To determine whether rTs-Pmy bound to complement C1q, plates were coated with different amounts of human C1q (0, 0.5, 1.0, 1.5 μg) and BSA (2 μg) in 100 μl of coating buffer (100 mM Na2CO3/NaHCO3, pH 9.6) at 4°C overnight. After washing three times with 1× phosphate buffered saline (PBS) pH 7.4 containing 0.05% Tween-20 (PBST), the plates were blocked with PBS containing 2% BSA for 2 h at 37°C. The different amounts of rTs-Pmy (0, 1, 2, 3, 4 μg) in 100 μl of 20 mM Tris-HCl, 50 mM NaCl and 1 mM CaCl2, pH 7.4 were added for 1 h at 37°C. The plates were washed three times with PBST, the binding of rTs-Pmy to human C1q was determined with anti-Ts-Pmy monoclonal antibody 9G3 (1:2,500 in 1% BSA/PBS). HRP-conjugated goat anti-mouse IgG (BD Biosciences, USA; 1:10,000 in 1% BSA/PBS) was used as the secondary antibody and o-phenylendiamine dihydrochloride (OPD, Sigma, USA) was used as the substrate. The absorbance of the supernatant was measured at 450 nm with a MultiskanGO plate reader (Thermo, USA).
O phenylendiamine dihydrochloride opd
Manufactured by Merck Group
Sourced in United States
O-phenylendiamine dihydrochloride (OPD) is a chemical compound commonly used as a laboratory reagent. It is a colorless to light brown crystalline solid. OPD is primarily used as a substrate in enzyme-linked immunosorbent assay (ELISA) techniques, where it serves as a chromogenic indicator.
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Lab products found in correlation
Hrp conjugated goat anti mouse igg, by BD (3 mentions)
Multiskan go plate reader, by Thermo Fisher Scientific (1 mentions)
Immulon 2hb plate, by Thermo Fisher Scientific (1 mentions)
Multiscan fc reader, by Thermo Fisher Scientific (1 mentions)
Mouse anti his mab, by Tiangen Biotech (1 mentions)
Elisa reader, by Thermo Fisher Scientific (1 mentions)
Human c1q, by Merck Group (1 mentions)
Hrp conjugated streptavidin, by Abcam (1 mentions)
4 protocols using o phenylendiamine dihydrochloride opd
1
C1q Binding Assay of rTs-Pmy
2
ELISA-based Malaria Serology Protocol
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Malaria serology was performed with an in-house screening ELISA for detection of specific Plasmodium spp. antibodies. P. falciparum antigen (strain NF54) was coated in 0.05 M sodium carbonate buffer, pH 9.6, to Immulon 2HB plates (, Thermo Scientific, Wohlen, Switzerland,). After washing, diluted sera were added to the plates and incubated for 15 min at 37 °C. After additional washing steps, horseradish peroxidase conjugated goat-anti-human-IgG (KPL, 474-1006, BioConcept Ltd, Allschwil, Switzerland) was added. Plates were incubated for 15 min at 37 °C, subsequently washed, and o-Phenylendiamine Dihydrochloride (OPD, Sigma, Buchs, Switzerland) was added. Reaction was stopped with 8 M H2SO4, and absorption was read with a Multiscan FC reader (ThermoScientific, Wohlen, Switzerland) at 492 nm. All sera giving positive or equivocal results were additionally tested with an in-house confirmatory Malaria IFA.
3
C1q Binding Assay for rTs-CRT
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Microtiter plates were coated with different amounts of human C1q (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 µg) (Merck) in 100 μL/well of carbonate buffer (100 mM Na2CO3/NaHCO3, pH 9.6) overnight at 4°C. Control wells were coated with the same amounts of BSA. Following three washes with PBS + 0.05% Tween-20 (PBST), the wells were blocked with 200 µL of 3% BSA in PBS at 37°C for 2 h. After being washed, 0–1.5 µg of rTs-CRT in 100 µL of 20 mM Tris–HCl, pH 7.4, 50 mM NaCl and 1 mM CaCl2 were added to each well and incubated for 2 h at 37°C. Then, mouse anti-His mAb (1:10,000, TIANGEN) and HRP-conjugated goat anti-mouse IgG (1:10,000) (BD Biosciences, San Jose, CA, USA) were added and incubated for 1 h at 37°C. After the final washing, the substrate o-phenylendiamine dihydrochloride (OPD, Sigma) was added. The absorbance was measured at 450 nm with an ELISA reader (Thermo).
4
ELISA-based C1q Binding Assay for Recombinant Proteins
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An ELISA-based assay was also used to determine the C1q binding activity of TsPmy and its fragments according to the method as previously described [28 (link)]. Briefly, a 96-well ELISA plate was coated with human C1q at 5.0 ug/ml in carbonate buffer (100 mM Na2CO3/NaHCO3, pH 9.6), blocked with 5.0% (w/v) BSA in PBS at 37 °C for 2 h, then incubated with different concentrations of recombinant TsPmy fragments at 25 °C for 1 h. Anti-His mAb at 1:1000 in PBS/1% BSA was used to probe recombinant proteins, and HRP-conjugated goat anti-mouse IgG (BD Biosciences, Franklin Lakes, NJ, USA; 1:5000 in PBS/1% BSA) was used as secondary antibody. For the peptide binding assay, synthesized peptide P2 was labeled with biotin and the C1q coated plate was incubated with different concentrations of biotinylated peptide P2 (B-P2). HRP-conjugated streptavidin (Abcam, Cambridge, UK; 1:10,000 in PBS/1% BSA) served as the detection antibody. After adding the substrate o-phenylendiamine dihydrochloride (OPD, Sigma-Aldrich, St. Louis, MO, USA), absorbance was measured at 450 nm with a plate reader (Thermo Fisher, Life Technologies). BSA coated on the same plate was used as a negative control.
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