One-step method for establishing 8-azaguanine-resistant hybridomas suitable for the preparation of triomas

Kontseková, Eva; Novák, Michal; Máciková, I; Kontsek, Peter

doi:10.1016/0022-1759(91)90333-b

Cited by 6 publications

(5 citation statements)

References 8 publications

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“…ELISA positive hybridoma cultures were further subcloned in soft agar according to the procedure described previously. 33 After purified hybridoma clones were obtained, the antibodies isotypes were determined by ELISA using a mouse Ig isotyping kit (ISO-2, SIGMA).…”

Section: Methodsmentioning

confidence: 99%

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

et al. 2022

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Background The emergence of new SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) that harbor mutations in the viral S protein raised concern about activity of current vaccines and therapeutic antibodies. Independent studies have shown that mutant variants are partially or completely resistant against some of the therapeutic antibodies authorized for emergency use. Methods We employed hybridoma technology, ELISA-based and cell-based S-ACE2 interaction assays combined with authentic virus neutralization assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize the new variants of SARS-CoV-2. Findings AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). In addition, AX677 is able to bind Omicron Spike protein just like the wild type Spike. The combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. Prophylactic administration of AX290 and AX677, either individually or in combination, effectively reduced viral burden and inflammation in the lungs, and prevented disease in a mouse model of SARS-CoV-2 infection. Interpretation The virus-neutralizing properties were fully reproduced in chimeric mouse-human versions of the antibodies, which may represent a promising tool for COVID-19 therapy. Funding The study was funded by AXON Neuroscience SE and AXON COVIDAX a.s.

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Section: Methodsmentioning

confidence: 99%

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

et al. 2022

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“…Three days before the spleen extraction, mice were injected intraperitoneally with 20 μg of the same antigens in PBS. Spleen cells from immunized mice were fused with NS0 myeloma cells according to the method of Kontsekova et al 39 . Splenocytes were mixed with NS0 myeloma cells (ratio 5:1) and fused for 1 minute in 1 ml of 50% polyethylene glycol (PEG) 1550 (Serva) in serum free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% dimethyl sulfoxide (DMSO).…”

Section: Methodsmentioning

confidence: 99%

Section: Preparation Of Hybridoma Cell Lines Producing Monoclonal Antibodies Specific To Sars-cov-2 Spike Protein and Rbdmentioning

confidence: 99%

“…Readouts with an absorbance value of at least twice the value of the negative controls (PBS) were considered positive. ELISA positive hybridoma cultures were further subcloned in soft agar according to the procedure described previously 39 . After purified hybridoma clones were obtained, the antibodies isotypes were determined by ELISA using a mouse Ig isotyping kit (ISO-2, SIGMA).…”

Section: Elisa For Screening Of Monoclonal Antibodiesmentioning

confidence: 99%

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Second-Generation Antibodies Neutralize Emerging SARS-CoV-2 Variants of Concern

Kováčech

Fialová

Filipčík

et al. 2021

Preprint

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Recently emerged SARS-CoV-2 variants show resistance to some antibodies that were authorized for emergency use. We employed hybridoma technology combined with authentic virus assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize new variants of SARS-CoV-2. AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 and B.1.351. Finally, the combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. The neutralizing properties were fully reproduced in chimeric mouse-human versions, which may represent a promising tool for COVID-19 therapy.

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“…Preparation of Double Mutant HRP 16 a) Selection for Azaquanine-resistant (Agr) cells: Hybridomas HRP 16 resistant to 20 ug/ml 8-azaquanine (Serva) were derived using the one-step cloning method as described (8).…”

Section: Hybridomasmentioning

confidence: 99%

Quadroma-Secreted Bi(Interferon Alpha 2—Peroxidase) Specific Antibody Suitable for One-Step Immunoassay

Kontseková

Kolcunova²,

Kontsek³

1992

Hybridoma

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A mouse hybridoma (quadroma) was prepared by fusing hybridomas producing monoclonal antibody of G1-isotype to human interferon-alpha 2 with hybridomas producing monoclonal antibody of G2a-isotype against horseradish peroxidase. The established quadroma line secreted immunoglobulins of both G1/G2a-isotypes which manifested parental and bispecific binding characteristics. Culture supernatant containing the bifunctional antibody cross-linking interferon and peroxidase was used for a one-step immunoassay. The developed sandwich ELISA was able to detect the human interferon-alpha 2 at a concentration of 10 units/ml (0, 1 ng/ml) within 2-3 hours.

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One-step method for establishing 8-azaguanine-resistant hybridomas suitable for the preparation of triomas

Cited by 6 publications

References 8 publications

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

Second-Generation Antibodies Neutralize Emerging SARS-CoV-2 Variants of Concern

Quadroma-Secreted Bi(Interferon Alpha 2—Peroxidase) Specific Antibody Suitable for One-Step Immunoassay

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