Cloned heavy- and light-chain plasmids derived from RBD-specific IgM+ B cells were initially screened in small-scale transfections. 293T cells (ATCC) were plated at 80% confluency and transfected with 0.5 μg each of paired heavy- and light-chain plasmids using polyethylenimine. 16 h later, medium was replaced with serum free-medium, and after 3–4 d, supernatants were harvested and cellular debris was removed by centrifugation. Antibody expression levels were determined using human IgG or IgM ELISA Antibody Pair Kit (Stemcell Technologies) according to the manufacturer’s instructions. RBD specificity was determined by ELISA as previously described (Rodda et al., 2021 (link)). Briefly, 96-well high-bind plates (Corning) were coated with 2 μg/ml SARS-CoV-2 RBD protein overnight at 4°C, washed with PBS and 0.05% Tween 20 (PBS-T), blocked with 3% milk in PBS-T, and incubated with serially diluted culture supernatants for 2 h at room temperature. Plates were washed, and bound antibodies were detected using anti-human IgG-HRP or anti-human IgM-HRP (Jackson ImmunoResearch) at a 1:3,000 dilution followed by 1× 3,3′,5,5″-tetramethylbenzidine (Invitrogen) and 1 M HCl. OD was measured on a spectrophotometer at 450 and 570 nm, and data were analyzed in Prism (v9.01; GraphPad).
Anti human igm hrp
Manufactured by Jackson ImmunoResearch
Sourced in Panama
Anti-human IgM HRP is a laboratory reagent used to detect and quantify human IgM antibodies in various immunoassays. It is composed of horseradish peroxidase (HRP) conjugated to antibodies specific for the human IgM immunoglobulin.
Automatically generated - may contain errors
Lab products found in correlation
Anti human igg hrp, by Jackson ImmunoResearch (3 mentions)
Tetramethylbenzidine, by Thermo Fisher Scientific (1 mentions)
96 well high bind plates, by Corning (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Mouse laminin, by Merck Group (1 mentions)
Calf thymus single stranded dna ssdna, by Merck Group (1 mentions)
Anti m13 hrp, by GE Healthcare (1 mentions)
Thyroglobulin, by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
Single stranded dna ssdna, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Thermo Fisher Scientific (1 mentions)
Cytation 5 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
Tween 20, by Merck Group (1 mentions)
5 protocols using anti human igm hrp
1
Screening RBD-specific IgM+ B Cell Antibodies
2
RVFV Antibody Detection Assay
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Maxisorp plates (Nalgene-Nunc, Rochester, NY) were coated with RVFV lysate prepared from infected Vero-E6 cells as previously described [24 (link)], diluted 1:2000 in PBS, and allowed to adsorb overnight at 4°C. Plates were blocked in 5% milk in PBS with 0.1% Tween-20 (PBST) with 5% FBS for 1 hour at 37°C. Patient samples and controls were serially diluted in blocking buffer and incubated on plates for 2 hours at 37°C. Plates were washed three times in PBST then incubated for 1 hour at 37°C with anti-human IgG HRP or anti-human IgM HRP (Jackson ImmunoResearch Inc, West Grove, PA) diluted 1:5000 in blocking solution. Plates were washed three times in PBST before incubation in TMB substrate (KPL, Milford, MA) for 10 minutes. Reactions were stopped with TMB stop solution, and plates were read at 450nm. Data were analyzed using Excel (Microsoft Corp, Redmond, WA) and Prism (GraphPad Software Inc, La Jolla, CA).
3
Quantifying Xenoantibody Responses by ELISA
4
Natural Antibody ELISA Assay
5
Anti-NET Enzyme-Linked Immunosorbent Assay
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A high-binding 96-well EIA/RIA plate (Greiner) was coated overnight at 4°C with MNase-digested NETs diluted to a concentration of 5 μg/mL in 0.05M bicarbonate buffer (coating buffer). The plate was then washed once with 0.05% Tween 20 (MilliporeSigma) in PBS (wash buffer) and blocked with 4% BSA (MilliporeSigma) in PBS (blocking buffer) for 2 hours at 37°C. Serum samples were diluted to 1% in blocking buffer, added to the plate, and incubated for 90 minutes at 37°C. The plate was washed 5 times with wash buffer and then incubated for 90 minutes at 37°C with either anti–human IgG-HRP or anti–human IgM-HRP (Jackson ImmunoResearch, 109-035-008, 109-035-129) diluted 1:10,000 in blocking buffer. The plate was washed 5 more times with wash buffer and was developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Invitrogen). The reaction was stopped by 2N sulfuric acid solution, and the absorbance was measured at a wavelength of 450 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Each sample was tested against a corresponding control in which no NETs antigen was plated. This created an individual background value for each sample, which was subtracted from the OD of NET-coated wells to obtain the final result. The schematic illustration of anti-NET ELISA in Figure 1 was created with BioRender.com.
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