scholarly journals Immunocytochemical detection of serotonin with monoclonal antibodies.

1981 ◽  
Vol 29 (12) ◽  
pp. 1425-1430 ◽  
Author(s):  
A Consolazione ◽  
C Milstein ◽  
B Wright ◽  
A C Cuello
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Brain Tissue ◽  
Heavy Chain ◽  
Light Chains ◽  
Immunocytochemical Detection ◽  
Binding Antibody ◽  
High Concentrations

The development of the monoclonal antibody YC5/45 HLK (YC5/HLK) against a 5HT-bovine seroalbumin immunogen and its application for immunocytochemistry is described. The YC5/HLK antibody is the product of a rat x rat hybrid myeloma, producing a heavy chain and two light chains. In hemagglutination tests, the antibody cross-reacts to entirety with dopamine, serotonin, and tryptamine at high concentrations. The serotonin-albumin conjugate is 20,000 times more effective in displacing the binding antibody, while albumin itself goes unrecognized by the antibody. In fixed preparations of brain tissue, immunofluorescence is observed only in neurons known to contain serotonin, while no reaction is observed in dopamine-rich neurons. All immunofluorescence is extinguished by the use of agents that inhibit the biosynthesis of 5HT, but not of the catecholamines.

scholarly journals Antigen preservation tests for immunocytochemical detection of cytoskeletal proteins: influence of aldehyde fixatives.

1989 ◽  
Vol 37 (5) ◽  
pp. 675-681 ◽  
Author(s):  
B M Riederer
Keyword(s):  
Monoclonal Antibodies ◽  
Brain Tissue ◽  
Polyclonal Antibody ◽  
Cytoskeletal Proteins ◽  
Immunocytochemical Localization ◽  
Neurofilament Proteins ◽  
Tissue Samples ◽  
Immunocytochemical Detection ◽  
Immunochemical Detection ◽  
Brain Spectrin

The effects of aldehyde fixatives on immunochemical detection of cytoskeletal proteins were demonstrated by applying several quantitative assays to evaluate antigen conservation. Immunologically detectable brain spectrin (240/235) was measured by dot-immunobinding and quantitative immunodot assay using a polyclonal antibody. Paraformaldehyde fixation led to a 43-66% reduction in brain spectrin (240/235) immunodetection, and increasing glutaraldehyde concentrations decreased the immunological detection even more. Quantitative cryosection immunoassay and immunocytochemical localization confirmed the aldehyde sensitivity of brain spectrin (240/235). Brain spectrin (240/235) immunoreactivity decreased with increasing protein crosslinking and was dependent on glutaraldehyde concentration and post-fixation period. The assays were also used to test for conservation of antigenicity of neurofilament proteins by two monoclonal antibodies. Neurofilament detection was abolished in brain tissue after aldehyde fixation. The described methods allow screening within 24 hr of many fixation conditions by use of purified proteins as well as brain tissue samples, and allow an estimate of fixative influence on the conservation of protein antigenicity.

Monoclonal antibodies against plasma protease inhibitors: II. Production and characterization of 25 monoclonal antibodies against human α1-antitrypsin. Correlation between antigenic structure and functional sites

1984 ◽  
Vol 4 (2) ◽  
pp. 139-147 ◽  
Author(s):  
P. Hérion ◽  
D. Siberdt ◽  
M. Francotte ◽  
J. Urbain ◽  
A. Bollen
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Protease Inhibitors ◽  
Antigenic Structure ◽  
Light Chains ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Sites ◽  
Heavy Chains ◽  
Low Levels

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.

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

1985 ◽  
Vol 101 (6) ◽  
pp. 2047-2054 ◽  
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.

scholarly journals Clathrin assembly involves a light chain-binding region.

1987 ◽  
Vol 105 (5) ◽  
pp. 2011-2019 ◽  
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.

Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian skeletal muscles

Development ◽  
1989 ◽  
Vol 107 (4) ◽  
pp. 771-784 ◽  
Author(s):  
A.J. Harris ◽  
M.J. Duxson ◽  
R.B. Fitzsimons ◽  
F. Rieger
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Light Microscopy ◽  
Heavy Chain ◽  
Skeletal Muscles ◽  
Single Pulse ◽  
Myotube Formation ◽  
Central Nuclei ◽  
Slow Myosin Heavy Chain ◽  
Thymidine Autoradiography

Myotubes were isolated from enzymically disaggregated embryonic muscles and examined with light microscopy. Primary myotubes were seen as classic myotubes with chains of central nuclei within a tube of myofilaments, whereas secondary myotubes had a smaller diameter and more widely spaced nuclei. Primary myotubes could also be distinguished from secondary myotubes by their specific reaction with two monoclonal antibodies (MAbs) against adult slow myosin heavy chain (MHC). Myonuclei were birth dated with [3H]thymidine autoradiography or with 2-bromo-5′-deoxyuridine (BrdU) detected with a commercial monoclonal antibody. After a single pulse of label during the 1–2 day period when primary myotubes were forming, some primary myotubes had many myonuclei labelled, usually in adjacent groups, while in others no nuclei were labelled. If a pulse of label was administered after this time labelled myonuclei appeared in most secondary myotubes, while primary myotubes received few new nuclei. Labelled and unlabelled myonuclei were not grouped in the secondary myotubes, but were randomly interspersed. We conclude that primary myotubes form by a nearly synchronous fusion of myoblasts with similar birthdates. In contrast, secondary myotubes form in a progressive fashion, myoblasts with asynchronous birthdates fusing laterally with secondary myotubes at random positions along their length. These later-differentiating myoblasts do not fuse with primary myotubes, despite being closely apposed to their surface. Furthermore, they do not generally fuse with each other, as secondary myotube formation is initiated only in the region of the primary myotube endplate.

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

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.

Aggregation to Thrombin and Collagen of Platelets from a Glanzmann Thrombasthenic Patient Lacking Glycoproteins IIb and IIIa

1989 ◽  
Vol 62 (03) ◽  
pp. 962-967 ◽  
Author(s):  
Lilian McGregor ◽  
Michel Hanss ◽  
Amal Sayegh ◽  
Juan J Calvette ◽  
Marie-Christine Trzeciak ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Membrane Glycoproteins ◽  
Albumin Solution ◽  
Western Blots ◽  
Glycoprotein Complex ◽  
Antigen Present ◽  
High Concentrations ◽  
Induced Aggregation

SummaryThe aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 μg/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 μM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 μg/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained twodimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on Ilia), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.

Insights on Monoclonal Antibodies to Kininogens' Heavy Chain Which Influence Kininogens' Binding to Platelets

1992 ◽  
Vol 68 (02) ◽  
pp. 143-148 ◽  
Author(s):  
Yongping Jiang ◽  
Weerasak Nawarawong ◽  
Frank J Meloni ◽  
Alvin H Schmaier
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Immunosorbent Assay ◽  
Heavy Chain ◽  
Gel Filtration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Molecular Weight ◽  
High Concentrations ◽  
Sepharose 4B

SummaryPurified domains of low molecular weight kininogen (LK) can be used directly to determine the epitopes of monoclonal antibodies (mAbs) that have been shown to influence kininogen function. LK, purified from plasma by carboxymethyl-papain-Sepharose 4B affinity chromatography and kaolin adsorption, was digested by trypsin and chymotrypsin. The domains of LK were then separated by gel filtration followed by carboxymethyl-papain-Sepharose 4B affinity chromatography. Using the purified domains of LK’s heavy chain, the regions on kininogens' heavy chain which various monoclonal antibodies are directed to were determined by enzyme-linked immunosorbent assay and immunoblotting. MAb 2B5 which neutralized kininogens' ability to inhibit calpain cross-reacted with domains 2 and 3. MAb HKH8 which reacted with kininogens' domain 1 and 2 was found to inhibit 125I-HK binding to platelets. At two-fold molar excess, mAb HKH8 was a better inhibitor of 125I-HK binding to platelets than higher concentrations, where the antibody was shown to cause increased binding to platelets. Alternatively, HKH8 F(ab')2 completely inhibited 125I-HK binding to platelets even at high concentrations of antibody. These studies indicate that purified domains of kininogens' heavy chain can be used to rapidly localize epitopes for antibodies. Further, mAb HKH8 should be a valuable probe to understand the mechanisms of kininogens' binding to platelets.

Efficacy and specificity of monoclonal antibodies to progesterone in preventing the establishment of pregnancy in the mouse

1988 ◽  
Vol 118 (1) ◽  
pp. 69-80 ◽  
Author(s):  
S. T. Ellis ◽  
R. B. Heap ◽  
A. R. Butchart ◽  
V. Rider ◽  
N. E. Richardson ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Effective Dose ◽  
Half Life ◽  
Slow Rate ◽  
Affinity Constant ◽  
High Affinity ◽  
Mouse Immunoglobulin ◽  
High Concentrations ◽  
Ig G

ABSTRACT Anti-progesterone monoclonal antibody prevents the establishment of pregnancy in BALB/c mice by the prevention of implantation when injected i.p. 32 h after mating. To determine the specificity of this effect, mice were injected with immune and non-immune purified mouse immunoglobulins. The results show that anti-implantation efficacy was due to high-affinity antibody which bound progesterone since two further mouse immunoglobulin (Ig) G1 preparations, mouse IgA and mouse IgM which failed to bind the steroid, had no effect on pregnancy rates. From a panel of anti-progesterone monoclonal antibodies, six with a high affinity (affinity constant, 0·24–0·80 litres/nmol) and specificity for progesterone were selected for additional studies. Anti-implantation efficacy for five antibodies was similar, with a 50% effective dose within the range of 0·8–2·0 nmol. Antibody reached high concentrations in plasma within 12 h after i.p. injection, and declined with a half-life of about 80 h. Purified F(ab′)2 fragments of antibody also bound progesterone, but were less effective than the native molecule in blocking pregnancy. The results show that implantation in the mouse can be blocked by a high-affinity antibody that binds progesterone and which is removed from the blood at a slow rate. J. Endocr. (1988) 118, 69–80

scholarly journals An antifibrin monoclonal antibody useful in immunoscintigraphic detection of thrombi

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 708-714 ◽  
Author(s):  
MN Wasser ◽  
PW Koppert ◽  
JW Arndt ◽  
JJ Emeis ◽  
RI Feitsma ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Degradation Product ◽  
Spleen Cells ◽  
Fibrinogen Degradation Product ◽  
Myeloma Cells ◽  
High Concentrations ◽  
Preformed Plasma

Abstract Balb/c mice were immunized with human plasmin-generated fibrinogen degradation product Y. Spleen cells were fused with P3X63-Ag8.653 myeloma cells. A clone (Y22) was found that produces monoclonal antibodies (MoAbs) with a strong reactivity with human fibrin and only a weak reactivity with fibrinogen in an enzyme immunoassay (EIA). Y22 also reacts with fibrin of rabbits, rats, sheep, and dogs. The antibodies are of the IgG1 kappa-type and appear to be directed against a conformation-dependent epitope in the D-domain of fibrin. Experiments with 99mTc-labeled Y22 in vitro show that Y22 binds rapidly to forming clots. 99mTc-Y22 also binds to preformed plasma clots in a plasma milieu, even in the presence of high concentrations of heparin. Clot localization experiments in rabbits and rats confirm the high fibrin specificity and the potential of 99mTc-Y22 for thrombus imaging in vivo.

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