G is the spike protein that generates antibody response. Hence, G protein-based ELISA method was used to check CHPV antibodies in serum sample. Serum samples from all donors were checked for IgG anti-CHPV antibodies as described previously [9 (link)]. Briefly, infected sf9 supernatants containing (rGp) of CHPV were diluted 1:10 in 50 mM carbonate buffer (pH 9.5) and were coated as 100 µl/well and kept for 2 h at 37 °C. Then, blocking solution was added and kept for 30 min at 37 °C. Following washing with wash buffer, NHS was diluted to 1:25 in blocking solution and 100 µl/well was added to wells. A 1:25 dilution of pre-immune serum served as negative control. Horseradish peroxidase conjugated goat anti-human IgG (Sigma chemicals, St Louis, MO, USA) at 1:10,000 dilution was added to each well and incubated for 30 min at 37 °C. Following the addition of tetramethylbenzidine substrate and incubation in dark for 8–10 min, reaction was stopped and absorbance was measured at 492 nm. A serum sample was considered to be reactive when optimal density (OD) was ≥ cut-off value (mean OD value for three negative controls multiplied by 3). Serum negative for IgG anti-CHPV antibodies was only included in the study.
Horseradish peroxidase conjugated goat anti human igg
Manufactured by Merck Group
Sourced in United States, Germany
Horseradish peroxidase-conjugated goat anti-human IgG is a laboratory reagent used for the detection and quantification of human immunoglobulin G (IgG) in various immunoassays. The product consists of goat-derived antibodies specific to human IgG that are conjugated to the enzyme horseradish peroxidase. This enzyme can catalyze a colorimetric reaction, allowing for the visualization and measurement of bound human IgG.
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Lab products found in correlation
Elx800, by Agilent Technologies (3 mentions)
Flat bottom 96 well microplate, by Corning (2 mentions)
Hitrap protein a hp column, by GE Healthcare (1 mentions)
293f cells, by Thermo Fisher Scientific (1 mentions)
Elisa microtiter plate, by Thermo Fisher Scientific (1 mentions)
Goat anti bovine igg, by Merck Group (1 mentions)
Wash buffer, by R&D Systems (1 mentions)
Elisa plate, by Corning (1 mentions)
Mini protean 2 apparatus, by Bio-Rad (1 mentions)
Semi dry system, by Bio-Rad (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Peroxidase substrate solution, by LGC (1 mentions)
Multiskan fc photometer, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Casein, by Merck Group (1 mentions)
14 protocols using horseradish peroxidase conjugated goat anti human igg
1
CHPV IgG Antibody Detection by ELISA
2
Production of Fc-Fused sdAbs
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The sequences of selected sdAbs were cloned into a mammalian expression vector under the control of hEF1-HTLV promotor and fused with N-terminal interleukin-2 signal peptide and C-terminal Fc region, comprising the CH2 and CH3 domains of human IgG1 heavy chain and the hinge region. Maxiprepped plasmids were transiently transfected into 293-F cells (Thermofisher) and the cells were further cultured in suspension for 6 days before harvesting antibody-containing supernatant. Fc-fused sdAbs were prepared with prepacked HiTrap® Protein A HP column (GE Healthcare). The produced Fc-fusion protein was analyzed by SDS-PAGE and the Western blot using standard protocols for dimerization, yield and purity measurement. The primary antibody used for Western blot was a horseradish peroxidase conjugated goat anti-human IgG (Sigma-Aldrich, St. Louis, MO, USA).
3
ELISA Assay for SARS-CoV-2 Antibody Detection
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ELISA protocols were developed for detecting IgG and IgM against S, N, and receptor binding domain (RBD) of SARS-CoV-2 as described previously (Ren et al., 2021 (link)). The purified full-length N protein, extracellular domain of S proteins, and RBD protein (Cat: 40589-V08B1 and 40592-V08H, Sino Biological, Beijing, China) were used as coating antigens, respectively. Horseradish peroxidase-conjugated goat anti-human IgG (Cat: A0710, Sigma-Aldrich, St. Louis, MO, USA) was diluted to 1:60,000 working solution and used as the second antibody. The optimal coating concentration of antigen and optimal plasma dilutions were 10 ng/well and 1:400, respectively. The cutoff values of IgM and IgG were 0.1 and 0.3 for N, 0.13 and 0.21 for S, and 0.1 and 0.3 for RBD, respectively, as described in previous study (Ren et al., 2021 (link)).
4
Onchocerciasis Antibody Detection by ELISA
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Analysis of serum IgG antibody levels was performed by ELISA as described previously by Mpagi et al. [55 (link)] with modifications to fit analysis with cattle sera. ELISA microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 μl of 200 ng/well O. ochengi or O. volvulus galectins, and O. ochengi and O. volvulus somatic extracts were used as positive controls. After overnight incubation, plates were washed four-times with PBS 0.05% Tween 20 (pH 7.2). Excess reactive sites were blocked for 2 h at room temperature with 200 μl of PBS/5% BSA for human sera and with PBS/5% (w/v) skimmed milk for cattle plasma analyses. Furthermore, individual human sera (n = 44: 35 males and 9 females) from over 10 years O. volvulus-infected individuals diluted at 1:1000, 1:3000, and 1:9000 or sera from 2 years old O. ochengi infected cattle (n = 9: four males and five females) diluted at 1:50, 1:100, and 1:200 were used. Sera from two European cattle not exposed to Onchocerca spp. (EC) from the University of Veterinary Medicine (Hannover, Germany) and healthy Europeans (naïve, n = 12: four males and eight females) individuals were included as negative controls. The secondary antibodies used were, horseradish peroxidase-conjugated goat anti-human IgG and goat anti-bovine IgG (Sigma, St. Louis, USA), respectively, each diluted at 1:5000. Plates were processed as described above.
5
ELISA Binding Assay for Antibody-Antigen Interactions
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To test the binding of antibodies to SOSIP.664 trimers or gp120 monomers, 96-well ELISA plates (Corning) were coated with 5 μg/ml of G. nivalis lectin (Sigma; L8275-5MG) at 4 °C overnight. Plates were washed three times with 1× wash buffer (R&D Systems) and blocked with 0.2% casein in PBS. The same washing procedure was carried out after each incubation. The SOSIP.664 trimer or gp120 monomers was added at 1 or 2 μg/ml and incubated at RT for 1 h. Serial dilutions of WT or chimeric antibodies were added to duplicate wells for 1 h at RT, followed by incubation with horseradish peroxidase-conjugated goat anti-human IgG (Sigma; A8419 or Jackson ImmunoResearch; 109-035-008). Substrate reagent and stop solution (R&D Systems) were used to reveal the signal. Light absorption at 450 nm was measured with a luminometer (PerkinElmer). All samples were tested in duplicate.
6
ELISA for Diagnosing Human Cystic Echinococcosis
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E. granulosus recombinant B8/1 antigen (rEgAgB8/1) was produced in our previous study which had a sensitivity of 93% and specificity of 92% for the diagnosis of human CE [7 (link)]. ELISA with the recombinant antigen was performed in flat-bottom 96-well microplates. The microplate was coated (100 μL/well) with a concentration of 5 μg/mL of the recombinant antigen in 0.1 M carbonate/bicarbonate (coating) buffer at 4°C overnight. The next day, after washing the microplate with PBST (phosphate-buffered saline, containing 0.05% Tween 20), wells were blocked with 3% skimmed milk for 2 h at room temperature (RT). Serum samples (1/100 dilution in PBST) were added to each well and incubated for 1 h at RT. Then, the plates were washed as before, and 100 μL of a predetermined dilution (1/4000) of the horseradish peroxidase-conjugated goat anti-human IgG (Sigma, USA) was added to the wells and incubated at RT for an hour. The plate was washed as before and incubated with a substrate solution containing 0.4 mg/mL OPD and 0.3% H2O2 in 0.1 M citrate buffer (100 μL/well) for 30 min in darkness at RT. The absorbance at 450 nm was measured, using a microplate reader (ELX800, BioTek, USA). In each run, positive and negative control sera were included. Finally, the cutoff point was set at 2SD from the mean of control samples.
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E. granulosus recombinant B8/1 antigen (rEgAgB8/1) was produced in our previous study which had a sensitivity of 93% and specificity of 92% for the diagnosis of human CE [7 (link)]. ELISA with the recombinant antigen was performed in flat-bottom 96-well microplates. The microplate was coated (100 μL/well) with a concentration of 5 μg/mL of the recombinant antigen in 0.1 M carbonate/bicarbonate (coating) buffer at 4°C overnight. The next day, after washing the microplate with PBST (phosphate-buffered saline, containing 0.05% Tween 20), wells were blocked with 3% skimmed milk for 2 h at room temperature (RT). Serum samples (1/100 dilution in PBST) were added to each well and incubated for 1 h at RT. Then, the plates were washed as before, and 100 μL of a predetermined dilution (1/4000) of the horseradish peroxidase-conjugated goat anti-human IgG (Sigma, USA) was added to the wells and incubated at RT for an hour. The plate was washed as before and incubated with a substrate solution containing 0.4 mg/mL OPD and 0.3% H2O2 in 0.1 M citrate buffer (100 μL/well) for 30 min in darkness at RT. The absorbance at 450 nm was measured, using a microplate reader (ELX800, BioTek, USA). In each run, positive and negative control sera were included. Finally, the cutoff point was set at 2SD from the mean of control samples.
7
Sandwich ELISA for Antibody Quantification
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The antibody titer in cell supernatants was measured using a sandwich-ELISA assay. The 96-well plates were coated with rabbit anti-human IgG-Fc gamma secondary (Pierce, Thermo Fisher Scientific, US) which was diluted 1:16000 in carbonate/bicarbonate buffer, pH 9.6. After an overnight incubation at 4°C, the plates were washed twice with PBS containing 0.05% tween-20 (PBST) and then blocked with 150 μl of BSA 0.5% (Roche, Germany) for one hour at 37°C. Following three washes with PBST, the plates were coated with 100 μl cells supernatants for one hour at 37°C. After three-step washes, horseradish peroxidase-conjugated goat anti-human IgG (Sigma, Germany) was diluted 1:20,000 in PBS, and 100 μl per well was added. The plates were incubated at 37°C for one hour. After washing the plates with PBST for three times, 100 μl 3,3’,5,5’-tetramethylbenzidine (TMB) (Sigma, USA) was added and developed at room temperature for fifteen minutes. The reaction was stopped by adding 100 μl H2SO4 (2N). The reaction absorbance was read at 450 nm, and protein concentration was estimated based on the human IgG1 standard curve.
8
SDS-PAGE and Western Blot Analysis
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Purified antibodies were subjected to electrophoresis on 12% SDS-PAGE gels using a Mini-PROTEAN II apparatus (Bio-Rad, USA). Twenty five microliters of each fraction were denatured and reduced in SDS sample buffer and finally loaded on the gel. The gel was stained with coomassie brilliant-blue. Following separation of samples on SDS-PAGE, western blot was performed. Samples were transferred to nitrocellulose membrane employing a semi-dry system (BioRad, USA). After an overnight incubation of the membrane with skim milk 5%, the membrane was washed three times with PBST. Later, it was incubated with 1:1000 diluted horseradish peroxidase-conjugated goat anti-human IgG (Sigma, Germany) for two hours and then washed three times with PBST. Finally, the color was developed in 3,3’-diaminobenzidine tetrahydrochloride (DAB) (Sigma, Germany) for 3–5 min. Relative intensities of the protein bands for each lane were quantified using the Quantity One software (BioRad). Furin cleavage efficiency was calculated based on the following formula: cleavage efficiency = cleaved form/(cleaved form+ uncleaved form) [24 (link)].
9
ELISA for Anti-Toxocara IgG Detection
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About a 3 mL, venous blood sample was collected from all participants in both groups. The serum was separated and tested for IgG anti-Toxocara antibodies by ELISA method using the T. canis larval excretory-secretory (E/S) antigens prepared using the method previously described (22 (link)). For the ELISA method, flat-bottom 96-well microplate (corning, USA) was coated with 5 μg/mL of Toxocara E/S antigen overnight at 4°C. Plate was washed five times with washing buffer (PBST, 0.05% Tween 20 in PBS). Blocking was performed with 5% skimmed milk in PBST for 1 hour at room temperature.
The plate was washed again and 100 μL of serum sample (1/100 dilution in PBST) was added to each well and incubated for 1 hour. After washing, 100 μL of horseradish peroxidase-conjugated goat anti-human IgG (Sigma, USA, 1/4000 dilution in PBST) was added to each well in the plate and incubated in 37 °C for 1 hour. After washing, the plate was incubated with 100 μL/well of the substrate (0.4 mg/mL OPD, 0.3% H2O2 in 0.1 M citrate buffer, pH=5.6) for 20 minutes to visualize the reaction. The optical density (OD) values were measured at a wavelength of 490 nm, using an ELISA plate reader (ELx800, Bio-Tek, USA). Positive and negative controls sera were run in each test and a cut-off point was calculated by the mean of negative controls OD value plus two standard deviations.
The plate was washed again and 100 μL of serum sample (1/100 dilution in PBST) was added to each well and incubated for 1 hour. After washing, 100 μL of horseradish peroxidase-conjugated goat anti-human IgG (Sigma, USA, 1/4000 dilution in PBST) was added to each well in the plate and incubated in 37 °C for 1 hour. After washing, the plate was incubated with 100 μL/well of the substrate (0.4 mg/mL OPD, 0.3% H2O2 in 0.1 M citrate buffer, pH=5.6) for 20 minutes to visualize the reaction. The optical density (OD) values were measured at a wavelength of 490 nm, using an ELISA plate reader (ELx800, Bio-Tek, USA). Positive and negative controls sera were run in each test and a cut-off point was calculated by the mean of negative controls OD value plus two standard deviations.
10
ELISA for Anti-Leishmania Antibody Detection
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ELISA was performed to detect anti-Leishmania antibodies in sera samples [19 (link)]. The flat-bottom 96-well microplates (Corning, USA) were coated with 100 μL/well of purified antigen at a concentration of 5 μg/mL in 0.1 M carbonate/bicarbonate (pH 9.6) buffer by overnight incubation at 4°C. Unbound antigens were removed by washing the plate five times in phosphate buffered saline-Tween 20 (PBST, pH 7.4 containing 0.05% Tween 20). Blocking was performed with 200 μL of 3% nonfat skimmed milk in PBST for 2 hours at room temperature. Then, the wells were washed, 5 times with washing buffer. Diluted serum samples (1 : 100 in PBST) were applied to the plates and incubated for 1 hour at room temperature. The plates were washed as before and 100 μL of 1 : 4000 dilution of horseradish peroxidase-conjugated goat anti-human IgG (Sigma, USA) was added to the plates and incubated for 1.5 hours at room temperature. The plates were then washed as before and incubated with substrate (100 μL/well of 0.4 mg/mL OPD, 0.3% H2O2 in 0.1 M citrate buffer, pH 5) for 20 minutes. The reaction was stopped by using 1 N H2SO4. The absorbance at 490 nm was checked with a microplate reader (Bio-Tek, ELx800). Positive sera from VL-confirmed cases were applied in each plate. The cutoff point was fixed at 2SD above the mean of control samples.
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