Immunization of BALB/c mice and generation of hybridomas producing anti-HBc mAbs were carried out as previously described [28 (link)]. Briefly, purified His-precore/core was injected into BALB/c mice using keyhole limpet hemocyanin as a carrier protein. Four weeks later, lymphocytes were isolated and fused to myeloma cell. Isotype determination was then performed using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit according to the manufacturer’s protocol (Roche Diagnostics, Basel, Switzerland). Purification of mAbs from hybridoma supernatant was performed using a Spin column based Antibody Purification Kit (Protein G) (Cosmo Bio Co., Ltd., Tokyo, Japan). The protein concentration was calculated using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific).
Antibody purification kit protein g
Manufactured by Cosmo Bio
Sourced in Japan
The Antibody Purification Kit (Protein G) is a laboratory equipment used for the purification of antibodies from biological samples. The kit utilizes Protein G, a bacterial cell wall protein that binds to the Fc region of immunoglobulins, to selectively capture and isolate antibodies. This process allows for the efficient separation and concentration of antibodies from complex mixtures, such as cell culture supernatants or serum samples.
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4 protocols using antibody purification kit protein g
1
Generation of Anti-HBc Monoclonal Antibodies
2
IgG Purification from Sera
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IgG was purified from sera by using HiTRAP Protein G HP columns (Amersham Biosciences, Roosendaal, The Netherlands) or by Spin column based Antibody Purification Kit (Protein G) (Cosmo Bio, Tokyo, Japan). The concentration of purified IgG was determined by measuring the optical density (OD) at 280 nm. Purified IgG was stored at −20 °C until use.
3
SARS-CoV-2 Neutralizing Antibody Assay
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Forty-nine samples from 40 participants and 40 samples from 19 participants were evaluated for coagulation analysis and neutralizing antibody analysis, respectively, before freezing and after thawing. Several samples were drawn from identical participants repetitively on other days.
The amount of fibrinogen and the activity of factor VIII were measured at a commercial laboratory (Bio Medical Laboratory, Tokyo, Japan).
Neutralizing antibody activity was measured by the National Center for Global Health and Medicine Research Institute (NCGM-RI), as reported previously [11] (link). In short, IgG fractions were purified from convalescent plasma by using a Spin column-based Antibody Purification Kit (Protein G) (Cosmo Bio, Tokyo, Japan). The mixture of purified-IgG and the wild-type SARS-CoV-205–2 N (PANGO lineage B) were preincubated for 20 min at 37 °C and inoculated to the TMPRSS2-overexpressing VeroE6 cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml kanamycin, and 1 mg/ml G418. After culturing the cells for 3 days, the levels of cytopathic effect observed in SARS-CoV-2-exposed cells were determined using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The purified IgG concentration resulting in 50 % inhibition of the cytopathic effect was defined as a 50 % inhibition concentration (IC50).
The amount of fibrinogen and the activity of factor VIII were measured at a commercial laboratory (Bio Medical Laboratory, Tokyo, Japan).
Neutralizing antibody activity was measured by the National Center for Global Health and Medicine Research Institute (NCGM-RI), as reported previously [11] (link). In short, IgG fractions were purified from convalescent plasma by using a Spin column-based Antibody Purification Kit (Protein G) (Cosmo Bio, Tokyo, Japan). The mixture of purified-IgG and the wild-type SARS-CoV-205–2 N (PANGO lineage B) were preincubated for 20 min at 37 °C and inoculated to the TMPRSS2-overexpressing VeroE6 cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml kanamycin, and 1 mg/ml G418. After culturing the cells for 3 days, the levels of cytopathic effect observed in SARS-CoV-2-exposed cells were determined using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The purified IgG concentration resulting in 50 % inhibition of the cytopathic effect was defined as a 50 % inhibition concentration (IC50).
4
Evaluating IgG Neutralization Against Diverse Phages
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Mouse immunization was performed in accordance with a previous report with some modifications60 (link). In brief, mice were injected with ΦSA012 (1011 pfu/head) subcutaneously (s.c.). Two weeks after the first inoculation (14 days post-inoculation [dpi]), the mice were injected with ΦSA012 (1011 pfu/head) s.c. again. Blood samples collected at 14 dpi and 28 dpi were centrifuged at 900 g for 25 min at 4 °C, and then sera were stored at − 30 °C until use. IgG was purified from serum samples collected at 28 dpi using a spin column-based Antibody Purification Kit–Protein G (Cosmo Bio, Tokyo, Japan) according to the manufacturer’s protocols. Purified IgG concentrations were determined by enzyme-linked immunosorbent assay (ELISA) as described below.
The neutralization activity of purified IgG was measured against ΦSA012, ΦSA039, ΦMR003, ΦS12-3, ΦR18, T1, T4, and T7 in vitro. In brief, phages (108 pfu/mL or 5 × 1010 pfu/mL) were mixed with purified IgG and SM buffer and incubated for 2 h at room temperature. Thereafter, aliquots were used for plaque assays or monitoring lytic activity using a plate reader as described above.
The neutralization activity of purified IgG was measured against ΦSA012, ΦSA039, ΦMR003, ΦS12-3, ΦR18, T1, T4, and T7 in vitro. In brief, phages (108 pfu/mL or 5 × 1010 pfu/mL) were mixed with purified IgG and SM buffer and incubated for 2 h at room temperature. Thereafter, aliquots were used for plaque assays or monitoring lytic activity using a plate reader as described above.
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