The binding of mFcALF2 to four species of bacteria, including E. coli, V. alginolyticus, B. licheniformis and S. epidermidis, was examined by indirected ELISA according to the method described previously [28 (link)]. The freshly cultured bacteria were collected and washed with PBS three times. Then the bacteria were resuspended by coating buffer (Na2CO3 1.59 g/L, NaHCO3 2.93 g/L, pH 9.6) to 108 cfu/mL. A 96-well plate was coated with 100 μL of bacteria suspension at 4 °C overnight. Then the wells were washed and blocked with 5% nonfat milk in Tris-buffered saline (TBS) buffer at 37 °C for 2 h. After three washes with TBS, 100 μL of mFcALF2 (32 μM) were added and incubated at 37 °C for 2 h. The wells were washed three times, and 100 μL HRP-conjugated anti-His Tag mouse monoclonal antibody (1/2000 diluted in TBS) was added. After incubation at 37 °C for 2 h and washing as described above, the reactivity was measured using 100 μL soluble TMB substrate solution (TianGen, Beijing, China). The absorbance was measured at 405 nm. The assay was performed in triplicates in three independent experiments.
Tmb substrate solution
Manufactured by Tiangen Biotech
Sourced in China
TMB substrate solution is a colorimetric reagent used in enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the presence of target analytes. The solution contains the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), which undergoes an enzymatic reaction catalyzed by the enzyme conjugate, resulting in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Elisa plate reader, by Bio-Rad (1 mentions)
Elx808, by Agilent Technologies (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
96 well plate, by Corning (1 mentions)
Horseradish peroxidase hrp conjugated goat anti human igg, by ZSGB-BIO (1 mentions)
96 well elisa plate, by Thermo Fisher Scientific (1 mentions)
Stop solution, by Solarbio (1 mentions)
Vegf165, by GenScript (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Skim milk, by Solarbio (1 mentions)
Automated elisa plate reader, by Bio-Rad (1 mentions)
Pbst buffer, by Solarbio (1 mentions)
Microplate absorbance reader, by Bio-Rad (1 mentions)
11 protocols using tmb substrate solution
1
Binding of mFcALF2 to Bacterial Species
2
Porcine Antibody ELISA Assay
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Sera were tested by indirect ELISA at 35 dpv and 47 dpv. Briefly, the peptide epitope B
(SHIQLIYNL) and epitope 7 (QWGRL) were synthesized by Shanghai Sangon Biological
Engineering and Technology Co., Ltd., and coated on high binding capacity microplate
(Corning Inc., Corning, NY, USA) at 10 µg/ml diluted with 50 mM carbonate buffer (pH 9.6)
at 4°C overnight. Following washing with PBST buffer (PBS containing 0.5% Tween-20, pH
7.4), wells were blocked using 2% bovine serum albumin at 4°C overnight. One hundred
microliters of sera samples were added to each well at a 1:100 dilution in PBS containing
1.2 mg/ml E. coli lysate and was subsequently incubated at 37°C for 1 hr.
After washing five times at an interval of 1 min by PBST buffer, a 100 µl volume of
HRP-labeled goat anti-porcine IgG (KPL, Milford, MA, USA) secondary antibody (Sigma,
Burlington, MA, USA) was added at 1:20,000 dilutions, following which the plates were
further incubated at 37°C for 1 hr. The wells were washed as before described and then
incubated with a 100 µl volume of TMB substrate solution (Tiangen, Beijing, China) at room
temperature for 10 min in the dark. The reaction was stopped by adding a 100 µl volume of
stop solution (2 M H2SO4). The absorbance at 450 nm was recorded
using an ELISA plate reader (Bio-Rad).
(SHIQLIYNL) and epitope 7 (QWGRL) were synthesized by Shanghai Sangon Biological
Engineering and Technology Co., Ltd., and coated on high binding capacity microplate
(Corning Inc., Corning, NY, USA) at 10 µg/ml diluted with 50 mM carbonate buffer (pH 9.6)
at 4°C overnight. Following washing with PBST buffer (PBS containing 0.5% Tween-20, pH
7.4), wells were blocked using 2% bovine serum albumin at 4°C overnight. One hundred
microliters of sera samples were added to each well at a 1:100 dilution in PBS containing
1.2 mg/ml E. coli lysate and was subsequently incubated at 37°C for 1 hr.
After washing five times at an interval of 1 min by PBST buffer, a 100 µl volume of
HRP-labeled goat anti-porcine IgG (KPL, Milford, MA, USA) secondary antibody (Sigma,
Burlington, MA, USA) was added at 1:20,000 dilutions, following which the plates were
further incubated at 37°C for 1 hr. The wells were washed as before described and then
incubated with a 100 µl volume of TMB substrate solution (Tiangen, Beijing, China) at room
temperature for 10 min in the dark. The reaction was stopped by adding a 100 µl volume of
stop solution (2 M H2SO4). The absorbance at 450 nm was recorded
using an ELISA plate reader (Bio-Rad).
3
Indirect ELISA for pB602L Protein
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Indirect ELISA was performed as described previously [12 (link)]. In brief, purified recombinant pB602L protein diluted in Tris-HCl buffer (pH 8.5) to a final concentration of 2.5 μg/mL was coated onto 96-well ELISA plates (100 μl/well) and incubated overnight at 4 ℃. The supernatant was discarded, and the plate was washed three times with phosphate-buffered saline (PBS) (pH 7.2-7.4) containing 0.5% Tween-20 (PBST). After blocking with 5% Difco skim milk for 1 h at room temperature, the monoclonal antibodies purified from ascites were added to the ELISA plate (100 μl/well), which was incubated at 37 ℃ for 30 min. After six washes with PBST, a rabbit anti-mouse IgG horseradish peroxidase (HRP) conjugate (whole molecule) (Sigma-Aldrich, USA) was added as a secondary antibody at a dilution of 1:2000 and incubated at 37 ℃ for 30 min, followed by six washes with PBST. TMB substrate solution (TianGen Biotech, China) was added to the plate (100 μl/well), which was incubated in the dark at room temperature for 20 min. The reaction was terminated by addition of 2M H2SO4 (50 μl/well), and the optical density was measured at 450 nm using a microplate reader (ELx808, BioTek, USA).
4
Quantitative Aβ Peptide Immunodetection
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For indirect ELISA, the wells of a 96-well plate (Corning Inc., USA) were coated with 0.5 μg/well of Aβ42 overnight at 4°C. The plate was blocked with 5% skimmed milk, and then incubated with primary antibodies (1F12, AuNP-1F12, or 1F12-MNPs) and the secondary antibody HRP-conjugated goat anti-mouse IgG (H + L) in an order. The 96-well plate was washed four times with PBS-T in each step. The immunoreaction was visualized by TMB substrate solution (Tiangen Biotech, Beijing, China) and detected with an Epoch Microplate Spectrophotometer (Bio Tek, USA) at 450 nm.
For competitive ELISA, 96-well plates were coated with 0.5 μg Aβ42 in each well and blocked with 5% skimmed milk. Triplicates of biotinylated 1F12 or 2C6 (250 ng/mL) were mixed with serially diluted Aβ preparations of Aβ40, Aβ42Ms, and Aβ42Os diluted in PBS with the final concentration from 25 μM to 5 pM. After 1 h pre-incubation at 4°C in 1.5 mL tubes, the antibody-antigen mixtures were incubated on the Aβ42 antigen-coated plates for 1 h at 25°C. After incubating with streptavidin-coupled poly-HRP, the immunoreaction was visualized and detected as above described.
For competitive ELISA, 96-well plates were coated with 0.5 μg Aβ42 in each well and blocked with 5% skimmed milk. Triplicates of biotinylated 1F12 or 2C6 (250 ng/mL) were mixed with serially diluted Aβ preparations of Aβ40, Aβ42Ms, and Aβ42Os diluted in PBS with the final concentration from 25 μM to 5 pM. After 1 h pre-incubation at 4°C in 1.5 mL tubes, the antibody-antigen mixtures were incubated on the Aβ42 antigen-coated plates for 1 h at 25°C. After incubating with streptavidin-coupled poly-HRP, the immunoreaction was visualized and detected as above described.
5
ELISA for VP1 and VP2 IgG Detection
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For VP1- and VP2-specific IgG tests, 96-well ELISA plates (Thermo Fisher, USA) were coated with 2 μg/mL purified VP1 or VP2 protein at 4 °C overnight. Plates were blocked using 1% BSA in PBST with 0.05% Tween-20 for 2 h at 37 °C. Then, 1:100 diluted serum was added in PBST containing 0.5% BSA and incubated for 1 h at 37 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (ZSGB-Bio, China) for 1 h at room temperature. PBST was used to wash between each step. Finally, 100 μL of TMB substrate solution (TIANGEN, China) was added for color development for 15 min, and then an equal volume stop solution (Sangon Biotech, China) was added to terminate the reactions. The absorbance was determined at 450 nm wavelength via a microplate spectrophotometer (Thermo Scientific Varioskan LUX, USA). For the peptide-specific IgG test, the procedure was similar to the one described above, except that the coated antigen was 5 M peptides, the serum dilution was 1:25, and the secondary antibody was diluted to 1:5000.
6
ELISA for Anti-VEGF Protein Quantification
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ELISA was used to determine the EC50 binding ability of recombinant proteins to VEGF165 and to quantify the concentration of anti‐VEGF proteins in samples from the aqueous chamber, vitreous humor and ocular tissue. Briefly, a 96‐well plate was coated overnight with 100 μL of 300 ng/mL VEGF165 (GenScript, Nanjing, China), followed by washing with 150 μL PBST three times, and immobilization with 5% non‐fat milk solution (Solarbio, Beijing, China) for 1 h at room temperature. Diluted proteins were added to the wells and incubated for 1 h at room temperature. Subsequently, 100 μL of antibodies (His‐tag antibody HRP‐66005, Proteintech, Wuhan, China, for N1H and N2H‐9GS; HRP, Goat Anti‐Human IgG, Abbkine, Wuhan, China, for nanoFc) were added to the plates and incubated for 1 h at room temperature to capture recombinant proteins. Lastly, 100 μL of TMB substrate solution (Tiangen, Beijing, China) and 50 μL of stop solution (Solarbio, Beijing, China) were sequentially added to the wells. The results were read at OD450 using a microplate reader (SpectraMax Gemini XPS/EM Microplate Readers, Molecular Devices, USA).
7
Quantifying Antibody Titers in Immunized Mice
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Enzyme-linked immunosorbent assay (ELISA) was used to measure the sera antibody titers of immunized BALB/c mice. The 96-well plate was coated with 100 μL of 10 μg/mL rETX in 0.05 M carbonate-buffered saline solution overnight, and then the plate was blocked with 200 μL of 5% bovine serum albumin (BSA) for 2 h at 37 °C. After being incubated with 100 μL of 10-fold serial diluted serum for 1 h at 37 °C and washed three times by PBST (0.01M PBS, 0.05% Tween-20), the plate was incubated with HRP-coupled goat anti-mouse IgG (1:50000) at 37 °C for 1 h. Then the plate was washed three times with PBST and added the TMB substrate solution (TIANGEN Biotechnology) to each plate, and finally 2 M H2SO4 was used to stop the reaction. The absorbance was determined at 450 nm by a microplate reader (Molecular Device).
8
ELISA-Based Binding Assay for Bacterial Interactions
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ELISA was performed as previously reported [23 (link)]. In brief, Gram-negative (Vibrio anguillarum, Vibrio harveyi, Pseudomonas fluorescens, Edwardsiella piscicida) and Gram-positive (Streptococcus iniae, Micrococcus luteus, Bacillus subtilis) bacteria were grown as described previously [24 (link)] and resuspended in coating buffer (0.159% Na2CO3, 0.293% NaHCO3, pH 9.6). Lipopolysaccharide (LPS) and peptidoglycan (PGN) (InvivoGen, San Diego, CA, USA) were diluted in coating buffer to a final concentration of 0.1 mg/mL. The bacterial resuspension or the LPS/PGN dilution was added into 96-well microtiter plates, and the plates were placed at 4 °C overnight. After blocking with 5% skim milk for 1 h, the plates were washed three times with PBST. Different concentrations (1, 2, 4, 8, 16, or 32 μg/mL) of AjCASPX2, CARDAjCASPX2, or Trx were added to the plates. The plates were incubated at 25 °C for 2 h and washed as above. The plates were then incubated with mouse anti-His tag antibody and HRP-conjugated goat anti-mouse IgG successively. The plates were washed five times with PBST, and TMB substrate solution (TIANGEN, Beijing, China) was added to the plates. The plates were analyzed with a microplate reader (BioTek Instruments, Winooski, VT, USA).
9
Bacterial Binding Assay by ELISA
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Protein binding to the bacteria and bacterial components was determined by ELISA as described previously [21 (link)]. Briefly, bacteria (108 CFU/mL) or bacterial cell wall components (PGN, LTA, or LPS, 200 μg/mL) were each added to a 96-well microtiter plate. The plate was incubated at 4 °C for 12–16 h, followed by washing in PBST (PBS with 0.05% Tween 20) three times. Two hundred microliters of 5% skim milk (Solarbio, Beijing, China) dissolved in PBST was added to the plate. After incubation at 37 °C for 2 h, the plate was washed as above. Two hundred microliters of Crus2, Crus2DC, or PBS (control) was added to the plate. After incubation at 37 °C for 1 h, the plate was washed as above. The bound protein was detected by adding anti-His antibody conjugated with HRP (1/1000 dilution) (ABclonal, Hubei, China) into the plate. After incubation at 37 °C for 1 h, the plate was washed five times with PBST. The TMB substrate solution (Tiangen, Beijing, China) was added to the plate for color development. ELISA stop solution was then added to the plate. The absorbance at 450 nm was measured using a multifunctional microplate reader. The binding index = OD450 of protein/OD450 of PBS.
10
Horseradish Peroxidase Labeling of mAbs
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mAbs were labeled with horseradish peroxidase (HRP, Sigma, Missouri, USA) by the sodium periodate oxidation method described by Kanpp [19] (link). The titers of the labeled mAbs were determined by indirect ELISA. The 96-well microtiter plates (Maxisorp Nunc, Denmark) were coated with purified capture mAbs diluted in sodium carbonate buffer. TMUV was added as antigen and the plates were incubated for 30 min at 37°C. Then mAbs conjugated with horseradish peroxidase (detection antibody) were added. The unbound conjugates were washed off after incubation, and 3, 3', 5, 5'-Tetramethylbenzidine (TMB) substrate solution (TIANGEN, Beijing, China) was added to each well. Incubation was carried out for 30 min and the reaction was stopped by adding 3M H2SO4. Plates were read at 450 nm on an automated ELISA plate reader (Bio-Rad, USA). The best pairing antibodies were obtained according to the recorded result.
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