A NEW FIBRIN-SPECIFIC ANTIBODY DISCRIMINATING BETWEEN FIBRIN AND FIBRINOGEN IS DIRECTED AGAINST THE SYNTHETIC PEPTIDE LEU-ILE-ASP-GLY-LYS-MET

1987 ◽  
Author(s):  
U Scheefers-Borchel ◽  
G Muller-Berghaus
Keyword(s):  
Monoclonal Antibodies ◽  
Specific Antibody ◽  
Synthetic Peptide ◽  
Degradation Products ◽  
Coagulation Disorders ◽  
Soluble Fibrin ◽  
Blood Constituents ◽  
C Terminus ◽  
Covalently Linked ◽  
Synthetic Hexapeptide

To determine soluble fibrin in blood of patients with coagulation disorders we produced monoclonal antibodies which distinct fibrin from fibrinogen and other blood constituents. Fibrin-specific monoclonal antibodies were obtained by immunizing mice with the synthetic hexapeptide Leu-Ile-Asp-Gly-Lys-Met which was covalently linked to KLH via its C-terminus. Several of the monoclonal antibodies which reacted with the hexapeptide also reacted with batroxobin-induced desAA-fibrin and thrombin-induced desAABB-fibrin, but not with fibrinogen. No reaction was observed with plasmin-induced fibrinogenolytic and fibrinolytic degradation products, respectively. The epitope recognized by these fibrin-specific antibodies is located on the αchain of fibrin and is not accessible for an antibody in native fibrinogen. One monoclonal antibody (B/H11) was used to quantify the amount of soluble fibrin in plasma of patients with a variety of coagulation disorders. This antibody could also be used to develop an ELISA based on two different fibrin-specific monoclonal antibodies. For this assay anti-fbn 17 (Scheefers- Borchel et al., Proc. Natl. Acad. Sci. USA 82: 7091, 1985) was coated onto ELISA plates. After adding plasma which contained soluble fibrin, the fibrin bound was detected by the second fibrin-specific antibody B/H^ to which biotin was covalently linked. The second antibody was probed by the addition of peroxydase conjugated streptavidin and the substrate ABTS for peroxydase. This test can be used to detect fibrin at concentrations as low as 70 ng/ml. With this assay system, it is possible to measure the amount of soluble fibrin present in plasma samples without the interference of fibrinogen which is associated with soluble fibrin.

MONOCLONAL ANTIBODIES OF THE IgM AND IgG CLASS SPECIFIC FOR CROSSLINKED FIBRIN DEGRADATION PRODUCTS

1987 ◽  
Author(s):  
P J Gaffney ◽  
L J Creighton ◽  
A Curry ◽  
B MacMahon ◽  
R Thorpe
Keyword(s):  
Monoclonal Antibodies ◽  
Thrombolytic Therapy ◽  
Epitope Mapping ◽  
Degradation Products ◽  
Subcutaneous Route ◽  
Fibrin Degradation Products ◽  
Conformational Epitopes ◽  
Immunoglobulin Class Switching ◽  
Unique Specificity ◽  
D Dimer

Monoclonal antibodies (mabs) to crosslinked fibrin degradation products (XL-FDP) having the general formula D/Y[X]nY/D (known as X-oligomer) and D-D (known as D dimer) have been raised in balb/C mice by both a novel mtrasplenic and a conventional subcutaneous route of immunisation and by combinations of both these procedures. Mabs to X-oligomers (NIBn 52 and NIBn 123) obtained by an intrasplenic procedure have been demonstrated to crossreact only with X-oligomer in a 2-site ELISA procedure and not with D dimer or whole fibrinogen and have been shown to be of value m the examination of clinical material obtained from patients with various types of thrombosis and have also been useful in monitoring the efficacy of thrombolytic therapy. The X-oligomer mabs are immunoglobulins of the M class. It was demonstrated that their unique specificity for conformational epitopes on the large X-oligomer fragments does not reside in the IgM structure since alterative immunisation procedures have been used to generate mabs of the IgG class which have the same specificity. Using immunoglobulin class switching in culture rather than during immunisation was suggested by certain cell lines which produced both IgM and IgG specific for X-oligomer. This latter point needs rigorous validation.Immunoglobulin G type mabs to highly purified D dimer were raised by conventional subcutaneous immunisation of balb/C mice. One of these, NIBn-11, was found to crossreact with PVC-immobilised X-oligomer and D dimer but not with fibrinogen. However NIBn-11 did not bind to D dimer in a 2-site ELISA procedure while crossreactmg quite avidly with X-oligomer. This suggests that the D dimer epitope to which NIBn-11 is directed is expressed in some conformations and not m others and that these conformations are always expressed in the complex X-oligomer group of fragments. These mabs, whilst of value in measuring certain unique fibrin fragments m plasma, are useful in the epitope mapping of fibrinogen/fibrin and their plasmm-mediated

Novel leukaemia markers

1995 ◽  
Vol 15 (6) ◽  
pp. 463-468
Author(s):  
Kingsley J. Micklem
Keyword(s):  
Monoclonal Antibodies ◽  
T Cell ◽  
Recombinant Protein ◽  
Follicular Lymphoma ◽  
B Lymphocytes ◽  
Synthetic Peptide ◽  
Paraffin Sections ◽  
Hematologic Disorders ◽  
Polyclonal Antisera ◽  
Chromosomal Defects

Using synthetic peptide or recombinant protein as immunising antigens we have produced monoclonal antibodies and polyclonal antisera directed against targets of particular interest in leukaemia diagnosis. In this way we have prepared reagents which recognise all T or all B lymphocytes in routinely fixed paraffin sections which are unique in this respect. We have also produced monoclonal antibodies to molecules potentially involved in specific neoplastic transformations, implicated by virtue of the involvement of their genes in chromosomal defects in these neoplasms. In particular, we have produced antibodies recognising bcl-2, involved in follicular lymphoma, tal-1, involved in T-cell acute leukaemias and HRX involved in a variety of hematologic disorders. The application of these reagents to diagnosis has so far proved useful. In addition their use outside the field of leukaemia diagnosis has proved to be even more important in some cases.

scholarly journals Novel Split Intein fortrans-Splicing Synthetic Peptide onto C Terminus of Protein

2009 ◽  
Vol 284 (10) ◽  
pp. 6194-6199 ◽  
Author(s):  
Julia H. Appleby ◽  
Kaisong Zhou ◽  
Gerrit Volkmann ◽  
Xiang-Qin Liu
Keyword(s):  
Synthetic Peptide ◽  
Split Intein ◽  
C Terminus

Detection of fos protein during osteogenesis by monoclonal antibodies

1988 ◽  
Vol 8 (5) ◽  
pp. 2251-2256
Author(s):  
P De Togni ◽  
H Niman ◽  
V Raymond ◽  
P Sawchenko ◽  
I M Verma
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Indirect Immunofluorescence ◽  
Protein Complex ◽  
Synthetic Peptide ◽  
Immunohistochemical Staining ◽  
Nuclear Staining ◽  
Rat Embryos ◽  
Fos Protein ◽  
Modified Forms

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

scholarly journals The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

2021 ◽  
Vol 12 ◽  
Author(s):  
Pia Gattinger ◽  
Shiva Izadi ◽  
Clemens Grünwald-Gruber ◽  
Somanath Kallolimath ◽  
Alexandra Castilho
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Degradation Products ◽  
Therapeutic Proteins ◽  
Cost Effective ◽  
Full Length ◽  
Peptide Sequence ◽  
Reporter Protein ◽  
The Stability

The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

scholarly journals Chimeric Fusion (F) and Attachment (G) Glycoprotein Antigen Delivery by mRNA as a Candidate Nipah Vaccine

2021 ◽  
Vol 12 ◽  
Author(s):  
Rebecca J. Loomis ◽  
Anthony T. DiPiazza ◽  
Samantha Falcone ◽  
Tracy J. Ruckwardt ◽  
Kaitlyn M. Morabito ◽  
...  
Keyword(s):  
Nipah Virus ◽  
Neutralizing Antibody ◽  
T Cell Response ◽  
Antigen Delivery ◽  
Design Elements ◽  
C Terminus ◽  
Glycoprotein Antigen ◽  
Pandemic Threat ◽  
Covalently Linked ◽  
Selection Of

Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development.

scholarly journals Prevalence of antibodies against a cyclic peptide mimicking the FG loop of the human papillomavirus type 16 capsid among Tunisian women

2020 ◽  
Author(s):  
Elham Hassen ◽  
Devendra Bansal ◽  
Randa Ghdira ◽  
Anouar Chaieb ◽  
Hedi Khairi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Papillomavirus ◽  
Sex Workers ◽  
Synthetic Peptide ◽  
Cyclic Peptides ◽  
Cyclic Peptide ◽  
Low Grade ◽  
Squamous Intraepithelial Lesions ◽  
Intraepithelial Lesions ◽  
Peptide Antibodies

Abstract Background In the past decade, cervical cancer has gone from being the second to the fourth most common cancer in women worldwide, but remains the second most common in developing countries. This cancer is most commonly caused by high-risk types of human papillomavirus (HPV), mainly type 16 (HPV16), which are sexually transmitted. This study aimed to investigate the usefulness of a cyclic synthetic peptide designed from the major L1 capsid protein of HPV16 for detecting anti-HPV16 antibodies. Methods We designed and synthetized a peptide that corresponds to the full sequence of the surface-exposed FG loop. We tested the antigenicity of the linear and the cyclic peptides against HPV16 L1 monoclonal antibodies. We used ELISA to detect anti-peptide antibodies in sera and cervical secretions of 179 Tunisian women, and we applied polymerase chain reaction and direct sequencing methods to detect and genotype HPV DNA. Results Both the linear and the cyclic peptides were recognized by the same neutralizing monoclonal antibodies, but the cyclic peptide was more reactive with human sera. The prevalence of the anti-peptide antibodies in sera was higher in women with low-grade squamous intraepithelial lesions (LGSIL) than in women with high-grade squamous intraepithelial lesions (HGSIL) (44% and 15%, respectively). This contrasts with HPV16 DNA prevalence. Compared to women from the general population, systemic IgG prevalence was significantly higher among sex workers (25%; P=0.002) and women with LGSIL (44%; P=0.001). In addition, systemic IgA and cervical IgG prevalence was higher among sex workers only (p=0.002 and P=0.001 respectively). We did not observe anti-peptide IgG antibodies in women with a current HPV16 infection.Conclusion Anti-peptide IgG in sera or in cervical secretions could be markers of an effective natural immunization against HPV16. This may open novel perspectives for monitoring vaccinated women and for the design of synthetic peptide-based vaccines.

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