IgG reactivity against recombinant proteins was measured by means of enzyme-linked immunosorbent assay (ELISA) as described elsewhere [20 (link)]. Briefly, plasma samples (1:400) followed by horseradish peroxidase–conjugated rabbit anti-human IgG (1:3000; Dako) were added to 96-well flat-bottom microtiter plates (Nunc MaxiSorp; Thermo Fisher Scientific) previously coated with recombinant proteins. Bound antibodies were detected by adding TMB PLUS2 (Eco-Tek), and the reaction stopped with 0.2 mol/L sulfuric acid. The optical density was read at 450 nm (HiPo MPP-96 microplate reader; Molecular Devices), and the specific antibody levels were calculated in arbitrary units, as described elsewhere [20 (link)]. Plasma samples from Danish adults without malaria exposure and a pool of Ghanaian adults with previous P. falciparum infection were included as negative and positive controls, respectively. Negative cutoff values were calculated as the mean arbitrary unit values plus 2 standard deviations obtained with the negative control samples.
Horseradish peroxidase conjugated rabbit anti human igg
Manufactured by Agilent Technologies
Sourced in United States
Horseradish peroxidase-conjugated rabbit anti-human IgG is a laboratory reagent that consists of rabbit-derived antibodies directed against human immunoglobulin G (IgG), which are conjugated to the enzyme horseradish peroxidase. This product is commonly used in various immunoassay techniques, such as enzyme-linked immunosorbent assays (ELISA), to detect and quantify the presence of human IgG in biological samples.
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Lab products found in correlation
Nunc maxisorp, by Thermo Fisher Scientific (5 mentions)
Microtiter plate, by Greiner (2 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Nunc immuno maxisorp, by Thermo Fisher Scientific (1 mentions)
Spectracount, by Hewlett-Packard (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by Agilent Technologies (1 mentions)
Dulbecco s phosphate buffered saline, by Lonza (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Abcam (1 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
Duo set antibody pairs, by R&D Systems (1 mentions)
Polyclonal rabbit anti human igg, by Agilent Technologies (1 mentions)
Magellan software, by Tecan (1 mentions)
Rabbit anti human igg, by Merck Group (1 mentions)
Mouse anti human igg2, by Merck Group (1 mentions)
14 protocols using horseradish peroxidase conjugated rabbit anti human igg
1
ELISA Assay for IgG Reactivity
2
ELISA-based Antibody Quantification
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Immunoglobulin G reactivity against recombinant proteins was measured by enzyme-linked immunosorbent assay as described elsewhere [31 (link)]. In brief, 96-well, flat-bottom, microtiter plates (Nunc MaxiSorp; Thermo Fisher Scientific) were coated with 100ng/well recombinant protein in Dulbecco’s phosphate-buffered saline ([PBS] Sigma-Aldrich) and incubated overnight at 4°C. After blocking (washing buffer with 1% Ig-free bovine serum albumin [BSA]), plasma samples (1:400) were added in duplicate, followed by horseradish peroxidase-conjugated rabbit antihuman IgG (1:3000; Dako). Bound antibodies were detected by adding TMB PLUS2 (Eco-Tek), and the reaction was stopped by adding 0.2 M H2SO4. The optical density (OD) was read at 450nm (VERSAmax microplate reader; Molecular Devices), and the specific antibody levels were calculated in arbitrary units (AUs) using the equation 100 × [(ODSAMPLE-ODBLANK)/(ODPOS.CTRL-ODBLANK)], essentially as described elsewhere [31 (link)]. Negative cutoff values were calculated as the mean AU values plus 2 standard deviations (SD) obtained with the negative control samples described above. Individuals were considered responders if their specific antibody level was higher than the cutoff. The breadth of antibody response was defined as the number of antigens recognized by an individual [36 (link)].
3
Detecting Anti-IFI16 Antibodies via ELISA
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To detect anti-IFI16 antibodies, polystyrene micro-well plates (Nunc-Immuno MaxiSorp; Nunc) were coated with a solution of recombinant IFI16 in PBS and, after blocking, sera were added in duplicate. After washing, horseradish peroxidase-conjugated rabbit anti-human IgG (Dako Cytomation, Carpinteria, CA, USA) was added. Following the addition of the substrate (TMB; KPL), absorbance was measured at 450 nm using a microplate reader (SpectraCount, Packard). The background reactivity of the reference mixture was subtracted to calculate the results. A standard curve was constructed by serially diluting IgG from an anti-IFI16-positive patient serum [16 (link), 17 (link)].
4
IgG Reactivity and ELISA Quantification
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IgG reactivity against FV2BIC, FV2CHO, FV6BIC, and PvDBP was measured in duplicate by an ELISA as described previously (18 (link), 28 (link), 54 (link)). Briefly, 96-well flat-bottom microtiter plates (Nunc MaxiSorp) were coated with recombinant protein in Dulbecco's phosphate-buffered saline (Lonza). After blocking and washing, human plasma samples (1:400), human and mouse monoclonal antibodies (0.08 to 10 μg/ml), or rabbit serum (1:20 to 1:2,560) was added, followed by horseradish peroxidase-conjugated rabbit anti-human IgG (1:3,000; Dako), goat anti-mouse IgG (1:1,000; ThermoFisher), or goat anti-rabbit IgG (1:3,000; Dako). Bound antibodies were detected by adding tetramethylbenzidine (TMB PLUS2; Eco-Tek), and the reaction was stopped by the addition of 0.2 M H2SO4 to the mixture. The optical density (OD) was read at 450 nm, and the specific antibody levels were calculated in arbitrary units (AU), as described previously (18 (link)). Negative cutoff values were calculated as the mean AU values plus 2 standard deviations (SD) obtained with the Danish control samples (set 6) (Table 1 ).
5
Autoantibody Detection by ELISA Assay
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Autoantibodies were detected by ELISA according to previously described methods [30 (link)]. Briefly, TAAs plus a fusion tag only (BirA-6xHis) control were coated on to the wells of microtiter plates at either two (160 nM and 50 nM, Discovery study) or five concentrations (160 nM, 50 nM, 16 nM, 5 nM and 1.6 nM, Confirmation study) in duplicate. All patient specimens were diluted 1 in 110 in blocking buffer and allowed to react with the immobilised TAAs. After plate washing, the presence of IgG AAbs was detected using horseradish peroxidase-conjugated rabbit anti-human IgG (Dako), 3,3′,5,5′-tetramethylbenzidine chromogenic substrate (Merck) and absorbance/optical density (OD) measured at 650 nm wavelength. Circulating AFP was measured using a commercially available ELISA (Aviva Systems Biology, OKBA00002).
6
Autoantibody Detection via ELISA
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Autoantibodies were detected by ELISA according to previously described methods [34] (link). The BirA control was included to allow subtraction of any assay signal due to nonspecific binding.
In brief 41 TAAs/antigenic fragments (see supporting information), and 2 assay controls (BirA and buffer only) were adsorbed to ELISA plates in duplicate and at 2 concentrations of antigen (100 nM and 50 nM). These proteins were then incubated with serum samples from cancer, high-risk and healthy control cohorts (Table 1 ) diluted 1 in 110 in blocking buffer. The presence of an IgG response to the antigens was detected with horseradish peroxidase-conjugated rabbit anti-human IgG (Dako) and 3,3′,5,5′-tetramethylbenzidine, as previously described [29] . All assays were conducted on a semi-automated robotic system and cancer, high-risk and healthy control samples were interspersed. Incubations with anti-His monoclonal antibody (AbCam) and, where available antigen-specific monoclonal antibodies (Sigma, AbCam, Santa-Cruz), were carried out to validate antigen plate coating. SDS-PAGE analysis of TAA plate-coating solutions was also carried out to verify plate layouts and protein dilutions.
In brief 41 TAAs/antigenic fragments (see supporting information), and 2 assay controls (BirA and buffer only) were adsorbed to ELISA plates in duplicate and at 2 concentrations of antigen (100 nM and 50 nM). These proteins were then incubated with serum samples from cancer, high-risk and healthy control cohorts (
7
Quantification of VAR2CSA-specific IgG by ELISA
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Levels of VAR2CSA-specific IgG were assessed by ELISA as previously described68 (link). In brief, 96-well flat-bottom microtiter plates (Nunc MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4 °C with full-length VAR2CSA (100 ng/well in PBS). Monoclonal antibody (0.08–10 μg/mL) or plasma samples (1:400) were added in duplicate, followed by washing and horseradish peroxidase-conjugated rabbit anti-human IgG (1:3,000; Dako). Bound antibodies were detected by adding TMB PLUS2 (Eco-Tek), and the reaction stopped by the addition of 0.2 M H2SO4. The optical density (OD) was read at 450 nm and the specific antibody levels were calculated in arbitrary units (arb. units), using the equation 100 × [(ODSAMPLE − ODBLANK)/(ODPOS.CTRL − ODBLANK)].
8
Serological Analysis of Malaria Antigens
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Samples were assayed for anti-malarial IgG antibody responses to P. falciparum and P. vivax merozoite surface protein (MSP-119) and apical membrane antigen 1 (AMA-1) recombinant protein [11 (link)] by enzyme-linked immunosorbent assay (ELISA) at the Amhara Region Health Research Laboratory Centre. Three-millimetre diameter circles were punched from DBS and reconstituted in PBS buffer as previously described [10 (link), 17 (link)]. Plates were coated with antigens at a concentration of 0.5 μg/mL overnight. Eluates from test samples were added at a dilution of 1:1000 and species-specific positive controls sera were added at serial dilutions on each plate to generate standard curves. After overnight incubation, horseradish-peroxidase-conjugated rabbit-anti-human IgG (DAKO, USA) secondary antibody was added followed by substrate Tetramethylbenzidine (TMB) (SurModics, USA) as color detection. Optical density (OD) values were read at 450 nm after 15 min incubation with the substrate.
9
IgG Antibody Quantification Against Malaria Proteins
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Plasma IgG levels against four recombinant P. falciparum proteins and a crude parasite lysate were measured using indirect ELISA, as previously described (Lopez-Perez et al., 2021 (link)). Briefly, 96-well flat-bottom microtitre plates (Nunc MaxiSorp; Thermo Fisher Scientific) were coated with 50 μL of recombinant protein (0.5 μg/mL) or crude antigen lysate (5 × 105 IEs/mL) in PBS and incubated overnight at 4 °C. After blocking with 1% BSA/PBS, plasma samples (1:300) were added in duplicate, followed by horseradish peroxidase-conjugated rabbit anti-human IgG (1:3000; Dako, Denmark). Bound plasma antibodies were detected by adding TMB (abcam, UK), stopping the reaction with 0.2 M H2SO4. The optical density (OD) was read at 450 nm (Varioskan LUX; Thermo Fisher, USA). The specific antibody levels were calculated in arbitrary units (AU), using the equation [(ODSAMPLE - ODBLANK)/ (ODPOSITIVE CONTROL - ODBLANK)] × 100 (Guitard et al., 2008 (link)). Plasma samples from malaria-unexposed Danish individuals and malaria-exposed Ghanaian children were included as negative and positive controls, respectively.
10
Anti-titin antibody ELISA protocol
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An in-house ELISA was used for the detection of anti-titin antibodies mostly as previously described [12 (link)]. In detail, 96-well plates (Immulon 2 HB) were coated with the titin recombinant fragments (or synthetic peptides) or BSA, in 0.01 M carbonate buffer pH 9.6 (0.5 μg peptide per well) for 90 min at RT. Then, they were washed 4 times with PBS-Tween 0.05% and once with PBS. The plates were blocked with 4% BSA in PBS for 1 h at RT, before being washed again as previously. Next, 100 μL of diluted sera (1/100 in 4% BSA in PBS) were added and incubated for 90 min at RT. Then, horseradish peroxidase-conjugated rabbit anti-human IgG (Dako), diluted 1/5000 in 4% BSA in PBS was added and incubated for 90 min, RT, followed by washes and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The reaction was stopped with 0.3 M sulphuric acid after 10 min and color development was quantified at 450 nm.
For the antibody competition assays, sera (at dilution 1/100 in 4% BSA in PBS) were treated with 5 μg of a titin fragment for 90 min, followed by their incubation with the immobilized titin fragment in the ELISA plate for the subsequent ELISA procedure as above.
For the antibody competition assays, sera (at dilution 1/100 in 4% BSA in PBS) were treated with 5 μg of a titin fragment for 90 min, followed by their incubation with the immobilized titin fragment in the ELISA plate for the subsequent ELISA procedure as above.
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