C3 in heparinized human plasma was measured after samples were diluted (PBST with1% BSA), detected using a sandwich ELISA (GenWay Biotech; San Diego, CA), according to the manufacturer's instructions. Briefly, Immulon ELISA plates were coated with 500ng/ml chicken IgY anti-human C3 in PBS overnight at 4°C. Plates were washed with PBST three times and blocked in 1% BSA in PBS for one hour at room temperature. Plasma samples and standards were diluted and added to the plates. After 2 hours of incubation at room temperature, plates were washed with PBST for three times. 20ng/ml HRP-conjugated chicken IgY anti- human C3 was added to detect C3. After one-hour incubation at room temperature, plates were washed five times with PBST and developed by BD OptEIA TMB substrate (BD; Franklin Lakes, NJ). Final readout was detected by absorbance at 450nm. Concentration of C3a in plasma samples was detected using human C3a ELISA kit (BD; Franklin Lakes, NJ) according to the manufacturer's instruction.
Opteia tmb substrate
Manufactured by BD
Sourced in United States
OptEIA TMB substrate is a laboratory reagent used in enzyme-linked immunosorbent assays (ELISA) to detect and quantify specific analytes. It serves as a chromogenic substrate for the horseradish peroxidase (HRP) enzyme, which catalyzes a color-producing reaction. The resulting color change can be measured using a spectrophotometer, providing a quantitative readout proportional to the amount of target analyte present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Nunc immuno maxisorp plates, by Thermo Fisher Scientific (5 mentions)
Imark microplate reader, by Bio-Rad (4 mentions)
Spectramax 190 microplate reader, by Molecular Devices (3 mentions)
Sandwich elisa, by GenWay Biotech (2 mentions)
Human c3a elisa kit, by BD (2 mentions)
D3664 5x2mg, by Merck Group (2 mentions)
Biotin, by Southern Biotech (1 mentions)
Streptavidin alkaline phosphatase, by Merck Group (1 mentions)
Anti mouse ig h l, by Southern Biotech (1 mentions)
Msips4w10, by Merck Group (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Sunrisetm, by Tecan (1 mentions)
Anti mouse igg hrp, by Bio-Rad (1 mentions)
Dsdna, by Merck Group (1 mentions)
Igg2c hrp, by Southern Biotech (1 mentions)
Protamine sulphate, by Merck Group (1 mentions)
Human igg elisa quantitation kit, by Fortis Life Sciences (1 mentions)
Goat anti human igg hrp, by Thermo Fisher Scientific (1 mentions)
Maxisorp immunoplate, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg hrp, by Southern Biotech (1 mentions)
24 protocols using opteia tmb substrate
1
Quantifying Human Plasma C3 Levels
2
Quantifying Antibody Responses via ELISA and ELISpot
3
Quantifying Human Plasma C3 Levels
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C3 in heparinized human plasma was measured after samples were diluted (PBST with1% BSA), detected using a sandwich ELISA (GenWay Biotech; San Diego, CA), according to the manufacturer's instructions. Briefly, Immulon ELISA plates were coated with 500ng/ml chicken IgY anti-human C3 in PBS overnight at 4°C. Plates were washed with PBST three times and blocked in 1% BSA in PBS for one hour at room temperature. Plasma samples and standards were diluted and added to the plates. After 2 hours of incubation at room temperature, plates were washed with PBST for three times. 20ng/ml HRP-conjugated chicken IgY anti- human C3 was added to detect C3. After one-hour incubation at room temperature, plates were washed five times with PBST and developed by BD OptEIA TMB substrate (BD; Franklin Lakes, NJ). Final readout was detected by absorbance at 450nm. Concentration of C3a in plasma samples was detected using human C3a ELISA kit (BD; Franklin Lakes, NJ) according to the manufacturer's instruction.
4
Evaluating SPy_2191 Antigen's Vaccine Potential
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To evaluate SPy_2191 antigen’s vaccine potential, total serum IgG levels were measured on day 0 (preimmune), 14, 28, and 42, by indirect enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well high-binding, polystyrene microtiter plates (NuncMaxiSorp™) were coated with SPy_2191 at a concentration of 500 ng per well, in coating buffer containing 50 mM sodium bicarbonate (pH 9.6). The plate was incubated overnight at 4 °C, followed by blocking with 150 μl of 2% bovine serum albumin (BSA) for 2 h at 37 °C. Serial dilution of the sera was prepared in PBS (102–106) and 0.1 ml of each dilution was added to the SPy_2191 precoated wells in triplicates and further incubated for 1 h at 37 °C. The plate was then washed four times with 1×PBST (1×PBS containing 0.05% tween-20) and incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG at a dilution of 1:5000, in PBS containing 2% BSA. After washing four times with 1×PBST, the microtiter plate was incubated in dark with BD OptEIA TMB substrate (BD Bioscience) for 20–30 min. The reaction was stopped by adding 1 N HCl before measuring the absorbance at 450 nm using 570 nm as the reference wavelength, in a TECAN SunriseTM microplate reader. The endpoint titre was defined as the highest sera dilution that gave an absorbance > mean+3 SD absorbance obtained at 1:100 dilution of the only PBS-immunized mice group.
5
Leishmania-specific IgG Antibody Detection
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Sera were prepared from naïve, immunized but either infected or uninfected mice for detection of Leishmania-specific IgG antibodies by indirect ELISA using biotin-labelled anti-mouse IgG2a and IgG1 (BD Biosciences) and developing the reaction by BD OptEIA™ TMB substrate [35] . Virulent Leishmania culture was resuspended in 20 mm Tris (pH 7•5), 150 mm NaCl, 1 mm each of EDTA and EGTA, 10% glycerol and 1% NP-40, diluted in sample buffer and probed with sera from empty or cloned vector primed mice in varying dilutions as indicated and detected with 1:5000 diluted HRP anti-mouse IgG (Bio-Rad Laboratories).
6
Autoantigen ELISA and Microarray Protocols
7
Quantifying Anti-dsDNA IgG Autoantibodies in Lupus
8
Quantifying Antibody Responses via ELISA
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96-well Nunc-Immuno MaxiSorp plates (Thermo Fisher Scientific) were precoated overnight at 4°C with 2 μg/ml anti-IgM, anti-IgG, anti-IgG1, and anti-IgG2C (Southern Biotech) or 50 µg/ml NP(5)-BSA and NP(30)-BSA (Biosearch Technologies). Plates were blocked for 1 h with 2% BSA in PBS before addition of diluted serum (1:6,250, 1:31,250, 1:156,250) for 2 h at RT or overnight at 4°C. Specific antibodies were detected by using goat anti–mouse IgM, IgG, IgG1, IgG3, or rat anti-IgG2C–horseradish peroxidase (1:2,000 dilution; Southern Biotech), and peroxidase reactions were developed by using OptEIA TMB substrate (BD Biosciences) and stopped with sulfuric acid. Absorbance at 450 nm was read by using a SpectraMax 190 microplate reader (Molecular Devices). 450-nm absorbance was corrected by subtraction of 570-nm absorbance.
9
Quantification of Serum and Supernatant IL-6 and Autoantibodies
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Serum and supernatant IL-6 levels were measured with the mouse IL-6 ELISA Ready-SET-Go! kit (88-7064-22; eBioscience). For specific autoAb ELISAs, 96 well Nunc-Immuno MaxiSorp plates (Thermo Fisher Scientific) were precoated overnight at 4°C with calf thymus dsDNA (100 µg/ml; D3664–5X2MG; Sigma-Aldrich) or Sm/RNP (5 µg/ml; ATR01-10; Arotec Diagnostics). Plates were blocked for 1 h with 1% BSA in PBS before addition of diluted serum for 2 h. Specific antibodies were detected using goat anti–mouse IgM–, IgG–, or IgG2c–horseradish peroxidase (1:2,000 dilution; SouthernBiotech), and peroxidase reactions were developed using OptEIA TMB substrate (BD) and stopped with sulfuric acid. Absorbance at 450 nm was read using a SpectraMax 190 microplate reader (Molecular Devices).
10
Quantifying Serum Insulin Antibodies
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The capture mouse anti-insulin antibody (1 μg/mL insulin) (clone 3A6; Novus Biologicals) was used to coat 96-well ELISA plates overnight at 4°C. Plates were washed and then blocked with 5% FBS for 1 h at RT. Following blocking, serum samples were added for 2 h at 37°C. Plates were then washed, and a detection mouse anti-insulin antibody (1 μg/mL) (clone 8E2-HRP; Novus Biologicals) was added for 2 h at 4°C. Antibody binding was visualized using OptEIA TMB substrate (BD Biosciences). The reaction was stopped using a diluted phosphoric acid, and absorbance was read at 450 nm.
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