BKPyV DNA was measured with an internal controlled quantitative in‐house quantitative polymerase chain reaction (PCR), amplifying 131 bp on the VP2, with a detection limit of log 2 cp/ml as previously described by Gard et al.18 Briefly, samples were extracted according to the manufactures instructions, using 190 μl sample with the addition of 10 μl seal herpes virus (PhHV), as internal control.19 Primer sequences were described by Gard et al.18 All PCR reactions were performed with 20 μl DNA and 30 μl PCR mix, containing 2× Universal Mastermix (Thermo Fisher, USA), 5 mg/ml bovine plasma albumin (Roche Diagnostics, Germany), 300 nM primers, 100 nM probes, and DNAse/RNAse‐free water (Sigma, The Netherlands). The ABI PRISM 7500 (Life Technologies, USA) was used for the detection and amplification using the thermal profile: 50°C for 2 min, 95°C for 10 min followed by 42 cycles of 95°C for 15 s and 60°C for 1 min.
Bovine plasma albumin
Manufactured by Roche
Sourced in Germany
Bovine plasma albumin is a protein derived from the blood plasma of cattle. It is a commonly used component in various laboratory applications, providing a standardized source of protein for cell culture, diagnostic assays, and other experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Universal buffer, by Roche (2 mentions)
Dnase rnase free water, by Merck Group (1 mentions)
Universal master mix, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
2 2 azino bis 3 ethylbenzthiazoline 6 sulfonic acid abts, by Merck Group (1 mentions)
3 protocols using bovine plasma albumin
1
Quantitative PCR for BKPyV DNA
2
Antibody Production and Characterization
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Chemicals, reagents & equipment Roche Diagnostics GmbH (Penzberg, Germany) produced all antibodies. They were aliquoted and stored at -80 • C. Murine monoclonal antibodies against human IgG and cynomolgus IgG were generated from mice immunized with purified human IgG or cynomolgus IgG. Antibodies from hybridoma supernatants were purified by protein A chromatography. Conjugates of cynomolgus monkey IgG with human IgG were generated in house [11] . Biotin (Bi)-and digoxigenin (Dig)-labeling was performed in house. Horse radish peroxidase (HRP)-labeled polyclonal anti-Dig antibody Fab fragments (pAb-Dig-S-Fab-HRP), phosphate-buffered saline (PBS), 2,2 -azinobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS; ready to use solution) and Tween 20, were from Sigma Aldrich (MO, USA). Bovine plasma albumin and Roche Universal buffer were from Roche Diagnostics GmbH. Low Cross Buffer R was from Candor Bioscience GmbH, Wangen, Germany. Cynomolgus monkey serum (individual and pooled) was from Sera Laboratory International, Ltd (Haywords Heath, UK). Streptavidin (SA)-coated microtiter plates (SA-MTP) were from Microcoat Biotechnologie GmbH (Bernried, Germany). Antibodies and standards for the Ella cartridges were provided by Roche and labeled, immobilized and used by ProteinSimple, Biotechne, MN, USA, to produce cartridges for the two assays.
3
Monoclonal Antibody Generation and Characterization
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The murine monoclonal antibodies against cynomolgus monkey IgG and minipig IgG were generated from mice immunized with purified cynomolgus monkey or minipig IgG. Antibodies from hybridoma supernatants were purified by protein A chromatography. Conjugates of cynomolgus monkey or minipig IgG with human IgG and anti-human kappa light chain M1.7.10 were generated in house. All antibodies were produced by Roche Diagnostics GmbH, Penzberg, Germany, and were stored in aliquots at -80 • C until use. Biotin (Bi)and digoxigenin (Dig)-labeling was performed by Roche Diagnostics GmbH. Polyclonal sheep antibody Fab fragments against Dig coupled with HRP (pAb-Dig-S-Fab-HRP), phosphate-buffered saline (PBS), Tween 20, 2,2´-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS; ready to use solution), bovine plasma albumin and Roche Universal buffer were obtained from Roche Diagnostics GmbH. Low Cross Buffer R was from Candor Bioscience GmbH (Wangen, Germany). Individual and pooled minipig sodium citrate, theophylline, adenosine and dipyridamol (CTAD) plasma, as well as individual and pooled cynomolgus monkey CTAD plasma for in vitro studies was obtained from Sera Laboratory International Ltd (Haywords Heath, Sussex, UK). Streptavidin-coated microtiter plates (SA-MTPs) were from Microcoat Biotechnologie GmbH (Bernried, Germany).
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