Isolation and characterization of high affinity and highly stable anti-Chikungunya virus antibodies using ALTHEA Gold Libraries™

Abstract Background More than three million infections were attributed to Chikungunya virus (CHIKV) in the 2014–2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis. Methods Chikungunya virions isolated from serum of a patient in Veracruz, México, were purified and characterized via electron microscopy, SDS-PAGE and binding to diverse well-characterized anti-CHIKV monoclonal antibodies. UV-inactivated CHIKV particles were used as selector in a solid-phase panning coupled with ELISA-based screening and Next-Generation Sequencing to discover specific and high affinity anti-CHIKV antibodies from ALTHEA Gold Libraries™. Results The CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of its capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained showed high expression yield, monomeric content over 95% after a single-step Protein A purification, and importantly, a thermal stability above 75oC. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2–40.0 µγ/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum. Conclusions The newly discovered antibodies offer valuable tools for characterization of CHIKV isolates and development of robust diagnostic tools for CHIKV infection surveillance. Application of ALTHEA Gold Libraries™ in combination with viral particles other than CHIKV could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks such as SARS-CoV-2 (COVID-19).

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Isolation and characterization of high affinity and highly stable anti-Chikungunya virus antibodies using ALTHEA Gold Libraries™

10.21203/rs.3.rs-34990/v2 ◽

2020 ◽

Author(s):

Martha Pedraza-Escalona ◽

Omar Guzmán-Bringas ◽

Ivan Arrieta-Oliva ◽

Keyla Gómez-Castellano ◽

Juana Salinas-Trujano ◽

...

Keyword(s):

Chikungunya Virus ◽

Dynamic Range ◽

Sandwich Elisa ◽

Diagnostic Methods ◽

Diagnostic Tools ◽

Chikv Infection ◽

High Affinity ◽

Isolation And Characterization ◽

Sds Page

Abstract Background: More than three million infections were attributed to Chikungunya virus (CHIKV) in the 2014-2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis. Methods: To isolate highly specific and high affinity anti-CHIKV, Chikungunya virions were isolated from serum of a patient in Veracruz, México. After purification and characterization via electron microscopy, SDS-PAGE and binding to well-characterized anti-CHIKV antibodies, UV-inactivated particles were utilized as selector in a solid-phase panning in combination with ALTHEA Gold Libraries™, as source of antibodies. The screening was based on ELISA and Next-Generation Sequencing. Results: The CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of CHIKV capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained, on the other hand, showed high expression yield, > 95% monomeric content after a single-step Protein A purification, and importantly, had a thermal stability above 75oC. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2 – 40.0 mg/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum.Conclusions: The newly discovered antibodies offer valuable tools for characterization of CHIKV isolates and development of robust diagnostic tools for CHIKV infection surveillance. Therefore, the strategy here followed using whole viral particles and ALTHEA Gold Libraries™ as universal source of antibodies could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks.

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Isolation and characterization of high affinity and highly stable anti-Chikungunya virus antibodies using ALTHEA Gold Libraries™

BMC Infectious Diseases ◽

10.1186/s12879-021-06717-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

M. Pedraza-Escalona ◽

O. Guzmán-Bringas ◽

H. I. Arrieta-Oliva ◽

K. Gómez-Castellano ◽

J. Salinas-Trujano ◽

...

Keyword(s):

Chikungunya Virus ◽

Dynamic Range ◽

Solid Phase ◽

Sandwich Elisa ◽

Diagnostic Methods ◽

Virus Protein ◽

High Affinity ◽

Isolation And Characterization ◽

Sds Page

Abstract Background More than 3 million infections were attributed to Chikungunya virus (CHIKV) in the 2014–2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis. Methods To isolate highly specific and high affinity anti-CHIKV, Chikungunya virions were isolated from serum of a patient in Veracruz, México. After purification and characterization via electron microscopy, SDS-PAGE and binding to well-characterized anti-CHIKV antibodies, UV-inactivated particles were utilized as selector in a solid-phase panning in combination with ALTHEA Gold Libraries™, as source of antibodies. The screening was based on ELISA and Next-Generation Sequencing. Results The CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of CHIKV capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained, on the other hand, showed high expression yield, > 95% monomeric content after a single-step Protein A purification, and importantly, had a thermal stability above 75 °C. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2–40.0 mg/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum. Conclusions The newly discovered antibodies offer valuable tools for characterization of CHIKV isolates. Therefore, the strategy here followed using whole viral particles and ALTHEA Gold Libraries™ could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks.

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Isolation and characterization of low and high affinity goat antibodies directed to single antigenic sites on human hemoglobin

Molecular Immunology ◽

10.1016/0161-5890(76)90130-9 ◽

1976 ◽

Vol 13 (11) ◽

pp. 921-927

Author(s):

A Tan-Wilson

Keyword(s):

Human Hemoglobin ◽

High Affinity ◽

Isolation And Characterization ◽

Antigenic Sites

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Overproduction of IL-6 and Type-I IFN in a Lethal Case of Chikungunya Virus Infection in an Elderly Man During the 2017 Italian Outbreak

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy276 ◽

2018 ◽

Vol 5 (11) ◽

Cited By ~ 5

Author(s):

Francesca Colavita ◽

Serena Vita ◽

Eleonora Lalle ◽

Fabrizio Carletti ◽

Licia Bordi ◽

...

Keyword(s):

Chikungunya Virus ◽

Strong Increase ◽

Type I ◽

Type I Ifn ◽

Chikv Infection ◽

Clinical Presentations ◽

Abnormal Pattern ◽

Disease Analysis

Abstract Chikungunya fever is caused by Chikungunya virus (CHIKV) and is generally considered a self-limiting disease. However, severe clinical presentations with a high mortality rate have been reported in association with underlying medical conditions. This study reports the molecular characterization of the virus and an abnormal pattern of circulating cytokines in a unique lethal CHIKV case during the 2017 outbreak in Italy, which involved an elderly patient with underlying cardiac disease. Analysis of inflammatory cytokines revealed a strong increase of interferon (IFN)-α and IFN-β, as well as interleukin-6, suggesting a possible role of type-I IFN in the cytokine storm, which may be correlated with unfavorable prognosis of CHIKV infection.

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Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA

Journal of Immunological Methods ◽

10.1016/j.jim.2008.03.003 ◽

2008 ◽

Vol 336 (1) ◽

pp. 1-8 ◽

Cited By ~ 70

Author(s):

Larry H. Stanker ◽

Paul Merrill ◽

Miles C. Scotcher ◽

Luisa W. Cheng

Keyword(s):

Monoclonal Antibodies ◽

Botulinum Toxin ◽

Botulinum Toxin Type A ◽

Sandwich Elisa ◽

Type A ◽

Botulinum Toxin Type ◽

Partial Characterization ◽

High Affinity

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Isolation and characterization of membrane proteins responsible for attachment of polyribosomes to rough microsomal fraction of rat liver

Biochemical Journal ◽

10.1042/bj1640053 ◽

1977 ◽

Vol 164 (1) ◽

pp. 53-66 ◽

Cited By ~ 7

Author(s):

S Fujita ◽

F Ogata ◽

J Nakamura ◽

S Omata ◽

H Sugano

Keyword(s):

Rat Liver ◽

Protein Fraction ◽

Microsomal Fraction ◽

Binding Capacity ◽

Gel Filtration ◽

High Affinity ◽

Isolation And Characterization ◽

Microsomal Membranes ◽

Equilibrium Centrifugation

A protein fraction which has a high affinity for polyribosomes was isolated from rough microsomal membranes of rat liver. The mode of polyribosome binding to this fraction (R-fraction) was studied by using CsCl equilibrium centrifugation and compared with that for stripped rough microsomal membranes. The following were found. (1) The polyribosome-binding cpacity of the R-fraction was heat-labile and sensitive to trypsin, and was suppressed by increasing KCl concentration and addition of 0.1 mM-aurintricarboxylic acid. (2) Of the four subfractions obtained by gel filtration of the R-fraction on a Sephadex G-200, only the R1-fraction, eluted at the void volume, showed a high affinity for polyribosomes. The polyribosome-binding capacity of the R1-fraction decreased with time on storage at 4 degrees C. (3) The R1-fraction contained three major proteins with mol. wts. 108,000, 99,000 and 65,000.

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TCR-based therapy for multiple myeloma and other B-cell malignancies targeting intracellular transcription factor BOB1

Blood ◽

10.1182/blood-2016-09-737536 ◽

2017 ◽

Vol 129 (10) ◽

pp. 1284-1295 ◽

Cited By ~ 17

Author(s):

Lorenz Jahn ◽

Pleun Hombrink ◽

Renate S. Hagedoorn ◽

Michel G. D. Kester ◽

Dirk M. van der Steen ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcription Factor ◽

B Cell ◽

B Cell Malignancies ◽

High Affinity ◽

Isolation And Characterization ◽

Key Points

Key Points Isolation and characterization of a high-affinity TCR targeting the intracellular B cell–specific transcription factor BOB1. T cells expressing a BOB1-specific TCR lysed and eradicated primary multiple myeloma and other B-cell malignancies in vitro and in vivo.

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Isolation and characterization of the rat β-subunit gene of the high affinity receptor for immunoglobulin E

Molecular Immunology ◽

10.1016/0161-5890(96)84614-1 ◽

1996 ◽

Vol 33 (10) ◽

pp. 881-890

Author(s):

Bruce A. Witthuhn ◽

Charles Faust

Keyword(s):

Immunoglobulin E ◽

Β Subunit ◽

Subunit Gene ◽

High Affinity ◽

Isolation And Characterization ◽

High Affinity Receptor ◽

Affinity Receptor

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Development of Nanobodies against Mal de Río Cuarto virus major viroplasm protein P9-1 for diagnostic sandwich ELISA and immunodetection

10.21203/rs.3.rs-831410/v1 ◽

2021 ◽

Author(s):

Gabriela Llauger ◽

Demián Monti ◽

Matías Adúriz ◽

Ema Romão ◽

Analía Delina Dumón ◽

...

Keyword(s):

Sandwich Elisa ◽

Elisa Test ◽

Host Proteins ◽

Maize Breeding ◽

Thin Sections ◽

Isolation And Characterization ◽

The Family ◽

Fundamental Research ◽

Nanomolar Range

Abstract Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI: 95.21–99.98) and specificity (100%, 95% CI: 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.

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