Exercise 29: Cloning and Sequencing cDNAs of Mouse Variable Regions of H and L Chains of Monoclonal Antibodies

2021 ◽  
pp. 447-461
Author(s):  
Tobili Y. Sam-Yellowe
Keyword(s):  
Monoclonal Antibodies ◽  
Cloning And Sequencing ◽  
Variable Regions

scholarly journals Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Patamalai Boonserm ◽  
Songchan Puthong ◽  
Thanaporn Wichai ◽  
Sajee Noitang ◽  
Pongsak Khunrae ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
3D Structure ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Variable Regions ◽  
Complementarity Determining Regions ◽  
Crucial Part ◽  
Major Amino Acid

AbstractIt is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22–100%) had a broader range of cross-reactivity than anti-nor 155 (62–100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

scholarly journals Development of Opsonic Mouse Monoclonal Antibodies against Multidrug-Resistant Enterococci

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Ermioni Kalfopoulou ◽  
Diana Laverde ◽  
Karmela Miklic ◽  
Felipe Romero-Saavedra ◽  
Suzana Malic ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Selection Process ◽  
Multidrug Resistant ◽  
Expression Vectors ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Strains ◽  
Variable Regions ◽  
Content Type

ABSTRACTMultidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide ofEnterococcus faecalis, and the secreted antigen A (SagA), an immunogenic protein fromEnterococcus faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing againstE. faecalistype 2, and SagA.01 showed 40% killing againstE. faecium11231/6. In addition, both MAbs showed cross-reactivity toward otherE. faecalisandE. faeciumstrains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.

Sequences of the variable regions of three monoclonal antibodies specific for histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system

1991 ◽  
Vol 69 (4) ◽  
pp. 297-302 ◽  
Author(s):  
Teresa Steeves ◽  
M. Michele Barry ◽  
Harry W. Duckworth ◽  
E. Bruce Waygood ◽  
Jeremy S. Lee
Keyword(s):  
Monoclonal Antibodies ◽  
Gene Sequencing ◽  
Gene Families ◽  
Gene Sequences ◽  
Phosphotransferase System ◽  
Variable Regions ◽  
Gene Usage ◽  
Vh Gene

The variable regions of three monoclonal antibodies, Jel 42, Jel 44, and Jel 324, specific for the histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system have been sequenced from their respective mRNAs. The Vh gene families were deduced from the percent homology to the concensus gene sequences and the J gene and D gene usage was also analysed.Key words: monoclonal antibodies, gene sequencing.

Engineering Fully Human Monoclonal Antibodies from Murine Variable Regions

2010 ◽  
Vol 396 (5) ◽  
pp. 1474-1490 ◽  
Author(s):  
Matthew J. Bernett ◽  
Sher Karki ◽  
Gregory L. Moore ◽  
Irene W.L. Leung ◽  
Hsing Chen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Monoclonal Antibodies ◽  
Variable Regions

Monoclonal Antibodies to Human T - Cell Receptor Variable ? Regions

1995 ◽  
Vol 756 (1 T-Cell Recept) ◽  
pp. 106-109 ◽  
Author(s):  
A. NECKER ◽  
H. MICHALAKI ◽  
M. C. RAYNAL ◽  
C. ROCHER ◽  
A. DIU ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Variable Regions ◽  
Human T Cell

scholarly journals Properties and Therapeutic Efficacy of Broadly Reactive Chimeric and Humanized H5-Specific Monoclonal Antibodies against H5N1 Influenza Viruses

2011 ◽  
Vol 55 (4) ◽  
pp. 1349-1357 ◽  
Author(s):  
Qingbing Zheng ◽  
Lin Xia ◽  
Wai Lan Wu ◽  
Zhenhua Zheng ◽  
Yongting Huo ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Virus Infection ◽  
H5n1 Virus ◽  
Influenza Viruses ◽  
Chimeric Antibody ◽  
Dependent Manner ◽  
Variable Regions ◽  
H5n1 Influenza ◽  
Therapeutic Regime ◽  
Humanized Antibodies

ABSTRACTHighly pathogenic H5N1 virus infection causes severe disease and a high rate of fatality in humans. Development of humanized monoclonal antibodies may provide an efficient therapeutic regime for H5N1 virus infection. In the present study, broadly cross-reactive monoclonal antibodies (MAbs) derived from mice were humanized to minimize immunogenicity. One chimeric antibody (cAb) and seven humanized antibodies (hAbs) were constructed. These antibodies retained broad-spectrum reactivity to H5N1 viruses, binding to recombinant H5-subtype HA1 molecules expressed in CHO cells in a dose-dependent manner and exhibiting similar reactivities against antigenically distinct H5N1 viruses in hemagglutination inhibition (HI) assays. One humanized antibody, 37 hAb, showed HI and neutralization activities comparable to that of the parental murine antibody, 13D4 MAb, while the other six antibodies were less reactive to H5N1 viruses. Analysis of amino acid sequences in the variable region frameworks of the seven humanized antibodies found that Q5 and Y27 in the VH region are highly conserved murine residues. Comparison of the three-dimensional structures derived from the variable regions of MAbs 37 hAb, H1202-34, and 13D4 revealed that residue substitutions at sites 70 and 46 may be the major cause for the observed differences in binding affinity. Examination of the chimeric antibody and one of the humanized antibodies, 37 hAb, showed that both antibodies offered postinfection protection against lethal challenge with antigenically diverse H5N1 viruses in the mouse model. Chimeric and humanized antibodies which retain the broadly reactive and protective properties of murine H5-specific monoclonal antibodies have great potential for use in the treatment of human H5N1 infection.

scholarly journals Sequence Variation in the porA Gene of a Clone of Neisseria meningitidis during Epidemic Spread

2000 ◽  
Vol 7 (3) ◽  
pp. 390-395 ◽  
Author(s):  
J. Jelfs ◽  
R. Munro ◽  
E. Wedege ◽  
D. A. Caugant
Keyword(s):  
Monoclonal Antibodies ◽  
Neisseria Meningitidis ◽  
Vaccine Development ◽  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Epidemic Spread ◽  
Insertion Element ◽  
Variable Regions ◽  
Geographic Origins

ABSTRACT The ET-15 clone within the electrophoretic type (ET)-37 complex ofNeisseria meningitidis was first detected in Canada in 1986 and has since been associated with outbreaks of meningococcal disease in many parts of the world. While the majority of the strains of the ET-37 complex are serosubtype P1.5,2, serosubtype determination of ET-15 strains may often be incomplete, with either only one or none of the two variable regions (VRs) of the serosubtype PorA outer membrane protein reacting with monoclonal antibodies. DNA sequence analysis of the porA gene from ET-15 strains with one or both unidentified serosubtype determinants was undertaken to identify the genetic basis of the lack of reaction with the monoclonal antibodies. Fourteen different porA alleles were identified among 38 ET-15 strains from various geographic origins. The sequences corresponding to subtypes P1.5a,10d, P1.5,2, P1.5,10d, P1.5a,10k, and P1.5a,10a were identified in 18, 11, 2, 2, and 1 isolate, respectively. Of the remaining four strains, which all were nonserosubtypeable, two had a stop codon within the VR1 and the VR2, respectively, while in the other two the porA gene was interrupted by the insertion element, IS1301. Of the strains with P1.5,2 sequence, one had a stop codon between the VR1 and VR2, one had a four-amino-acid deletion outside the VR2, and another showed no expression of PorA on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results reveal that numerous genetic events have occurred in theporA gene of the ET-15 clone in the short time of its epidemic spread. The magnitude of microevolutionary mechanisms available in meningococci and the remarkable genetic flexibility of these bacteria need to be considered in relation to PorA vaccine development.

scholarly journals Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

2015 ◽  
Vol 89 (7) ◽  
pp. 3610-3618 ◽  
Author(s):  
Wenqian He ◽  
Caitlin E. Mullarkey ◽  
J. Andrew Duty ◽  
Thomas M. Moran ◽  
Peter Palese ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Monoclonal Antibodies ◽  
Virus Vaccine ◽  
Neutralizing Antibodies ◽  
Influenza A ◽  
Human Peripheral Blood ◽  
Influenza Virus Vaccine ◽  
Influenza Viruses ◽  
Broadly Neutralizing Antibodies ◽  
Variable Regions

ABSTRACTCurrent influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferiorin vitrobut was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design.IMPORTANCEInfluenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly neutralizing antibodies has been recently discovered and may be the key to developing a “universal” influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly neutralizing antibodies in polyclonal contexts will be essential for guiding design of “universal” influenza virus vaccines and therapeutics.

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