Immulon 4hbx plate

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Immulon 4HBX plates are a type of microplate used in enzyme-linked immunosorbent assay (ELISA) applications. The plates feature a high-binding surface to facilitate the immobilization of target analytes, such as proteins or antibodies, for subsequent detection and quantification.

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Lab products found in correlation

Dtx 880 multimode detector system, by Beckman Coulter (4 mentions) Tmb substrate chromogen, by Agilent Technologies (3 mentions) Spectramax plus spectrophotometer, by Molecular Devices (3 mentions) 1 step ultra tmb elisa, by Thermo Fisher Scientific (3 mentions) Ultra tmb elisa, by Thermo Fisher Scientific (3 mentions) Avidin horseradish peroxidase, by BD (2 mentions) Biotinylated goat anti mouse total igg, by Jackson ImmunoResearch (2 mentions) Sigmafast opd, by Merck Group (2 mentions) Peroxidase conjugated streptavidin, by Jackson ImmunoResearch (2 mentions) Anti il 10 clone jes5 2a5, by BD (2 mentions) Biotinylated anti il 10 clone jes5 16e3, by Jackson ImmunoResearch (2 mentions) Maxisorp plate, by Thermo Fisher Scientific (2 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Infinite m200 pro, by Tecan (1 mentions) Goat anti mouse igg horseradish peroxidase, by Thermo Fisher Scientific (1 mentions) Synergy h1 hybrid multi mode reader, by Agilent Technologies (1 mentions) Spectramax i3 plate reader, by Molecular Devices (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Spectramax 190, by Molecular Devices (1 mentions) Np bsa, by Biosearch Technologies (1 mentions)

Close spelling variants of this product

Immulon 4HBX (49 citations) Immulon 4HBX 96-well plates (15 citations) Immulon 4HBX microtiter plates (9 citations) Immulon 4HBX 96-well microtiter plates (6 citations) Immulon 4HBX flat bottom microtiter plates (5 citations) Immulon clear 4HBX 96-well plates (2 citations)

41 protocols using immulon 4hbx plate

ELISA for Anti-HIV Gag Antibodies

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Immulon 4HBX plates (Thermo Scientific) were coated overnight at 4°C with HIV Gag protein diluted in 0.1 M Carbonate Buffer (pH 9.8). Plates were then blocked with 1% FBS/PBS, and plasma from Gag immunized mice was applied in serial 10-fold dilutions in ELISA diluent (0.1% FBS/PBS) and incubated for 1 h at 37°C. Biotinylated Goat-anti-mouse total IgG (Jackson Immuno-Research, Westgrove, Pennsylvania) was added and incubated for 1 h at 37°C. For detection, Avidin-Horse Radish Peroxidase (BD) was added and incubated for 30 minutes at room temperature, followed by the addition of TMB substrate-chromogen (Dako, Glostrup, Denmark) and a 2N sulfuric acid stop solution. Washing was performed between steps with PBS + 0.05% Tween 20. Plates were read on a SpectraMax Plus spectrophotometer (Molecular Devices) at 450 nm. Data was analyzed in Prism to generate a non-linear regression fit to estimate EC50 (midpoint titer) or EC05 (endpoint titers).

Lynn G.M., Laga R., Darrah P.A., Ishizuka A.S., Balaci A.J., Dulcey A.E., Pechar M., Pola R., Gerner M.Y., Yamamoto A., Buechler C.R., Quinn K.M., Smelkinson M.G., Vanek O., Cawood R., Hills T., Vasalatiy O., Kastenmuller K., Francica J.R., Stutts L., Tom J.K., Ryu K.A., Esser-Kahn A.P., Etrych T., Fisher K.D., Seymour L.W, & Seder R.A. (2015). In vivo characterization of the physicochemical properties of TLR agonist delivery that enhance vaccine immunogenicity. Nature biotechnology, 33(11), 1201-1210.

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Quantification of Galactosaminoglycan in Fungal Cultures

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GAG was quantified in fungal culture supernatants using a modified version of our previously described ELLA [32 (link)]. N-acetylated or de-N-acetylated GAG-containing A. fumigatus culture supernatants were serially diluted in TRIS-buffered saline (TBS, Alfa Aesar, Haverhill, MA, USA) in 96-well Immulon 4HBX plates (Thermo Scientific, Waltham, MA, USA). N-acetylated GAG-containing culture supernatants were de-N-acetylated with 130 nM rAgd3 for 1 h at room temperature. Plates were washed 3 times with TBS with 0.05% Tween 20 (T20, BioShop, Burlington, ON, Canada), stained with lectin solution (30 nM biotinylated soybean agglutinin (Vector Labs, Burlingame, CA, USA) and a 1:700 dilution of avidin conjugated to horseradish peroxidase (Invitrogen)) at room temperature for 1 h. Plates were washed 3 times with TBS-T20 and developed with 3,3′,5,5′-tetramethybenzidine (TMB) substrate ultrasensitive solution (Millipore, Burlington, VT, USA) and 1 M H₂SO₄ (BioShop, Burlington, ON, Canada) stop solution. Absorbance was measured at 450 nm (Tecan Infinite M200PRO, Tecan, Mannedorf, Switzerland).

Ostapska H., Le Mauff F., Gravelat F.N., Snarr B.D., Bamford N.C., Van Loon J.C., McKay G., Nguyen D., Howell P.L, & Sheppard D.C. (2022). Co-Operative Biofilm Interactions between Aspergillus fumigatus and Pseudomonas aeruginosa through Secreted Galactosaminogalactan Exopolysaccharide. Journal of Fungi, 8(4), 336.

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ELISA for C. difficile Toxin Detection

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Immulon 4 HBX plates (Thermo Fisher Scientific) were coated with 100 ng/well of either C. difficile A or B toxoids (List Biological Labs, Inc.) in 1X phosphate-buffered saline (PBS) overnight. Wells were washed and blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature (RT) for 2 h. After washing with TBST, half-log dilutions of each serum sample were plated in triplicate and incubated for 3 h at RT. Wells were washed and goat anti-mouse IgG-horseradish peroxidase (Thermo Fisher Scientific) was added to each well. Plates were incubated for 2 h at RT. Wells were washed and 1 step Ultra TMB ELISA (Thermo Fisher Scientific) was added to each well. When color developed, 2M H₂SO₄ was added. OD450 was determined with a BioTek Synergy H1 Hybrid Multi-Mode Reader. Reciprocal titers were statistically defined based on 95% confidence interval defined previously [31 (link)].

Matchett W.E., Anguiano-Zarate S., Malewana G.B., Mudrick H., Weldy M., Evert C., Khoruts A., Sadowsky M, & Barry M.A. (2020). A Replicating Single-Cycle Adenovirus Vaccine Effective against Clostridium difficile. Vaccines, 8(3), 470.

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ELISA for Detecting Influenza Antibodies

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Purified soluble Cal/09 HA and cH6/1 comprised of the HA head domain of A/Mallard/Sweden/81/02 H6N1 and the stalk domain of PR8 with a C-terminal T4 trimerization domain and a 6-His tag was generated using a baculovirus expression system as previously described.¹⁴ (link) Clear flat-bottom 96-well Immulon 4 HBX plates (ThermoFisher) were coated with 50 μL of 2 μg/mL of Cal/09 HA or cH6/1 diluted in ELISA coating buffer (50 mM Na₂CO₃, 50 mM NaHCO₃, pH 9.4) overnight at 4°C. After the incubation, the plates were blocked for 1 hour with 100 μL of blocking buffer (5% milk powder in PBS with 5% Tween-20 (PBS-T)). Serum serially diluted in blocking buffer was then added to the wells and incubated for 2 hours at room temperature. After the incubation, the plate was washed 3 times with PBS-T. Anti-human IgG (Fab specific) – peroxidase-conjugated antibody (Sigma) diluted 1:5000 in blocking buffer was added to the wells and incubated for 1 hour at room temperature. After 3 additional washes with PBS-T, 50μL of reconstituted SIGMAFAST OPD (Sigma) was added to each well. The reaction was stopped 10 minutes later by adding 50 μL of 3M HCl to each well, and the absorbance at 490nm was read using a SpectraMax i3 plate reader (Molecular Devices).

Zhang A., Chaudhari H., Agung Y., D’Agostino M.R., Ang J.C., Tugg Y, & Miller M.S. (2022). Hemagglutinin stalk-binding antibodies enhance effectiveness of neuraminidase inhibitors against influenza via Fc-dependent effector functions. Cell Reports Medicine, 3(8), 100718.

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Quantitative ELISA for Influenza Antibodies

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Immulon 4HBX plates (Thermo Fisher Scientific) were coated with beta propiolactone–inactivated PR8 allantoic fluid (0.4 HAU/μl) or A/California/07/2009 HA (2 μg/ml) (NR-44074, BEI Resources) diluted in PBS overnight at 4°C. Plates were blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich) in PBS for 2 hours at room temperature and then washed with distilled water. Primary antibodies or sera were diluted in 1% BSA in PBS and incubated in the plates for 2 hours at room temperature. Plates were washed, and secondary antibody (goat anti-mouse IgG, IgG1, or IgG2a, human-adsorbed and conjugated to alkaline phosphatase; SouthernBiotech) was diluted 1:1000 in 1% BSA in PBS and incubated in the plates for 1 hour at room temperature. Plates were washed and developed with p-nitrophenyl phosphate (PNPP) (1 mg/ml) for 1 hour at room temperature and read on a SpectraMAX 190 (Molecular Devices). Data are reported as absorbance (OD₄₅₀).

Willis E., Pardi N., Parkhouse K., Mui B.L., Tam Y.K., Weissman D, & Hensley S.E. (2020). Nucleoside-modified mRNA vaccination partially overcomes maternal antibody inhibition of de novo immune responses in mice. Science translational medicine, 12(525), eaav5701.

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NP-Specific IgG1 Quantification by ELISA

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Levels of NP-specific serum IgG1 were determined by ELISA. Immulon 4HBX plates (Thermo Fisher Scientific) were coated overnight at 4°C with 5μg NP-BSA/mL (Biosearch Technologies), in 50μl/well. Plates were then blocked with 10% FBS, and incubated with serial dilutions of sera, followed by a peroxidase-coupled anti-IgG1 conjugate (Southern Biotech) and SureBlue TMB or ABTS substrate (KPL).

Zaretsky A.G., Konradt C., Dépis F., Wing J.B., Goenka R., Atria D.G., Silver J.S., Cho S., Wolf A.I., Quinn W.J., Engiles J.B., Brown D.C., Beiting D., Erikson J., Allman D., Cancro M.P., Sakaguchi S., Lu L., Benoist C.O, & Hunter C.A. (2017). T Regulatory Cells Support Plasma Cell Populations in the Bone Marrow. Cell reports, 18(8), 1906-1916.

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Quantifying Vaccine-Induced Antibody Responses

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Abs in sera from vaccinated mice were quantified by enzyme-linked immunosorbent assay (ELISA). cH3N2 vaccine (0.4 HAU/ul), cH3N8 vaccine (0.4 HAU/ul) or recombinant cH3 from cH3N2 virus (5 ug/ml; Influenza Reagent Resource, FR-1478) were diluted in PBS and adsorbed on Immulon 4HBX plates (Thermo Scientific, Waltham, MA) overnight at 4 °C. For chimeric HA ELISAs, recombinant chimeric HA protein was diluted in bicarbonate buffer to 2 ug/ml and adsorbed on Immunlon 4HBX plates overnight at 4 °C. Plates were blocked for 2 hr with 3% BSA in PBS at room temperature and then washed with distilled water. Sera were serially diluted in 1% BSA and incubated on the plates for 2 hrs at room temperature. Plates were washed with distilled water and anti-mouse IgG conjugated to alkaline phosphatase diluted in 1% BSA (Southern Biotech, Birmingham, AL) was incubated for 1 hr at room temperature. Plates were washed with distilled water and PNPP (p-nitrophenyl phosphate; Thermo Scientific, Waltham, MA) was added and optical densities were measured using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA).

Willis E., Parkhouse K., Krammer F, & Hensley S.E. (2016). Canine H3N8 influenza vaccines partially protect mice against the canine H3N2 strain currently circulating in the United States. Vaccine, 34(46), 5483-5487.

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Cytokine ELISA Protocols

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For IL-10 ELISAs, Immulon 4HBX plates (Thermo Fisher Scientific, Waltham, MA) were coated with anti-IL-10 (clone JES5-2A5) (BD Pharmingen San Diego, CA), blocked in 5% FBS in PBS, and loaded with samples. Biotinylated anti-IL-10 (clone JES5-16E3) was used for detection followed by peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA), SureBlue (KPL, Gaithersburg, MD) and TMB Stop Solution (KPL). For IFN-γ ELISAs, plates were coated with anti- IFN-γ (clone AN-18) (eBioscience) and loaded with samples. Biotinylated anti- IFN-γ (clone R4-6A2) (eBioscience) was used for detection, followed by peroxidase-conjugated streptavidin and ABTS (KPL). For IL-12p40 ELISAs, plates were coated with anti-IL-12p40 (clone C17.8), followed by detection using biotinylated anti-IL-12p40 (clone C15.6).

Wagage S., John B., Krock B.L., Hall A.O., Randall L.M., Karp C.L., Simon M.C, & Hunter C.A. (2014). The aryl hydrocarbon receptor promotes IL-10 production by natural killer cells. Journal of immunology (Baltimore, Md. : 1950), 192(4), 1661-1670.

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Quantitative Immunoglobulin Isotype ELISA

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Immulon-4 HBX plates (Thermo-Scientific) were coated with 200 ng/well with goat, anti-mouse kappa light chain (Bethyl) and incubated overnight at 4°C. The following day, the plates were blocked with Superblock (Pierce) for 2 h at room temperature, and serial dilutions of IgM, IgG1, IgG2b, IgG2c and IgG3 standards (Bethyl) were added using PBS as a diluent and incubated for 1.5 h. The plate was washed six times with PBS containing 0.25% Tween-20, then HRP-labeled conjugates were added and incubated for 1.5 h at room temperature. The conjugate concentrations were: IgM, 1:2000 (Bethyl); IgG1, 1:1000 (Bethyl); IgG2b, 1:2000 (Bethyl), IgG2c, 1:2000 (Southern Biotechnology); IgG3, 1:10,000 (Bethyl). After incubating for 1.5 h at room temperature, the plates were washed six times with PBS containing 0.25% Tween-20, and developed by adding TMB substrate (BioFX) and stopped with 0.3M sulfuric acid. Plates were read at 450 nm using a Victor X5 plate reader. Ig concentrations were interpolated from linear regression curves generated with the standards using GraphPad Prism 8.0.

Barrett B.S., Nguyen D.H., Xu J., Guo K., Shetty S., Jones S.T., Mickens K.L., Shepard C., Roers A., Behrendt R., Wu L., Kim B, & Santiago M.L. (2021). SAMHD1 promotes the antiretroviral adaptive immune response in mice exposed to lipopolysaccharide. Journal of immunology (Baltimore, Md. : 1950), 208(2), 444-453.

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ELISA for Lyme Disease Antibody Detection

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B. burgdorferi whole-cell lysate was prepared as previously described (53 (link)). Total protein in the sonicate was quantified on the basis of the absorbance at 280 nm using a NanoDrop1000 spectrophotometer (Thermo Scientific), and aliquots were stored at −20°C. The protein was diluted to 5 µg/mL, and 50 µL per well was placed in Immulon 4HBX plates (Thermo Scientific) and coated overnight at 4°C. After blocking with 3% BSA, serially diluted serum was added in PBS with 2% BSA and incubated for 2 h at RT. Goat anti-mouse IgG conjugated to HRP (cat #A16084, Invitrogen) or goat anti-mouse IgM conjugated to HRP (cat #62–6820, Invitrogen) was diluted to 1:5,000 or 1:2,000, respectively, in 1% BSA and incubated for 1 h at RT. After incubation, antigen-specific antibodies were detected using TMB substrate as described by the manufacturer (Thermo Scientific), and the reaction was stopped with 2-M H₂SO₄. Antibody endpoint titers for IgG were determined as the highest serum dilution corresponding to a cutoff of 1.0 Abs₄₅₀. Antibody endpoint titers for IgM were determined as the highest serum dilution corresponding to a cutoff of 0.2 Abs₄₅₀.

Williams M.T., Zhang Y., Pulse M.E., Berg R.E, & Allen M.S. (2024). Suppression of host humoral immunity by Borrelia burgdorferi varies over the course of infection. Infection and Immunity, 92(4), e00018-24.

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