.05% Tween-20). Next, antibody titrations diluted in 1% BSA, 0.05% Tween-20 in 1X PBS were added to plates and incubated for 1 h at 37 °C before washing three times with wash buffer. Antigen-bound antibodies were detected using alkaline phosphatase-conjugated anti-human IgG (Jackson ImmunoResearch, Cat # 109-055-098) diluted 1:1000 in 1% BSA, 0.05% Tween-20 in 1X PBS for 1 h at 37 °C. Plates were then washed three times and developed with 25 µl of alkaline phosphatase staining buffer (pH 9.8) for 15 min. Absorbance was measured at 405 nm using a spectrophotometer (VersaMax). Experiments were performed in duplicate using the same IgG preparations, and the area under the curve was calculated after subtracting background absorbance from a no-antibody control.
5
Quantification of HIV-specific Antibody-Secreting Cells
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The frequency of HIV-specific ASC in total PBMC were determined by ELISpot similar to as previously described.65 (link),66 (link) Briefly, sterile 96-well PVDF membrane plates (MilliporeSigma, USA) were coated overnight at 4°C with 50 μL of 5 μg/ml HIV1 Env MN.B D11 gp120 or A244.AE D11 gp120 (NIH AIDS Reagent Program), 6 μg/ml FluZone (2006-2007) or 1 μg/ml anti-human IgG (Jackson Immunoresearch, West Grove, PA) in PBS. Plates were blocked with RPMI1640 (Corning, VA, USA) media with 10% fetal bovine serum (Atlanta Biologicals, GA, USA) for 2 h at 37°C. Then 100,000 and 500,000 PBMCs (for Ag specific wells) or 10,000 and 20,000 PBMCs (for total IgG-specific wells) in a final volume of 200 μL were added per well in triplicate. The plates were incubated for ∼40 hours at 37°C in 5% CO2 and then washed with PBS containing 0.1% Tween 20. Bound antibodies were detected with 50 μL of 1 μg/ml alkaline phosphatase-conjugated anti-human IgG (diluted in PBS containing 0.1% Tween 20 and 1% BSA) antibody (Jackson Immunoresearch, West Grove, PA) for 2 hours at 37°C in 5% CO2 and then developed with VECTOR Blue, Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA). The spots per well were counted using the CTL immunospot reader (Cellular Technologies Ltd., Shaker Heights, OH, USA).
6
Quantifying Influenza-Specific Antibody Responses
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The frequency of influenza antigen-specific antibody-secreting cells (ASCs) was measured by ELISpot assay as previously described (64 (link)). Briefly, ELISpot assay plates were coated overnight with either recombinant NA (5 μg/ml B/Florida/04/2009 or B/Hong Kong/330/2001 virus; BEI Resources, Manassas, VA, USA) or Fluzone IIV vaccine (6 μg/ml, Sanofi Pasteur Inc., Swiftwater, PA, USA) and incubated at 37°C for ∼40 h with 500,000 or 100,000 PBMC. Bound antibodies were detected with alkaline phosphatase-conjugated anti-human IgG (Jackson Immunoresearch) (1 μg/ml). Spots in each well were counted using a CTL immunospot reader (Cellular Technologies Ltd., Shaker Heights, OH, USA).
7
Measuring P. gingivalis and F. nucleatum Antibodies
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P. gingivalis strain 381 and F. nucleatum, which were used as antigens in our experiment, were cultured anaerobically and grown in GAM broth (Nissui, Tokyo, Japan) at 37 °C. The levels of antibodies specific for P. gingivalis and F. nucleatum in the serum specimens were determined by a modified ELISA. The bacterial cells were grown to an optical density of 0.1 at 660 nm and extracted. The P. gingivalis or F. nucleatum antigens were coated onto 96-well assay plates (Corning, Cambridge, MA, USA) using 100 ng/well of sonicated extracts. Diluted serum samples (1:50) were applied to the wells precoated with antigen as described above and were incubated at 4 °C for 16 h. Bound human IgG was detected with an alkaline phosphatase-conjugated anti-human IgG (Jackson ImmunoResearch, West Grove, PA) followed by development with o-phenylenediamine (OPD) (Sigma, St. Louis, MO, USA). The reproducibility of the ELISAs was validated by testing and analyzing the intra- and interplate differences. The antibody levels were expressed as absorbance units.
The Il-6 serum levels were examined using a highly specific quantitative sandwich ELISA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The optical density was measured at 450 nm using a microplate reader. The concentrations of IL-6 were calculated on the basis of a standard curve.
The Il-6 serum levels were examined using a highly specific quantitative sandwich ELISA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The optical density was measured at 450 nm using a microplate reader. The concentrations of IL-6 were calculated on the basis of a standard curve.
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