Antigenic and Functional Characterization Of Rinderpest Virus Envelope Proteins Using Monoclonal Antibodies

Background. Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues. Materials and methods. Mice were immunized with a multi-antigen peptide corresponding to segment 65-75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase. Results. On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use. Conclusions. In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.

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Epitope mapping and functional characterization of monoclonal antibodies specific for the alpha subunit of Escherichia coli RNA polymerase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)31565-x ◽

1994 ◽

Vol 269 (38) ◽

pp. 23655-23660

Author(s):

K.A. Sharif ◽

N. Fujita ◽

R. Jin ◽

K. Igarashi ◽

A. Ishihama ◽

...

Keyword(s):

Escherichia Coli ◽

Monoclonal Antibodies ◽

Rna Polymerase ◽

Epitope Mapping ◽

Functional Characterization ◽

Alpha Subunit

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Characterization of Functional Hepatitis C Virus Envelope Glycoproteins

Journal of Virology ◽

10.1128/jvi.78.6.2994-3002.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 2994-3002 ◽

Cited By ~ 157

Author(s):

Anne Op De Beeck ◽

Cécile Voisset ◽

Birke Bartosch ◽

Yann Ciczora ◽

Laurence Cocquerel ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Conformational Changes ◽

Structural Changes ◽

Envelope Proteins ◽

Viral Envelope ◽

Envelope Glycoproteins ◽

Functional Envelope

ABSTRACT Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

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The alpha/B.1.1.7 SARS-CoV-2 variant exhibits significantly higher affinity for ACE-2 and requires lower inoculation doses to cause disease in K18-hACE2 mice

eLife ◽

10.7554/elife.70002 ◽

2021 ◽

Vol 10 ◽

Author(s):

Rafael Bayarri-Olmos ◽

Laust Bruun Johnsen ◽

Manja Idorn ◽

Line S Reinert ◽

Anne Rosbjerg ◽

...

Keyword(s):

Monoclonal Antibodies ◽

United Kingdom ◽

Disease Progression ◽

Low Dose ◽

Transmission Rate ◽

Functional Characterization ◽

Fitness Advantage ◽

The United Kingdom ◽

A Minor

The alpha/B.1.1.7 SARS-CoV-2 lineage emerged in autumn 2020 in the United Kingdom and transmitted rapidly until winter 2021 when it was responsible for most new COVID-19 cases in many European countries. The incidence domination was likely due to a fitness advantage that could be driven by the RBD residue change (N501Y), which also emerged independently in other Variants of Concern such as the beta/B.1.351 and gamma/P.1 strains. Here we present a functional characterization of the alpha/B.1.1.7 variant and show an eight-fold affinity increase towards human ACE-2. In accordance with this, transgenic hACE-2 mice showed a faster disease progression and severity after infection with a low dose of B.1.1.7, compared to an early 2020 SARS-CoV-2 isolate. When challenged with sera from convalescent individuals or anti-RBD monoclonal antibodies, the N501Y variant showed a minor, but significant elevated evasion potential of ACE-2/RBD antibody neutralization. The data suggest that the single asparagine to tyrosine substitution remarkable rise in affinity may be responsible for the higher transmission rate and severity of the B.1.1.7 variant.

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