The enzyme-linked immunosorbent assay [ELISA] technique was used for detection of CHI3L1 autoAbs in serum samples by using recombinant human CHI3L1 as solid-phase antigen. Briefly, 2 µg/ml CHI3L1, diluted in bicarbonate buffer [pH 9.5], was coated on microtitre plates [Greiner Bio-one] overnight at 4°C. After blocking with 2% bovine serum albumin at room temperature for 1 h, the plates were incubated with serum samples [diluted 1:100 in sample buffer] for 1 h at room temperature. After washing, horseradish peroxidase-conjugated anti-human IgA, IgG [Dianova] or anti-human IgA secretory component [antibodies-online] were added. Plates were developed with ready-to-use TMB substrate [Seramun Diagnostik] and stopped with 0.25 M sulphuric acid after 10 min. The optical density [OD] was read at 450 nm/620 nm in a microplate reader [Sunrise, Tecan Trading].
Microtitre plates
Manufactured by Greiner
Sourced in Germany
Microtitre plates are flat-bottomed plates with multiple wells used for various laboratory applications. They provide a standardized format for performing small-scale experiments, tests, or assays. The plates are typically made of plastic and are available in different well sizes and configurations to accommodate different sample volumes and experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Potassium phosphate tribasic, by Merck Group (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Acetone, by Thermo Fisher Scientific (2 mentions)
Toluene, by Thermo Fisher Scientific (2 mentions)
Dichloromethane, by Thermo Fisher Scientific (2 mentions)
Mesitylene, by Merck Group (2 mentions)
Diethyl ether, by Thermo Fisher Scientific (2 mentions)
Triethylamine, by Thermo Fisher Scientific (2 mentions)
N 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride, by Merck Group (2 mentions)
Carbon disulfide, by Merck Group (2 mentions)
4 4 azobis 4 cyanovaleric acid, by Merck Group (2 mentions)
4 dimethylaminopyridine, by Thermo Fisher Scientific (2 mentions)
Concanavalin a (cona), by Vector Laboratories (2 mentions)
Dodecanethiol, by Merck Group (2 mentions)
Pentafluorophenol, by Thermo Fisher Scientific (2 mentions)
Soybean agglutinin, by Vector Laboratories (2 mentions)
N hydroxethyl acrylamide, by Merck Group (2 mentions)
2 bromo 2 methylpropionic acid, by Thermo Fisher Scientific (2 mentions)
Ricinus communis agglutinin 120, by Vector Laboratories (2 mentions)
Microplate reader, by Tecan (1 mentions)
9 protocols using microtitre plates
1
Quantitative ELISA for CHI3L1 Autoantibodies
2
Synthesis and Characterization of Glyco-Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals were used as supplied unless otherwise stated. Acetone, dichloromethane, toluene, methanol, diethyl ether were purchased from Fischer Scientific at laboratory grade. Dodecane thiol (≥98%), potassium phosphate tribasic (≥98%), carbon disulfide (99%), N-hydroxethyl acrylamide (97%), 4,4′-azobis(4-cyanovaleric acid) (98%), mesitylene (reagent grade), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (≥98%) were all purchased from Sigma-Aldrich. 2-Bromo-2-methylpropionic acid (98%), 4-(dimethylamino)pyridine (99%), pentafluorophenol (99%), triethylamine (99%) were purchased from Acros. Microtitre plates were purchased from Greiner Bio-one. 10 mmol HEPES buffer containing 0.05 M NaCl, 0.1 mM CaCl2 and 0.01 mM MnCl2 (pH 7.5, HEPES) was prepared in 200 mL of milliQ water (with a resistance > 19 mOhms). 60 nm gold nanoparticles were obtained from BBI solutions. Concanavalin A and soybean agglutinin and Ricinus communis agglutinin-120 were purchased from Vector Labs.
3
Quantifying mAb Binding Efficacy to PfHRP2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To understand the binding efficacy of these mAbs, a malaria PfHRP2-specific mAb C1-13 (kindly provided by Dr Martin Bubb, National Products Institute, South Africa) was used as positive control in this study. mAb C1-13 is a commercial mouse IgG1 mAb with binding specificity against PfHRP2, that has been reported in previous studies [23 (link),27 (link)].
Microtitre plates (Greiner Bio-One, Germany) were coated with 100 μL of diluted rPfHRP2 protein (1 μg/mL) and blocked with 2% MPBS. After three wash steps with 0.1% PBST, the blocked wells were incubated with 100 μL of two-fold serial diluted mAbs D2 and F9, and control antibody C1-13, starting at 20 ng/mL for 1 hour at room temperature. Plates were washed with 0.1% PBST. One-hundred μL of secondary detection antibody was added, using goat anti-human antibody-conjugated HRP (Cat #:A8667) (Sigma-Aldrich; MO, USA), diluted 1:5,000 in 2% MPBS for D2 and F9 wells, or secondary goat anti-mouse antibody-conjugated HRP (Cat #: 115-035-003) (Jackson, USA), diluted 1:5,000 in 2% MPBS for C1-13 wells. Plates were incubated at room temperature for 1 hour. After washing with 0.1% PBST, 100 μL of freshly prepared OPD solution (Sigma, USA) was added to each well, and absorbance read at a wavelength of 405 nm using ELISA plate reader (Biotek Synergy HT, USA).
Microtitre plates (Greiner Bio-One, Germany) were coated with 100 μL of diluted rPfHRP2 protein (1 μg/mL) and blocked with 2% MPBS. After three wash steps with 0.1% PBST, the blocked wells were incubated with 100 μL of two-fold serial diluted mAbs D2 and F9, and control antibody C1-13, starting at 20 ng/mL for 1 hour at room temperature. Plates were washed with 0.1% PBST. One-hundred μL of secondary detection antibody was added, using goat anti-human antibody-conjugated HRP (Cat #:A8667) (Sigma-Aldrich; MO, USA), diluted 1:5,000 in 2% MPBS for D2 and F9 wells, or secondary goat anti-mouse antibody-conjugated HRP (Cat #: 115-035-003) (Jackson, USA), diluted 1:5,000 in 2% MPBS for C1-13 wells. Plates were incubated at room temperature for 1 hour. After washing with 0.1% PBST, 100 μL of freshly prepared OPD solution (Sigma, USA) was added to each well, and absorbance read at a wavelength of 405 nm using ELISA plate reader (Biotek Synergy HT, USA).
4
Biofilm Formation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microorganisms were cultured to mid-exponential phase, harvested by centrifugation, and adjusted to OD650 = 0.1. Biofilms of recovered isolates were established in 96 well microtitre plates (Greiner) in 50 μl NB, by inoculating each well with 5 μl of equilibrated cells. Plates were incubated at 37°C for 48 h. Biomass was determined as described above.
5
Measurement of Collagen VII Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure the serum levels of rabbit and mouse collagen VII-specific antibodies, ELISA was performed as described, with minor modifications [20 (link),23 (link),36 (link)]. Briefly, microtitre plates (Greiner Bio-One, Frickenhausen, Germany) were coated, overnight with 200 or 500 ng of recombinant full-length collagen VII protein in 0.1 M bicarbonate buffer (pH 9.6). After blocking, wells were incubated with a 250-fold or a 50-fold dilution of mouse sera. Bound Abs were detected using horseradish peroxidase (HRP)-conjugated 3000 times or 2000 times diluted secondary Abs specific for rabbit (Novus Biologicals, Littleton, CO, USA) and mouse IgG (Invitrogen, Molecular Probes®) respectively, and orthophenylene diamine (Dako, Hamburg, Germany) as a chromophore (492 nm). For IgG subclass identification, the incubation with the 100 times diluted mouse sera was followed by incubation with 250 times diluted biotinylated monoclonal rat antimouse IgG1, IgG2a, IgG2b and IgG3 Abs (all from BD Pharmingen, Heidelberg, Germany), and HRP-conjugated streptavidine (Dianova, Hamburg, Germany). The immunoreactions were visualized as described above.
6
Hirudin-anticoagulated Blood Cytokine Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole blood (10 ml) was collected from healthy volunteers (ethical approval obtained from National Institute for Biological Standards and Control’s Human Materials Advisory Committee) by venepuncture from an antecubital vein into a sterile syringe. Blood was anticoagulated with recombinant hirudin (Pharmion, UK) to a final concentration of 30 μg/ml. Blood was processed for experiments within 30 minutes of collection.
Whole blood was distributed into microtitre plates (Greiner Bio-one, Frickenhausen, Germany) to which increasing concentrations of histones (Type III-S, Sigma, Gillingham, UK) were added. The microtitre plates were placed on an orbital shaker (500 rpm) in a humidified CO2 incubator and incubated for either 6 or 24 hours. After incubation, the blood was spun, and the supernatant was collected for cytokine analysis. The cellular component was washed four times using Hank’s balanced salt solution before being frozen for tissue factor analysis.
Whole blood was distributed into microtitre plates (Greiner Bio-one, Frickenhausen, Germany) to which increasing concentrations of histones (Type III-S, Sigma, Gillingham, UK) were added. The microtitre plates were placed on an orbital shaker (500 rpm) in a humidified CO2 incubator and incubated for either 6 or 24 hours. After incubation, the blood was spun, and the supernatant was collected for cytokine analysis. The cellular component was washed four times using Hank’s balanced salt solution before being frozen for tissue factor analysis.
7
Quantifying Autoantibodies in Serum
8
Synthesis and Characterization of Glyco-Nanoparticles
9
Antibacterial Activity of Plant Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Minimum inhibitory concentration (MIC) values for antibacterial activity of the plant extracts were determined using the microdilution bioassay in 96-well (Greiner Bio-one GmbH, Germany) microtitre plates (Eloff, 1998 (link)) as detailed by Ndhlala et al. (2009) (link). The pathogens used in the tests were, two Gram-negative (Escherichia coli ATCC 11775 and Klebsiella pneumoniae ATCC 13883), and two Gram-positive bacteria (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600). Neomycin (Sigma–Aldrich) was used as positive control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!