RESUMEN
The leaves of the Mitragynine speciosia tree (also known as Kratom) have long been chewed, smoked, or brewed into a tea by people in Southeastern Asian countries, such as Malaysia and Thailand. Just this past year, the plant Kratom gained popularity in the United States as a "legal opioid" and scheduling it as a drug of abuse is currently pending. The primary alkaloid found in Kratom is a µ-opioid receptor agonist, mitragynine, whose structure contains a promising scaffold for immunopharmacological use. Although Kratom is regarded as a safe opioid alternative, here we report the LD50 values determined for its two main psychoactive alkaloids, mitragynine and 7-hydroxymitragynine, as comparable to heroin in mice when administered intravenously. Given Kratom's recent emergence in the U.S., there is currently no diagnostic test available for law enforcement or health professionals, so we sought to design such an assay. Mitragynine was used as a starting point for hapten design, resulting in a hapten with an ether linker extending from the C9 position of the alkaloid. Bacterial flagellin (FliC) was chosen as a carrier protein for active immunization in mice, yielding 32 potential monoclonal antibodies (mAbs) for assay development. Antimitragynine mAbs in the range of micro- to nanomolar affinities were uncovered and their utility in producing a convenient lateral flow detection assay of human fluid samples was examined. Antibodies were screened for binding to mitragynine, 7-hydroxymitragynine, and performance in lateral flow assays. Two monoclonal antibodies were subcloned and further purified with 93 and 362 nM affinity to mitragynine. Test strip assays were optimized with a detection cut off of 0.5 µg/mL for mitragynine in buffer and urine (reflecting projected clinically relevant levels of drug in urine), which could be beneficial to law enforcement agencies and health professionals as the opioid epidemic in America continues to evolve.
Asunto(s)
Mitragyna/química , Extractos Vegetales/análisis , Hojas de la Planta/química , Alcaloides de Triptamina Secologanina/análisis , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/biosíntesis , Inyecciones Intravenosas , Ratones , Conformación Molecular , Extractos Vegetales/administración & dosificación , Extractos Vegetales/toxicidad , Alcaloides de Triptamina Secologanina/administración & dosificación , Alcaloides de Triptamina Secologanina/toxicidad , Resonancia por Plasmón de SuperficieRESUMEN
The exchange of information within and among bacterial populations using small diffusible molecules has been termed "quorum sensing" (QS). Due to the extracellular distribution of the QS autoinducer molecules and the evolutionary highly conserved nature of signaling components, microbial QS systems represent an excellent target for anti-infective immunotherapy. Recently, we have described the generation of quorum quenching monoclonal antibodies (mAbs) against acyl homoserine lactones (AHL) used by Pseudomonas aeruginosa as well as Staphylococcal autoinducing peptides (AIP). These mAbs suppressed QS signaling in bacteria and neutralized AHL-mediated cytotoxic effects in vitro, as well as protected animals in Staphylococcus aureus infection models.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Percepción de Quorum , Acil-Butirolactonas/inmunología , Animales , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/biosíntesis , Western Blotting , Bovinos , Línea Celular Tumoral , Células Clonales , Ensayo de Inmunoadsorción Enzimática , Proteínas Hemolisinas/biosíntesis , Hibridomas/inmunología , Inmunización , Inmunoconjugados/química , Inmunoconjugados/inmunología , Ratones , Péptidos Cíclicos , Pseudomonas aeruginosa/citología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Piocianina/biosíntesis , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/citología , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismoRESUMEN
The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr(86)) of murine tumor necrosis factor-alpha (mTNF-alpha) to p-nitrophenylalanine (pNO(2)Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr(86) --> Phe mutant. The antibodies generated against the pNO(2)Phe are highly cross-reactive with native mTNF-alpha and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.
Asunto(s)
Sustitución de Aminoácidos , Formación de Anticuerpos/efectos de los fármacos , Mutación Missense , Autotolerancia/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Enfermedades Transmisibles/genética , Enfermedades Transmisibles/inmunología , Endotoxemia/inducido químicamente , Endotoxemia/tratamiento farmacológico , Endotoxemia/genética , Endotoxemia/inmunología , Epítopos/genética , Epítopos/inmunología , Epítopos/farmacología , Inmunoquímica , Lipopolisacáridos/toxicidad , Masculino , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Nitrofenoles/inmunología , Nitrofenoles/farmacología , Autotolerancia/genética , Autotolerancia/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Vacunas/genética , Vacunas/inmunologíaRESUMEN
Endotoxin or lipopolysaccharide (LPS) contamination in proteins expressed by Gram-negative bacteria is a major drawback associated with protein expression. Endotoxin intoxication in humans and animals above a certain threshold level can result in a fatal immune response. Reduction in endotoxin levels is therefore essential before proteins can be used in in vivo studies or sold as pharmaceutical products. Affinity chromatography employing the peptide Polymyxin B (PMB) as an affinity ligand is one way in which endotoxin contamination has been addressed; this is, however, a costly process. We describe the synthesis of a novel affinity ligand based on the structure of the drug pentamidine, which can be applied effectively in endotoxin removal. The synthetic route to this ligand is straightforward and inexpensive, while the ligand can be readily immobilized onto activated sepharose beads. Thus, we demonstrate that these pentamidine affinity beads bind endotoxin/LPS with comparable capacity to PMB affinity systems, that the beads can be recycled efficiently and economically without loss of binding capacity, and application of the functionalized beads for endotoxin removal in an authentic contaminated antibody sample.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía de Afinidad , Endotoxinas/metabolismo , Pentamidina/farmacología , Polimixina B/farmacología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Endotoxinas/antagonistas & inhibidores , Endotoxinas/aislamiento & purificación , Humanos , Ligandos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/metabolismo , Pentamidina/química , Polimixina B/química , Unión Proteica , Sefarosa/química , Sefarosa/metabolismoRESUMEN
Antibodies that selectively bind to N-formylmethionyl leucyl phenylalanine (fMLF, also known as fMLP) have been generated. These antibodies bound to fMLF with higher affinity than to non-formylated peptide MLF: the differences in the binding energies between fMLF and MLF were 1.4->2.1 kcal/mol.
Asunto(s)
Anticuerpos/química , Química Farmacéutica/métodos , N-Formilmetionina Leucil-Fenilalanina/química , Péptidos/química , Animales , Bovinos , Diseño de Fármacos , Humanos , Inmunoglobulina G/química , Cinética , Modelos Químicos , Conformación Molecular , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Unión Proteica , Receptores de Formil Péptido/química , Albúmina Sérica Bovina/químicaRESUMEN
A squaric monoester monoamide motif was employed as an effective reactive immunogen for the discovery of monoclonal antibodies with reactive residue(s) in their combining sites. Two antibodies, 2D4 and 3C8, were uncovered that enhance paraoxon hydrolysis over background. Kinetic analysis of these antibodies was performed and interestingly both undergo a single turnover event due to covalent modification within the antibody combining site. Because antibodies 2D4 and 3C8 result in covalent attachment and thus inactivation of paraoxon, they could be useful probes for investigating paraoxon intoxication.
Asunto(s)
Anticuerpos Catalíticos/farmacología , Ciclobutanos/inmunología , Haptenos/química , Paraoxon/inmunología , Vacunación , Amidas , Animales , Anticuerpos Catalíticos/biosíntesis , Sitios de Unión de Anticuerpos , Ciclobutanos/administración & dosificación , Ciclobutanos/síntesis química , Ésteres , Haptenos/administración & dosificación , Haptenos/inmunología , Hidrólisis , Cinética , Ratones , Ratones Endogámicos BALB C , Paraoxon/antagonistas & inhibidores , Paraoxon/química , Plaguicidas/antagonistas & inhibidores , Plaguicidas/química , Plaguicidas/inmunología , Relación Estructura-ActividadRESUMEN
We have generated antibody FTB8E9 by immunization with the designed hapten to catalyze the aminolysis reaction of a chloropyrimidine derivative.
Asunto(s)
Anticuerpos/metabolismo , Pirimidinas/metabolismo , Anticuerpos/química , Catálisis , Pirimidinas/químicaRESUMEN
Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. Previous studies have demonstrated that targeting foreign antigens to DC leads to enhanced antigen (Ag)-specific responses in vivo. However, the utility of this strategy for the generation of MAbs has not been investigated. To address this question we immunized mice with IgG-peptide conjugates prepared with the hamster anti-murine CD11c MAb N418. Synthetic peptides corresponding to two different exposed regions of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a human C-type lectin, were conjugated to N418 using thiol-based chemistry. The N418 MAb served as the targeting molecule and synthetic peptides as the Ag (MAb-Ag). A rapid and peptide specific serum IgG response was produced by Day 7 when the synthetic peptides were linked to the N418 MAb, compared to peptide co-delivered with the N418 without linkage. Spleen cells from N418-peptide immunized mice were fused on Day 10, and three IgG1/k monoclonal antibodies (MAbs) were selected to one of the peptide epitopes (MID-peptide). One of the MAbs, Novik 2, bound to two forms of recombinant DC-SIGN protein in enzyme-linked immunosorbent assay (ELISA), and was specifically inhibited by the MID-peptide in solution. Two of these MAbs show specific binding to DC-SIGN expressed by cultured human primary DC. We conclude that in vivo DC targeting enhances the immunogenicity of synthetic peptides and is an effective method for the rapid generation of MAbs to predetermined epitopes.