1 step ultra tmb elisa

The murine plasma IL-1β was measured by DuoSet ELISA kit (DY401-05, R&D systems) according to the manufacturer’s instructions. 1-Step™ Ultra TMB-ELISA (Thermo scientific, cat# 34028) was used for final horseradish peroxidase reaction and terminated by 1 M sulfuric acid.

Immunolon 4 HBX microtiter 96-well strips (Thermo Fisher, Waltham, MA, USA) were coated with 200 ng/well of recombinant HA protein from BEI Resources, Manassas, VA, USA NIAID (A/California/04/2009, NR-15749; A/PR/8/34, NR-19240; A/Brisbane/59/07, NR-28607; A/New Caledonia/20/99, NR-48873) in bicarbonate/carbonate buffer overnight at 4 °C. Plates were blocked with 2% bovine serum albumin (BSA) in PBS and incubated for two hours at RT. Sera was serial diluted two-fold in PBS with 1% BSA starting with a 1:100 dilution in 100 μL per well. After two hours of incubation at RT, plates were washed 4× with phosphate buffered saline with Tween 20 (PBST) and 2× with PBS before incubation with 1:5000 goat anti-mouse-HRP antibody (sc-47724; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in 1% BSA at RT for one hour. Plates were washed 4× with PBST and 2× with PBS prior to development with 1-Step Ultra TMB-ELISA (Thermo Fisher, Waltham, MA, USA). Development was stopped with the addition of 2M sulfuric acid. The OD450 was measured using SpectraMax i3x Multi-Mode microplate reader (Molecular Devices, San Jose, CA, USA). Responses ≥2 times the negative control absorbance were considered positive and used to calculate the geometric mean titer (GMT).

Ninety-Six well plates (Nunc, Waltham, MA, USA) were coated with anti-Tau caspase cleaved antibody (C3, Millipore) at a final concentration of 6 μg ml⁻¹, for 48 h at 4 °C. After washing 3 ×, the plates were blocked for 1 h at room temperature using StartingBlock Blocking buffer (Thermo Scientific). Plates were washed 5 × and 50 μl (1 μg μl⁻¹) of brain S2 fraction were added to the wells with 50 μl of DA9-HRP detection antibody. Plates were incubated O/N shaking at 4 °C and then washed 9 × in wash buffer. 1-Step ULTRA TMB-ELISA (Thermo Scientific) was added for 30′ at room temperature before stopping the reaction with 100 μl 2 m H₂SO₄. Plates were read with an Infinite m200 plate reader (Tecan, Morrisville, NC, USA) at 450 nm. Recombinant human Tau-441 was purchased from rPeptide (Watkinsville, GA, USA).

The concentration of active mAb (i.e., capable of recognizing TNFα) was determined by an ELISA procedure, as previously described by González et al. (2011 (link)). Briefly, samples form mAb producer CHO cell cultures were centrifuged at 5,000 rpm for 5 min to remove cells and cellular debris. Supernatants were diluted using PBS 1X to a 1:1 ratio. In 96 well plates (Maxisorp; NUNC, New York, NY, USA), each of the following solutions were added sequentially: 100 μL/well of a 5 μg/mL TNFα (BioSource™; Invitrogen), 300 μL/well of SuperBlock^®T20 (Thermo Scientific; Pierce, Rockford, IL, USA), 100 μL/well of samples or standards [standards were prepared by serial dilution from commercially available Infliximab (Remicade^®; Schering-Plough, Innishannon, Ireland)], 100 μL/well of anti-human IgG-HRP conjugate at a 1:30,000 dilution (Thermo Scientific; Pierce), and 100 μL/well of 1-step Ultra TMB-ELISA (Thermo Scientific; Pierce). In between the addition of the different solutions, individual wells were washed three times with 300 μL/well of PBS-0.05 % Tween-20 solution (10 mM phosphate, 0.15 M NaCl, pH 7.2 ± 0.2). The TNFα solution was incubated at 4 °C overnight. The rest of the incubation steps were done at room temperature for 1 h. The enzymatic reaction was stopped by adding 50 μL/well of 1 M H₂SO₄. Plates were read at 450 nm.

Immunolon 4 HBX microtiter 96-well strips (VWR) were coated with 150 ng per well of recombinant ZIKV E protein (Cat #:MBS596088; MyBioSource) or recombinant H1 hemagglutinin of A/Puerto Rico/8/1934 (H1N1) (NR-19240, BEI) in bicarbonate/carbonate coating buffer overnight at 4 °C. The plates were blocked with 2% BSA in PBS for 2 hours at room temperature (RT). Sera was serially diluted in 1% BSA in PBS and incubated for 2 hours at RT. The plates were washed 6X with PBST and incubated with goat anti-mouse-HRP antibody (1:5000; Thermo Fisher) in 1% BSA in PBS for 1 hour at RT. After washing 4X with PBST and 2X with PBS, the plate was developed with 1-Step Ultra TMB-ELISA (Thermo Fisher) and the reaction was stopped with 2 M sulfuric acid. The OD450 was detected using a SpectraMax i3x Multi-Mode microplate reader (Molecular Devices) and the endpoint titer was determined as signal that was two and a half times background values.

Peptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific) at 1 μg/ml. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific), the plates were incubated with culture supernatant with subsequent 1:2000 diluted peroxidase-conjugated anti-mouse IgG (Dako). The enzymatic reaction was conducted with 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories). These reactions were performed with a volume of 50–100 μl at 37 °C.

Proteins were immobilized on Nunc Maxisorp 96-well immuno plates (Thermo Fisher Scientific, Inc.) at 1 μg/mL for 30 min. After blocking with 1% bovine-serum albumin (BSA) in 0.05% Tween 20/phosphate buffered saline (PBS, Nacalai Tesque, Inc.), the plates were incubated with culture supernatant followed by 1:2000 diluted peroxidase-conjugated anti-mouse immunoglobulins (Agilent Technologies, Inc., Santa Clara, CA). The enzymatic reaction was produced with a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).