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

Analysis of binding residues in monoclonal antibody with high affinity for the head domain of the rat P2X4 receptor

2020 ◽  
Author(s):  
Tatsuhiro Igawa ◽  
Shuhei Kishikawa ◽  
Yoshito Abe ◽  
Makoto Tsuda ◽  
Kazuhide Inoue ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Neuropathic Pain ◽  
Monoclonal Antibodies ◽  
Dissociation Constants ◽  
Site Directed Mutagenesis ◽  
P2x4 Receptor ◽  
High Affinity ◽  
Head Domain ◽  
Binding Residues ◽  
Nanomolar Range

Abstract P2X4 receptor is known to be involved in neuropathic pain. In order to detect the expression of P2X4 receptor on microglia at the time of onset of neuropathic pain, one approach consists on the preparation of the monoclonal antibodies with both selective binding and high affinity. We have recently established a monoclonal antibody (named 12-10H) which had high affinity to rat P2X4 receptor expressed in 1321N1 cells. The dissociation constants of the complex between the monoclonal antibodies obtained so far and the head domain (HD) in the rat P2X4 receptor were in the nanomolar range. To improve the affinity by rational mutations, we need to know the precious location of the binding site in these monoclonal antibodies. Here, we have analysed and identified the binding residues in the monoclonal antibody (12-10H) with high affinity for the HD of the rat P2X4 receptor by site-directed mutagenesis.

Theraputic Monoclonal Antibody Interference In Immunofixation Electrophoresis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4996-4996 ◽  
Author(s):  
Elizabeth A. Guancial ◽  
Vinay S. Mahajan ◽  
Ronald P. McCaffrey ◽  
Neal Lindeman
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immune Thrombocytopenic Purpura ◽  
Half Life ◽  
Monoclonal Gammopathy ◽  
Chimeric Antibody ◽  
Thrombocytopenic Purpura ◽  
Steroid Resistant ◽  
Monoclonal Protein ◽  
Anti Cd20

Abstract Abstract 4996 The application of monoclonal antibodies represents an increasingly powerful weapon in the arsenal against hematologic diseases. With the advent of new therapies come previously unrecognized laboratory interference that if not properly identified could have clinical repercussions. We encountered two patients, neither of whom had a history of plasma cell dyscrasia, who underwent treatment with rituximab for immune thrombocytopenic purpura (ITP) and were discovered to have a monoclonal IgG kappa band on serum protein electrophoresis (SPEP) followed by immunofixation (IFX). Both patients had severe, steroid-resistant ITP and received rituximab on an empiric basis, at a weekly dose of 375 mg/m2 IV. In Patient #1, an evaluation for secondary causes of thrombocytopenia included SPEP and IFX. The IFX yielded a faint, atypical IgG kappa band that migrated to the far cathodal zone of the gel. No band was seen on SPEP alone. Since the patient was treated with rituximab the day before these studies were performed, we considered the possibility that the IgG kappa band was due to circulating rituximab. Rituximab is a chimeric antibody composed of human IgG1 kappa chain constant regions and heavy- and light-chain variable regions from a murine anti-CD20 antibody. The elimination half-life of this therapeutic antibody increases with subsequent doses and is approximately 60 hours for the first dose and 174 hours for the fourth dose (range 26 to 442 hours). An aliquot of rituximab was obtained from pharmacy and analyzed by SPEP/IFX, showing an IgG kappa monoclonal protein band identical to the M-protein in the patient's serum. Repeat assessment of serum from this patient obtained immediately prior to her fourth treatment with rituximab demonstrated that the IgG kappa paraprotein was less intense on IFX compared to the first IFX. Several hours after the fourth rituximab infusion was completed, a more intense band corresponding to the IgG kappa paraprotein was detected again on IFX. In Patient #2, who also received rituximab for steroid-resistant ITP, a similar circulating rituximab-related protein was detected on IFX performed one hour after his fourth rituximab infusion. IFX performed on a sample obtained immediately before treatment did not demonstrate a paraprotein, and testing one week after treatment showed that the band had resolved. We believe that awareness of the presence of a faint monoclonal protein by SPEP/IFX following recent administration of rituximab is important to avoid unnecessary further evaluation for a pathologic monoclonal gammopathy. Alternatively, if suspicion exists, SPEP/IFX should be conducted prior to the initiation of treatment with rituximab or after the treatment course is completed in order to avoid uncertainty. Furthermore, it is important to be cognizant of this finding in patients on maintenance rituximab for an indolent lymphoma and an associated paraprotein who may undergo interval monitoring of the monoclonal gammopathy. It is possible that a similar phenomenon may be seen with other therapeutic monoclonal antibodies with a dosing regimen and half-life that is similar to rituximab, such as cetuximab, a monoclonal antibody directed against the epidermal growth factor receptor. Finally, the presence of a characteristic band on immunofixation may allow for the qualitative assessment of the presence of a therapeutic monoclonal antibody in the serum. Further studies are necessary to determine the sensitivity and specificity of such a laboratory test. Disclosures: Off Label Use: Rituximab is an anti-CD20 chimeric antibody and is used for the treatment of immune thrombocytopenic purpura.

scholarly journals Binding characteristics of antibodies to the TSH receptor

1998 ◽  
Vol 20 (2) ◽  
pp. 233-244 ◽  
Author(s):  
Y Oda ◽  
J Sanders ◽  
S Roberts ◽  
M Maruyama ◽  
R Kato ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Tsh Receptor ◽  
Affinity Constant ◽  
Translation System ◽  
Scatchard Analysis ◽  
Facs Analysis ◽  
Binding Characteristics ◽  
Fab Fragments

We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

scholarly journals Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 383
Author(s):  
Yanan Wang ◽  
Xiaofei Wang ◽  
Haitang Zhang ◽  
Hanna Fotina ◽  
Jinqing Jiang
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Limit Of Detection ◽  
Broad Class ◽  
Rapid Test ◽  
Competitive Elisa ◽  
Affinity Constant ◽  
High Affinity ◽  
Ic50 Values ◽  
Hybridoma Cell Lines

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 102 in supernatants and (1.28 to 5.12) × 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 109 and 6.54 × 109 L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

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.

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.

scholarly journals Towards Dewetting Monoclonal Antibodies for Therapeutical Purposes

2019 ◽  
Author(s):  
Arturo Tozzi
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Ion Channels ◽  
Influenza A ◽  
Water Molecules ◽  
Transient Phenomenon ◽  
Water Flows ◽  
High Affinity ◽  
Channel Blockage ◽  
Severe Impairment

Dewetting transition - a concept borrowed from fluid mechanics -  is a physiological process which takes place inside the hydrophobic pores of ion channels.  This transient phenomenon causes a metastable state which forbids water molecules to cross the microscopic receptors’ cavities.  This leads to a decrease of conductance, a closure of the hole and, subsequently, severe impairment of cellular performance.  We suggest that  artificially-provoked dewetting transition in ion channels’ hydrophobic pores could stand for a molecular candidate to erase detrimental organisms, such as viruses, bacteria and cancer cells.  We describe a novel type of high-affinity monoclonal antibody, which: a) targets specific trans-membrane receptor structures of harmful or redundant cells; b) is equipped with lipophilic and/or hydrophobic fragments that prevent physiological water flows inside ion channels.  Therefore, we achieve an artificial dewetting transition inside receptors’ cavities which causes transmembrane ionic flows discontinuity, channel blockage and subsequent damage of morbid cells.  As an example, we describe dewetting monoclonal antibodies targeting the M2 channel of the Influenza A virus: they might prevent water to enter the pores, thus leading to virion impairment.

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

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.

Engineered human immunoglobulin anti-yellow fever virus monoclonal antibodies to treat severe yellow fever

2020 ◽  
Vol 27 (7) ◽  
Author(s):  
W Y Leong
Keyword(s):  
Clinical Trial ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Yellow Fever ◽  
Clinical Benefit ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Human Immunoglobulin ◽  
Potential Clinical Benefit ◽  
Ig G

Teaser A new engineered human immunoglobulin (Ig)G anti-yellow fever virus monoclonal antibody suggested potential clinical benefit in Phase 1a and 1b clinical trial.

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