scholarly journals Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens

Genes ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 38
Author(s):  
Takehiro Mukae ◽  
Sho Okumura ◽  
Takuma Watanobe ◽  
Kyoko Yoshii ◽  
Takahiro Tagami ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Capacity ◽  
Specific Binding ◽  
Egg White ◽  
Peptide Sequence ◽  
Efficient Production ◽  
Antigen Binding ◽  
Transgenic Chicken ◽  
Bioreactor System ◽  
Transgenic Chickens

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4–1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.

INHIBITORY EFFECTS OF ITP SERA ON BINDING OF ANTIPLATELET GLYCOPROTEIN (GP) IIb/IIIa MONOCLONAL ANTIBODIES TO HUMAN UMBILICAL VASCULAR ENDOTHELIAL CELLS (HUVE)

1987 ◽  
Author(s):  
T Nakajima ◽  
T Koyama ◽  
Y Nishida ◽  
H Tanaka ◽  
E Kakishita ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vascular Endothelial Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Cell Pellet ◽  
A Cell ◽  
Molecular Masses ◽  
The Difference

Some ITP patients have specific autoantibodies to platelet GP IIb/IIIa. On the other hand, HUVE were shown to synthesize platelet GP IIb/IIIa like substances. Therefore, we studied the binding of ITP sera to HUVE by showing the inhibitory effect of ITP sera on the binding of anti-platelet GP IIb/IIIa monoclonal antibodies to HUVE. HUVE were cultured according to the method of Jaffe et al. 125-I-anti-platelet GP IIb/IIIa monoclonal antibody (125-I-Anti-GP) (40.3 mCi/mg), 40 yl, was added to a cell suspension of HUVE (1.5 × 104/500 μl) in a plastic RIA tube. After incubation for 30 min. at 4°C and centrifugation of 10,000 xg for 3 min., the radioactivity of the cell pellet was measured. Specific binding was determined by determining the difference between cell-bound radioactivity in the absence and presence of an excess amount of unlabelled ligand at 100 x concentrations. Scatchard analysis using 125-I-Anti-GP showed that the maximum binding capacity was 8 × 104/cell and Kd was 40.2 nM. The binding rate of 125-I-Anti-GP to HUVE treated with ITP (high PAIgG) sera (n=6) was 15.2±3.3% compared with 24.0±7.5%, observed for HUVE treated with normal sera (n=10). Treatment of ITP sera to HUVE significantly lowered the binding of 125-I-Anti-GP to HUVE (P<0.05). A combined analysis of SDS-PAGE and Western blotting of washed platelet and endothelial cell lysates shows that two proteins from each cells had similar or identical molecular masses to GP IIb/IIIa.These findings show that there are GP IIb/IIIa on the HUVE, ITP sera from our patients may have antibodies to HUVE GP IIb/IIIa and that anti-platelet GP IIb/IIIa antibodies in the ITP sera may bound not only to some platelets, but also to the HUVE

Immunocytochemical verification of the insulin receptor's specificity in the nuclear envelope of Tetrahymena. Comparison with receptors of the plasma membrane

1994 ◽  
Vol 14 (1) ◽  
pp. 25-31 ◽  
Author(s):  
G. Csaba ◽  
Hargita Hegyesi
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Hormone Receptors ◽  
Binding Capacity ◽  
Specific Binding ◽  
Antibody Technique ◽  
Binding Characteristics ◽  
Immunocytochemical Method ◽  
Higher Rank

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.

scholarly journals piggyBac Transposition and the Expression of Human Cystatin C in Transgenic Chickens

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1554
Author(s):  
Seo Woo Kim ◽  
Jeong Hyo Lee ◽  
Ji Seon Han ◽  
Seung Pyo Shin ◽  
Tae Sub Park
Keyword(s):  
Cystatin C ◽  
Mass Production ◽  
Animal Cell ◽  
Egg White ◽  
Efficient Production ◽  
Animal Cells ◽  
Bioactive Substances ◽  
Human Cystatin C ◽  
Transgenic Chickens ◽  
Human Cystatin

A bioreactor can be used for mass production of therapeutic proteins and other bioactive substances. Although various methods have been developed using microorganisms and animal cells, advanced strategies are needed for the efficient production of biofunctional proteins. In microorganisms, post-translational glycosylation and modification are not performed properly, while animal cell systems require more time and expense. To overcome these problems, new methods using products from transgenic animals have been considered, such as genetically modified cow’s milk and hen’s eggs. In this study, based on a non-viral piggyBac transposition system, we generated transgenic bioreactor chickens that produced human cystatin C (hCST3). There were no differences in the phenotype or histochemical structure of the wild-type and hCST3-expressing transgenic chickens. Subsequently, we analyzed the hCST3 expression in transgenic chickens, mainly in muscle and egg white, which could be major deposition warehouses for hCST3 protein. In both muscle and egg white, we detected high hCST3 expression by ELISA and Western blotting. hCST3 proteins were efficiently purified from muscle and egg white of transgenic chickens using a His-tag purification system. These data show that transgenic chickens can be efficiently used as a bioreactor for the mass production of bioactive materials.

scholarly journals Presence of IgT-C and I-A subregion-encoded determinants on distinct chains of monoclonal antigen-specific augmenting factor derived from a T cell hybridoma.

1983 ◽  
Vol 157 (6) ◽  
pp. 2110-2120 ◽  
Author(s):  
P B Nakajima ◽  
A Ochi ◽  
F L Owen ◽  
T Tada
Keyword(s):  
T Cells ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
Polypeptide Chain ◽  
Binding Capacity ◽  
Chromosome 12 ◽  
Antigen Binding ◽  
T Cell Factor

Monoclonal antibodies specific for mouse T cell alloantigens, Tindd and Tsud, linked to the Igh-1 locus on chromosome 12, were used to directly define the antigen-binding molecule produced by a cloned hybridoma. The T cell hybridoma, FL10, was established from antigen-binding T cells of A/J mice. FL10 produces an antigen-specific augmenting T cell factor (TaF) that bears a unique I region-controlled determinant (I-A) and has antigen-binding capacity. The Tindd, but not the Tsud, determinant was detected on the surface of FL10. The presence of both Tindd and I-A subregion-controlled determinants on FL10-derived TaF was directly demonstrated by the adsorption of TaF with immunoadsorbents prepared with monoclonal antibodies. The Igh-1-linked T cell alloantigen, Tsud, was not found on TaF. Further experiments indicated that Tindd is present on the antigen-binding polypeptide chain and not on the second chain bearing the I-A determinant. Despite the presence of the Tindd determinant on hybridoma-derived TaF, augmentation induced by TaF was restricted by the H-2 type of the responding mice and not by the Igh-1 allotype.

Concanavalin A-induced Aggregation of Human Blood Platelets

1975 ◽  
Vol 33 (02) ◽  
pp. 354-360 ◽  
Author(s):  
Heinrich Patscheke ◽  
Reinhard Brossmer
Keyword(s):  
Concanavalin A ◽  
Binding Capacity ◽  
Specific Binding ◽  
Blood Platelets ◽  
Residual Effect ◽  
Independent Effect ◽  
Con A ◽  
Experimental Conditions ◽  
Main Effect ◽  
Induced Aggregation

SummaryConcanavalin A (CON A) causes platelets to aggregate. A Ca++-independent effect of CON A could be separated from a main effect which depends on Ca++. The main effect probably is a consequence of the CON A-induced platelet release reaction and therefore is platelet-specific. The weak residual effect observed in the presence of Na2EDTA may be due to a similar mechanism as has been demonstrated for CON A-induced aggregations of several other normal and malignant transformed animal cells.Na2EDTA did not inhibit the carbohydrate-specific binding capacity of CON A. Therefore, Na2EDTA appears not to demineralize the CON A molecules under these experimental conditions.α-methyl-D-glucoside inhibits the Ca++-independent as well as the Ca++-dependent effect of CON A.Pretreatment by neuraminidase stimulated the platelet aggregation induced by CON A. It is possible that removal of terminal sialic acid residues makes additional receptors accessible for the binding of CON A.

Conformation as the Therapeutic Target for Neurodegenerative Diseases

2017 ◽  
Vol 14 (4) ◽  
pp. 393-402 ◽  
Author(s):  
Rajaraman Krishnan ◽  
Franz Hefti ◽  
Haim Tsubery ◽  
Michal Lulu ◽  
Ming Proschitsky ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neurodegenerative Diseases ◽  
Protein Misfolding ◽  
Specific Binding ◽  
Drug Candidate ◽  
High Affinity ◽  
Novel Approach ◽  
Multiple Protein ◽  
Clinical Benefits ◽  
Effective Drugs

Therapeutic strategies that target pathways of protein misfolding and the toxicity of intermediates along these pathways are mainly at discovery and early development stages, with the exception of monoclonal antibodies that have mainly failed to produce convincing clinical benefits in late stage trials. The clinical failures represent potentially critical lessons for future neurodegenerative disease drug development. More effective drugs may be achieved by pursuing the following two strategies. First, conformational targeting of aggregates of misfolded proteins, rather than less specific binding that includes monomer subunits, which vastly outnumber the toxic targets. Second, since neurodegenerative diseases frequently include more than one potential protein pathology, generic targeting of aggregates by shape might also be a crucial feature of a drug candidate. Incorporating both of these critical features into a viable drug candidate along with high affinity binding has not been achieved with small molecule approaches or with antibody fragments. Monoclonal antibodies developed so far are not broadly acting through conformational recognition. Using GAIM (General Amyloid Interaction Motif) represents a novel approach that incorporates high affinity conformational recognition for multiple protein assemblies, as well as recognition of an array of assemblies along the misfolding pathway between oligomers and fibers. A GAIM-Ig fusion, NPT088, is nearing clinical testing.

scholarly journals In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Merricka C. Livingstone ◽  
Alexis A. Bitzer ◽  
Alish Giri ◽  
Kun Luo ◽  
Rajeshwer S. Sankhala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Disease Burden ◽  
Vaccine Candidate ◽  
Specific Binding ◽  
Circumsporozoite Protein ◽  
Target Epitope ◽  
Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

scholarly journals Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9

2021 ◽  
Vol 11 (10) ◽  
pp. 4659
Author(s):  
Eun-Jung Kim ◽  
Gyu-Min Im ◽  
Chang-Soo Lee ◽  
Yun-Gon Kim ◽  
Byoung Joon Ko ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Calcium Binding ◽  
Nucleotide Sequences ◽  
Antibody Fragments ◽  
High Yield ◽  
Antigen Binding ◽  
Hybridoma Cell Culture ◽  
Inflammatory Protein ◽  
Inflammatory Processes ◽  
Yield And Purity

The calcium-binding protein S100A9 regulates inflammatory processes and the immune response. It is overexpressed in a variety of inflammatory and oncologic conditions. In this study, we produced a recombinant human S100A9 (hS100A9) antigen with high yield and purity and used it to generate a hybridoma cell culture-based monoclonal anti-hS100A9 antibody. We selected five anti-hS100A9 antibodies from cell supernatants that showed high antigen binding efficiency and identified the nucleotide sequences of three antibodies: two with high effective concentration values and one with the lowest value. The antigen and antibody development procedures described herein are useful for producing large amounts of monoclonal antibodies against hS100A9 and other antigens of interest. The nucleotide sequences of the anti-hS100A9 monoclonal antibody revealed herein will be helpful in the generation of recombinant antibodies or antibody fragments against hS100A9.

scholarly journals Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding

Antibodies ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 31
Author(s):  
Ann Christina Bergmann ◽  
Cecilie Kyllesbech ◽  
Rimantas Slibinskas ◽  
Evaldas Ciplys ◽  
Peter Højrup ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Epitope Mapping ◽  
Biological Function ◽  
Specific Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potential Therapeutic Target ◽  
Charged Amino Acids ◽  
Chaperone Protein ◽  
Terminal Amino

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

Sign in / Sign up

Export Citation Format

Share Document