Dissociation and fractionation of heavy and light chains from IgG monoclonal antibodies

Twenty-five hybridomas secreting monoclonal antibodies against human α1-antitrypsim have been produced by the cell-fusion techmque (Kóhler and Milstein, 1976). All antibodies are specific for α1-antitrypsim and carry γ1-antitrypsim heavy chains and κ light chains. Inhibition experiments showed that these monoclonal antibodies define three independent antigenic regions on the α1-antitrypsim molecule; one of these domains appears to be involved in the interaction between α1-antitrypsim and trypsin. In addition, one monoclonal antibody, AATY39, was used to develop an enzyme-linked immunosorbent assay capable of detecting low levels of α1-antitrypsim in the range of 1 to 2 ng/ml.

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Monoclonal antibodies against plasma protease inhibitors: I. Production and characterization of 23 monoclonal antibodies against human α2

Bioscience Reports ◽

10.1007/bf01120822 ◽

1984 ◽

Vol 4 (1) ◽

pp. 39-48 ◽

Cited By ~ 5

Author(s):

P. Herion ◽

D. Siberdt ◽

G. Garduno Soto ◽

J. Urbain ◽

A. Bollen

Keyword(s):

Monoclonal Antibodies ◽

Protease Inhibitors ◽

Light Chains ◽

Enzyme Linked Immunosorbent Assay ◽

High Avidity ◽

Fusion Technique ◽

Epitope Specificity ◽

Fast Acting ◽

Inhibition Pattern

23 hybridomas secreting monoclonal antibodies against human α2, the fast-acting inhibitor of plasmin present in plasma, have been produced by the cell-fusion technique. Isotyping of the monoclonal antibodies has revealed that 14 monoclonal antibodies belong to the class IgG1, 6 to the class IgG2a, and 3 to the class tgG2b. All light chains belong to the κ group. The specificity and relative avidity of these monoclonals have been determined Using an indirect enzyme-linked immunosorbent assay. 13 monoclonals exhibit a relatively high avidity for α2, 5 are of intermediate avidity, and 5 of low avidity. The epitope specificity of these 23 rnonoclonal antibodies, originating from a single mouse, have been examined in inhibition experiments. A group of 10 monoclonal antibodies exhibit a very similar inhibition pattern. Partial inhibition effects displayed by 10 other antibodies define partially overlapping antigenic regions. The binding of these antibodies seems to produce a conformational change in the α2 molecule, reducing the binding of two other antibodies. The last antibody defines an independent epitope.

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Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2047 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2047-2054 ◽

Cited By ~ 65

Author(s):

F M Brodsky

Keyword(s):

Monoclonal Antibodies ◽

Western Blot ◽

Heavy Chain ◽

The Other ◽

Light Chains ◽

Antigenic Determinants ◽

Binding Studies ◽

Myeloma Cells ◽

Antigenic Sites ◽

Immunogenic Proteins

Three monoclonal antibodies that react with previously undefined antigenic determinants on the clathrin molecule have been produced and characterized. They were isolated from a fusion between myeloma cells and popliteal lymphocytes from SJL mice that had received footpad injections of human brain clathrin. This protocol was chosen to favor the production of antibodies to poorly immunogenic proteins and thereby increase the repertoire of anti-clathrin monoclonal antibodies. One antibody (X16) reacts preferentially with the heavier of the two clathrin light chains (LCa) when it is not associated with heavy chain. This specificity is different from that of the anti-LCa antibody, CVC.6, which has preferential reactivity with heavy chain-associated LCa. In addition, X16 and CVC.6 bound simultaneously to LCa, confirming that they react with different sites. The other two antibodies produced, X19 and X22, react with two different determinants on the clathrin heavy chain, based on immunoprecipitation, Western blot, and binding studies. Competitive binding studies with anti-clathrin monoclonal antibodies showed that they define a total of five distinct antigenic determinants on bovine clathrin.

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Production and characterization of monoclonal antibodies specific for the functional domains of poly(ADP-ribose) polymerase

Biochemistry and Cell Biology ◽

10.1139/o86-051 ◽

1986 ◽

Vol 64 (4) ◽

pp. 368-376 ◽

Cited By ~ 13

Author(s):

Daniel Lamarre ◽

Brian Talbot ◽

Yvan Leduc ◽

Sylviane Muller ◽

Guy Poirier

Keyword(s):

Monoclonal Antibodies ◽

Dna Binding ◽

Binding Site ◽

Limited Proteolysis ◽

Antigenic Site ◽

Light Chains ◽

Immunoglobulin Light Chains ◽

Substrate Binding Site ◽

Dna Binding Site

Monoclonal antibodies were developed against poly(ADP-ribose) polymerase and analyzed for their reactivity against the NAD+- and DNA-binding fragments. Two fusions were performed to obtain hybridomas and the resulting anti-poly(ADP-ribose) polymerase antibodies were further screened by characterization of their immunoglobulin light chains. Five different hybridomas were isolated which produced different immunoglobulin light chains, all of which were specific for poly(ADP-ribose) polymerase. The specificities of these antibodies were determined by immunoblotting against the purified poly(ADP-ribose) polymerase, its autodegradation fragments, and the fragments prepared by limited proteolysis with chymotrypsin and papain. These fragments have been suggested to contain the NAD+-binding site, the DNA-binding site, and the automodification site, respectively. All the monoclonal antibodies reacted with the 116 kdalton (kDa) band corresponding to the purified enzyme. Four antibodies reacted exclusively with antigenic site(s) on the 46-kDa fragment which contains the DNA-binding site. A fifth antibody reacted exclusively with a clearly different antigenic site on the 74- and 54-kDa fragments which possess the NAD+ (substrate) binding site. The immunoreactivity with the major autodegradation products (69- and 46-kDa fragments) of the purified enzyme confirms this difference between the two groups of antibodies. The 22-kDa fragment corresponding to the automodification site does not show any immunoreactivity with the antibodies.

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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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A Mechanism of Gold Nanoparticle Aggregation by Immunoglobulin G Preparation

Applied Sciences ◽

10.3390/app10020475 ◽

2020 ◽

Vol 10 (2) ◽

pp. 475

Author(s):

Dmitriy V. Sotnikov ◽

Irina V. Safenkova ◽

Anatoly V. Zherdev ◽

Vadim G. Avdienko ◽

Irina V. Kozlova ◽

...

Keyword(s):

Molecular Weight ◽

Gold Nanoparticles ◽

Monoclonal Antibodies ◽

Flow Field ◽

Light Chains ◽

Field Flow Fractionation ◽

Nanoparticle Aggregation ◽

Antibody Preparation ◽

Gold Nanoparticle Aggregation ◽

Common Problems

Conjugates of gold nanoparticles (GNPs) and antibodies are widely used in various fields of biochemistry and microbiology. However, the procedure for obtaining such conjugates remains precarious, and the properties of conjugates differ significantly for different antibody clones. One of the most common problems is the aggregation of GNPs in the course of their conjugation with antibodies. This article considers an example of the conjugation of monoclonal antibodies with non-stable aggregating product. The composition of the antibody preparation was studied using electrophoresis, asymmetrical flow field-flow fractionation, and ultracentrifugation. It was shown that the component that causes the aggregation of the GNPs is the light chains of immunoglobulins that appear due to the spontaneous decay of the antibodies. After separation of the fraction with a molecular weight of less than 30 kDa, stable conjugates of antibodies with GNPs were obtained. The high functional activity of the obtained conjugates was confirmed by immunochromatography.

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