scholarly journals The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Kristopher R. Genschmer ◽  
Cintia F. M. Vadesilho ◽  
Larry S. McDaniel ◽  
Sang-Sang Park ◽  
Yvette Hale ◽  
...  
Keyword(s):  
Amino Acid ◽  
The Other ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Assay ◽  
Passive Protection ◽  
Protective Antibody ◽  
Killing Assay ◽  
Rich Domain ◽  
Modified Surface

ABSTRACT Pneumococcal surface protein A (PspA) elicits antibody protective against lethal challenge by Streptococcus pneumoniae and is a candidate noncapsular antigen for inclusion in vaccines. Evaluation of immunity to PspA in human trials would be greatly facilitated by an in vitro functional assay able to distinguish protective from nonprotective antibodies to PspA. Mouse monoclonal antibodies (MAbs) to PspA can mediate killing by human granulocytes in the modified surface killing assay (MSKA). To determine if the MSKA can distinguish between protective and nonprotective MAbs, we examined seven MAbs to PspA. All bound recombinant PspA, as detected by enzyme-linked immunosorbent assay and Western blotting; four gave strong passive protection against fatal challenge, two were nonprotective, and the seventh one only delayed death. The four that were able to provide strong passive protection were also most able to enhance killing in the MSKA, the two that were not protective in mice were not effective in the MSKA, and the MAb that was only weakly protective in mice was weakly effective in the MSKA (P < 0.001). One of the four most protective MAbs tested reacted to the proline-rich domain of PspA. Two of the other most protective MAbs and the weakly protective MAb reacted with a fragment from PspA’s α-helical domain (αHD), containing amino acids (aa) 148 to 247 from the N terminus of PspA. The fourth highly protective MAb recognized none of the overlapping 81- or 100-aa fragments of PspA. The two nonprotective MAbs recognized a more N-terminal αHD fragment (aa 48 to 147). IMPORTANCE The most important finding of this study is that the MSKA can be used as an in vitro functional assay. Such an assay will be critical for the development of PspA-containing vaccines. The other important findings relate to the locations and nature of the protection-eliciting epitopes of PspA. There are limited prior data on the locations of protection-eliciting PspA epitopes, but those data along with the data presented here make it clear that there is not a single epitope or domain of PspA that can elicit protective antibody and there exists at least one region of the αHD which seldom elicits protective antibody. Moreover, these data, in concert with prior data, strongly make the case that protective epitopes in the αHD are highly conformational (≥100-amino-acid fragments of the αHD are required), whereas at least some protection-eliciting epitopes in the proline-rich domain are encoded by ≤15-amino-acid sequences.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Serologic Results in >1000 Patients With Suspected Heparin-Induced Thrombocytopenia

2007 ◽  
Vol 14 (4) ◽  
pp. 410-414 ◽  
Author(s):  
Suresh G. Shelat ◽  
Anne Tomaski ◽  
Eleanor S. Pollak
Keyword(s):  
Laboratory Diagnosis ◽  
Serotonin Release ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Life Threatening ◽  
Release Assay ◽  
Pathogenic Antibodies

Heparin-induced thrombocytopenia (HIT) can lead to life-threatening and limb-threatening thrombosis. HIT is thought to be initiated by the interaction of pathogenic antibodies toward a complex platelet factor 4 (PF4) and heparin (PF4:H), which can activate platelets and predispose to thrombosis. As such, the laboratory diagnosis of HIT includes antigenic and functional assays to detect antibodies directed at PF4:H complexes. We performed a retrospective analysis of 1017 consecutive samples tested by serotonin-release assay and by enzyme-linked immunosorbent assay (ELISA). Most samples showed no serologic evidence of HIT, whereas 4% to 5% of samples demonstrated both antigenic and functional serological evidence for HIT. Approximately 12% to 18% of samples showed immunologic evidence of anti-PF4:H antibodies but without functional evidence of serotonin release in vitro. Interestingly, a small minority of samples (0.7%) caused serotonin release but were negative in the ELISA. The results are presented using cutoff values established at our hospital and for the ELISA manufacturer. This study provides a pretest probability of the serologic results from an antigenic assay (ELISA) and a functional assay (serotonin-release assay) in patients clinically suspected of having HIT.

scholarly journals Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody

1999 ◽  
Vol 65 (8) ◽  
pp. 3279-3286 ◽  
Author(s):  
Qiaoping Yuan ◽  
James J. Pestka ◽  
Brandon M. Hespenheide ◽  
Leslie A. Kuhn ◽  
John E. Linz ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Synthetic Peptide ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Translation System

ABSTRACT Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.

The mode of action of homocysteine on mouse brain glutamic decarboxylase and γ-aminobutyrate aminotransferase

1977 ◽  
Vol 55 (9) ◽  
pp. 1013-1018 ◽  
Author(s):  
G. Tunnicliff ◽  
T. T. Ngo
Keyword(s):  
Amino Acid ◽  
Mouse Brain ◽  
Mode Of Action ◽  
Acid Metabolism ◽  
Competitive Inhibition ◽  
The Other ◽  
Aminobutyric Acid ◽  
Aminobutyrate Aminotransferase ◽  
Dual Action

In the belief that homocysteine-induced convulsions might be related to alterations in brain γ-aminobutyric acid metabolism, we have studied the action of this amino acid on the activity of glutamic decarboxylase (GAD, EC 4.1.1.15) and γ-aminobutyrate aminotransferase (EC 2.6.1.19)of mouse brain in vitro. DL-homocysteine competitively inhibited GAD with respect to both L-glutamate and pyridoxal 5′-phosphate. The respective Ki's were 3.8 mM and 0.3 mM, The activity of GABA-T also was altered in the presence of DL-homocysteine. A competitive inhibition (Ki = 6 mM) was observed with γ-aminobutyric acid, and an uncompetitive inhibition with respect to pyridoxal 5′-phosphate and α-ketoglutarate. These results are explained in terms of a dual action of homocysteine on each of the enzymes: one involving a competition for substrate binding site and the other involving the formation of an inactive inhibitor – cofactor complex. The significance of the inhibition of these enzymes of γ-aminobutyric acid metabolism is discussed in relation to the convulsant action of homocysteine.

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

scholarly journals Correlation of In Vitro Susceptibilities to Newer Quinolones of Naturally Occurring Quinolone-Resistant Neisseria gonorrhoeae Strains with Changes in GyrA and ParC

2001 ◽  
Vol 45 (3) ◽  
pp. 734-738 ◽  
Author(s):  
Tiffany R. Shultz ◽  
John W. Tapsall ◽  
Peter A. White
Keyword(s):  
Amino Acid ◽  
Neisseria Gonorrhoeae ◽  
Southeast Asia ◽  
Point Mutations ◽  
The Other ◽  
Naturally Occurring ◽  
Ciprofloxacin Resistance ◽  
Amino Acid Mutations ◽  
Sydney Australia

ABSTRACT The in vitro activities of ciprofloxacin, trovafloxacin, moxifloxacin, and grepafloxacin against 174 strains of Neisseria gonorrhoeae isolated in Sydney, Australia, were determined. The strains included 84 quinolone-less-sensitive and -resistant N. gonorrhoeae (QRNG) strains for which ciprofloxacin MICs were in the range of 0.12 to 16 μg/ml. The QRNG included strains isolated from patients whose infections were acquired in a number of countries, mostly in Southeast Asia. The gyrA and parCquinolone resistance-determining regions (QRDR) of 18 selected QRNG strains were sequenced, and the amino acid mutations observed were related to the MICs obtained. The activities of moxifloxacin and grepafloxacin against QRNG were comparable to that of ciprofloxacin. Trovafloxacin was more active than the other quinolones against some but not all of the QRNG strains. Increments in ciprofloxacin resistance occurred in a step-wise manner with point mutations initiated ingyrA resulting in amino acid alterations Ser91-to-Phe, Ser91-to-Tyr, Asp95-to-Gly, and Asp95-to-Asn. Single gyrAchanges correlated with ciprofloxacin MICs in the range 0.12 to 1 μg/ml. The Ser91 changes in GyrA were associated with higher MICs and further QRDR changes. QRNG strains for which ciprofloxacin MICs were greater than 1 μg/ml had both gyrA and parCQRDR point mutations. ParC alterations were seen in these isolates only in the presence of GyrA changes and comprised amino acid changes Asp86-to-Asn, Ser87-to-Asn, Ser87-to-Arg, Ser88-to-Pro, Glu91-to-Lys, and Glu91-to-Gln. QRNG strains for which MICs were in the higher ranges had double GyrA mutations, but again only with accompanying ParC alterations. Not only did the nature and combination of GyrA and ParC changes influence the incremental increases in ciprofloxacin MICs, but they seemingly also altered the differential activity of trovafloxacin. Our findings suggest that the newer quinolones of the type examined are unlikely to be useful replacements for ciprofloxacin in the treatment of gonorrhea, particularly where ciprofloxacin MICs are high or where resistance is widespread.

scholarly journals Amino Acid Substitutions in GyrA of Burkholderia glumae Are Implicated in Not Only Oxolinic Acid Resistance but Also Fitness on Rice Plants

2006 ◽  
Vol 73 (4) ◽  
pp. 1114-1119 ◽  
Author(s):  
Yukiko Maeda ◽  
Akinori Kiba ◽  
Kouhei Ohnishi ◽  
Yasufumi Hikichi
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Oxolinic Acid ◽  
The Other ◽  
Amino Acid Substitutions ◽  
Burkholderia Glumae ◽  
Field Isolates ◽  
Rice Plants ◽  
Resistant Mutants

ABSTRACT Oxolinic acid (OA) resistance in field isolates of Burkholderia glumae, a causal agent of bacterial grain rot, is dependent on an amino acid substitution at position 83 in GyrA (GyrA83). In the present study, among spontaneous in vitro mutants from the OA-sensitive B. glumae strain Pg-10, we selected OA-resistant mutants that emerged at a rate of 5.7 � 10−10. Nucleotide sequence analysis of the quinolone resistance-determining region in GyrA showed that Gly81Cys, Gly81Asp, Asp82Gly, Ser83Arg, Asp87Gly, and Asp87Asn are observed in these OA-resistant mutants. The introduction of each amino acid substitution into Pg-10 resulted in OA resistance, similar to what was observed for mutants with the responsible amino acid substitution. In vitro growth of recombinants with Asp82Gly was delayed significantly compared to that of Pg-10; however, that of the other recombinants did not differ significantly. The inoculation of each recombinant into rice spikelets did not result in disease. In inoculated rice spikelets, recombinants with Ser83Arg grew less than Pg-10 during flowering, and growth of the other recombinants was reduced significantly. On the other hand, the reduced growth of recombinants with Ser83Arg in spikelets was compensated for under OA treatment, resulting in disease. These results suggest that amino acid substitutions in GyrA of B. glumae are implicated in not only OA resistance but also fitness on rice plants. Therefore, GyrA83 substitution is thought to be responsible for OA resistance in B. glumae field isolates.

scholarly journals O-Polysaccharide Epitopic Heterogeneity at the Surface of Brucella spp. Studied by Enzyme-Linked Immunosorbent Assay and Flow Cytometry

1998 ◽  
Vol 5 (6) ◽  
pp. 862-870 ◽  
Author(s):  
Axel Cloeckaert ◽  
Vincent Weynants ◽  
Jacques Godfroid ◽  
Jean-Michel Verger ◽  
Maggy Grayon ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
The Other ◽  
Antigenic Structure ◽  
Enzyme Linked Immunosorbent Assay ◽  
Major Goal ◽  
Passive Protection ◽  
Epitope Specificity ◽  
New Information ◽  
High Degree

ABSTRACT Smooth Brucella strains are classified into three serotypes, i.e., A+M−, A−M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface ofBrucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucellaspecies and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees byBrucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

scholarly journals Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders

Pharmaceutics ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 642 ◽  
Author(s):  
Hana Petroková ◽  
Josef Mašek ◽  
Milan Kuchař ◽  
Andrea Vítečková Wünschová ◽  
Jana Štikarová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Large Scale ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ribosome Display ◽  
Amino Acid Peptide ◽  
His Tag ◽  
Insoluble Form ◽  
Protein Binders ◽  
Protein Variants

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

scholarly journals Correlation betweenIn VitroComplement Deposition and Passive Mouse Protection of Anti-Pneumococcal Surface Protein A Monoclonal Antibodies

2014 ◽  
Vol 22 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Naeem Khan ◽  
Raies Ahmad Qadri ◽  
Devinder Sehgal
Keyword(s):  
Monoclonal Antibodies ◽  
Bactericidal Activity ◽  
Age Groups ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Vaccine ◽  
Hyperimmune Serum ◽  
Passive Protection ◽  
Clade 1

ABSTRACTThe shortcomings of the licensed polysaccharide-based pneumococcal vaccine are driving efforts toward development of a protein-based vaccine that is serotype independent and effective in all age groups. An opsonophagocytic killing assay (OPKA) is used to evaluate the antibody response against polysaccharide-based pneumococcal vaccines. However, the OPKA is not reliable for noncapsular antigens. Thus, there is a need to develop anin vitrosurrogate for protection for protein vaccine candidates like pneumococcal surface antigen A (PspA). PspA is a serologically variable cell surface virulence factor. Based on its sequence, PspA has been classified into families 1 (clade 1 and 2), 2 (clades 3, 4 and 5), and 3 (clade 6). Here, we report the characterization of 18 IgG anti-PspA monoclonal antibodies (anti-PspAhkR36AMAbs) generated from mice immunized with heat-killed strain R36A (clade 2). An enzyme-linked immunosorbent assay (ELISA)-based analysis of the reactivity of the MAbs with recombinant PspAs from the 6 clades indicated that they were family 1 specific. This was confirmed by flow cytometry using a hyperimmune serum generated against PspA from R36A. Eight MAbs that bind at least one clade 1- and clade 2-expressing strain were evaluated for complement deposition, bactericidal activity, and passive protection. The anti-PspAhkR36AMAb-dependent deposition of complement on pneumococci showed a positive correlation with passive protection against strain WU2 (r= 0.8783,P= 0.0041). All of our protective MAbs showed bactericidal activity; however, not all MAbs that exhibited bactericidal activity conferred protectionin vivo. The protective MAbs described here can be used to identify conserved protection eliciting B cell epitopes for engineering a superior PspA-based vaccine.

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