Cytogam

Manufactured by CSL Behring

Sourced in United States

Cytogam is a sterile cytomegalovirus (CMV) immune globulin intravenous (human) solution used for the prevention of CMV disease associated with kidney or heart transplantation. It is a blood-derived product manufactured by CSL Behring.

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Lab products found in correlation

24 well plate, by BD (2 mentions) Giemsa, by Merck Group (1 mentions) Thymoglobulin, by Sanofi (1 mentions) Zenapax, by Roche (1 mentions) Simulect, by Novartis (1 mentions) Rabbit complement, by Merck Group (1 mentions) Arpe 19, by American Type Culture Collection (1 mentions) Mrc 5, by American Type Culture Collection (1 mentions) Privigen, by CSL Behring (1 mentions) Hizentra, by CSL Behring (1 mentions) Igg2κ, by Merck Group (1 mentions) Sandoglobulin, by CSL Behring (1 mentions) Rhophylac, by CSL Behring (1 mentions) Streptavidin alexa 633, by Thermo Fisher Scientific (1 mentions)

9 protocols using cytogam

Virus Neutralization Assay in MRC5 Cells

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MRC5 cells were seeded in duplicate with DMEM at a density of 5 × 10⁴ cells per well in a 24-well plate (BD-Falcon, Franklin Lakes, NJ). The next day cells were infected with virus that was pre-incubated with a range of mAb concentrations (MOI 0.002). Following infection (2 h at 37 °C), cells were washed 2x with DMEM and wells were refilled with 3% DMEM containing DMSO supplemented with 10 μg ml⁻¹ Cytogam (CSL Behring, King of Prussia, PA). At 7–10 dpi, cells were fixed in 4% paraformaldehyde (20 min at 4 °C), and stained with Giemsa (Harleco, EMD Millipore; 1 h at 37 °C). Wells were washed with dH₂O 5 × and plaques were blind counted using phase contrast microscopy.

Gardner T.J., Stein K.R., Duty J.A., Schwarz T.M., Noriega V.M., Kraus T., Moran T.M, & Tortorella D. (2016). Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein. Nature Communications, 7, 13627.

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Evaluating HCMV Inhibitors in MRC5 Cells

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MRC5 cells were seeded in duplicate with DMEM at a density of 5×10⁴ cells/well in a 24-well plate (BD-Falcon, Franklin Lakes, NJ). The next day cells were treated with 0.1% DMSO, 12μM ganciclovir (APP pharmaceuticals, Schaumberg, IL), or 5nM convallatoxin for one hour prior to infection with AD169 WT (MOI 0.002) (1hr at 37 °C). Following infection, cells were washed 2× with DMEM and wells were refilled with 3% DMEM containing DMSO, ganciclovir or convallatoxin and supplemented with 10μg/mL Cytogam (CSL Behring, King of Prussia, PA). At 5dpi, cells were fixed in 4% PFA (20 minutes at 4 °C), and stained with Giemsa (Harleco, EMD Millipore) (1hr at 37 °C). Wells were washed with dH₂0 5× and plaques were blind counted using phase contrast microscopy.

Gardner T.J., Cohen T., Redmann V., Lau Z., Felsenfeld D, & Tortorella D. (2014). Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle. Antiviral research, 113, 49-61.

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Induction and Immunosuppression for Kidney Transplant

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Induction therapy consisted of daclizumab (2 mg/kg intraoperatively and 1 mg/kg at 2, 4, 6, and 8 weeks, Zenapax®, Roche), antithymocyte globulin (1.5 mg/kg, 1 intraoperative and 4 daily doses, Thymoglobulin®, Genzyme), or basiliximab (20 mg intraoperatively and on post-operative day #4, Simulect, Novartis).
Triple-drug immunosuppression consisted of tacrolimus, mycophenolate mofetil, and steroids. After achieving a tacrolimus level of 8–12 ng/ml, the prednisone dose was lowered to 20 mg/day and after the first month, tapered to 5 mg/day by 3 months post-transplant. HLA-incompatible and ABO-incompatible transplant recipients also underwent peri-operative desensitization with plasmapheresis (1 volume exchange every other day) followed by administration of intravenous immunoglobulin (100mg/kg, Cytogam®, CSL Behring), as has been previously described (9 (link)).

Orandi B.J., Alachkar N., Kraus E.S., Naqvi F., Lonze B.E., Lees L., Van Arendonk K.J., Wickliffe C., Bagnasco S.M., Zachary A., Segev D.L, & Montgomery R.A. (2015). Presentation and Outcomes of C4d-Negative Antibody-Mediated Rejection After Kidney Transplantation. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(1), 213-220.

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HCMV Neutralization Assay Protocol

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Neutralization assays were performed similarly to Stegmann et al. [38 (link)] by serially diluting Fc-fused PDGFRα proteins or anti-HCMV immunoglobulin (Cytogam, CSL Behring) in PBS (vehicle) and incubating diluted proteins with HCMV virions for 1 h at 37°C. Virion-protein mixtures were adsorbed onto target cells for 2 h at 37°C / 5% CO₂, washed once with PBS, and infected cells were allowed to recover for 18 h. The ability of each treatment to neutralize HCMV infection was measured using indirect-immunofluorescence by determining the number of cells expressing viral IE1, using anti-IE1 clone 1B12 [83 (link)] (generously provided by Thomas Shenk, Princeton University) and counterstaining nuclei with DAPI. A multiplicity of infection of 1 was used for AD169 infections, and a multiplicity of infection of 0.5 was used for TB40/E infections (based on stock titers acquired on MRC-5 fibroblasts).

Park J., Gill K.S., Aghajani A.A., Heredia J.D., Choi H., Oberstein A, & Procko E. (2020). Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology. PLoS Pathogens, 16(6), e1008647.

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Antibody-mediated HCMV Neutralization Assay

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The Chinese hamster ovary (CHO) cell line, DG44 was purchased from Invitrogen and maintained in CD DG44 medium. MRC-5 and ARPE-19 cell lines were purchased from ATCC, and cultured using EMEM or DMEM/F-12K medium, respectively, both supplemented with 10% fetal bovine serum. Purified monomeric recombinant HCMV gB protein was purchased from Sino Biologicals, Inc. (Beijing, P. R. China). HCMV strain AD169^WT131 was provided by Drs. Xiao Wang and Haruhiko Murata (Food and Drug Administration) and strain AD169 was purchased from ATCC. HCMV clinical isolates UXC, CSL5001, 38532 and 39621 were provided by Dr. Michael McVoy (Virginia Commonwealth University). HCMV strain AD169WT¹³¹ was propagated in ARPE-19 cells, HCMV strain AD169 and clinical isolates were propagated in MRC-5 cells. Cyto-Gam was a gift from CSL Behring to Dr. Michael McVoy. Rabbit complement was purchased from Sigma-Aldrich. Monoclonal mouse IgGl anti-gB antibody 2F12 was purchased from Virusys corporation (Taneytown, MD), and monoclonal mouse IgGl anti-gB antibody LS-C64457 was purchased from LifeSpan BioSciences, Inc. (Seattle, WA).

Cui X., Cao Z., Wang S., Lee R.B., Wang X., Murata H., Adler S.P., McVoy M.A, & Snapper C.M. (2018). Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response ,. Vaccine, 36(37), 5580-5590.

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Characterization of IgG Subclass Composition

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The IgG preparations used in this study included Sandoglobulin, Privigen, Hizentra, Cytogam and Rhophylac (CSL Behring AG), Gamunex (Talecris Biotherapeutics) and Intratect (Biotest). Sandoglobulin and Privigen (IgG content ≥98%) contained ≤2% and ≤0.01% IgA, respectively, and only traces of IgM, IgD, and IgE (as reported by CSL Behring). The IgG subclass composition of Sandoglobulin was analyzed by nephelometry (Quest Diagnostics) and contained principally IgG₁ (61%) and IgG₂ (32%), with small quantities of IgG₃ (5%) and IgG₄ (2%). Privigen contained 66% IgG₁, 29% IgG₂, 3% IgG₃, and 3% IgG₄ (as reported by CSL Behring). To remove the stabilizing sucrose component of Sandoglobulin, dialysis was performed as previously described (62 (link)). Further information on the immunoglobulin preparations is provided in table S2.
A control IgG preparation (IgG control mix) was made by mixing 2 monoclonal human myeloma proteins, IgG₁λ (67%) and IgG₂κ (33%), purchased from Sigma-Aldrich. This process resulted in a κ/λ ratio of 0.5, which is well within the range found in normal serum (0.26–1.65). Anti-human IgG monoclonal antibody (clone HP-6043-Biot), recognizing all IgG subtypes, and Streptavidin-Alexa633 were purchased from Invitrogen (Invitrogen, Life Technologies).

Schneider C., Smith D.F., Cummings R.D., Boligan K.F., Hamilton R.G., Bochner B.S., Miescher S., Simon H.U., Pashov A., Vassilev T, & von Gunten S. (2015). The human IgG anti-carbohydrate repertoire exhibits a universal architecture and contains specificity for microbial attachment sites. Science translational medicine, 7(269), 269ra1.

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Antibody Neutralization of Endotheliotropic HCMV

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The endotheliotropic HCMV clinical isolate VR1814 (GenBank Sequence Accession: GU179289) was kindly provided by Dr. Elena Percivalle (Fondazione IRCCS Policlinico San Matteo, Pavia, Italy).
Purified normal immunoglobulin G (IgG) Hizentra (Hizentra^®; CSL Behring, King of Prussia, PA, USA), CytoGam (CytoGam^®; CSL Behring, King of Prussia, PA, USA), and a panel of human monoclonal antibodies including anti-gB #1 6B4, anti-gB #2 2B11, anti-gH 11B12 and anti-pentamer 8I21 were kindly provided by Pfizer Inc. at Pearl River, NY, USA. Two concentrations of antibodies were used. The low concentrations were selected based on the IC90 values for VR1814 in fibroblasts previously described by Macagno et al. [21 (link)]: CytoGam (640 μg/mL for urine or 1280 μg/mL for saliva), 10 μg/mL for anti-gB #1, 7.5 μg/mL for anti-gB#2, 35 μg/mL for anti-gH, and 25 μg/mL for anti-pentamer. The high concentrations of immunoglobulin and antibodies were chosen based on the concentrations used by Cui et al. [16 (link)]: CytoGam (1280 μg/mL for urine or 2100 μg/mL for saliva) and 50 μg/mL for anti-gB #1, anti-gB #2, anti-gH, and anti-pentamer.

Yu J., Hasing M.E., Preiksaitis J.K, & Pang X. (2024). Evaluation of a Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)-Based Microneutralization Assay for Assessing Clinical Human Cytomegalovirus-Neutralizing Antibody Activity. Microorganisms, 12(4), 742.

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Preparation and Validation of CytoGam® Solution

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CytoGam^® was purchased from the manufacturer (CSL Behring, King of Prussia, PA). The 50 mg/ml stock was adjusted to 10 mg/ml with culture medium to approximate the concentration of IgG in human sera. Human sera were obtained from normal healthy adults and assayed for CMV seropositivity by gB-ELISA. Research conducted with human sera was approved by the Virginia Commonwealth University Committee for the Conduct of Human Research.

McVoy M.A., Lee R., Saccoccio F.M., Hartikka J., Smith L.R., Mahajan R., Ben Wang J., Cui X, & Adler S.P. (2015). A Cytomegalovirus DNA Vaccine Induces Antibodies that Block Viral Entry into Fibroblasts and Epithelial Cells. Vaccine, 33(51), 7328-7336.

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Antiviral Assay for HCMV Inhibitors

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MRC5 cells were seeded in triplicate with DMEM at a density of 5 × 10⁴ cells/well in a 24-well plate. The next day, cells were pretreated 1 h with: 0.1% DMSO; 12 μM ganciclovir; 0.5, 5, 50, and 500 nM of podofilox, colchicine, or nocodozole. Following AD169_IE2-YFP infection (MOI 0.1) for 2 h with the indicated drugs, the cells were washed twice with DMEM and incubated with media containing 10 μg/mL of Cytogam (CSL Behring, King of Prussia, PA, USA). At 5 d p.i., cells were fixed in 4% PFA and stained with Giemsa (Harleco, EMD Millipore, Bellerica, MA, USA). Wells were washed with dH₂O and plaques were counted using phase contrast microscopy.

Cohen T., Schwarz T.M., Vigant F., Gardner T.J., Hernandez R.E., Lee B, & Tortorella D. (2016). The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus. Viruses, 8(10), 295.

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