Antibodies against B. pertussis antigens mutant Ptx, Prn, FHA, Fim2/3, BrkA, Vag8, and OMV were measured using a MIA. Conjugation of antigens and OMVs to magnetic beads of different bead regions (Luminex, Austin, TX, USA) was performed using the Bio-Plex Amine Coupling kit (Bio-Rad, Hercules, CA, USA) and was described previously [20 (link)]. Serum was diluted 1:100 for IgG (subclass) and 1:1000 for anti-OMV IgG in PBS containing 0.1% Tween 20 and 3% bovine serum albumin (Sigma, Zwijndrecht, The Netherlands) and added to the conjugated beads in a 1:1 ratio. Subsequently, samples were incubated with R-Phycoerythrin (RPE)-conjugated anti-mouse IgG (1:200), IgG1 (1:200), IgG2a (1:40), IgG2b (1:200), and IgG3 (1:200) (Southern Biotech, Birmingham, AL, USA). Data was acquired with a Bio-Plex 200, processed using Bio-Plex Manager software (v6.1, Bio-Rad Laboratories, Hercules, CA, USA), corrected for the background signal, and presented as fluorescence intensities (FI). The limit of detection of each analyte was set at 15% of the highest FI-value in the dataset.
Bio plex amine coupling kit
Manufactured by Bio-Rad
Sourced in United States, France
The Bio-Plex Amine Coupling Kit is a laboratory product designed for the covalent coupling of amine-containing molecules to Bio-Plex magnetic beads. The kit includes the necessary reagents and protocols to facilitate this coupling process.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex pro magnetic cooh beads, by Bio-Rad (4 mentions)
Bio plex 200 system, by Bio-Rad (3 mentions)
Magplex c microsphere, by DiaSorin (2 mentions)
Hulamixer, by Thermo Fisher Scientific (2 mentions)
Storage buffer, by Bio-Rad (2 mentions)
Igg2b, by Southern Biotech (1 mentions)
Igg2a, by Southern Biotech (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bio plex manager software, by Bio-Rad (1 mentions)
Bio plex 200, by Bio-Rad (1 mentions)
Bio plex manager software version 6, by Bio-Rad (1 mentions)
Flexmap 3d reader, by Bio-Rad (1 mentions)
Duoset, by R&D Systems (1 mentions)
Magplex microsphere, by DiaSorin (1 mentions)
Streptavidin phycoerythrin sa pe, by Bio-Rad (1 mentions)
Rna extraction kit, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Dnase 1, by Worthington (1 mentions)
Mab098, by R&D Systems (1 mentions)
Bio plex 200, by DiaSorin (1 mentions)
19 protocols using bio plex amine coupling kit
1
Bead-based Multiplex Assay for Pertussis Antibodies
2
Multiplex Cytokine and Complement Profiling in Perfusate
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To determine the concentrations of porcine-specific cytokines [interleukin (IL)-6, IL-1β, IL-10, tumor necrosis factor alpha (TNF-α)], basic fibroblast growth factor (bFGF), as well as the complement activation marker soluble (s)C5b-9 in perfusate samples, a multiplex xMAP technology (Luminex) assay was performed according to a custom-made protocol developed by our group35 (link). In brief, microbeads (Luminex) were coupled with respective capture antibodies using the Bio-Plex amine coupling kit (Bio-Rad). Coupled beads were then incubated with samples, followed by biotinylated detection antibodies and Streptavidin-R-PE (922721, Qiagen, Hilden, Germany). Measurement and data analysis were performed with a Flexmap 3D reader and the Bio-Plex Manager software version 6.1 (Bio-Rad). Concentrations of human C5a were detected by ELISA using a commercially available kit (DuoSet, R&D Systems, Minneapolis, USA).
3
Influenza HA Antigen Coupling Protocol
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Purified recombinant HA proteins for each influenza strain/subtype were covalently coupled to Bio-plex Pro Magnetic COOH Beads (Bio-Rad, Hercules, CA) using the Bio-Plex Amine Coupling Kit (Bio-Rad, Hercules, CA) (Table 1 ) according to the manufacture’s instructions. Purified HA proteins (12 μg) were individually bound to activated COOH beads (3.75×106 beads per coupling reaction) for 2 hours in the dark separately, and unreactive sites on the beads surface blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. Protein binding efficiency was tested with anti-HA subtype specific rabbit polyclonal Abs and a goat anti-rabbit PE-conjugated detection antibody (SouthernBiotech, Birmingham, AL). Coupled beads were counted using a hemocytometer and stored at 4°C in the dark.
4
SARS-CoV-2 Protein Coupling on Luminex Beads
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Luminex beads used for the serological binding assays were prepared by covalent coupling of SARS-CoV-2 proteins with MagPlex beads using the manufacture’s protocol with a Bio-Plex Amine Coupling kit (Bio-Rad, France). Briefly, 1 ml of MagPlex-C microspheres (Luminex) were washed with wash buffer and then resuspended in activation buffer containing a freshly prepared solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (S-NHS), (Thermo Fisher Scientific, USA). Activated beads were washed in phosphate-buffered saline (PBS) followed by the addition of 50 μg of protein antigen. The coupling reaction was performed at 4°C overnight with bead agitation using a Hula-Mixer (Thermo Fisher). Beads were then washed with PBS, resuspended in blocking buffer, and then incubated for 30 min with agitation at room temperature. Following a final PBS washing step, beads were resuspended in 1.5 ml of storage buffer and kept protected from light in an opaque tube at 4°C. Each of the SARS-CoV-2 proteins was coupled with different colored MagPlex beads so that tests could be performed with a single protein bead per well or in a multiplexed Luminex serological binding assay.
5
Antigen Coupling to Microspheres
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Recombinant antigens were covalently coupled to a spectrally distinct microsphere (Magplex Microsphere, Luminex Corp., Austin, TX, USA). Briefly, 2 µg of each antigen was coupled to 1.25 × 106 beads using a BioPlex amine coupling kit (Bio-Rad) according to the manufacturer’s procedure.
6
Comprehensive RNA Extraction and Protein Analysis
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RNA extraction kits (Ambion) and primers (Applied Biosystems) were purchased from Thermo Scientific (Waltham, MA). The iScript cDNA synthesis kit was purchased from Bio-Rad (Hercules, CA). DNase I was purchased from Worthington Biochemical (Lakewood, NJ). Magnetic MagPlex beads and bead conjugation kits (Bio-Plex Amine Coupling kit) were purchased from Bio-Rad (Hercules, CA). Streptavidin-phycoerythrin (SA-PE) was purchased from Bio-Rad (Hercules, CA). The following primary antibodies were used: monoclonal anti-VGLUT2 (clone EPR21085) from Abcam (Cambridge, MA), biotinylated anti-VGLUT2 from Lifespan Biosciences (Seattle, WA), monoclonal anti α-tubulin (DM1A) from Cell Signaling Technologies (Danvers, MA).
7
Multiplex Luminex Assay for Angiogenic Factors
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Vitreous samples of patients with wAMD, diabetic retinopathy, retinal vein occlusion, and macular hole were collected by Dr. M. Koss, University of Heidelberg (Koss et al, 2011 ). Collection of samples was performed after local full ethics committee approval (57/08) in accordance with the European Guidelines for Good Clinical Practice and the Declaration of Helsinki. Informed consent was obtained from each patient before the start of therapy. Samples were stored at −70°C and transferred to Roche for the analysis of ANG‐1 and ANG‐2 using a multiplex Luminex kit produced in‐house using the following reagents: anti‐ANG‐1 capture (R&D, MAB9231) and anti‐ANG‐1 detection (Novus Biologicals, NB110‐85464), anti‐ANG‐2 capture (R&D, MAB098) and anti‐ANG‐2 detection (R&D, BAM0981). Capture antibodies were coupled to Luminex beads using the Bio‐Plex Amine Coupling Kit (Bio‐Rad 171406001). Vitreous samples diluted one‐third were incubated with capture antibody bead for 2 h. After washing the beads, biotinylated detector antibodies were added and incubated with the beads for 1 h. Streptavidin‐conjugated fluorescent protein, R‐phycoerythrin (BD, 554061), was then added and incubated for 30 min. After washing, the beads were analyzed using a Luminex 100 detection system.
8
Mapping CHIKV CP Epitopes with Peptides
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A series of 20-mer peptides, with 10-residue overlaps, encompassing the entire CHIKV CP were designed (Table 2 ) and commercially synthesized (Mocell Biotech, Hong Kong). Each peptide was coupled to carboxylated polystyrene beads using the Bio-Plex amine coupling kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The coupled beads were then multiplexed by diluting in microsphere immunoassay (MIA) buffer (1% BSA, 0.05% ProClin 300 (Supelco, Bellefonte PA, USA) in PBS) and distributed to give approximately 2500 of each beadset (~100 µL) per well of a MultiScreen filter plate (Merck Millipore, Billerica, MA, USA). Each anti-CHIKV CP mAb was then tested against the multiplexed beadsets by incubation for 45 min with shaking at room temperature. The wells were then washed three times with PBS/T wash buffer, prior to the addition of R-Phycoerythrin donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA). Following that, the wells were incubated and washed as previously, after which the beads were resuspended in MIA buffer. The Bio-Plex 200 (Luminex, Bio-Rad, Austin, TX, USA) was then utilized to read the plate, measuring the mean fluorescence intensity for each beadset in their respective wells. mAb recognition of peptides was also assessed by ELISA and dot blot as previously described [29 (link)].
9
Recombinant Protein Coupling to Fluorescent Beads
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Extracellular domain of recombinant human MOG1-125 (rMOG) (Eurogentec, France) expressed in E. coli, human albumin (LFB, France), and nontoxic tetanus toxin C-fragment (Sigma, France) proteins were coupled to fluorescent Bio-Plex COOH beads (Bio-Rad, France) as described [21 (link)]. The carboxyl groups of fluorescent COOH beads were activated by EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (Fisher Scientific, France) and S-NHS (sulfo-N-hydroxysulfosuccinimide) (Fisher Scientific); then the Bio-Plex amine coupling kit (Bio-Rad) was used to couple proteins to the activated COOH beads. The coupling reaction was systematically checked through flow cytometry using the appropriate antibodies. For MOG, we checked that the anti-MOG 8.18C5 mouse antibody, shown to react with the folded MOG pattern [24 (link)], effectively recognizes the MOG1-125 covalently coated beads. In addition, we checked that these MOG1-125 coated beads are also recognized by B cells from transgenic 8.18C5 antibody mice [21 (link)].
10
Coupling Proteins and Peptides to Luminex Beads
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We described in detail in our previous works the protocol for coupling proteins and peptides to Luminex microsphere beads [12 (link),13 (link)]. In brief, recombinant spike proteins (1 μg/1.25 × 106 beads) and nucleocapsid (2 μg/1.25 × 106 beads) were covalently coupled on carboxyl functionalized fluorescent magnetic beads (Luminex Corp., Austin, TX) with the BioPlex amine coupling kit (Bio-Rad Laboratories, Marnes-la-Coquette, France) according to the manufacturer’s instructions. For each recombinant protein-coupled bead set, we used 2000 beads/μl of assay buffer. Preliminary experiments on different plasma dilutions (1/100−1/1000) showed that the dilution 1/200 gave the best signal to noise ratio. Diluted samples were incubated with coupled beads for 16 h at 4 °C. Reactions were revealed after incubation with a biotin-labeled anti-human IgG and streptavidin-R-phycoerythrin conjugate. Antigen-antibody reactions were read on BioPlex-200 equipment (Bio-Rad, Marnes-la-Coquette. France) and the results were expressed as median fluorescence intensity (MFI) per 100 beads. To determine IgG titers of a subset of samples against the different antigens tested, we performed a 2-fold serial dilution of these samples from 1/100 to 1/12,800 and tested them as described above. The titer was the highest value of reciprocal dilution factor given a signal above the cut-off.
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