RESUMEN
Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.
Asunto(s)
Cisteína/metabolismo , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de Electrospray/métodos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Secuencia de Aminoácidos , Cisteína/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos de Péptidos/química , Unión Proteica , Dominios Proteicos , Subunidades de ProteínaRESUMEN
We describe an immunodeficient adult with Ogilvie's syndrome preceding a disseminated papulovesicular skin rash in whom varicella-zoster virus infection was demonstrated by PCR assay in cutaneous and colonic biopsy specimens. In view of the significant morbidity and mortality that this condition carries, early and accurate molecular diagnosis and timely treatment are strongly recommended.
Asunto(s)
Varicela/complicaciones , Seudoobstrucción Colónica/diagnóstico , Seudoobstrucción Colónica/patología , Herpesvirus Humano 3/aislamiento & purificación , Seudoobstrucción Colónica/virología , Humanos , Huésped Inmunocomprometido , Masculino , Persona de Mediana Edad , Radiografía Abdominal , Tomografía Computarizada por Rayos XRESUMEN
Vascular Endothelial Growth Factor (VEGF) is a key driver of the neovascularization and vascular permeability that leads to the loss of visual acuity of eye diseases like wet age-related macular degeneration, diabetic macular edema, and retinopathy of premature. Among the several anti-VEGF therapies under investigation for the treatment of neovascular eye diseases, our group has developed the vaccine candidate CIGB-247-V that uses a mutated form of human VEGF as antigen. In this work we evaluated if the vaccine could prevent or attenuate VEGF-induced retinal neovascularization in the course of a rabbit eye neovascularization model, based on direct intravitreal injection of human VEGF. Our experimental findings have shown that anti-VEGF IgG antibodies induced by the vaccine were available in the retina blood circulation, and could neutralize in situ the neovascularization effect of VEGF. CIGB-247-V vaccination proved to effectively reduce retinal neovascularization caused by intravitreal VEGF injection. Altogether, these results open the way for human studies of the vaccine in neovascular eye syndromes, and inform on the potential mechanisms involved in its effect.
Asunto(s)
Modelos Animales de Enfermedad , Neovascularización Retiniana/prevención & control , Vacunación , Vacunas Sintéticas/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Angiografía con Fluoresceína , Inmunoglobulina G/sangre , Inyecciones Subcutáneas , Inyecciones Intravítreas , Conejos , Proteínas Recombinantes de Fusión/inmunología , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/diagnóstico , Vasos Retinianos/patología , Factor A de Crecimiento Endotelial Vascular/toxicidad , Cuerpo Vítreo/inmunologíaRESUMEN
A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.
Asunto(s)
Anticuerpos Monoclonales Humanizados/biosíntesis , Anticuerpos Monoclonales Humanizados/farmacología , Receptores ErbB/antagonistas & inhibidores , Inmunoglobulinas/biosíntesis , Nicotiana/genética , Nicotiana/metabolismo , Planticuerpos/farmacología , Animales , Antineoplásicos/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Agricultura Molecular/métodos , Fosforilación/efectos de los fármacos , Planticuerpos/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes , Tirosina/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) is currently a major public health problem. The development of pulmonary fibrosis secondary to acute respiratory distress syndrome (ARDS) is one of the expected sequelae. In this case series, we describe five instances of the use of anakinra in late-phase COVID-19 pneumonia in hospitalized patients with pulmonary fibrosis and refractory respiratory failure fulfilling ARDS criteria. The study demonstrates that anakinra has promising efficacy and safety in late-phase COVID-19 infection in patients with ARDS and refractory hypoxaemia, and suggests its potential application as antifibrotic therapy in these patients. LEARNING POINTS: Up to one third of patients with severe COVID-19 pneumonia progress to acute respiratory distress syndrome (ARDS).Pulmonary fibrosis is a known consequence of ARDS.Our study shows promising results regarding the efficacy and safety of anakinra used in late-phase COVID-19 infection in patients with pulmonary fibrosis secondary to ARDS.
RESUMEN
BACKGROUND: The role of vitamin D status in COVID-19 patients is a matter of debate. OBJECTIVES: To assess serum 25-hydroxyvitamin D (25OHD) levels in hospitalized patients with COVID-19 and to analyze the possible influence of vitamin D status on disease severity. METHODS: Retrospective case-control study of 216 COVID-19 patients and 197 population-based controls. Serum 25OHD levels were measured in both groups. The association of serum 25OHD levels with COVID-19 severity (admission to the intensive care unit, requirements for mechanical ventilation, or mortality) was also evaluated. RESULTS: Of the 216 patients, 19 were on vitamin D supplements and were analyzed separately. In COVID-19 patients, meanâ ±â standard deviation 25OHD levels were 13.8â ±â 7.2 ng/mL, compared with 20.9â ±â 7.4 ng/mL in controls (Pâ <â .0001). 25OHD values were lower in men than in women. Vitamin D deficiency was found in 82.2% of COVID-19 cases and 47.2% of population-based controls (Pâ <â .0001). 25OHD inversely correlates with serum ferritin (Pâ =â .013) and D-dimer levels (Pâ =â .027). Vitamin D-deficient COVID-19 patients had a greater prevalence of hypertension and cardiovascular diseases, raised serum ferritin and troponin levels, as well as a longer length of hospital stay than those with serum 25OHD levels ≥20 ng/mL. No causal relationship was found between vitamin D deficiency and COVID-19 severity as a combined endpoint or as its separate components. CONCLUSIONS: 25OHD levels are lower in hospitalized COVID-19 patients than in population-based controls and these patients had a higher prevalence of deficiency. We did not find any relationship between vitamin D concentrations or vitamin deficiency and the severity of the disease.
Asunto(s)
COVID-19/sangre , Deficiencia de Vitamina D/epidemiología , Vitamina D/análogos & derivados , Anciano , COVID-19/diagnóstico , COVID-19/mortalidad , COVID-19/terapia , Estudios de Casos y Controles , Femenino , Mortalidad Hospitalaria , Humanos , Tiempo de Internación/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Vitamina D/sangre , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/diagnósticoRESUMEN
BACKGROUND: The role of vitamin D status in COVID-19 patients is a matter of debate. OBJECTIVES: To assess serum 25-hydroxyvitamin D (25OHD) levels in hospitalized patients with COVID-19 and to analyze the possible influence of vitamin D status on disease severity. METHODS: Retrospective case-control study of 216 COVID-19 patients and 197 population-based controls. Serum 25OHD levels were measured in both groups. The association of serum 25OHD levels with COVID-19 severity (admission to the intensive care unit, requirements for mechanical ventilation, or mortality) was also evaluated. RESULTS: Of the 216 patients, 19 were on vitamin D supplements and were analyzed separately. In COVID-19 patients, meanâ ±â standard deviation 25OHD levels were 13.8â ±â 7.2 ng/mL, compared with 20.9â ±â 7.4 ng/mL in controls (Pâ <â .0001). 25OHD values were lower in men than in women. Vitamin D deficiency was found in 82.2% of COVID-19 cases and 47.2% of population-based controls (Pâ <â .0001). 25OHD inversely correlates with serum ferritin (Pâ =â .013) and D-dimer levels (Pâ =â .027). Vitamin D-deficient COVID-19 patients had a greater prevalence of hypertension and cardiovascular diseases, raised serum ferritin and troponin levels, as well as a longer length of hospital stay than those with serum 25OHD levels ≥20 ng/mL. No causal relationship was found between vitamin D deficiency and COVID-19 severity as a combined endpoint or as its separate components. CONCLUSIONS: 25OHD levels are lower in hospitalized COVID-19 patients than in population-based controls and these patients had a higher prevalence of deficiency. We did not find any relationship between vitamin D concentrations or vitamin deficiency and the severity of the disease.
Asunto(s)
COVID-19/diagnóstico , Vitamina D/sangre , Anciano , COVID-19/mortalidad , COVID-19/patología , COVID-19/terapia , Estudios de Casos y Controles , Femenino , Mortalidad Hospitalaria , Hospitalización/estadística & datos numéricos , Humanos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Tiempo de Internación/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Mortalidad , Pronóstico , Respiración Artificial/estadística & datos numéricos , Estudios Retrospectivos , SARS-CoV-2/patogenicidad , Índice de Severidad de la Enfermedad , España/epidemiología , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/mortalidad , Deficiencia de Vitamina D/terapiaRESUMEN
Because of the wide use and high demand in medicine, monoclonal antibodies are among the main recombinant pharmaceuticals at present, although present limitations of the productive platforms for monoclonal antibodies are driving the improvement of the large-scale technologies and the development of alternative expression systems. This has drawn the attention on plants as expression system for monoclonal antibodies and related derivatives, owning the capacity of plants to properly express and process eukaryotic proteins with biological activity resembling that of the natural proteins. In this chapter, the procedures from the isolation of the monoclonal antibody genes to the biochemical and biological characterization of the plant-expressed monoclonal antibody are described.
Asunto(s)
Nicotiana/genética , Planticuerpos/genética , Caulimovirus/genética , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Heberprovac is a GnRH based vaccine candidate containing 2.4 mg of the GnRHm1-TT peptide as the main active principle; 245 µg of the very small size proteoliposomes adjuvant (VSSP); and 350 µL of Montanide ISA 51 VG oil adjuvant. The aim of this study was to assess the safety and tolerance of the Heberprovac in advanced prostate cancer patients as well as its capacity to induce anti-GnRH antibodies, the subsequent effects on serum levels of testosterone and PSA and the patient overall survival. The study included eight patients with histologically-proven advanced prostate cancer with indication for hormonal therapy, who received seven intramuscular immunizations with Heberprovac within 18 weeks. Anti-GnRH antibody titers, testosterone and PSA levels, as well as clinical parameters were recorded and evaluated. The vaccine was well tolerated. Significant reductions in serum levels of testosterone and PSA were seen after four immunizations. Castrate levels of testosterone were observed in all patients at the end of the immunization schedule, which remained at the lowest level for at least 20 months. In a 10-year follow-up three out of six patients who completed the entire trial survived. In contrast only one out eight patients survived in the same period in a matched randomly selected group receiving standard anti-hormonal treatment. Heberprovac vaccination showed a good security profile, as well as immunological, biochemical and, most importantly, clinical benefit. The vaccinated group displayed survival advantage compared with the reference group that received standard treatment. These results warrant further clinical trials with Heberprovac involving a larger cohort.
RESUMEN
Following the clinical success of Bevacizumab, a humanized monoclonal antibody that affects the interaction between vascular endothelial growth factor (VEGF) and its receptors, blocking tumor-induced angiogenesis has become one of the most important targets for the development of new cancer therapeutic drugs and procedures. Among the latter, therapeutic vaccination using VEGF as antigen presents itself as very attractive, with the potential of generating not only a growth factor blocking antibody response but also a cellular response against tumor cells and stromal elements, which appear to be a major source of tumor VEGF. In this paper, we report the development of a protein vaccine candidate, based on a human modified VEGF antigen that is expressed at high levels in E. coli. With respect to controls, immunization experiments in C57BL/6 mice using weekly doses of this antigen and three adjuvants of different chemical natures show that time for tumor development after subcutaneous injection of Melanoma B16-F10 cells increases, tumors that develop grow slower, and overall animal survival is higher. Immunization also prevents tumor development in some mice, making them resistant to second tumor challenges. Vaccination of mice with the human modified VEGF recombinant antigen produces antibodies against the human antigen and the homologous mouse VEGF molecule. We also show that sera from immunized mice block human VEGF-induced HUVEC proliferation. Finally, a possible contribution of T cell cytotoxicity to the overall anti-tumor effect is suggested from the results of vaccination experiments where CD8+ lymphocytes were impaired using neutralizing rat antibodies.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos/inmunología , Inmunoterapia Activa , Neoplasias/inmunología , Neoplasias/terapia , Proteínas Recombinantes/inmunología , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos , Conducta Animal/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Escherichia coli , Humanos , Sueros Inmunes , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Trasplante de Neoplasias , Neoplasias/patología , Proteínas Recombinantes/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cancer is one of the most prevalent diseases worldwide, which explains why biological therapies for cancer are forecast to make up 35% of total recombinant pharmaceuticals by 2010. Because of the high demand for cancer drugs, the need to lower production costs and the constraints of present production technologies for recombinant pharmaceuticals (such as the difficulties involved in culturing bacteria, yeast and mammalian cells), attention has recently been focused on recombinant expression of pharmaceutical anti-cancer proteins in plants. This review aims to provide an update on the most recent publications about anti-cancer recombinant pharmaceuticals expressed in plants, as well as on the relevant technical issues, potential and prospects of this emerging production system.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Productos Biológicos/uso terapéutico , Extractos Vegetales/uso terapéutico , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Productos Biológicos/metabolismo , Humanos , Extractos Vegetales/metabolismoRESUMEN
CIGB 247 is a novel cancer therapeutic vaccine based on human vascular endothelial growth factor (VEGF) variant molecule as antigen, in combination with a bacterial adjuvant. This vaccine candidate has previously demonstrated efficacy and safety in mice, rats, rabbits and non-human primates. In the present study we evaluated the effects on the clinical, hematological and biochemical parameters of CIGB 247 vaccine in Chlorocebus aethiops monkeys. Three groups of monkeys were immunized with three doses of vaccine formulation to measure physiological values of clinical, hematological and serum biochemical parameters. Monkeys' body weight and temperature were kept stable and close to standard values throughout the study. Variations in the levels of red blood cells and hemoglobin were observed among the different groups for all injected doses, but these hematological parameters recovered normal values at the end of the study. On the other hand, biochemical parameters such as the total bilirubin and total protein counts showed variations along the study, while they were not associated with the test substance. In summary, no negative effects on clinical, hematological and biochemical parameters were detected. Together, our results put forward the potential and support the safety of the CIGB 247 vaccine candidate for use in clinical applications. The data presented here can be used to estimate a human dosing regimen from preclinical data.
Asunto(s)
Vacunas contra el Cáncer/toxicidad , Animales , Chlorocebus aethiops , Eritrocitos/efectos de los fármacos , Hemoglobinas/metabolismo , Masculino , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
CIGB-247 is a cancer therapeutic vaccine, based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, in combination with the adjuvant VSSP, a bacterially-derived adjuvant. The vaccine have demonstrated efficacy in several murine malignancy models. These studies supported the rationale for a phase I clinical trial where safety, tolerance, and immunogenicity of CIGB-247 was studied in patients with advanced solid tumors at three antigen dose level. Surviving individuals of this clinical trial were eligible to receive off-trial voluntary re-immunizations. The present work is focus in the immunological follow up of these patients after approximately three years of immunizations, without additional oncological treatments. Long term vaccination was feasible and safe. Our results indicated that after sustained vaccination most of the patients conserved their seroconversion status. The specific anti-VEGF IgG titer diminished, but in all the cases keeps values up from the pre-vaccination levels. Continued vaccination was also important to produce a gradual shift in the anti-VEGF IgG response from IgG1 to Ig4. Outstanding, our results indicated that long-term off-trial vaccination could be associated with the maintaining of one reserve of antibodies able to interfere with the VEGF/Receptor interaction and the production of IFNγ secretion in CD8+ cells. The results derived from the study of this series of patients suggest that long term therapeutic vaccination is a feasible strategy, and highlight the importance of continuing the clinical development program of this novel cancer therapeutic vaccine candidate. We also highlight the future clinical applications of CIGB-247 in cancer and explain knowledge gaps that future studies may address. Registration number and name of trial registry: RPCEC00000102. Cuban Public Clinical Trial Registry (WHO accepted Primary Registry). Available from: http://registroclinico.sld.cu/.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Inmunidad Celular , Inmunidad Humoral , Inmunoterapia Activa , Neoplasias/terapia , Factor A de Crecimiento Endotelial Vascular/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/efectos adversos , Terapias Complementarias , Femenino , Estudios de Seguimiento , Humanos , Inmunización/métodos , Esquemas de Inmunización , Inmunogenicidad Vacunal , Inmunoglobulina G/sangre , Interferón gamma/inmunología , Interferón gamma/metabolismo , Masculino , Neoplasias/inmunología , Vacunación , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
During a 13-month period, 513 patients who were scheduled to undergo cardiac surgery were prospectively observed for surgical site infection during hospitalization after surgery and for 1 month after hospital discharge. Fifty-three patients showed evidence of surgical site infection (during hospitalization for 31 patients and after discharge for 22). Multivariate analysis identified that risk factors for surgical site infection differed between infections that occurred during hospitalization and those that occurred after discharge.
Asunto(s)
Procedimientos Quirúrgicos Cardíacos/efectos adversos , Infección de la Herida Quirúrgica/epidemiología , Anciano , Femenino , Hospitalización , Hospitales Comunitarios , Hospitales de Enseñanza , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Riesgo , España/epidemiología , Infección de la Herida Quirúrgica/etiologíaRESUMEN
STUDY OBJECTIVES: To assess the incidence and risk factors for nosocomial infection after lung surgery. DESIGN: Prospective cohort study. SETTING: Service of thoracic surgery of an acute-care teaching hospital in Santander, Spain. PATIENTS: Between June 1, 1999, and January 31, 2001, all consecutive patients undergoing lung surgery were prospectively followed up for 1 month after discharge from the hospital to assess the development of nosocomial infection, the primary outcome of the study. INTERVENTIONS: During the hospitalization period, patients were visited on a daily basis. Postdischarge surveillance was based on visits to the surgeon. MEASUREMENTS AND RESULTS: We studied 295 patients (84% men; mean age, 60.9 years), 89% of whom underwent resection operations. Ninety episodes of nosocomial infection were diagnosed in 76 patients, including pneumonia (n = 10), lower respiratory tract infection (n = 47), wound infection (n = 16; one third were detected after hospital discharge), urinary tract infection (n = 9), and bacteremia (n = 8; three fourths were catheter-related bacteremia). Twenty patients had severe infections (pneumonia or empyema), with a mortality rate of 60%. COPD (adjusted odds ratio [OR], 2.70; 95% confidence interval [CI], 1.52 to 4.84), duration of surgery with an increased risk for each additional minute (Mantel-Haenzel chi(2) test for trend, p = 0.037), and ICU admission (OR, 3.69; 95% CI, 1.94 to 7.06) were independent risk factors for nosocomial infection. The use of an epidural catheter was a protective factor (OR, 0.45; 95% CI, 0.22 to 0.95). There were no differences according to the use of amoxicillin/clavulanate or cefotaxime for surgical prophylaxis. CONCLUSIONS: Nosocomial infections are common after lung surgery. One third of wound infections were detected after hospital discharge. The profile of a high-risk patient includes COPD as underlying disease, prolonged operative time, and postoperative ICU admission.
Asunto(s)
Infección Hospitalaria/epidemiología , Complicaciones Posoperatorias/epidemiología , Procedimientos Quirúrgicos Pulmonares/efectos adversos , Infecciones Bacterianas/clasificación , Infecciones Bacterianas/epidemiología , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Neumonía/epidemiología , Procedimientos Quirúrgicos Pulmonares/mortalidad , Infecciones del Sistema Respiratorio/epidemiología , Factores de Riesgo , España , Infección de la Herida Quirúrgica/epidemiologíaRESUMEN
BACKGROUND: Prostate-specific antigen (PSA) has been used for the diagnosis and follow up of prostate cancer (PCa). METHODS: Mouse monoclonal antibodies (MAbs) were generated against human prostate-specific antigen (PSA) for the development of a sensitive total PSA (t-PSA) assay. Two MAbs, denoted CB-PSA.4 and CB-PSA.9, with affinities of 3.7 x 10(9) and 4.7 x 10(10) l/mol, respectively, were used to develop an enzyme-linked immunosorbent assay (ELISA) for quantifying serum t-PSA concentration. RESULTS: The detection limit (DL) of the assay was 0.1 microg/l (n=20, mean of "zero" standard+3S.D.), and the recovery of t-PSA was 96-103%. The within-run and between-day coefficients of variation (CV) ranged from 2.1% to 3.2%, and from 2.8% to 6.3% for PSA concentrations of 10 and 1 microg/l, respectively. The equimolar detection of t-PSA and free-PSA was demonstrated by two different methods, one consisted in the comparative evaluation of a sera panel (n=9) with our enzyme-linked immunosorbent assay (ELISA) and four commercial total PSA assays and the concordance with CIS bio total PSA assay. The assay had a linear range of 0.12 to 25 microg/l. CONCLUSIONS: The analytical performance characteristics of our PSA ELISA suggest that it will provide clinically useful PSA results, particularly when diagnostic algorithms are used.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Antígeno Prostático Específico/sangre , Animales , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/diagnóstico , alfa 1-Antiquimotripsina/análisis , alfa 1-Antiquimotripsina/metabolismoRESUMEN
No disponible
Asunto(s)
Humanos , Femenino , Anciano , Infecciones Bacterianas/diagnóstico , Gammaproteobacteria , EspañaRESUMEN
We have previously reported the isolation of a novel single-chain variable fragment (scFv) against vascular endothelial growth factor (VEGF), from a phage-displayed human antibody repertoire. This scFv, denominated 2H1, was shown to block the binding of VEGF to its receptor but exhibited a moderate binding affinity. Here, we describe the affinity maturation of the 2H1 scFv. Two phage-displayed libraries were constructed by diversification of the third complementarity-determining regions (CDRs) of the light (VL) and heavy (VH) chain variable domains of 2H1 using parsimonious mutagenesis. A competitive phage-selection strategy in the presence of the parental scFv as a competitor was used to eliminate low affinity binders. High affinity variants were retrieved from both libraries. An optimized VL variant was designed and constructed by combining recurrent replacements found among selected variants in a single molecule, resulting in an additional affinity increase. Further affinity improvements were achieved by combining this optimized VL with the best VH variants. The final variant obtained here, L3H6, showed an overall affinity improvement of 18-fold over the parental scFv and exhibited an enhanced potency to block the binding of VEGF to its receptor. Using phage display and extensive mutagenesis of VEGF, we determined the fine specificity of L3H6. This functional mapping revealed a novel neutralizing epitope on human VEGF defined by the residues Y25, T71, E72, N100, K101, E103 and R105. The conformational epitope recognized by L3H6 was recapitulated by grafting human VEGF residues into the mouse molecule, providing further confirmation of the nature of the identified epitope.