Magplex avidin microsphere

Manufactured by DiaSorin

MagPlex-Avidin microspheres are magnetic beads coated with avidin, a protein that has a high affinity for biotin. These microspheres are used as a versatile platform for various bioassay and bioanalytical applications that involve the capture, separation, and detection of biotinylated molecules.

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Lab products found in correlation

Magpix system, by DiaSorin (3 mentions) 200 analyzer, by DiaSorin (1 mentions) Anti his tag antibody, by Abcam (1 mentions) Elecsys, by Roche (1 mentions) Pcag gfp, by Addgene (1 mentions) Magpix reader, by DiaSorin (1 mentions) Expi293f system, by Thermo Fisher Scientific (1 mentions) Bira kit, by Avidity (1 mentions)

9 protocols using magplex avidin microsphere

Multiplex Peptide Assay using Luminex

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Biotinylated peptides and the compound that serves as negative control (NC) were immobilized as described before [19 (link)], using 5 × 10⁵ avidin coated paramagnetic beads (MagPlex-Avidin microspheres; Luminex, Den Bosch, The Netherlands). The biotinylated compounds were used at a final concentration of 4000 nM. After binding of these compounds to avidin-coated beads, the beads were washed and blocked with biotin [19 (link)].
In total, 10 spectrally distinct bead sets were used to assemble three different bead mixes. Each bead mix contained a bead set with the NC and up to 9 bead sets with peptides, as follows. Bead mix 1 contained 8 bead sets, loaded with resp. pCga001-003, pCga008-011, and NC; bead mix two contained 9 bead sets, loaded with resp. pCps001, 002, 009, 010, 013–016, and NC; bead mix three contained 10 bead sets, loaded with resp. pCab001-005, pCav001-004, and NC. The three bead mixes together constitute the Luminex suspension array when employed in parallel. By doing so, it was possible to run a cost-efficient multiplex assay for 24 peptides and internal controls with only 10 different beads.

van der Wal F.J., Achterberg R.P., van der Goot J.A., Dinkla A., Bossers-de Vries R., van Solt-Smits C., Bossers A, & Heijne M. (2023). Proof of concept for multiplex detection of antibodies against Chlamydia species in chicken serum using a bead-based suspension array with peptides as antigens. Veterinary Research, 54, 31.

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Multiplex Surrogate Virus Neutralization Tests

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Two different methods of performing surrogate virus neutralization tests (sVNTs) were used in this study. The singleplex sVNTs for SARS-CoV-1 and SARS-CoV-2 have been described previously^{6 (link)} and are briefly described in the Methods section of the Supplementary Appendix, available with the full text of this article at NEJM.org. The sVNT kit for SARS-CoV-2 has been commercialized under the trade name cPass (GenScript), with Food and Drug Administration emergency use authorization granted in November 2020.
For multiplex sVNTs, we adapted the sVNT using the Luminex platform as described previously.^{7 (link)} AviTag-biotinylated receptor-binding domain (RBD) proteins from 10 different sarbecoviruses were coated on MagPlex-Avidin microspheres (Luminex) at 5 μg per 1 million beads (see the Methods section of the Supplementary Appendix). RBD-coated microspheres (600 beads per antigen) were preincubated with serum at a final dilution of 1:20 or greater for 1 hour at 37°C with 800 rpm agitation. After 1 hour of incubation, 50 μl of phycoerythrin (PE)–conjugated human angiotensin-converting enzyme 2 (ACE2) (hACE2; 1 μg per milliliter; GenScript) was added to the well and incubated for 30 minutes at 37°C with agitation, followed by two washes with 1% bovine serum albumin in phosphate-buffered saline (PBS). The final readings were acquired with the use of the MAGPIX system (Luminex).

Tan C.W., Chia W.N., Young B.E., Zhu F., Lim B.L., Sia W.R., Thein T.L., Chen M.I., Leo Y.S., Lye D.C, & Wang L.F. (2021). Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors. The New England Journal of Medicine.

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Multiplex Serological Profiling of DENV Antibodies

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Antibody responses to envelope protein domain 3 (EDIII) and nonstructural 1 protein (NS1) of DENV serotypes 1–4 were measured in a random subset of participants (n = 295, Supplemental Table 2) using Luminex multiplex assay as previously described³¹. Briefly, biotinylated EDIII antigens and biotinylated bovine serum albumin (BSA) were coupled to unique MagPlex^®-Avidin Microspheres (Luminex) while His-tagged NS1 antigens (The Native Antigen Company) were coupled following immobilization of anti-His tag antibody (abcam) onto unique avidin-coated microspheres. The panel of EDIII, NS1 and BSA conjugated microspheres were mixed in equal ratios and plated at 2,500 beads per antigen in 50μL/well in 96 well plates. Diluted human serum (1:500) was incubated with antigen-conjugated microspheres for one hour at 37°C, 700rpm. Later, immune complexes were incubated with goat anti-human IgG Fc multispecies SP ads-PE antibody (Southern Biotech) following three washes. Antibody responses were detected using a Luminex 200 analyzer and expressed as median fluorescence intensity after subtracting the non-specific antibody binding signal (to BSA). Selected samples from healthy donors and well-characterized DENV and ZIKV seropositive individuals were run on multiple assay plates to verify assay performance and assess inter-assay variability.

Katzelnick L., Odio C., Daag J., Crisostomo M.V., Voirin C., Escoto A.C., Adams C., Hein L.D., Aogo R., Mpingabo P., Rodriguez G.R., Firdous S., Fernandez M.A., White L., Agrupis K.A., Deen J., de Silva A, & Ylade M. (2024). Dengue virus IgG and serotype-specific neutralizing antibody titers measured with standard and mature viruses are associated with protection. Research Square.

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Multiplex Serology Profiling of SARS-CoV-2 Variants

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Biotinylated SARS-CoV-2, SARS-CoV, RaTG13, RmYN02, GX-P5L, and SL-ZC45 RBD were coated onto MagPlex Avidin microspheres (Luminex) at final concentration of 800 nM in PBS with 1% BSA. Luminex assays were performed by pre-incubation of 1:100 diluted sera with 1250 beads/antigen for 1 h at 37 °C followed by 1:1000 diluted PE-conjugated anti-rabbit IgG (Thermo Scientific) or PE-conjugated anti-human IgG (eBioscience) for 1 h at 37 °C. The level of antibody binding was determined using Luminex MAGPIX system. The readings were normalized against readings obtained from anti-6XHis tag antibody (at final dilution of 1:600) (Thermo Scientific). Panel of COVID-19 and SARS sera used in this study were from Singapore PROTECT study and SARS recalled sampling in 2020 (ethics approval number: NHG DSRB E 2020/00091), both are approved by National Health Group Singapore, with written informed consent for both the use and reporting in the research.

Wacharapluesadee S., Tan C.W., Maneeorn P., Duengkae P., Zhu F., Joyjinda Y., Kaewpom T., Chia W.N., Ampoot W., Lim B.L., Worachotsueptrakun K., Chen V.C., Sirichan N., Ruchisrisarod C., Rodpan A., Noradechanon K., Phaichana T., Jantarat N., Thongnumchaima B., Tu C., Crameri G., Stokes M.M., Hemachudha T, & Wang L.F. (2021). Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia. Nature Communications, 12, 972.

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Multiplex sVNT Bead-Based Assay

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Multiplex sVNT were established as previously described^{23 (link)}. Briefly, AviTag-biotinylated RBD proteins was coated on MagPlex-Avidin microspheres (Luminex) at 5 μg per 1 million beads. RBD-coated beads (600 per antigen) were pre-incubated with testing serum at final dilution of 1:20, 1:80, 1:320, 1:1280) for 15 min at 37°C with agitation, followed by addition of 50 μl of PE conjugated human ACE2 (2 mg/ml; Genscript) and incubated for an additional 15 minutes at 37°C with agitation. After two washes with 1 % BSA in PBS, the final readings were acquired using the MAGPIX system (Luminex) following manufacturer’s instruction.

Wang L.F., Tan C.W., Chia W.N., Zhu F., Young B., Chantasrisawad N., Hwa S.H., Yeoh A.Y., Lim B.L., Yap W.C., Pada S.K., Tan S.Y., Jantarabenjakul W., Chen S., Zhang J., Mah Y.Y., Chen V., Chen M., Wacharapluesadee S., Putcharoen O, & Lye D. (2022). Differential escape of neutralizing antibodies by SARS-CoV-2 Omicron and pre-emergent sarbecoviruses. Research Square.

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Multiplex sVNT for Vaccine Breakthrough

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Multiplex sVNT was performed using enzymatic biotinylated RBD‐coated MagPlex Avidin microsphere (Luminex). Briefly, RBD‐coated microspheres (600 beads/antigen) were pre‐incubated with diluted plasma (1:400) (n = 54 vaccine breakthrough, n = 86 close contact) or memory B cell culture supernatant (1:2) (n = 48 vaccine breakthrough, n = 86 close contact) for 1h at 37°C with 800 rpm agitation. PE‐conjugated ACE2 at 1,000 ng/ml were added into beads followed by 30‐min incubation at 37°C with 800 rpm agitation. Beads were washed twice with 1% BSA‐PBS prior to signal acquisition using MAGPIX Luminex system. Single‐point data were collected.

Tay M.Z., Rouers A., Fong S., Goh Y.S., Chan Y., Chang Z.W., Xu W., Tan C.W., Chia W.N., Torres‐Ruesta A., Amrun S.N., Huang Y., Hor P.X., Loh C.Y., Yeo N.K., Wang B., Ngoh E.Z., Salleh S.N., Chavatte J., Lim A.J., Maurer‐Stroh S., Wang L., Lin R.V., Wang C., Tan S., Young B.E., Leo Y., Lye D.C., Renia L, & Ng L.F. (2022). Decreased memory B cell frequencies in COVID‐19 delta variant vaccine breakthrough infection. EMBO Molecular Medicine, 14(3), e15227.

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Multiplex SARS-CoV-2 Variant Neutralization Assay

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Serum samples were tested with a newly developed m-sVNT assay (27 (link)). Briefly, AviTag-biotinylated RBD proteins from ancestral SARS-CoV-2 and SARS-CoV, nine VOCs/VOIs (Alpha, Delta, Beta, Gamma, Delta Plus, Lambda, Mu, Omicron BA.1, and Omicron BA.2), and nine zoonotic sarbecoviruses (BANAL-52, BANAL-236, GD-1, RaTG13, GX-P5L, Rs2018B, LYRa11, RsSHC014, and WIV-1) were coated on a MagPlex Avidin microsphere (Luminex) at 5 μg/1 million beads. The RBD-coated microspheres (600 beads per antigen) were preincubated with mAbs at a starting concentration of 10,000 ng/ml serially diluted fourfold for 15 min at 37°C with 250 rpm agitation. After 15 min incubation, 50 μl of PE-conjugated huACE2 (2 μg/ml; GenScript) were added to the well and incubated for 15 min at 37°C with agitation, followed by two phosphate-buffered saline (PBS)–1% bovine serum albumin washes. The final readings were acquired using the MAGPIX system (Luminex Corporation).

Chia W.N., Tan C.W., Tan A.W., Young B., Starr T.N., Lopez E., Fibriansah G., Barr J., Cheng S., Yeoh A.Y., Yap W.C., Lim B.L., Ng T.S., Sia W.R., Zhu F., Chen S., Zhang J., Kwek M.S., Greaney A.J., Chen M., Au G.G., Paradkar P.N., Peiris M., Chung A.W., Bloom J.D., Lye D., Lok S, & Wang L.F. (2023). Potent pan huACE2-dependent sarbecovirus neutralizing monoclonal antibodies isolated from a BNT162b2-vaccinated SARS survivor. Science Advances, 9(30), eade3470.

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Multiplex SARS-CoV-2 Antibody Profiling

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Serum samples were tested with a newly developed multiplex-sVNT assay using the Luminex platform [8 (link)]. Briefly, AviTag-biotinylated RBD proteins from wild-type SARS-CoV-2 and 5 VOCs (Alpha, Beta, Gamma, Delta, Omicron) were coated on a MagPlex Avidin microsphere (Luminex) at 5 µg/1 million beads. RBD-coated microspheres (600 beads/antigen) were preincubated with serum at a final concentration of 1:20 or greater for 15 minutes at 37°C with 250 rpm agitation. After 15 minutes incubation, 50 µL of phycoerythrin-conjugated human angiotensin-converting enzyme 2 (GenScript 2 µg/mL) were added to the well and incubated for 15 minutes at 37°C with agitation, followed by 2 phosphate-buffered saline-1% bovine serum albumin washes. The final readings were acquired using the MAGPIX system.
Serological results were obtained using the Elecsys (Roche, Basel, Switzerland) anti–SARS-CoV-2 chemiluminescent immunoassays following the manufacturer’s instructions (anti-nucleocapsid [anti-N] and anti-spike protein RBD [anti-S]). Antibody titers in U/mL from the Elecsys anti-S assay are equivalent to the WHO standard binding antibody units per milliliter, with no conversion required [9 (link)].

Poh X.Y., Tan C.W., Lee I.R., Chavatte J.M., Fong S.W., Prince T., Hartley C., Yeoh A.Y., Rao S., Chia P.Y., Ong S.W., Lee T.H., Sadarangani S.P., Lin R.J., Lim C., Teo J., Lim D.R., Chia W., Hiscox J.A., Ng L.F., Ren E.C., Lin R.T., Renia L., Lye D.C., Wang L.F, & Young B.E. (2022). Antibody Response of Heterologous vs Homologous Messenger RNA Vaccine Boosters Against the Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant: Interim Results from the PRIBIVAC Study, a Randomized Clinical Trial. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 75(12), 2088-2096.

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SARS-CoV-2 RBD Antibody Quantification

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The S-RBD binding antibody in mouse sera was quantified using a multiplex microsphere-immunoassay (MMIA). AviTag-enzymatic biotinylated SARS-CoV-2 ancestral, Alpha, Beta, Gamma, Delta RBDs were custom-made by Genscript. The remaining RBDs were produced in-house by transfection of pCAGGS-expression plasmids (modified from pCAG-GFP, addgene, 11150) into the Expi293F system (ThermoFisher, A14527). The RBDs were purified using Nickel Sepharose columns (Cytiva, 17526802), and biotinylated using a BirA kit (Avidity, LLC, BirA500). The RBDs were coated on the Magplex avidin-microsphere (Luminex, MA-A012-01) at 25 μg per 5 million microspheres. The RBD-coated beads were pre-incubated with 1:100 diluted mouse sera for 1 h at 37 °C with agitation at 800 rpm. Following two 1% BSA PBS washes, the mouse IgG was immunostained with 1:1000 diluted PE-labelled anti-mouse IgG antibody (R&D systems, IC002P) for 1 h at 37 °C with agitation at 800 rpm. After two washes, the MFI signals were acquired using the Luminex MAGPIX reader.

O’Neill A., Mantri C.K., Tan C.W., Saron W.A., Nagaraj S.K., Kala M.P., Joy C.M., Rathore A.P., Tripathi S., Wang L.F, & St. John A.L. (2023). Mucosal SARS-CoV-2 vaccination of rodents elicits superior systemic T central memory function and cross-neutralising antibodies against variants of concern. eBioMedicine, 99, 104924.

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