scholarly journals A New Workflow to Generate Monoclonal Antibodies against Microorganisms

2021 ◽  
Vol 11 (20) ◽  
pp. 9359
Author(s):  
Markus Göthel ◽  
Martin Listek ◽  
Katrin Messerschmidt ◽  
Anja Schlör ◽  
Anja Hönow ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Legionella Pneumophila ◽  
Initial Study ◽  
Cross Reactivity ◽  
Cell Surface Receptor ◽  
Peptide Epitopes ◽  
E Coli ◽  
Efficient Detection ◽  
Surface Receptor ◽  
Fusion Cell

Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.

scholarly journals Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 365
Author(s):  
Fengli Liu ◽  
Yanxin Cao ◽  
Maokai Yan ◽  
Mengxu Sun ◽  
Qingshui Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Colloidal Gold ◽  
Preventive Intervention ◽  
Virus Detection ◽  
Cross Reactivity ◽  
Duck Enteritis Virus ◽  
Detection Sensitivity ◽  
Immunochromatographic Assay ◽  
Efficient Detection ◽  
Enteritis Virus

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

scholarly journals A Targeted Catalytic Nanobody (T-CAN) with Asparaginolytic Activity

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5637
Author(s):  
Maristella Maggi ◽  
Greta Pessino ◽  
Isabella Guardamagna ◽  
Leonardo Lonati ◽  
Cristina Pulimeno ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
Cell Surface Receptor ◽  
Single Domain Antibody ◽  
Antibody Drug Conjugate ◽  
E Coli ◽  
Novel Approach ◽  
Innovative Solutions ◽  
Surface Receptor ◽  
New Type

E. coli L-asparaginase is an amidohydrolase (EC 3.5.1.1) which has been successfully used for the treatment of Acute Lymphoblastic Leukemia for over 50 years. Despite its efficacy, its side effects, and especially its intrinsic immunogenicity, hamper its usage in a significant subset of cases, thus limiting therapeutic options. Innovative solutions to improve on these drawbacks have been attempted, but none of them have been truly successful so far. In this work, we fully replaced the enzyme scaffold, generating an active, miniaturized form of L-asparaginase by protein engineering of a camel single domain antibody, a class of antibodies known to have a limited immunogenicity in humans. We then targeted it onto tumor cells by an antibody scFv fragment directed onto the CD19 B-cell surface receptor expressed on ALL cells. We named this new type of nanobody-based antibody-drug conjugate “Targeted Catalytic Nanobody” (T-CAN). The new molecule retains the catalytic activity and the binding capability of the original modules and successfully targets CD19 expressing cells in vitro. Thanks to its theoretically reduced immunogenic potential compared to the original molecule, the T-CAN can represent a novel approach to tackle current limitations in L-asparaginase usage.

scholarly journals Selective inhibition of the growth of human erythroid bursts by monoclonal antibodies against transferrin or the transferrin receptor

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1631-1638 ◽  
Author(s):  
KM Shannon ◽  
JW Larrick ◽  
SA Fulcher ◽  
KB Burck ◽  
J Pacely ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transferrin Receptor ◽  
Thymidine Incorporation ◽  
Selective Inhibition ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Transferrin Receptors ◽  
Human Erythropoietin ◽  
Surface Receptor ◽  
Transferrin Receptor Expression

Abstract The relative requirements of colonies derived from erythroid (BFU-E) and myeloid (CFU-c) progenitors for transferrin were examined using monoclonal antibodies directed against the transferrin molecule (TF-6) or its cell surface receptor (TFR-A12, TFR1–2B). Growth of erythroid bursts was profoundly reduced at concentrations of all three antibodies that had no effect on CFU-c-derived colonies. When TFR1–2B was layered over cultures established one to seven days previously, further burst development was inhibited, and degeneration of early erythroid colonies was observed. Addition of erythropoietin augmented transferrin receptor expression on cells harvested after 1 to 2 weeks in culture and analyzed by flow cytometry. Recombinant human erythropoietin gave results comparable to those obtained in experiments using human urinary erythropoietin. Analysis of erythroblasts plucked directly from culture plates confirmed the presence of transferrin receptors on BFU-E-derived colonies. Thymidine incorporation was maximal early in the second week of culture and coincided with high transferrin receptor expression. These data demonstrate that transferrin must be available into the second week of culture to support the growth and differentiation of BFU- E-derived erythroid bursts, that the generation of erythroid colonies from BFU-E is more dependent on transferrin than myeloid colony formation from CFU-c, and that erythropoietin modulates the expression of transferrin receptors on growing bursts.

scholarly journals An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3127-3134
Author(s):  
MM Fiore ◽  
PF Neuenschwander ◽  
JH Morrissey
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Surface Receptor ◽  
Coagulation Cascade ◽  
Binding Assays ◽  
Cell Binding ◽  
Small Peptide ◽  
Direct Competition ◽  
Surface Receptor

Tissue factor (TF), the cell surface receptor and cofactor for factor VIIa (FVIIa), is considered the major physiologic trigger of the coagulation cascade. Most monoclonal antibodies to TF have been reported to inhibit TF activity by blocking association of FVII(a) with TF. Using solution-phase kinetic analyses, we have reexamined two strongly inhibitory anti-TF monoclonal antibodies (TF8–11D12 and TF9–9C3) previously reported to block FVII binding in cell-binding assays. Kinetic analysis of TF9–9C3 was consistent with direct competition with FVIIa for binding to TF. However, antibody TF8–11D12 did not block FVIIa binding to TF as measured by ability of the TF:FVIIa complex to cleave a small peptide substrate or by enhanced reactivity of FVIIa with a tripeptidyl-chloromethylketone. Interestingly, TF8–11D12 strongly inhibited cleavage of all three known macromolecular substrates (factors VII, IX, and X) of the TF:FVIIa complex. We hypothesize that TF8–11D12 blocks access of macromolecular substrates to the active site of FVIIa by steric hindrance. This study identifies a useful probe for TF function and provides insights into the inhibitory mechanism of an unusual class of antibody proposed for therapeutic intervention in thrombotic disease.

Do Lipocalins Play a Role in Mast Cell Physiology?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1645-1645
Author(s):  
Lennart E. Logdberg ◽  
Bo Akerstrom ◽  
Gregory A. Hair ◽  
Maria Allhorn ◽  
Tatyana Vikulina ◽  
...  
Keyword(s):  
Mast Cell ◽  
Monoclonal Antibodies ◽  
Mast Cells ◽  
Cell Surface ◽  
Gene Transcription ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Cell Physiology ◽  
Myeloma Protein ◽  
Surface Receptor

Abstract Introduction. Aiming to identify molecular properties of allergens responsible for their “intrinsic allergenicity”, we focus on a subset of xenogeneic lipocalins (LCs) that comprise the major mammalian respiratory allergens. These structurally and functionally homologous molecules likely possess conserved molecular motifs promoting IgE-dependent allergy. We hypothesize that such LC “allergenicity” depends on non-IgE interactions of LCs with components of the innate immune system. Herein we describe a possible basis for such interactions between LCs and mast cells. Materials and Methods. Two sources of human mast cells were used; primary cultures derived from peripheral blood CD34+ progenitor cells; or the LAD-2 cell line. Cells were cultured in serum-free medium with recombinant human stem cell factor (SCF; 100 ng/ml). Monoclonal antibodies to human gp330/megalin (MAb E11) were a kind gift of Prof. Lars Rask (Uppsala University, Sweden). Cell surface protein expression was assessed by flow cytometry and gene transcription was measured by real-time PCR. Results. Monoclonal antibodies to an endocytic cell surface receptor (megalin, also known as low-density lipoprotein receptor-related protein (LRP)-2) known to bind multiple LCs stained the mast cell lines. This putative expression of megalin by the mast cells corresponded to their transcription of megalin mRNA as shown as by PCR. Moreover, mast cell megalin gene transcription could be induced (≥ 1000-fold) by overnight culture with monomeric IgE myeloma protein (100 ng/ml), and such induction of the megalin message correlated with both an increase in cell surface expression of the molecule and the specific binding of a purified human LC (a-1 microglobulin). Conclusion. Megalin, an LC-binding cell surface receptor, appears to be constitutively expressed by both progenitor and mature mast cells, and its expression seems to be strongly upregulated by culture with monomeric IgE. This is consistent with a role for direct mast cell-LC interactions in the development of IgE-dependent allergy. In addition, and also of potential clinical relevance, endogenous LCs may play a functional role in normal mast cell physiology.

scholarly journals Selective inhibition of the growth of human erythroid bursts by monoclonal antibodies against transferrin or the transferrin receptor

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1631-1638
Author(s):  
KM Shannon ◽  
JW Larrick ◽  
SA Fulcher ◽  
KB Burck ◽  
J Pacely ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transferrin Receptor ◽  
Thymidine Incorporation ◽  
Selective Inhibition ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Transferrin Receptors ◽  
Human Erythropoietin ◽  
Surface Receptor ◽  
Transferrin Receptor Expression

The relative requirements of colonies derived from erythroid (BFU-E) and myeloid (CFU-c) progenitors for transferrin were examined using monoclonal antibodies directed against the transferrin molecule (TF-6) or its cell surface receptor (TFR-A12, TFR1–2B). Growth of erythroid bursts was profoundly reduced at concentrations of all three antibodies that had no effect on CFU-c-derived colonies. When TFR1–2B was layered over cultures established one to seven days previously, further burst development was inhibited, and degeneration of early erythroid colonies was observed. Addition of erythropoietin augmented transferrin receptor expression on cells harvested after 1 to 2 weeks in culture and analyzed by flow cytometry. Recombinant human erythropoietin gave results comparable to those obtained in experiments using human urinary erythropoietin. Analysis of erythroblasts plucked directly from culture plates confirmed the presence of transferrin receptors on BFU-E-derived colonies. Thymidine incorporation was maximal early in the second week of culture and coincided with high transferrin receptor expression. These data demonstrate that transferrin must be available into the second week of culture to support the growth and differentiation of BFU- E-derived erythroid bursts, that the generation of erythroid colonies from BFU-E is more dependent on transferrin than myeloid colony formation from CFU-c, and that erythropoietin modulates the expression of transferrin receptors on growing bursts.

scholarly journals An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3127-3134 ◽  
Author(s):  
MM Fiore ◽  
PF Neuenschwander ◽  
JH Morrissey
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Surface Receptor ◽  
Coagulation Cascade ◽  
Binding Assays ◽  
Cell Binding ◽  
Small Peptide ◽  
Direct Competition ◽  
Surface Receptor

Abstract Tissue factor (TF), the cell surface receptor and cofactor for factor VIIa (FVIIa), is considered the major physiologic trigger of the coagulation cascade. Most monoclonal antibodies to TF have been reported to inhibit TF activity by blocking association of FVII(a) with TF. Using solution-phase kinetic analyses, we have reexamined two strongly inhibitory anti-TF monoclonal antibodies (TF8–11D12 and TF9–9C3) previously reported to block FVII binding in cell-binding assays. Kinetic analysis of TF9–9C3 was consistent with direct competition with FVIIa for binding to TF. However, antibody TF8–11D12 did not block FVIIa binding to TF as measured by ability of the TF:FVIIa complex to cleave a small peptide substrate or by enhanced reactivity of FVIIa with a tripeptidyl-chloromethylketone. Interestingly, TF8–11D12 strongly inhibited cleavage of all three known macromolecular substrates (factors VII, IX, and X) of the TF:FVIIa complex. We hypothesize that TF8–11D12 blocks access of macromolecular substrates to the active site of FVIIa by steric hindrance. This study identifies a useful probe for TF function and provides insights into the inhibitory mechanism of an unusual class of antibody proposed for therapeutic intervention in thrombotic disease.

scholarly journals Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

2006 ◽  
Vol 80 (17) ◽  
pp. 8695-8704 ◽  
Author(s):  
Ania M. Owsianka ◽  
Judith M. Timms ◽  
Alexander W. Tarr ◽  
Richard J. P. Brown ◽  
Timothy P. Hickling ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Envelope Glycoprotein ◽  
Viral Envelope ◽  
Cell Surface Receptor ◽  
Conserved Residues ◽  
Mutant Proteins ◽  
Surface Receptor ◽  
Cd81 Binding

ABSTRACT Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.

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