2DE of biotinylated material was performed as reported in [15 (link)], proteins were transferred to nitrocellulose membrane and incubated for 12 hr at 4°C either with discovery Crc patient pooled sera or with control subject pooled sera (1:300 dilution), both from our previous study [15 (link)]. Immunodetection was revealed with HRP-conjugated anti-human-IgG (Southern Biotechnology) followed by ECL reaction and film exposure. Preparative 2DE was stained with Coomassie blue; gels or films were acquired with Laser-Densitometer (Molecular Dynamics), and spot patterns investigated using Progenesis-PG240 software (Nonlinear Dynamics).
Hrp conjugated anti human igg
Manufactured by Southern Biotech
Sourced in United States
HRP conjugated anti-human IgG is a laboratory reagent used to detect and quantify human immunoglobulin G (IgG) in various immunoassays. It consists of an anti-human IgG antibody conjugated to the enzyme horseradish peroxidase (HRP). This conjugate can be used to signal the presence of human IgG in samples, enabling its identification and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Laser densitometer, by GE Healthcare (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab39178, by Abcam (1 mentions)
Ab8813, by Abcam (1 mentions)
Ab39153, by Abcam (1 mentions)
Ab39163, by Abcam (1 mentions)
Can get signal 2, by Toyobo (1 mentions)
Opd substrate, by Merck Group (1 mentions)
Polysorp 96 well plates, by Corning (1 mentions)
Cpass kit, by GenScript (1 mentions)
H2so4, by Merck Group (1 mentions)
1 step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions)
Microlon plates, by Greiner (1 mentions)
7 protocols using hrp conjugated anti human igg
1
Immunoblotting Workflow for Biomarker Discovery
2
SARS-CoV-2 Antibody ELISA Protocol
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ELISAs were performed as previously described in Goenka et al13 (link) and Halliday et al18 , based on the methodology described previously1 (link). Spike, RBD and N-protein were each diluted in sterile PBS (Sigma) and MaxiSorp plates (NUNC) were coated with either 10 mg/mL (spike) or 20 mg/mL (RBD; N-protein) of protein overnight at 4 °C before use. Plates were blocked with a 1 h incubation in 3% Bovine Serum Albumin (BSA) (Sigma-Aldrich) in PBS with 0.1% Tween-20 (Sigma-Aldrich) (PBS-T) at room temperature. Serum samples were thawed on ice before use, tested in duplicate and diluted to a final volume of 100 µL per well at a pre-optimised dilution, either at 1 in 50 (IgA) or 1 in 450 dilution (IgG), in dilution buffer (1% BSA in PBS-T). All samples were tested on a single plate for each antigen and antibody isotype combination. Secondary antibodies were used as follows with the dilution factor indicated: HRP conjugated anti-human IgG (Southern Biotech: 1 in 25,000) and IgA (Sigma: 1 in 6,000- 10,000). SIGMA FAST TM OPD (o-phenylenediamine dihydrochloride) (Sigma-Aldrich) was used to develop plates and reactions were stopped after 30 min with 3 M HCl. ODs were read at 492 nm and 620 nm using the same reader used for salivary ELISAs.
3
Autoantibodies against ADAM10 in Colorectal Cancer
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Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative. Membrane strips were incubated for 2 hr at 20°C with IgG purified from Crc patients sera (50 μg/ml of IgG working dilution) and reactivity revealed with HRP-conjugated anti-human-IgG (Southern Biotechnology) followed by ECL reaction and film exposure. As controls, the reactivity of the rabbit anti-ADAM10 Ab (Abcam, ab39153) that recognizes both mature and immature ADAM10, rabbit anti-ADAM10 pro-domain Ab (Abcam, ab39178), rabbit anti-ADAM17 (Abcam, ab39163) and sheep anti-ceruloplasmin (Abcam, ab8813) were tested on the spotted proteins. Immunoreactivities were revealed as described above.
4
SARS-CoV-2 RBD IgG Quantification
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RBD IgG-specific enzyme-linked immunosorbent assay system was used to quantify the RBD IgG levels in plasma samples [20 (link)]. Briefly, 96-well Nunc-Immuno Plate F96 Maxisorp plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 2 μg/mL of recombinant RBD (amino acids: 331–529) overnight at 4 °C and then blocked with PBS/1% BSA. Heat-inactivated plasma and monoclonal antibodies of either COVA1-18 or CR3022 were added with serial dilution and incubated overnight at 4 °C. On the following days, HRP-conjugated Anti-human IgG (Southern Biotech, Birmingham, AL, USA) in Can Get Signal #2 (TOYOBO) was added and then HPR activity was visualized/detected by OPD substrate (Sigma, Kawasaki, Japan). RBD IgG titer in plasma was determined by reference antibody.
5
Serological Assay for LID-1 and ND-O-LID Antibodies
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Serum IgG antibodies to LID-1 were detected by ELISA. Polysorp 96-well plates (Corning Costar, NY, USA) were coated with 100 μl/well of 1 µg/mL LID-1 or with 100 μl/well of 0.25 µg/mL ND-O-LID. Blocking was performed with PBS-T 1% BSA. Serum samples diluted 1/200 in PBS-T-10% NGS were added in duplicate and incubated for 2 h at room temperature. Plates were washed and incubated for 1 h with HRP-conjugated anti-human IgG (Southern Biotech, Birmingham, AL, USA) for anti-LID-1 serology or for anti-ND-O-LID serology plates were incubated with anti-human IgG (Southerm Biotech, Birmingham, AL, USA) plus anti-human IgM (Immuno Chemicals, St. Louis, MO, USA). After washings, reactions were developed with peroxidase color substrate (KPL, Gaithersburg, MD, USA) and quenched by the addition of 1 N H2SO4. The optical density (OD) was determined (Bio-Rad microplate reader, Life Science, Hercules, CA, USA) at 450 nm. For anti-LID-1 serology, the cutoff was calculated as two times the SD of the OD of sera from healthy endemic controls, such that samples with OD > 0.3 were considered positive (15 (link)). As previously described, the anti-ND-O–LID serology threshold for positive responses was considered OD >0.923 (20 (link)). The results of serologic tests were expressed as the mean OD of duplicates.
6
Evaluating SARS-CoV-2 Spike Protein Variants
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The following proteins were purchased from Sino Biological (Beijing, China): SARS-CoV-2 recombinant Spike RBD wild type, RBD (S477N), RBD (Y453F), RBD (K458R), RBD (F342L), RBD (V367F), RBD (N354D), RBD (A435S), RBD (V483A), RBD (W436R), RBD (G476S), RBD (R408I), RBD (K417N), RBD (Y505C), RBD (N501Y), and human ACE2-histag. The different anti-SARS-CoV-2 Spike protein antibodies were purchased from Sino Biological, Active Motif (Carlsbad, USA), Biolegend (San Diego, CA), ACRO Biosystems (Newark, USA), and Absolute Antibody (Oxford, United Kingdom) (Table 1 ). HRP conjugated anti-human IgG was from Southern Biotech (Birmingham, AL, USA). Human ACE2-derived peptides described by Cao et al.9 (link) were custom ordered from Peptide 2.0 and the list is presented in Supplementary Table 1 .
Lumit SARS-CoV-2 Spike RBD:ACE2 immunoassay components were from Promega (Madison, USA) and they consist of 0.5 μM SARS-CoV-2 RBD-rabbit Fc (RBD-Fc), 0.5 μM human ACE2-mouse Fc (ACE2-Fc), Lumit Detection Substrate C, 10X Lumit Immunoassay Buffer C, Lumit anti-rabbit Ab-SmBiT and Lumit anti-mouse Ab-LgBiT. The Lumit anti-rabbit Ab-LgBiT and Lumit anti-mouse Ab-SmBiT used in some control experiments were also from Promega. c-Pass kit was purchased from Genscript (Piscataway, New Jersey).
Lumit SARS-CoV-2 Spike RBD:ACE2 immunoassay components were from Promega (Madison, USA) and they consist of 0.5 μM SARS-CoV-2 RBD-rabbit Fc (RBD-Fc), 0.5 μM human ACE2-mouse Fc (ACE2-Fc), Lumit Detection Substrate C, 10X Lumit Immunoassay Buffer C, Lumit anti-rabbit Ab-SmBiT and Lumit anti-mouse Ab-LgBiT. The Lumit anti-rabbit Ab-LgBiT and Lumit anti-mouse Ab-SmBiT used in some control experiments were also from Promega. c-Pass kit was purchased from Genscript (Piscataway, New Jersey).
7
SARS-CoV-2 Spike Protein Antibody ELISA
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ELISAs were performed as previously described in Goenka et al17 (link). Salivary antibodies specific for whole SARS-CoV-2 spike protein, for its RBD region and for the viral N-protein were detected with an ELISA based on methodology originally described for serum1 (link). Modifications were made following optimisation of assay parameters described below. Final assay conditions were as follows: antigens were diluted in PBS and MICROLON® plates (Greiner Bio-One) were coated with 10 mg/mL spike protein overnight at 4 °C. Saliva supernatants were assayed singly, diluted at either 1 in 10 (IgA) or 1 in 5 (IgG) to a final volume of 100 mL per well. Secondary antibodies were used as follows with the dilution factor indicated: HRP conjugated anti-human IgG (Southern Biotech: 1 in 15,000) and IgA (Sigma: 1 in 20,000). Plates were developed with 1-StepUltra TMB-ELISA Substrate Solution (Thermo Fisher) for 20 min and the reaction was quenched with 2 M H2SO4 (Merck). All incubations were temperature controlled at 24 °C. Optical density (OD) was read at 450 nm (to measure signal) and 570 nm (background) using a BMG FLUOstar OMEGA plate reader with MARS Data Analysis software. The OD readings at 450 nm for each well were subtracted from the OD at 570 nm then corrected for the average signal of blank wells from the same plate; ODs reported are an average of duplicate wells per sample.
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