scholarly journals A Novel Monoclonal Antibody against FbaA Can Inhibit the Binding of the Complement Regulatory Protein Factor H to Group A Streptococcus

2011 ◽  
Vol 18 (4) ◽  
pp. 552-558 ◽  
Author(s):  
Cuiqing Ma ◽  
Yiyang Guo ◽  
Haiyan Gu ◽  
Ling Zhang ◽  
Hainan Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Competitive Inhibition ◽  
Group A Streptococcus ◽  
Regulatory Protein ◽  
Factor H ◽  
Microbial Pathogens ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Protein Factor ◽  
Group A

ABSTRACTSome microbial pathogens utilize human complement regulatory proteins, such as factor H (FH) and factor H-like protein 1 (FHL-1), for immune evasion. FbaA is an FHL-1 and FH binding protein expressed on the surface of group A streptococcus (GAS), a common agent of pharyngeal, skin, and soft tissue infections. In this study, we prepared monoclonal antibodies (MAbs) against FbaA, assayed them for specificity, and located their binding domains in FbaA. We found an MAb called FbaA MAb2, which demonstrated the highest affinity to GAS among all of the MAbs. Based on the binding with component peptides, the detected epitope, which was specific for FbaA MAb2, was the amino acid residues 95 to 118 of FbaA; on the other hand, it did not bind with the truncated protein of the internally deleted residues of the segment from 95 to 118 of FbaA. Furthermore, the predominant amino acids specific for FbaA MAb2 screened by phage display epitope library were I, T, P, D, and L, corresponding to the amino acid residues 101, 103, 105, 106, and 110 of FbaA, respectively. The binding location of FbaA with FH and FHL-1 was a 16-amino-acid region corresponding to amino acid residues 97 to 112 of FbaA, which overlapped the FbaA MAb2 binding domain, as confirmed by competitive inhibition enzyme-linked immunosorbent assay and immunofluorescence microscopy. Based on the results of the invasion assay, FbaA MAb2 can inhibit the binding of FH to GAS.

scholarly journals Bactericidal Antibody Responses Induced by Meningococcal Recombinant Chimeric Factor H-Binding Protein Vaccines

2008 ◽  
Vol 76 (6) ◽  
pp. 2568-2575 ◽  
Author(s):  
Peter T. Beernink ◽  
Dan M. Granoff
Keyword(s):  
Bactericidal Activity ◽  
Vaccine Candidate ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Factor H ◽  
Enzyme Linked Immunosorbent Assay ◽  
Meningococcal Vaccine ◽  
Serum Antibodies ◽  
Classical Complement Pathway ◽  
Protein Factor

ABSTRACT Factor H-binding protein (fHbp) is a novel meningococcal vaccine candidate that elicits serum antibodies that activate classical complement pathway bacteriolysis and also inhibit binding of the complement down-regulatory protein, factor H, to the bacterial surface. One limitation of fHbp as a vaccine candidate is antigenic variability, since antibodies to fHbp in the variant 1 (v.1) antigenic group do not protect against strains expressing v.2 or v.3 proteins, and vice versa. We have identified amino acid residues of epitopes recognized by bactericidal anti-fHbp monoclonal antibodies prepared against fHbp from each of the variant groups. One epitope expressed by nearly all v.1 proteins mapped to the B domain, while epitopes expressed by fHbp v.2 or v.3 mapped to the C domain. The results provided the rationale for engineering chimeric fHbp molecules containing the A domain (which is conserved across all variant groups), a portion of the B domain of a v.1 protein, and the carboxyl-terminal portion of the B domain and the C domain of a v.2 protein. By enzyme-linked immunosorbent assay, the resulting recombinant chimeric proteins expressed epitopes from all three variant groups. In mice, the chimeric vaccines elicited serum antibodies with bactericidal activity against a panel of genetically diverse strains expressing fHbp v.1, v.2, or v.3. The data demonstrate the feasibility of preparing a meningococcal vaccine from a single recombinant protein that elicits broad bactericidal activity, including group B strains, which account for 50 percent of cases of meningococcal disease and for which there currently is no broadly protective vaccine.

scholarly journals M Protein of the Group A StreptococcusBinds to the Seventh Short Consensus Repeat of Human Complement Factor H

1998 ◽  
Vol 66 (4) ◽  
pp. 1427-1431 ◽  
Author(s):  
Timothy K. Blackmore ◽  
Vincent A. Fischetti ◽  
Tania A. Sadlon ◽  
Helena M. Ward ◽  
David L. Gordon
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Regulatory Protein ◽  
Factor H ◽  
M Protein ◽  
Protein Binding Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complement Factor ◽  
Heparin Binding ◽  
Protein Factor

ABSTRACT Streptococcus pyogenes evades complement by binding the complement-regulatory protein factor H (fH) via the central conserved C-repeat region of M protein. However, the corresponding binding region within fH has not previously been precisely localized. fH is composed of 20 conserved modules called short consensus repeats (SCRs), each of which contains approximately 60 amino acids. A series of fH truncated and deletion mutants were prepared, and their interaction with M6 protein was examined. The M protein binding site was initially localized to SCRs 6 to 15 as demonstrated by ligand dot blotting, chemical cross-linking, and enzyme-linked immunosorbent assay. SCR 7 was then shown to contain the M protein binding site, as a construct consisting of the first seven SCRs bound M protein but a construct containing the first six SCRs did not bind. In addition, deletion of SCR 7 from full-length fH abolished binding to M protein. SCR 7 is known to contain a heparin binding domain, and binding of fH to M6 protein was almost totally inhibited in the presence of 400 U of heparin per ml. These results localize the M6 protein binding site of fH to SCR 7 and indicate that it is in close proximity to the heparin binding site.

scholarly journals Immunochemical localization and amino acid sequences of crossreactive epitopes within the group A streptococcal M6 protein.

1986 ◽  
Vol 164 (4) ◽  
pp. 1226-1238 ◽  
Author(s):  
K F Jones ◽  
S A Khan ◽  
B W Erickson ◽  
S K Hollingshead ◽  
J R Scott ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequences ◽  
Vaccine Development ◽  
Competitive Inhibition ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Dot Blot ◽  
Amino Terminal ◽  
Group A

mAbs 10A11, 10B6, and 10F5, raised against the native group A streptococcal M6 protein, were examined for their crossreactivity with non-laboratory passaged clinical isolates, representing 58 M serotypes, by bacterial dot blot immunoassay. mAb 10A11 crossreacted with 9, mAb 10B6 with 30, and mAb 10F5 with 30 different non-M6 serotypes. To identify the epitopes for these antibodies, the native M6 protein was cleaved with pepsin or staphylococcal V8 protease. Resultant peptides were purified by HPLC, examined for binding to crossreactive mAbs in ELISA, and reactive peptides were subjected to amino acid sequence analysis. Peptides were aligned with the amino acid sequence of the entire M6 protein predicted by the DNA sequence of the M6 gene. Competitive inhibition studies using peptides synthesized on the basis of peptide and DNA sequences, in concert with selective blocking of amino acid residues, allowed for the further identification and placement of these crossreactive epitopes within the M6 molecule. The 10A11 epitope was located within the six amino acid residues at position 134-139, which repeat at positions 159-164 and 184-189 within the variable amino terminal half of the native molecule. The conserved 10B6 and 10F5 epitopes were positioned within a 15-amino-acid span at position 275-289, with the possibility that either epitope could have been repeated at residues 239-247. Chemical modification of amino acids within this sequence aided in the differentiation of these two epitopes. Such studies should aid in the recognition of a sequence(s) common to a greater number of M serotypes, which may be useful for future vaccine development or group A streptococcal identification.

scholarly journals Complement Regulatory Protein Factor H Is a Soluble Prion Receptor That Potentiates Peripheral Prion Pathogenesis

2017 ◽  
Vol 199 (11) ◽  
pp. 3821-3827 ◽  
Author(s):  
Sarah J. Kane ◽  
Taylor K. Farley ◽  
Elizabeth O. Gordon ◽  
Joshua Estep ◽  
Heather R. Bender ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Factor H ◽  
Complement Regulatory Protein ◽  
Protein Factor

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.

Arg127 of complement regulatory protein Factor H is important for its secretion from human fibroblasts

2009 ◽  
Vol 46 (14) ◽  
pp. 2869
Author(s):  
José Antonio Tavares Albuquerque ◽  
Dayseanne Araújo Falcão ◽  
Lourdes Isaac
Keyword(s):  
Human Fibroblasts ◽  
Regulatory Protein ◽  
Factor H ◽  
Complement Regulatory Protein ◽  
Protein Factor

Functional properties of the allotypes of mouse complement regulatory protein, factor H: Difference of compatibility of each allotype with human factor I

1993 ◽  
Vol 30 (9) ◽  
pp. 841-848 ◽  
Author(s):  
Okada Michiyo ◽  
Kojima Ayako ◽  
Takano Hiromi ◽  
Harada Yoshinobu ◽  
Nonaka Mayumi ◽  
...  
Keyword(s):  
Functional Properties ◽  
Human Factor ◽  
Regulatory Protein ◽  
Factor H ◽  
Complement Regulatory Protein ◽  
Factor I ◽  
Protein Factor

scholarly journals Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 273
Author(s):  
Caixia Zhang ◽  
Weiqi Zhang ◽  
Xiaoqian Tang ◽  
Qi Zhang ◽  
Wen Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ochratoxin A ◽  
Limit Of Detection ◽  
Basic Knowledge ◽  
Affinity Constant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Assay Sensitivity ◽  
Coating Antigen ◽  
Relative Standard

Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol−1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

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