Light chain modulates heavy chain conformation to change protection profile of monoclonal antibodies against influenza A viruses

SummaryFactor VIII-Light Chain (FVIII-LC) and FVIII-Heavy Chain (FVIII-HC) were purified from human plasma by the use of immunosorbents containing monoclonal antibodies or human inhibitor antibodies. FVIII-LC was subsequently isolated in essentially pure state by cation exchange chromatography. The preparations obtained contained 50 ng of protein for each unit of FVIII-LC antigen (FVIII-LC: Ag).Affinity purified FVIII-LC and FVIII-HC preparations containing less than 0.3% of the opposite subunit were added in FVIILC inhibition assay of hemophilia A inhibitor antibodies. FVIII-LC was able to fully block the inhibitor activity in 6 out of 7 hemophilia A plasmas and partially block the inhibitor activity of one plasma. FVIII-HC only blocked FVIILC inhibiting antibodies form the plasma that was not fully blocked by FVIII-LC. It is suggested that FVIII-LC can be used for immunotherapy of the patients whose FVIILC inhibiting antibodies are directed towards FVIII-LC.When FVIII-LC was coupled to Sepharose at a concentration of 4800 units of FVIII-LC: Ag per ml Sepharose, 0.2 ml of the immunosorbent was able to bind 900 Bethesda units from 100 ml hemophilia A inhibitor plasma. This opens the possibility to remove FVIII inhibitor antibodies from circulation by extracorporeal immunotherapy with FVIII-LC coupled to Sepharose.

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Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2055 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2055-2062 ◽

Cited By ~ 32

Author(s):

F M Brodsky

Keyword(s):

Monoclonal Antibodies ◽

Heavy Chain ◽

Light Chain ◽

Coated Vesicles ◽

Cell Lysates ◽

Over Time

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.

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Clathrin assembly involves a light chain-binding region.

The Journal of Cell Biology ◽

10.1083/jcb.105.5.2011 ◽

1987 ◽

Vol 105 (5) ◽

pp. 2011-2019 ◽

Cited By ~ 15

Author(s):

G S Blank ◽

F M Brodsky

Keyword(s):

Monoclonal Antibodies ◽

Binding Site ◽

Heavy Chain ◽

Light Chain ◽

Intermediate Filament ◽

Binding Sites ◽

Light Chains ◽

Binding Region ◽

Intermediate Filament Proteins ◽

Interaction Site

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

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Monoclonal antibodies targeting the influenza virus N6 neuraminidase

10.1101/2021.02.15.431355 ◽

2021 ◽

Author(s):

Shirin Strohmeier ◽

Fatima Amanat ◽

Juan Manuel Carreño ◽

Florian Krammer

Keyword(s):

Monoclonal Antibodies ◽

Influenza A ◽

Neutralization Assay ◽

Escape Mutant ◽

Influenza A Viruses ◽

Highly Pathogenic ◽

Lateral Ridge

AbstractInfluenza A viruses are a diverse species that include 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes. While the antigenicity of many HA subtypes is reasonably well studied, less is known about NA antigenicity, especially when it comes to non-human subtypes that only circulate in animal reservoirs. The N6 NA subtypes are mostly found in viruses infecting birds. However, they have also been identified in viruses that infect mammals, such as swine and seals. More recently, highly pathogenic H5N6 subtype viruses have caused rare infections and mortality in humans. Here, we generated murine mAbs to the N6 NA, characterized their breadth and antiviral properties in vitro and in vivo and mapped their epitopes by generating escape mutant viruses. We found that the antibodies had broad reactivity across the American and Eurasian N6 lineages, but relatively little binding and inhibition of the H5N6 NA. Several of the antibodies exhibited strong NA inhibition activity and some also showed activity in the antibody dependent cellular cytotoxicity reporter assay and neutralization assay. In addition, we generated escape mutant viruses for six monoclonal antibodies and found mutations on the lateral ridge of the NA. Lastly, we observed variable protection in H4N6 and H5N6 mouse challenge models when the antibodies were given prophylactically.ImportanceThe N6 NA has recently gained prominence due to the emergence of highly pathogenic H5N6 viruses. Currently, there is limited characterization of the antigenicity of avian N6 neuraminidase. Our data is an important first step towards a better understanding of the N6 NA antigenicity.

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Analysis by Monoclonal Antibodies of H3N2 Subtype of Influenza A Viruses in Japan and South East Asia During 1980-1982

Kansenshogaku zasshi ◽

10.11150/kansenshogakuzasshi1970.58.113 ◽

1984 ◽

Vol 58 (2) ◽

pp. 113-120

Author(s):

Yukio YAMAZI ◽

Hiroshi SUZUKI ◽

Eiji WATARI ◽

Yasue TAKEUCHI ◽

Akira OHYA ◽

...

Keyword(s):

Monoclonal Antibodies ◽

East Asia ◽

Influenza A ◽

South East Asia ◽

Influenza A Viruses ◽

H3n2 Subtype

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Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin.

The Journal of Cell Biology ◽

10.1083/jcb.100.4.1016 ◽

1985 ◽

Vol 100 (4) ◽

pp. 1016-1023 ◽

Cited By ~ 24

Author(s):

G Peltz ◽

J A Spudich ◽

P Parham

Keyword(s):

Monoclonal Antibodies ◽

Atpase Activity ◽

Heavy Chain ◽

Light Chain ◽

Binding Sites ◽

Myosin Molecule ◽

Dictyostelium Myosin ◽

Antigenic Sites ◽

Myosin Filaments ◽

A Site

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.

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Pathological Lesions in the Lungs of Ducks Infected with Influenza A Viruses

Veterinary Pathology ◽

10.1177/030098588902600101 ◽

1989 ◽

Vol 26 (1) ◽

pp. 1-5 ◽

Cited By ~ 38

Author(s):

A. J. Cooley ◽

H. Van Campen ◽

M. S. Philpott ◽

B. C. Easterday ◽

V. S. Hinshaw

Keyword(s):

Monoclonal Antibodies ◽

Respiratory Tract ◽

Influenza A ◽

Infectious Virus ◽

Histopathological Examination ◽

Clinical Signs ◽

Immunoperoxidase Staining ◽

Influenza A Viruses ◽

Pathological Lesions ◽

Viral Antigens

To determine histopathological damage in the respiratory tract, ducks were inoculated with five different influenza A viruses, including viruses virulent for other avian hosts. Lungs were collected for detection of virus and histopathological examination. Small amounts of infectious virus were recovered from lungs, and viral antigens were demonstrated by immunoperoxidase staining with monoclonal antibodies to the viral nucleoprotein. Although clinical signs were not detected, lungs of ducks infected with both virulent and avirulent viruses had mild pneumonia characterized by infiltrates of lymphocytes and macrophages. These findings show that although clinical signs are not evident, ducks may have damage to the respiratory tract during influenza.

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Study of Basic Local Alignment Search Tool (BLAST) and Multiple Sequence Alignment (Clustal- X) of Monoclonal mice/human antibodies

10.1101/2021.07.09.451785 ◽

2021 ◽

Author(s):

IVAN VITO FERRARI ◽

Paolo PATRIZIO

Keyword(s):

Amino Acids ◽

Monoclonal Antibodies ◽

Sequence Alignment ◽

Heavy Chain ◽

Light Chain ◽

Light Chains ◽

Local Alignment ◽

Multiple Sequence ◽

Heavy Chains ◽

Search Tool

In this work, we have focused on the study of the Basic Local Alignment Search Tool (BLAST) and Multiple Sequence Alignment (Clustal- X) of different monoclonal mice antibodies to understand better the multiple alignments of sequences. Our strategy was to compare the light chains of multiple monoclonal antibodies to each other, calculating their identity percentage and in which amino acid portion. (See below figure 2) Subsequently, the same survey of heavy chains was carried out with the same methodology. (See below figure 3) Finally, sequence alignment between the light chain of one antibody and the heavy chain of another antibody was studied to understand what happens if chains are exchanged between antibodies. (See below figure 4) From our results of BLAST estimation alignment, we have reported that the Light Chains (Ls) of Monoclonal Antibodies in Comparison have a sequence Homology of about 60-80% and they have a part identical in sequence zone in range 100-210 residues amino acids, except ID PDB 4ISV, which it turns out to have a 40% lower homology than the others antibodies. As far as, the heavy chains (Hs) of Monoclonal Antibodies are concerned, however they tend to have a less homology of sequences, compared to lights chains consideration, equal to 60%-70% and they have an identical part in the sequence zone between 150-210 residues amino acids; with the exception of ID PDB 3I9G-3W9D antibodies that have an equal homology at 50%. ( See supporting part) Summing up: about 70-80% identity among 2 light chains of 2 antibodies, 60-70% identity between 2 heavy chains of 2 antibodies, 30% identity between the two chains of a antibody and 30% if you compare the light chain of one antibody with the heavy chain of another antibody.

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