Detection of anti-GMZ2 total IgG was carried out by ELISA on samples collected at inclusion before the first vaccine administration (D0) and 28 days after administration of the third dose of the vaccine (D84). ELISA was performed as described by Esen et al. with minor modifications (39 (link)). These modifications consisted of dilution of plasma sample in PBS, 3% non-fat milk, 0.1% Tween 20, and use of peroxidase conjugated goat anti-human IgG (Invitrogen) at a 1:65,000 dilution in buffer. The concentration of sHLA-G was quantified by ELISA in plasma samples collected on D0 and on D84, using a commercially-available kit (LSBio LifeSpan BioSciences, Inc), that measures the sHLA-G1 and sHLA-G5 isoform(s). Each sample was measured in duplicate. All experiments were performed under the same laboratory conditions. Each ELISA plate contained sample collected on D0 and on D84. sHLA-G final concentration was determined by optical densities measured at 450 nm by a microplate reader (Thermo Multiskan, Model 355). All other steps of the sHLA-G ELISA protocol were followed as specified in the manufacturer’s instructions (https://www.lsbio.com/elisakits/manualpdf/ls-f5033.pdf ). In addition to these immunological assays, haematology and clinical biochemistry values were measured using calibrated auto analysers with peripheral venous blood samples collected into appropriate tubes at D0.
Peroxidase conjugated goat anti human igg
Manufactured by Thermo Fisher Scientific
Peroxidase-conjugated goat anti-human IgG is a secondary antibody reagent. It is used to detect and quantify human immunoglobulin G (IgG) in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti human ig, by Southern Biotech (3 mentions)
Tmb substrate, by Thermo Fisher Scientific (2 mentions)
Rabbit anti human igm, by Jackson ImmunoResearch (2 mentions)
96 well flat bottom plate, by Corning (2 mentions)
Synergy 2 reader, by Agilent Technologies (2 mentions)
Human igg standard, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine substrate, by Thermo Fisher Scientific (1 mentions)
5 protocols using peroxidase conjugated goat anti human igg
1
Immunological Response to Anti-GMZ2 Vaccine
2
Measuring Anti-GMZ2 IgG Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The anti-GMZ2 total IgG was measured by enzyme-linked immunosorbent assay (ELISA) on isolated plasma collected at D0 and at D84 as described by Esen et al. [5 (link)] with minor modifications. These modifications consisted of the dilution of the plasma sample in PBS, 3% non-fat milk, 0.1% Tween 20, and the use of peroxidase conjugated goat anti-human IgG (Invitrogen) at a 1:65,000 dilution [22 (link)]. As reference for the assay, European malaria-naive pooled sera was taken as negative control whereas the pooled sera from Gabonese adults was used as positive control.
3
Quantification of Immunoglobulin G by ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoglobulin G concentrations in supernatants were determined by ELISA using flat-bottom 96-well half-area plates (Corning, Tewksbury, USA) coated overnight at 4°C with goat anti-human Ig (1 mg/ml; Southern Biotech, Birmingham, USA). Plates were washed with PBS/0.05%Tween-20 to remove unbound antibody and blocked with PBS/5%FCS for 2 h at room temperature. Sample and a human IgG standard (Sigma-Aldrich/Merck, Darmstadt, Germany) were added for 1.5 h at room temperature. Subsequently, plates were washed with PBS/0.05%Tween-20 and bound IgG was detected by peroxidase-conjugated goat anti-human IgG (Thermo Fisher Scientific, Landsmeer, The Netherlands). TMB Substrate (Thermo Fisher Scientific) was used to reveal peroxidase activity. The reaction was stopped with H2SO4 and optical density was measured at 450 nm.
4
ELISA for Detecting Human Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flat-bottom 96-well plates (Corning) were coated overnight with goat anti-human Ig (1 mg/ml; Southern Biotech) at 4°C, washed with PBS/0.05%Tween-20, and blocked with PBS/5% FCS for 2 hours at room temperature. Samples were added for 1.5 hours. After washing, peroxidase-conjugated goat anti-human IgG (Thermo Fisher Scientific) or rabbit anti-human IgM (Jackson ImmunoResearch) was used to detect bound antibody. TMB Substrate (Thermo Fisher Scientific) was used to reveal peroxidase activity. Reactions were stopped with sulfuric acid and optical densities were measured at 450 nm using a BioTek Synergy 2 reader (Winooski). Concentrations were calculated using standard curves that were generated for each assay.
5
Quantification of IgM and IgG in Cultured Memory B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IgM and IgG levels were determined in supernatants of memory B cells cultured for 6 days using ELISA. After overnight coating with goat anti-human Ig (1 mg/ml; Southern Biotech, Birmingham, USA) at 4 °C, flat-bottom 96-well plates (Corning, Tewksbury, USA) were washed with PBS/0.05% Tween-20 and subsequently blocked with PBS/5%FCS for 2 h at RT. Samples were added for 1.5 h at room temperature. After washing, peroxidase-conjugated goat anti-human IgG (Thermo Fisher Scientific) or rabbit anti-human IgM (Jackson, Uden, The Netherlands) were used to detect bound antibody. 3,3′,5,5′-Tetramethylbenzidine substrate (Thermo Fisher Scientific) was used to reveal peroxidase activity. Reactions were stopped with sulfuric acid and optical densities were measured at 450 nm using a BioTek Synergy 2 reader (Winooski, USA). Concentrations were calculated using standard curves for IgM and IgG.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!