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1.
Methods Mol Biol ; 2261: 307-321, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33420998

RESUMEN

Immuno-PCR (IPCR) is a sensitive antigen detection by means of specific antibody-DNA conjugates. To ensure the successful conjugation of a protein (an antibody) with a reporter DNA, immuno-PCR method based on cDNA display (cD-IPCR) has been introduced. The cDNA display molecule is a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level, which is directly used for antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.


Asunto(s)
Aptámeros de Péptidos/inmunología , ADN Complementario/inmunología , Inmunoensayo , Inmunoconjugados/inmunología , Reacción en Cadena de la Polimerasa , Anticuerpos de Dominio Único/inmunología , Proteína Estafilocócica A/análisis , Aptámeros de Péptidos/genética , ADN Complementario/genética , Inmunoconjugados/genética , Anticuerpos de Dominio Único/genética
2.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 1056-1062, May-June, 2020. tab, graf
Artículo en Inglés | LILACS, VETINDEX | ID: biblio-1129773

RESUMEN

Utilizando um anticorpo monoclonal contra a aflatoxina B1 (AFB1) como ligante, foi identificado um mimotopo específico de aflatoxina B1 após se realizarem quatro ciclos de seleção biológica de 7-peptídeos aleatórios em biblioteca de fago exibida. O mimotopo é denominado P10, e sua sequência de aminoácidos é YRRHEKD. O soro imunológico de ratos Balb/c imunizados com P10 foi especificamente ligado à aflatoxina B1-albumina, indicando que o anticorpo era específico ao AFB1. Esses resultados sugerem que é possível desenvolver a vacina baseada em mimotopo associado à toxina.(AU)


Asunto(s)
Animales , Ratas , Vacunas Fúngicas/análisis , Aflatoxina B1 , Aptámeros de Péptidos/inmunología , Inmunogenicidad Vacunal , Ratones Endogámicos BALB C/inmunología
3.
J Agric Food Chem ; 67(17): 4958-4966, 2019 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-30966750

RESUMEN

The mud crab ( Scylla paramamosain) is widely consumed but can cause a severe food allergic reaction. To reduce allergenicity to arginine kinase (AK), site-directed mutagenesis was used to destroy disulfide bonds or mutate critical amino acids of conformational epitopes. Three hypoallergenic mutant AKs (mAK1, mAK2, and mAK3) were generated, with the immunoreactivity decreasing by 54.2, 40.1, and 71.4%, respectively. In comparison to recombinant AK (rAK), the structure of mAKs was clearly changed. Additionally, antisense peptides were designed on the basis of linear epitopes and pepsin-cutting sites of AK. Five peptide aptamers were screened by molecular docking and then analyzed by the immunoglobulin E inhibition enzyme-linked immunosorbent assay and human Laboratory of Allergic Diseases 2 mast cell degranulation assay. The peptide aptamers could significantly inhibit allergenicity of rAK and mAKs, and the inhibitory effect of peptide aptamer 3 was slightly better than the others. These results provide synergistic methods to reduce allergenicity to AK, which could be applied to other shellfish allergens.


Asunto(s)
Aptámeros de Péptidos/genética , Arginina Quinasa/genética , Arginina Quinasa/inmunología , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Braquiuros/inmunología , Hipersensibilidad a los Mariscos/inmunología , Adolescente , Adulto , Alérgenos/química , Alérgenos/genética , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Aptámeros de Péptidos/inmunología , Arginina Quinasa/química , Proteínas de Artrópodos/química , Braquiuros/enzimología , Braquiuros/genética , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Femenino , Humanos , Inmunoglobulina E/inmunología , Masculino , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Adulto Joven
4.
Circulation ; 135(17): 1651-1664, 2017 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-28438806

RESUMEN

Plasma biomarkers that reflect molecular states of the cardiovascular system are central for clinical decision making. Routinely used plasma biomarkers include troponins, natriuretic peptides, and lipoprotein particles, yet interrogate only a modest subset of pathways relevant to cardiovascular disease. Systematic profiling of a larger portion of circulating plasma proteins (the plasma proteome) will provide opportunities for unbiased discovery of novel markers to improve diagnostic or predictive accuracy. In addition, proteomic profiling may inform pathophysiological understanding and point to novel therapeutic targets. Obstacles for comprehensive proteomic profiling include the immense size and structural heterogeneity of the proteome, and the broad range of abundance levels, as well. Proteome-wide, untargeted profiling can be performed in tissues and cells with tandem mass spectrometry. However, applications to plasma are limited by the need for complex preanalytical sample preparation stages limiting sample throughput. Multiplexing of targeted methods based on capture and detection of specific proteins are therefore receiving increasing attention in plasma proteomics. Immunoaffinity assays are the workhorse for measuring individual proteins but have been limited for proteomic applications by long development times, cross-reactivity preventing multiplexing, specificity issues, and incomplete sensitivity to detect proteins in the lower range of the abundance spectrum (below picograms per milliliter). Emerging technologies to address these issues include nucleotide-labeled immunoassays and aptamer reagents that can be automated for efficient multiplexing of thousands of proteins at high sample throughput, coupling of affinity capture methods to mass spectrometry for improved specificity, and ultrasensitive detection systems to measure low-abundance proteins. In addition, proteomics can now be integrated with modern genomics tools to comprehensively relate proteomic profiles to genetic variants, which may both influence binding of affinity reagents and serve to validate the target specificity of affinity assays. The application of deep quantitative proteomic profiling to large cohorts has thus become increasingly feasible with emerging affinity methods. The aims of this article are to provide the broad readership of Circulation with a timely overview of emerging methods for affinity proteomics and recent progress in cardiovascular medicine based on such methods.


Asunto(s)
Proteínas Sanguíneas/análisis , Enfermedades Cardiovasculares/sangre , Ensayos Analíticos de Alto Rendimiento , Inmunoensayo , Proteoma , Proteómica/métodos , Animales , Anticuerpos/inmunología , Afinidad de Anticuerpos , Aptámeros de Péptidos/inmunología , Biomarcadores/sangre , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/inmunología , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/inmunología , Marcadores Genéticos , Humanos , Espectrometría de Masas , Valor Predictivo de las Pruebas , Pronóstico , Reproducibilidad de los Resultados
5.
Cancer Discov ; 7(7): 694-703, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28288993

RESUMEN

Disruption of the programmed cell death protein 1 (PD-1) pathway with immune checkpoint inhibitors represents a major breakthrough in the treatment of non-small cell lung cancer. We hypothesized that combined inhibition of C5a/C5aR1 and PD-1 signaling may have a synergistic antitumor effect. The RMP1-14 antibody was used to block PD-1, and an L-aptamer was used to inhibit signaling of complement C5a with its receptors. Using syngeneic models of lung cancer, we demonstrate that the combination of C5a and PD-1 blockade markedly reduces tumor growth and metastasis and leads to prolonged survival. This effect is accompanied by a negative association between the frequency of CD8 T cells and myeloid-derived suppressor cells within tumors, which may result in a more complete reversal of CD8 T-cell exhaustion. Our study provides support for the clinical evaluation of anti-PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer.Significance: Using a variety of preclinical models of lung cancer, we demonstrate that the blockade of C5a results in a substantial improvement in the efficacy of anti-PD-1 antibodies against lung cancer growth and metastasis. This study provides the preclinical rationale for the combined blockade of PD-1/PD-L1 and C5a to restore antitumor immune responses, inhibit tumor cell growth, and improve outcomes of patients with lung cancer. Cancer Discov; 7(7); 694-703. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 653.


Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Complemento C5a/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Aptámeros de Péptidos/inmunología , Aptámeros de Péptidos/uso terapéutico , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Proliferación Celular/efectos de los fármacos , Complemento C5a/inmunología , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Metástasis de la Neoplasia , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal
6.
Chem Commun (Camb) ; 50(58): 7787-9, 2014 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-24901743

RESUMEN

Distance-dependent signal intensity in immunoassay by attenuated total reflection-surface enhanced infrared absorption spectroscopy is demonstrated by controlling the distance of target proteins away from the enhancement substrate. Based on this optical near-field effect, sensitive detection of protein molecules with a detection limit of 0.6 nM and investigation of the kinetics and thermodynamics of protein-aptamer/antibody interactions can be achieved.


Asunto(s)
Anticuerpos/inmunología , Antígenos/inmunología , Aptámeros de Péptidos/inmunología , Inmunoensayo/métodos , Proteínas/análisis , Espectrofotometría Infrarroja/métodos , Selectina L/análisis , Termodinámica
7.
BMB Rep ; 47(3): 130-4, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24499668

RESUMEN

The combination of a high-affinity antibody to a hapten, and hapten-conjugated compounds, can provide an alternative to the direct chemical cross-linking of the antibody and compounds. An optimal hapten for in vitro use is one that is absent in biological systems. For in vivo applications, additional characteristics such as pharmacological safety and physiological inertness would be beneficial. Additionally, methods for cross-linking the hapten to various chemical compounds should be available. Cotinine, a major metabolite of nicotine, is considered advantageous in these aspects. A high-affinity anti-cotinine recombinant antibody has recently become available, and can be converted into various formats, including a bispecific antibody. The bispecific anti-cotinine antibody was successfully applied to immunoblot, enzyme immunoassay, immunoaffinity purification, and pre-targeted in vivo radioimmunoimaging. The anti-cotinine IgG molecule could be complexed with aptamers to form a novel affinity unit, and extended the in vivo half-life of aptamers, opening up the possibility of applying the same strategy to therapeutic peptides and chemical compounds.


Asunto(s)
Anticuerpos/química , Anticuerpos/inmunología , Cotinina/química , Cotinina/inmunología , Haptenos/química , Inmunoconjugados/química , Inmunoconjugados/inmunología , Animales , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Aptámeros de Péptidos/química , Aptámeros de Péptidos/inmunología , Haptenos/inmunología , Humanos , Radioinmunodetección
8.
Talanta ; 85(3): 1560-5, 2011 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-21807222

RESUMEN

In this study, polyaniline-multiwalled carbon nanotube film (PANi-MWCNT) has been polymerized on interdigitated platinum electrode arrays (IDA), fabricated by MEMS technology for the detection of human papillomavirus (HPV) infection, using immobilized peptide aptamers as affinity capture reagent. Label-free, electrochemical detection of the specific immune reaction between antigen peptide aptamer HPV-16-L1 (with a molecular weight of 1825 Da), the most common genotype in cytological normal women worldwide, and its specific antibody of HPV-16 (which is much bigger with molecular weight of ca. 150 kDa) on multifunctional PANi-MWCNT based arrays was reported. The most significant advantage of this technique consists of reagentless and multiple detection of antigen-antibody complex formation on well conducting IDA interface of PANi-MWCNT, without intermediate steps or any labeling reagents, as normally required in the previous works.


Asunto(s)
Compuestos de Anilina/química , Técnicas Electroquímicas/métodos , Nanotubos de Carbono/química , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Aptámeros de Péptidos/análisis , Aptámeros de Péptidos/química , Aptámeros de Péptidos/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Técnicas Electroquímicas/instrumentación , Electrodos , Femenino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/inmunología , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Nanotubos de Carbono/ultraestructura , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/inmunología , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/virología
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