scholarly journals Fine Specificity Epitope Analysis by HX-MS Identifies Contact Points on Ricin Toxin Recognized by Protective Monoclonal Antibodies

2018 ◽  
Author(s):  
Greta Van Slyke ◽  
Siva Krishna Angalakurthi ◽  
Ronald T. Toth ◽  
David J Vance ◽  
Yinghui Rong ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hydrogen Exchange ◽  
The Other ◽  
Back Side ◽  
Exchange Mass Spectrometry ◽  
Contact Points ◽  
Protein Toxin ◽  
Control And Prevention

AbstractRicin is a fast-acting protein toxin classified by the Centers for Disease Control and Prevention as a biothreat agent. In this report we describe five new mouse monoclonal antibodies (mAbs) directed against an immunodominant region, so-called epitope cluster II, on the surface of ricin’s ribosome-inactivating enzymatic subunit, RTA. The five mAbs were tested alongside four previously described cluster II-specific mAbs for their capacity to passively protect mice against 10 × LD50 ricin challenge by injection. Only three of the mAbs (LE4, PH12 and TB12) afforded protection over the seven-day study period. Neither binding affinity nor in vitro toxin-neutralizing activity could fully account for LE4, PH12 and TB12’s potent in vivo activity relative to the other six mAbs. However, epitope mapping studies by hydrogen exchange-mass spectrometry (HX-MS) revealed that LE4, PH12 and TB12 shared common contact points (i.e., “strong” protection by HX-MS) on RTA that encompassed residues 154-164 and 62-69, which correspond to RTA α-helices D-E and β-strands d-e, respectively, located on the back side of RTA relative to the active site. The other six mAbs recognized overlapping epitopes on RTA but none shared the same HX-MS profile as LE4, PH12 and TB12. A high-density competition ELISA with a panel of ricin-specific single domain camelid antibodies (VHHs) indicated that even though LE4, PH12 and TB12 make contact with similar secondary motifs, they ultimately approach RTA different from angles. These results underscore how subtle differences in epitope specificity have significant impacts on the antibody functionality in vivo and have important implications in the design of immune-based countermeasures against ricin.

scholarly journals Possible Future Monoclonal Antibody (mAb)-Based Therapy against Arbovirus Infections

2013 ◽  
Vol 2013 ◽  
pp. 1-21 ◽  
Author(s):  
Giuseppe Sautto ◽  
Nicasio Mancini ◽  
Giacomo Gorini ◽  
Massimo Clementi ◽  
Roberto Burioni
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Side Effects ◽  
Antiviral Activity ◽  
The Other ◽  
Viral Escape ◽  
Passive Immunotherapy ◽  
Medical Need

More than 150 arboviruses belonging to different families are known to infect humans, causing endemic infections as well as epidemic outbreaks. Effective vaccines to limit the occurrence of some of these infections have been licensed, while for the others several new immunogens are under development mostly for their improvements concerning safety and effectiveness profiles. On the other hand, specific and effective antiviral drugs are not yet available, posing an urgent medical need in particular for emergency cases. Neutralizing monoclonal antibodies (mAbs) have been demonstrated to be effective in the treatment of several infectious diseases as well as in preliminaryin vitroandin vivomodels of arbovirus-related infections. Given their specific antiviral activity as well-tolerated molecules with limited side effects, mAbs could represent a new therapeutic approach for the development of an effective treatment, as well as useful tools in the study of the host-virus interplay and in the development of more effective immunogens. However, before their use as candidate therapeutics, possible hurdles (e.g., Ab-dependent enhancement of infection, occurrence of viral escape variants) must be carefully evaluated. In this review are described the main arboviruses infecting humans and candidate mAbs to be possibly used in a future passive immunotherapy.

Monoclonal Antibodies to Human Fibrin: Interaction with other Animal Fibrins

1996 ◽  
Vol 75 (04) ◽  
pp. 595-599 ◽  
Author(s):  
Tracey Edgell ◽  
F McEvoy ◽  
P Webbon ◽  
P J Gaffney
Keyword(s):  
Monoclonal Antibodies ◽  
Dissociation Constant ◽  
Dissociation Constants ◽  
The Other ◽  
Limited Data ◽  
Thrombolytic Agents ◽  
Thrombus Imaging ◽  
Fibrin Clots

SummaryFour monoclonal antibodies have previously been raised in our laboratory for possible use in thrombus imaging and the targeting of thrombolytic agents. These antibodies were raised to various human fibrin-related immunogens and each antibody had been selected for its specificity towards fibrin and not fibrinogen. To study further these antibodies in animal circulation models both in vivo and in vitro, their selectivity towards human fibrin as opposed to other animal fibrins was examined. In this study dissociation constants for each antibody with each of six species fibrins (human, baboon, pig, dog, sheep, and rabbit) were estimated using both fibrin clots and monolayers. Some limited data were also obtained with Sepharose-fibrin. Of the antibodies two (denoted 12B3B10 and 12B3A11) are seen to bind almost exclusively to human fibrin with dissociation constants of about 8 × 10−10 M using fibrin clots and monolayers. These same two mabs bound to baboon fibrin with a dissociation constant of 2 × 10−9 M, while neither displayed significant levels of binding to the fibrins from dog, pig, sheep and rabbit. The other two antibodies investigated (1H10 and 5F3) were found to bind well to fibrins of human, baboon, pig and dog, with dissociation constants in the range of 1.4-4.2 × 10−9 M. However neither 1H10 nor 5F3 displayed significant recognition of sheep and rabbit fibrins. Both 1H10 and 5F3 were also found by means of competitive ELISA’s to retain their selectivity to baboon, dog and pig fibrins in the presence of their respective fibrinogens.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

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.

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 In Vitro and In Vivo Antibacterial Activities of DW-224a, a New Fluoronaphthyridone

2006 ◽  
Vol 50 (6) ◽  
pp. 2261-2264 ◽  
Author(s):  
Hee-Soo Park ◽  
Hyun-Joo Kim ◽  
Min-Jung Seol ◽  
Dong-Rack Choi ◽  
Eung-Chil Choi ◽  
...  
Keyword(s):  
Antibacterial Activities ◽  
The Other ◽  
Vitro Activity ◽  
Gram Negative Bacteria ◽  
In Vitro Activity ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

ABSTRACT DW-224a showed the most potent in vitro activity among the quinolone compounds tested against clinical isolates of gram-positive bacteria. Against gram-negative bacteria, DW-224a was slightly less active than the other fluoroquinolones. The in vivo activities of DW-224a against gram-positive bacteria were more potent than those of other quinolones.

scholarly journals Simulated diabetic ketoacidosis therapy in vitro elicits brain cell swelling via sodium-hydrogen exchange and anion transport

2015 ◽  
Vol 309 (4) ◽  
pp. E370-E379 ◽  
Author(s):  
Keeley L. Rose ◽  
Andrew J. Watson ◽  
Thomas A. Drysdale ◽  
Gediminas Cepinskas ◽  
Melissa Chan ◽  
...  
Keyword(s):  
Diabetic Ketoacidosis ◽  
Hydrogen Exchange ◽  
Brain Slices ◽  
Anion Transport ◽  
Brain Cell ◽  
Cell Swelling ◽  
Sodium Hydrogen ◽  
Disulphonic Acid

A common complication of type 1 diabetes mellitus is diabetic ketoacidosis (DKA), a state of severe insulin deficiency. A potentially harmful consequence of DKA therapy in children is cerebral edema (DKA-CE); however, the mechanisms of therapy-induced DKA-CE are unknown. Our aims were to identify the DKA treatment factors and membrane mechanisms that might contribute specifically to brain cell swelling. To this end, DKA was induced in juvenile mice with the administration of the pancreatic toxins streptozocin and alloxan. Brain slices were prepared and exposed to DKA-like conditions in vitro. Cell volume changes were imaged in response to simulated DKA therapy. Our experiments showed that cell swelling was elicited with isolated DKA treatment components, including alkalinization, insulin/alkalinization, and rapid reductions in osmolality. Methyl-isobutyl-amiloride, a nonselective inhibitor of sodium-hydrogen exchangers (NHEs), reduced cell swelling in brain slices elicited with simulated DKA therapy (in vitro) and decreased brain water content in juvenile DKA mice administered insulin and rehydration therapy (in vivo). Specific pharmacological inhibition of the NHE1 isoform with cariporide also inhibited cell swelling, but only in the presence of the anion transport (AT) inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid. DKA did not alter brain NHE1 isoform expression, suggesting that the cell swelling attributed to the NHE1 was activity dependent. In conclusion, our data raise the possibility that brain cell swelling can be elicited by DKA treatment factors and that it is mediated by NHEs and/or coactivation of NHE1 and AT.

scholarly journals Monoclonal antibodies specific to a Ca2+-bound form of lipocortin I distinguish its Ca2+-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2

1990 ◽  
Vol 269 (3) ◽  
pp. 709-715 ◽  
Author(s):  
H Hayashi ◽  
M K Owada ◽  
S Sonobe ◽  
K Domae ◽  
T Yamanouchi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Phospholipase A2 ◽  
Inhibitory Activity ◽  
Biological Effects ◽  
Free Form ◽  
E Coli ◽  
Phospholipid Binding ◽  
Ef Hand

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.

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