Purified CVA6-W of 1×104 TCID50 were diluted with the ELISA coating buffer (pH 9.6) (Solarbio, China) and added into each well of 96-well ELISA plate (Costar, USA). All plates were stored at 4°C temperature overnight and then washed with PBST (pH 7.2–7.4) (Solarbio, China). Then, 100 µl blocking buffer (5% bovine serum albumin [BSA] in PBST) (Solarbio, China) were added into each well and stored at 37°C for 1 h. After the plate was washed, the 10-fold serially diluted antisera in blocking buffer were added into the plate and incubated at 37°C for 1 h. The plates were washed three times with PBST and incubated with a 1:8,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG(H+L) (CWBIO, China) secondary antibody or HRP-conjugated goat anti-mouse IgM (Abcam, UK) at 37°C for 1 h. After the plate was washed four times, 100 µl TMB Single-Component Substrate solution (Solarbio, China) were added into each well of the plates for 10 min. Then 100 µl ELISA Stop Solution (Solarbio, China) were added into each well and the results were analyzed with an ELISA plate reader (Multiskan MK3; Thermo Scientific) at 450 nm.
96 well elisa plate
Manufactured by Corning
Sourced in United States
96-well ELISA plates are a common laboratory equipment used for enzyme-linked immunosorbent assays (ELISAs). These plates consist of a grid of 96 individual wells that can be used to perform multiple analyses simultaneously. The plates are designed to facilitate the detection and quantification of specific proteins, antibodies, or other analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan fc, by Thermo Fisher Scientific (4 mentions)
Tmb substrate, by Promega (3 mentions)
Tmb substrate set, by BioLegend (3 mentions)
H2so4, by Fujifilm (3 mentions)
Pnpp substrate, by Merck Group (2 mentions)
Elisa stop solution, by Solarbio (2 mentions)
Tween 20, by Merck Group (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Microplate reader, by Molecular Devices (2 mentions)
Elisa reader, by Bio-Rad (2 mentions)
Tween 20, by Fujifilm (2 mentions)
Goat anti mouse total igg or iga conjugated to horseradish peroxidase, by Jackson ImmunoResearch (2 mentions)
Elisa plate reader, by Gene Tech (2 mentions)
Blocking buffer, by Solarbio (1 mentions)
Elisa coating buffer, by Solarbio (1 mentions)
Multiskan mk3, by Thermo Fisher Scientific (1 mentions)
Tmb single component substrate solution, by Solarbio (1 mentions)
Avidin alkaline phosphatase, by Merck Group (1 mentions)
P nitrophenyl phosphate, by Merck Group (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Merck Group (1 mentions)
75 protocols using 96 well elisa plate
1
ELISA for Detecting CVA6-Specific Antibodies
2
Pan-Specific ELISA for Mouse Anti-Human Ad5 Antibodies
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A pan-specific (IgA, IgE, IgGs, IgM) ELISA for mouse anti-human Ad5 antibodies was performed, as previously described [19 (link),28 (link)]. A 96-well ELISA plate (Corning Costar, Acton, MA, USA) was coated with 5 × 109 viral particles of human Ad5 per well overnight at 4 °C in a 100 mM bicarbonate buffer at pH 9.6. The plate was then washed with TBS (Tris-buffered saline) and blocked with 3% BSA (bovine serum albumin) in TBS. Mouse bronchoalveolar lavage fluid (BALF) and serum diluted in 1:200 or 1:200,000 TBS, respectively, were added to the wells for overnight incubation. After washing with TBS, the plate was incubated with anti-mouse-Ig-biotin (BD Pharmingen, San Diego, CA, USA) and diluted in 1:5000 TBS for 3 hr at room temperature. The plate was washed again and incubated with avidin-alkaline phosphatase (Sigma-Aldrich, Oakville, ON, Canada), diluted 1:50,000 in TBS for 2 hr at room temperature. The plate was subsequently washed and incubated with 1 mg/mL p-nitrophenyl phosphate (Sigma-Aldrich, Oakville, ON, Canada) in 100 mM diethanolamine buffer at pH 9.8, containing 0.5 mM MgCl2, for 10 min at room temperature. The reaction was stopped by the addition of 25 µL of 0.2 M EDTA (ethylenediaminetetraacetic acid) and the optical density was read at 405 nm.
3
Antibody Titration for rSsEno Protein
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rSsEno IgG, IgG1, IgG2a and IgG3 antibody titration was conducted as described by Portuondo et al.19 (link) with some modifications. Briefly, a 96-well ELISA plate (Costar) was coated with 5 μg rSsEno/mL in PBS and incubated overnight at 4 °C. The plate was washed with washing buffer (0.1% Tween 20 in PBS) and then saturated for 1 h at RT with blocking buffer (5% dried skim milk in washing buffer). Next, dilutions (1:100 in blocking buffer) of the serum samples were added to each well and incubated for 2 h at RT. After washing, 100 µl/well of peroxidase-conjugated anti-mouse IgG (1/500) (Sigma) in blocking buffer was added and incubated at 37 °C for 1 h. For determination of the IgG1, IgG2a and IgG3 subclasses, ELISA plates coated as before were first incubated with an unconjugated rabbit anti-mouse IgG1, IgG2a or IgG3 (Bio-Rad) at 37 °C for 1 h and then with a peroxidase-conjugated goat anti-rabbit IgG (Sigma) overnight at 4 °C. After exhaustive washing, immune complexes were revealed by incubation with tetramethylbenzidine for 30 min at RT. The reaction was stopped by the addition of 50 µL/well 1 M H2SO4, and the absorbance was read with an ELISA reader (Multiskan ascent, Labsystem) at 450 nm.
4
ELISA for Detecting PCNA-NKp44 Interaction
5
Screening Phage Library for TeNT-Hc Binders
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After three rounds of panning, the phage clones were screened by an enzyme-linked immunosorbent assay (ELISA). The 96-well ELISA plate (Costar, Corning, NY, USA) was coated with 40 nM TeNT-Hc, and also coated with 130 nM Yersinia pestis capsular F1, 54 nM Yersinia pestis capsular V protein (F1 and V, purified in our lab) and 91 nM interferon-omega (IFN-ω, purified in our lab) individually as negative antigens.50 μL of phage particles at a concentration of approximately 8 μM in 3% Marvel™ PBS (pH 7.4) were added, and the binding of the phages to the immobilized antigen was performed at 37 °C for 2 h. The unbound phages were removed by washing extensively with PBST. The plate wells were detected by horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE Healthcare), followed by the addition of peroxidase substrate, o-phenylenediamine dihydrochloride (OPD). 2 M H2SO4 was used to stop the reaction, and the results were monitored at OD492/630 with a microplate reader (Multiskin MK3, Shanghai Labsystems, Shanghai, China). The particular clones that were bound to TeNT-Hc were selected, and the corresponding phage plasmids were sequenced.
6
Quantification of Anti-OCT4 IgG by ELISA
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Anti-OCT4 IgG was measured by an ELISA as previously reported20 . Briefly, each 96-well ELISA plate (Costar, Corning, NY, USA) was coated with 2 µg of synthetic OCT4 peptide at 4°C overnight. Plates were then washed with PBST (0.01 M PBS containing 0.05% Tween 20, pH 7.4) three times. Serum samples were detected at a 1:500 dilution for 2 h. Alkaline phosphatase-conjugated detection antibody for total IgG (Sigma-Aldrich) was added and incubated for 1 h at room temperature. Then, p-NPP substrate (Millipore) and stop solution (50 µL of 3 M NaOH) were added to each well, and the optical density (OD, 405 nm) was measured by a spectrophotometer (BioTek, Winooski, VT, USA).
7
Antibody-Specific Phage Panning Protocol
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Panning allows the selection of displayed oligopeptides based on specific affinity binding to serum antibodies. In order to reduce background signals, the serum used for panning was precleared from antibodies reactive with phage particles alone. Therefore, two wells of a 96-well ELISA plate (Costar) were coated with 4 × 1010 cfu Hyperphage in 300 μL phosphate buffer saline (PBS). The protein binding capacity of the well surface was saturated with 350 μL blocking solution (PBS supplemented with 2% skim milk powder (Roth) and 0.1% Tween 20) per well. The blocking solution was removed and the mouse sera (1:500 dilution in 300 μL blocking buffer) were incubated for 1 h in each of the two wells (total 2 h) with immobilized “Hyperphage” to preclear anti-phage serum antibodies.
The precleared serum was transferred into 2 wells (150 μL each) previously coated with anti-mouse (IgA, IgG and IgM specific) antibodies (antikoerper-online, ABIN376851) and saturated with blocking buffer in order to capture the serum antibodies used for the panning procedure (2 h at room temperature). Excess serum antibodies and other serum proteins were removed by washing 3 times with washing buffer (PBS supplemented with 0.05% Tween 20).
The precleared serum was transferred into 2 wells (150 μL each) previously coated with anti-mouse (IgA, IgG and IgM specific) antibodies (antikoerper-online, ABIN376851) and saturated with blocking buffer in order to capture the serum antibodies used for the panning procedure (2 h at room temperature). Excess serum antibodies and other serum proteins were removed by washing 3 times with washing buffer (PBS supplemented with 0.05% Tween 20).
8
ELISA for Anti-FcεR1α Autoantibody
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In-house enzyme-linked immunosorbent assay (ELISA) was developed for detecting anti-FcεR1α auto-antibody. A 96-well ELISA plate (Costar, Kennebunk, MC) was coated with 0.00035 mg/ml of human FcεR1alpha protein (Sino Biological, Wayne, PA). After the plate was kept overnight at 4°C, it was washed five times with PBS-Tween and blocked with 3% skim milk for 1 hour at 37°C. After washing again, a 50 μl serum sample diluted with 1% skim milk buffer to final dilution at 1 : 100 dilution was added into each well; then, the plate was incubated at 37°C for one hour. After incubation, the plate was washed; meanwhile, HRP-conjugated rabbit anti-hIgG (Dako, Santa Clara, CA) was freshly prepared final dilution at 1 : 1,000 and added to all sample wells. The plate was incubated at 37°C for one hour and washed after all. To measure the amount of anti-FcεR1α auto-antibody, the reaction was developed to detect color using 3,3′,5,5′-tetramethylbenzidine (Abcam, Cambridge, UK) in substrate solution at room temperature in a dark chamber for 10 min. The reaction was stopped with 1 N HCl, and optical density (OD) was read at 450 nm with a microplate spectrophotometer (Biotek, Winooski, VT). To quantify human IgG anti-FcεR1α autoAbs, human immunoglobulin G (hIgG) range from 0.25 to 0.0019 μg/ml was also used to establish a standard curve.
9
ELISA Quantification of Reconstituted Infliximab
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The IgG content of the reconstituted samples was determined with an ELISA. The 96-well ELISA plate (Costar, #3590) was coated overnight at 4°C with polyclonal goat-anti-human-IgG-Fc (5 ug/mL in PBS; Jackson) 100 μL per well. The plate was washed three times with wash-buffer (PBS containing 0.05% Tween 80) followed by blocking for 2 hours with blocking buffer (1% m/v fish skin gelatin, Roche). The control and reconstituted Infliximab samples were filtered with a 0.22 μm syringe filter (Whatman) and a dilution series in blocking buffer with a starting concentration of 500 ng/mL, diluted to 1 ng/mL (according to Nanodrop measurement), was added to the plate. For stressed samples, a starting concentration of 1000 ng/mL was used. After 5x washing, 100 μL of detection antibody goat-anti-human-IgG HRP conjugate (Jackson) was added and incubated for 1 hour at room temperature. After 2 washing 5x, bound HRP-conjugate was detected with tetramethyl benzidine (TMB) as the peroxidase substrate. The enzymatic reaction was allowed to proceed at room temperature for 3 min and stopped by adding 50 μL of 1 M sulfuric acid. The absorbance was measured at a wavelength of 450 nm using an Envision plate reader (Perkin Elmer). Based on different dilutions, the data was calculated back to the theoretical concentration of 5 mg/mL.
10
ELISA for Anti-OCT4 and Anti-Globo H IgG
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In order to assess humoral responses to anti-OCT4 IgG and anti-Globo H IgG levels, ELISA was performed to measure antigen-speci c total IgG, according to previously described (59) . Brie y, each 96-well ELISA plate (Costar, Corning, New York, USA) was coated with 2 μg synthetic OCT4 peptide and 2 μg Globo H and incubated at 4°C overnight. The plates were then washed with PBST (0.01 M PBS containing 0.05% Tween 20, pH 7.4) three times. Serum samples were detected at a different dilution for 2 h. Alkaline phosphate-conjugated detection antibody for total IgG (Sigma-Aldrich) was added and incubated for 1 h at room temperature. Then, p-NPP substrate (Millipore) and stop solution (50 μL of 3 M NaOH) were added to each well, and the optical density (OD, 405 nm) was measured using a spectrophotometer (BioTek, Winooski, VT, USA).
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