scholarly journals Modeling the Effect of Amino Acids and Copper on Monoclonal Antibody Productivity and Glycosylation: A Modular Approach

2020 ◽  
pp. 2000261
Author(s):  
Yu Luo ◽  
Robert J. Lovelett ◽  
J. Vincent Price ◽  
Devesh Radhakrishnan ◽  
Kristopher Barnthouse ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Modular Approach ◽  
Antibody Productivity

scholarly journals Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding

1989 ◽  
Vol 257 (2) ◽  
pp. 461-469 ◽  
Author(s):  
G E Morris
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Creatine Kinase ◽  
Specific Binding ◽  
Trypsin Digestion ◽  
Proteinase K ◽  
Enzyme Linked Immunosorbent Assay ◽  
Muscle Creatine Kinase ◽  
Western Blots ◽  
Methionine Residues

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K ‘nicks’ native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to ‘nicked’ CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.

scholarly journals Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Liping Huang ◽  
Zhenzhao Sun ◽  
Deli Xia ◽  
Yanwu Wei ◽  
Encheng Sun ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Neutralizing Antibodies ◽  
Vaccine Design ◽  
Binding Mode ◽  
Porcine Circovirus Type ◽  
Therapeutic Antibody ◽  
Capsid Proteins ◽  
Porcine Circovirus

ABSTRACT Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections. IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

scholarly journals Identification of an immunodominant functional domain on human CD36 antigen using human-mouse chimaeric proteins and homologue-replacement mutagenesis

1995 ◽  
Vol 305 (1) ◽  
pp. 221-224 ◽  
Author(s):  
L Daviet ◽  
R Buckland ◽  
M D Puente Navazo ◽  
J L McGregor
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Functional Domain ◽  
Oxidized Low Density Lipoprotein ◽  
Key Residues ◽  
Antibody Epitopes ◽  
Human And Mouse

The human CD36 antigen is a multifunctional membrane glycoprotein that acts as a receptor for thrombospondin, malaria-infected erythrocytes and oxidized low-density lipoprotein, as well as being implicated in the recognition of apoptotic neutrophils by macrophages. OKM5 and other anti-CD36 monoclonal antibodies have been shown to inhibit these CD36 adhesive functions, suggesting that the monoclonal-antibody epitopes and the domains that mediate these events are closely related. Analysis of a series of chimaeric exchanges between human and mouse CD36 shows that six anti-CD36 monoclonal antibodies (OKM5, FA6-152, L103, 5F1, SM phi and 10/5) recognize epitopes within the domain comprising amino acids 155-183. A seventh monoclonal antibody (13/10) binds to another domain that spans amino acids 30-76. Homologue-replacement mutagenesis performed within the human 155-183 immunodominant sequence identifies key residues for the binding of three functional monoclonal antibodies (OKM5, FA6-152 and L103). The fact that antibodies directed against the 155-183 domain can inhibit adhesion suggests that this domain is directly involved in CD36-ligand binding.

Epitope mapping and analysis of a growth-enhancing monoclonal antibody by limited tryptic digestion of porcine GH

1995 ◽  
Vol 145 (1) ◽  
pp. 169-174 ◽  
Author(s):  
H-M Shieh ◽  
R T Bass ◽  
B S Wang ◽  
M J Corbett ◽  
B L Buckwalter
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Helical Structure ◽  
Tryptic Digestion ◽  
Amino Acid Residues ◽  
Optimal Sequence ◽  
Epitope Region ◽  
Dose Dependent Fashion ◽  
Growth Promoting ◽  
Dose Dependent

Abstract In this study, the epitope of a murine PS-7·6 monoclonal antibody (mAb) which was raised against the recombinant porcine GH (pGH) and subsequently shown to enhance the growth-promoting activity of pGH in a hypophysectomized rat model, was mapped by the limited tryptic digestion of pGH. A pGH fragment corresponding to amino acid residues 70–95 was separated by reverse-phase HPLC and also immunoprecipitated by PS-7·6 mAb. This fragment was found in an RIA to compete with radiolabelled pGH for the binding of PS-7·6 mAb in a dose-dependent fashion. Several peptides covering this potential epitope region of pGH(70–95) were synthesized and assayed by competitive RIA. The results suggested that pGH(75–90) was the optimal sequence recognized by PS-7·6 mAb. Sequential alanine substitution of each residue of pGH(75–90) revealed that the side chains of Leu76, Ile83 and Leu87 were critical for binding to PS-7·6 mAb. Other residues could be replaced by alanine without substantially altering the binding affinity. The region of amino acids 75–95 comprises the C-terminal end of the second helix of pGH and the repeating pattern of i and i+3 (i+7) of the critical amino acids appears consistent with PS-7·6 mAb binding to the hydrophobic side of the helix. The sequence and the helical structure of the epitope of PS-7·6 mAb provide the basis for designing the effective peptide vaccines to enhance the growth performance of animals. Journal of Endocrinology (1995) 145, 169–174

Contrasting the Influence of Cationic Amino Acids on the Viscosity and Stability of a Highly Concentrated Monoclonal Antibody

2016 ◽  
Vol 34 (1) ◽  
pp. 193-207 ◽  
Author(s):  
Barton J. Dear ◽  
Jessica J. Hung ◽  
Thomas M. Truskett ◽  
Keith P. Johnston
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Cationic Amino Acids

scholarly journals Cytotoxic Necrotizing Factor Type 1-Neutralizing Monoclonal Antibody NG8 Recognizes Three Amino Acids in a C-Terminal Region of the Toxin and Reduces Toxin Binding to HEp-2 Cells

2008 ◽  
Vol 77 (1) ◽  
pp. 170-179 ◽  
Author(s):  
Kerian K. Grande ◽  
Karen C. Meysick ◽  
Susan B. Rasmussen ◽  
Alison D. O'Brien
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Dependent Manner ◽  
Triple Mutant ◽  
Potent Inducer ◽  
Cytotoxic Necrotizing Factor ◽  
Toxin Binding ◽  
Neutralizing Monoclonal Antibody ◽  
Factor Type

ABSTRACTCytotoxic necrotizing factor type 1 (CNF1) and CNF2 are toxins of pathogenicEscherichia colithat share 85% identity over 1,014 amino acids. Although both of these toxins modify GTPases, CNF1 is a more potent inducer of multinucleation in HEp-2 cells, binds more efficiently to HEp-2 cells, and, despite the conservation of amino acids (C866 and H881) required for enzymatic activity of the toxins, deamidates RhoA and Cdc42 better than CNF2. Here we exploited the differences between CNF1 and CNF2 to define the epitope on CNF1 to which the CNF1-specific neutralizing monoclonal antibody (MAb) (MAb NG8) binds and to determine the mechanism by which MAb NG8 neutralizes CNF1 activity on HEp-2 cells. For these purposes, we generated a panel of 21 site-directed mutants in which amino acids in CNF1 were exchanged for the amino acids in CNF2 between amino acids 546 and 869 and vice versa. This region of CNF1 not only is recognized by MAb NG8 but also is involved in binding of this toxin to HEp-2 cells. All the mutants retained the capacity to induce multinucleation of HEp-2 cells. However, the CNF1 double mutant with D591E and F593L mutations (CNF1D591E F593L) and the CNF1H661Qsingle mutant displayed drastically reduced reactivity with MAb NG8. A reverse chimeric triple mutant, CNF1E591D L593F Q661H, imparted MAb NG8 reactivity to CNF2. MAb NG8 neutralized CNF2E591D L593F Q661Hactivity in a dose-dependent manner and reduced the binding of this chimeric toxin to HEp-2 cells. Taken together, these results pinpoint three amino acids in CNF1 that are key amino acids for recognition by neutralizing MAb NG8 and further help define a region in CNF1 that is critical for full toxin binding to HEp-2 cells.

Use of a Plackett-Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells

2011 ◽  
Vol 27 (6) ◽  
pp. 1709-1717 ◽  
Author(s):  
I. J. González-Leal ◽  
L. M. Carrillo-Cocom ◽  
A. Ramírez-Medrano ◽  
F. López-Pacheco ◽  
D. Bulnes-Abundis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Antibody Production ◽  
Cho Cells ◽  
Statistical Design ◽  
Monoclonal Antibody Production
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