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Beta propiolactone bpl

Manufactured by Merck Group
Sourced in United States

Beta-propiolactone (BPL) is a colorless, oily liquid used as a chemical intermediate in various industrial and laboratory applications. It serves as a cross-linking agent and disinfectant. BPL is commonly used in the synthesis of other organic compounds and for sterilization purposes in laboratory settings.

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13 protocols using beta propiolactone bpl

1

Rabies Virus Production and Purification

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Once the viruses were recovered, they were grown on Vero CCL81 cells in viral production serum-free medium (VP-SFM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% Glutamax, 1% penicillin-streptomycin, and 1% HEPES buffer. Cells were infected at an MOI of 0.01 in either a T175 flask or 2-stack chambers (Corning, Corning, NY, USA). Supernatant collections occurred 5 days post-infection and every 3 days afterward, for a total of 17 days. For titration, RABVs were overlayed on VeroCCL81 cells with a starting dilution of 1:10 and diluted 10-fold in a 96-well plate in triplicate. After 48 h, cells were fixed with acetone and stained with RABV-N GFP stain to determine the foci-forming units (ffu)/mL using fluorescence microscopy.
Viruses were filtered through 0.45 µm PES membrane filters (Nalgene, Conway, NH, USA) and concentrated down to 50 mL for purification. Viruses were purified over 20% sucrose cushion and ultracentrifuged at 25,000 rpm in a SW32 rotor for 1.5 h. Viral particles were resuspended in TEN buffer + 2% sucrose and inactivated with 50 µL per mg of particles with β-propiolactone (BPL, Millipore Sigma, Cat# P5648, Burlington, VT, USA) in cold culture grade water. The level of inactivation was verified by inoculating Vero CCL81 cells over three passages with 10 µg of BPL-inactivated virions.
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2

Inactivated viral vaccine production

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To produce inactivated GP38+ GC-, GP38- GC+ , GP38+ GC+ , and Filorab1 vaccines, viral supernatant was concentrated, sucrose purified84 (link), and inactivated81 (link). Viral supernatants with the highest titers were pooled for each virus and concentrated at least 5x in an Amicon® 300 mL stirred cell concentrator using a 500 kDa exclusion PES membrane (MilliporeSigma®). Concentrated supernatants were then overlaid onto a 20% sucrose cushion and centrifuged at 76,755 x g for 2 h. Virions pellets were resuspended in TEN buffer (100 mM Tris base, 50 mM NaCl, 2 mM EDTA in ddH2O) with 2% sucrose and incubated overnight (O.N.) at 4 °C. β-propiolactone (BPL) (MilliporeSigma®) was added at a 1:2000 dilution for inactivation. Samples were left at 4 °C O.N. shaking and then incubated the following day at 37 °C for 30 min to hydrolyze the BPL. Virus inactivation was confirmed by inoculated supernatant with 10 µg of inactivated virions was passaged in T25 flasks of Vero cells; cells were fixed and stained with a 1:200 dilution of FITC anti-RABV-N (Fujirebio®, product #800–092) or monitored for cytopathic effect25 (link).
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3

Rabies Virus Immunization and Antibody Response

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Groups of 8–10 weeks old C57BL/J6 female mice (Jackson) were immunized intramuscularly (i.m) via gastrocnemius with 100 μl (50 μl/leg) of live or inactivated RABV or RABV-ED51-mBAFF as indicated in the figures. Inactivated RABV and inactivated RABV-ED51-mBAFF were prepared as follows: virus stocks were grown in OptiPRO Serum Free Media (Gibco) [4mM L-Glutamine, 1% PS], harvested, and cell debris was removed using Corning 0.45μm filter (430516; Corning). β-Propiolactone (BPL; P5648; Sigma) was added to viral supernatants (final concentration 0.05% BPL), and incubated overnight at 4ᵒC. Treated supernatants were purified using ultracentrifugation. Viral inactivation was confirmed by viral titer [37 (link)]. Total protein concentrations were quantified by BCA Protein Assay Kit as described by the manufacturer (Pierce). Blood from immunized mice was collected via retro-orbital at 5, 7, 10 days post-immunization. RABV G-specific IgM and IgG (and subclasses) antibody levels were determined by ELISA as described previously [36 (link)–39 (link)]. VNA titers were determined by RFFIT as described previously [36 (link), 37 (link), 40 (link)].
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4

Influenza Virus Propagation and Inactivation

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Virus stock was propagated in MDCK cells cultured in a T-175 flask (Greiner bio-one GmbH, Germany); then, cells were microscopically investigated daily. The virus-infected culture supernatant was clarified by centrifugation at 2500 rpm for 15 min at 4°C twice. The harvested virus was titrated by plaque titration assay. Harvested influenza A/H1N1, A/H3N2, rgH1N1, rgH3N2, rgH1N2, and rgH3N1 were inactivated with 0.1% β-propiolactone (BPL) (Sigma-Aldrich); then, the treated virus was incubated at 4°C for 2 days in a cooling shaking incubator. The β-propiolactone-treated viruses were tested for loss of viral infectivity by inoculating them into MDCK monolayers for up to 5 days. No cytopathic effect (CPE) was observed on MDCK cells that were inoculated with inactivated viruses. The inactivated viruses were further concentrated by ultracentrifugation (80,000×g, 4 °C, 1 h with 20% sucrose as a cushion). Total protein content was measured by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).
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5

Inactivated H9N2 virus vaccine protocol

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A/chicken/Shanghai/441/2009 (H9N2), designated SH441, was amplified in nine-day-old specific pathogen-free (SPF) chicken embryos (Beijing Merial Vital Laboratory Animal Technology Co., Ltd., Beijing, China). SH441 was titrated in SPF chicken embryos before it was used for infection or immunization. Allantoic fluids containing the SH441 virus were inactivated with 0.05% β-propiolactone (BPL; Sigma-Aldrich, St. Louis, MO, USA) at 4 °C for 12 h to be used as an inactivated vaccine and was confirmed by infecting in nine-day-old SPF chicken embryos for 3 days to check the HA titer.
Chickens and shelducks aged 10–12 weeks old were hatched from SPF embryos (chicken embryos were obtained from Beijing Merial Vital Laboratory Animal Technology Co., Beijing; duck embryos were obtained from Harbin Veterinary Research, Harbin) and housed in isolators until further analysis.
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6

Inactivation of LASSARAB and FILORAB1 Vaccines

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To produce inactivated LASSARAB and FILORAB138 (link) (kind gift of Drishya Kurup, Thomas Jefferson University) vaccines, viral supernatant were sucrose purified and inactivated37 (link). Briefly, viral supernatants were concentrated at least 10x by Amicon® stirred cell concentrator using a 500 kDa exclusion PES membrane (Millipore®) and centrifuged at 110,000 × g through a 20% sucrose cushion. Virion pellets were resuspended in 1 × DPBS (Corning®) containing 2% sucrose and betapropiolactone (BPL) (Sigma-Aldrich®) was added at a 1:2000 dilution for inactivation. Samples were left at 4 °C O/N shaking and next day BPL was hydrolized at 37 °C for 30 min.
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7

Zika Virus Strains Isolation and Characterization

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Two ZIKV strains were used in this study; ZIKV strain Uganda 976 (ZIKVAF) was kindly provided by Dr. Misa Korva (University of Ljubljana) (European Virus Archive number 007-EVAg1585) and Zika Suriname ZIKVNL00013 (ZIKVAS) was isolated from a female patient in Netherlands who traveled to Suriname (Van Der Eijk et al., 2016 (link)). Virus stocks used in this study were prepared on Vero cells. ZIKV Uganda 976 with total passage number (P) 6 and ZIKVAS P4 were used in the experiments. Viral titers were determined on Vero cells by calculating the 50% Tissue Culture Infective Dose (TCID50) using the Spearman-Kärber formula (Kärber, 1931 (link)) after determining the presence of cytopathic effects (CPE) after 5 days of incubation. As a control, infectious virus was inactivated using beta-propiolactone (BPL) (Sigma-Aldrich, United States, 1:4000 v/v) at 4°C for 24 h. Subsequently, BPL was inactivated by incubating for 1 h at 37°C. All virus stocks were stored at −80°C until use. A summary of isolation history of the ZIKV strains used in this study with related information is provided in Table 1. A phylogenetic tree of the virus strains used in this study was recently published (Anfasa et al., 2017 (link)).
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8

BRBV Neutralizing Antibody Generation

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All animal experiments were preformed according to the Institutional Animal Care and Use Committee (IACUC) at Washington University in St. Louis. Eleven- to 12-week-old female C57BL/6 mice were obtained from The Jackson Laboratory. Mice (n= 3) were immunized intramuscularly with 500 ng of beta-propiolactone (BPL; Sigma-Aldrich)-inactivated and purified BRBV complemented with Addavax (InvivoGen) (1:1 [vol/vol]) to generate BRBV GP-specific MAbs. Four weeks later, the mice were immunized intramuscularly with 5 μg of baculovirus-derived recombinant GP of BRBV complemented with Addavax (1:1 [vol/vol]). Sera were collected 1 day prior to each immunization and stored at −20°C. To generate positive-control sera, containing BRBV-specific neutralizing antibodies, we infected C57BL/6 mice with 104 infectious units of BRBV and collected sera at 22 days postinfection. Sera from five BRBV-infected mice were pooled to use as the positive control in the virus neutralization assays.
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9

Serum Antibody Detection for H7N9 Influenza

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The serum was treated with a receptor-destroying enzyme (RDE) (supplied and validated by APHA) and heat-treated at 56 °C for 30 min; seroconversion to AIV H7N9 was assessed using the hemagglutination inhibition assay, as previously described [18 (link)]. Beta-propiolactone (BPL) (97%, Sigma) inactivated homologous A/Anhui/1/13 [H7N9] antigen [20 (link)] was used to quantify hemagglutination inhibition titres (HITs). HITs ≤ 1/5 were considered negative.
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10

BPL Inactivation of PRRSV-1 Viral Stock

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After propagation of PRRSV-1 on AM, part of the viral stock was inactivated by adding beta-propiolactone (BPL) (Sigma-Aldrich) (1:4000 dilution) after incubation for 2 h at 37 °C in a 5% CO2 humidified atmosphere. Inactivated viral stock was clarified, purified (as described above), and stored at −80 °C. The efficiency of the inactivation process was tested by a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) targeting PRRSV-1 (see PCR section) on supernatant obtained after inoculation of the AM with the inactivated virus over three passages.
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