RESUMO
PURPOSE: Negative laboratory results make targeting microbial keratitis treatment difficult. We investigated factors associated with laboratory negativity in patients with microbial keratitis in the context of a transition to a new specimen collection method. METHODS: Microbial keratitis patients with associated laboratory tests were identified in the electronic health record of a tertiary care facility from August 2012 to April 2022. Patient demographics and laboratory results were obtained. Random sampling of 50% of charts was performed to assess the impact of the ocular history and pretreatment measures. The relationship between probability of negative laboratory results with demographics, ocular history, pretreatment measures, and utilization of a new specimen collection method (i.e. ESwab) was evaluated by multivariable logistic regression. RESULTS: Of 3395 microbial keratitis patients identified, 31% (n = 1051) had laboratory tests. Laboratory testing increased over time (slope = 2.5% per year, p < 0.001; 19.6% in 2013 to 42.2% in 2021). Laboratory negative rate increased over time (slope = 2.2% per year, p = 0.022; 48.5% in 2013 to 62.3% in 2021). Almost one-third of patients (31.2%, n = 164) were pretreated with steroids. Over two-thirds of patients were pretreated with antibiotics (69.5%, n = 367). 56.5% (n = 297) of patients were outside referrals. In multivariable regression, patients with corticosteroid pretreatment had lower odds of negative laboratory results (odds ratio [OR] = 0.49, p = 0.001). There were higher odds of negative laboratory results for every additional antibiotic prescribed to a patient prior to presentation (OR = 1.30, p = 0.006) and for specimens collected using ESwabs (OR = 1.69, p = 0.005). Age, prior eye trauma, outside referrals, and contact lens wear were not significantly associated with negative laboratory results. CONCLUSION: More microbial keratitis associated laboratory tests are being taken over time. Over 60% of tests were negative by 2022. Factors associated with negative laboratory test results included pretreatment with antibiotics and specimens collected with the new collection method.
Assuntos
Úlcera da Córnea , Infecções Oculares Bacterianas , Ceratite , Humanos , Úlcera da Córnea/tratamento farmacológico , Estudos Retrospectivos , Ceratite/tratamento farmacológico , Antibacterianos/uso terapêutico , Manejo de Espécimes , Fatores de Risco , Infecções Oculares Bacterianas/diagnóstico , Infecções Oculares Bacterianas/tratamento farmacológicoRESUMO
PURPOSE: Evaluate the effect of corneal and contact lens-related (CLR) culture results on visual acuity (VA) in patients with microbial keratitis (MK). METHODS: MK patients with corneal and CLR cultures were identified in the University of Michigan electronic health record from August 2012 to April 2022. Test results were classified as laboratory-positive or laboratory-negative. Linear regression was used to examine trends of VA and associations between changes in VA (differences of VA at 90-day and baseline VA) and corneal and CLR culture results, after adjustment for baseline VA. One-sample t-tests were used to test if the slope estimates were different from zero. RESULTS: MK patients (n = 50) were on average 49 years old (standard deviation = 20.9), 56% female, and 90% White. Positive corneal and CLR cultures were reported in 60% and 64% of patients, respectively, and 38% reported both. The agreement rate between corneal and CLR culture results was 30% (n = 15/50). LogMAR VA improved over time in patients with positive corneal and CLR cultures (Estimate=-0.19 per 10-day increase, p = 0.002), and in those with negative corneal and positive CLR cultures (Estimate= -0.17 per 10-day increase, p = 0.004). Compared to patients with negative corneal and CLR cultures, there was a trend toward improvement in VA for patients with positive corneal and CLR cultures (Estimate=-0.68, p = 0.068), and those with negative corneal and positive CLR cultures (Estimate= -0.74, p = 0.059), after adjusting for baseline VA. CONCLUSIONS: Positive CLR cultures are associated with significant improvement in VA over time. These additional cultures can provide guidance on appropriate antimicrobial selection, especially when corneal cultures are negative.
Assuntos
Lentes de Contato , Úlcera da Córnea , Infecções Oculares Bacterianas , Ceratite , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/tratamento farmacológico , Infecções Oculares Bacterianas/diagnóstico , Infecções Oculares Bacterianas/tratamento farmacológico , Estudos Retrospectivos , Ceratite/diagnóstico , Acuidade VisualRESUMO
PURPOSE: Microbial keratitis (MK) is a potentially blinding corneal disease caused by an array of microbial etiologies. However, the lack of early organism identification is a barrier to optimal care. We investigated clinician confidence in their diagnosis of organism type on initial presentation and the relationship between confidence and presenting features. METHODS: This research presents secondary data analysis of 72 patients from the Automated Quantitative Ulcer Analysis (AQUA) study. Cornea specialists reported their confidence in organism identification. Presenting sample characteristics were recorded including patient demographics, health history, infection morphology, symptoms, and circumstances of infection. The association between confidence and presenting characteristics was investigated with 2-sample t-tests, Wilcoxon tests, and Chi-square or Fisher's exact tests. RESULTS: Clinicians reported being "confident or very confident" in their diagnosis of the causal organism in MK infections for 39 patients (54%) and "not confident" for 33 patients (46%). Confidence was not significantly associated with patient demographics, morphologic features, or symptoms related to MK. MK cases where clinicians reported they were confident, versus not confident in their diagnosis, showed significantly smaller percentages of previous corneal disease (0% versus 15%, p = 0.017), were not seen by an outside provider first (69% versus 94%, p = 0.015), or had no prior labs drawn (8% versus 33%, p = 0.046), and a significantly larger percentage of cases wore contact lenses (54% versus 28%, p = 0.029). CONCLUSION: In almost half of MK cases, cornea specialists reported lack of confidence in identifying the infection type. Confidence was related to ocular history and circumstances of infection but not by observable signs and symptoms or patient demographics. Tools are needed to assist clinicians with early diagnosis of MK infection type to expedite care and healing.
Assuntos
Lentes de Contato , Doenças da Córnea , Ceratite , Humanos , Ceratite/diagnóstico , Córnea , CausalidadeRESUMO
CONTEXT.: The authors announce the launch of the Consortium for Analytic Standardization in Immunohistochemistry, funded with a grant from the National Cancer Institute. As with other laboratory testing, analytic standards are important for many different stakeholders: commercial vendors of instruments and reagents, biopharmaceutical firms, pathologists, scientists, clinical laboratories, external quality assurance organizations, and regulatory bodies. Analytic standards are customarily central to assay development, validation, and method transfer into routine assays and are critical quality assurance tools. OBJECTIVE.: To improve immunohistochemistry (IHC) test accuracy and reproducibility by integrating analytic standards into routine practice. To accomplish this mission, the consortium has 2 mandates: (1) to experimentally determine analytic sensitivity thresholds (lower and upper limits of detection) for selected IHC assays, and (2) to inform IHC stakeholders of what analytic standards are, why they are important, and how and for what purpose they are used. The consortium will then publish the data and offer analytic sensitivity recommendations where appropriate. These mandates will be conducted in collaboration and coordination with clinical laboratories, external quality assurance programs, and pathology organizations. DATA SOURCES.: Literature review and published external quality assurance data. CONCLUSIONS.: Integration of analytic standards is expected to (1) harmonize and standardize IHC assays; (2) improve IHC test accuracy and reproducibility, both within and between laboratories; and (3) dramatically simplify and improve methodology transfer for new IHC protocols from published literature or clinical trials to clinical IHC laboratories.
Assuntos
Serviços de Laboratório Clínico , Laboratórios , Humanos , Imuno-Histoquímica , National Cancer Institute (U.S.) , Reprodutibilidade dos Testes , Estados UnidosRESUMO
BACKGROUND: The antiepileptic medication vigabatrin has been associated with ocular toxicity, and close ophthalmic monitoring has been recommended; however, there is no clear consensus regarding the value and feasibility of such monitoring in children. We describe ophthalmic assessments in children in a real-world clinical setting, the incidence of vigabatrin-related ocular toxicity, and the utility of regular screening or ancillary testing in children taking vigabatrin. METHODS: The medical records of children taking vigabatrin with one or more ophthalmic assessments at Children's Hospital of Philadelphia or University of California, San Francisco, between May 2010 and May 2021, were reviewed retrospectively. Abnormalities on ophthalmic examination, visual field (VF), electroretinogram (ERG), and optical coherence tomography (OCT) were reviewed and categorized as attributable to vigabatrin, possibly attributable to vigabatrin, or not attributable to vigabatrin. RESULTS: A total of 1,281 assessments of 284 children (mean age, 2.09 years) were included. Of these, 283 (99.6%) had funduscopic examination(s), 37 (13.0%) had ERG, 19 (6.7%) had OCT, and 6 (2.1%) had formal VF. Rate of examinations and ERGs per child decreased over the 10-year study period. Two children (0.7%) had definite vigabatrin-related ocular toxicity, both identified on ERG. An additional 4 children (1.4%) had optic atrophy of unclear relation to vigabatrin, categorized as possible toxicity. The remaining 278 children did not have abnormal examination or testing findings attributable to vigabatrin. CONCLUSIONS: The incidence of vigabatrin-related ocular toxicity in children was low in our cohort. Ocular and neurologic comorbidities and limited examinations in children make identification of such toxicity challenging and the value of screening is unclear.
Assuntos
Anticonvulsivantes , Vigabatrina , Pré-Escolar , Humanos , Anticonvulsivantes/efeitos adversos , Eletrorretinografia , Estudos Retrospectivos , Neuropatia Óptica Tóxica , Vigabatrina/efeitos adversosRESUMO
Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH), we have synthesized a short trimeric coiled-coil peptide (TCC) that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg) from reaching the brain.
Assuntos
Afinidade de Anticorpos/imunologia , Nicotina/imunologia , Peptídeos/imunologia , Abandono do Hábito de Fumar/métodos , Tabagismo/terapia , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos/sangue , Anticorpos/imunologia , Encéfalo/metabolismo , Feminino , Haptenos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/agonistasRESUMO
A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, 3 also bound to full-length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.
Assuntos
Anticorpos Antibacterianos/química , Especificidade de Anticorpos , Toxinas Botulínicas Tipo A/química , Epitopos/química , Anticorpos de Cadeia Única/química , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/genética , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/metabolismo , Epitopos/genética , Epitopos/metabolismo , Humanos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genéticaRESUMO
In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and ß-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.
Assuntos
Cromatografia de Afinidade/métodos , Escherichia coli/genética , Cadeias kappa de Imunoglobulina/isolamento & purificação , Inteínas , Proteínas Recombinantes de Fusão/isolamento & purificação , Quitina/química , Quitina/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Humanos , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Mycobacterium tuberculosis/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (e.g., yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype--binding conferred by an antibody fragment--with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.
Assuntos
Anticorpos Monoclonais/biossíntese , Biomarcadores Tumorais/análise , Neoplasias/diagnóstico , Biblioteca de Peptídeos , Proteômica/métodos , Biomarcadores Tumorais/imunologia , Humanos , Neoplasias/imunologiaRESUMO
Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from non-immune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast-displayed and -secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast-displayed scFv that bind non-overlapping or non-competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast-displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.
Assuntos
Antígenos de Protozoários/análise , Citometria de Fluxo/métodos , Imunoensaio/métodos , Região Variável de Imunoglobulina/metabolismo , Soluções/química , Animais , Antígenos de Protozoários/imunologia , Região Variável de Imunoglobulina/imunologia , Ligação Proteica , Trypanosoma brucei brucei/imunologia , Leveduras/genéticaRESUMO
A fluorescence sandwich immunoassay using high-affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum neurotoxin serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-H(C)-fragment) as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. Detection to 31 pM with a total incubation time of 3 h was demonstrated. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the reactions were carried out in microcentrifuge tubes with an incubation time of 1 h. The beads were subsequently captured and concentrated in a rotating rod "renewable surface" flow cell equipped with a fiber optic system for fluorescence measurements. In PBS buffer, the BoNT/A-H(C)-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.
Assuntos
Anticorpos Antibacterianos/imunologia , Toxinas Botulínicas/análise , Imunoensaio/métodos , Pontos Quânticos , Toxinas Botulínicas/imunologia , Desenho de Equipamento , Fluorescência , Imunoensaio/instrumentação , Modelos Moleculares , Sensibilidade e EspecificidadeRESUMO
A renewable surface biosensor for rapid detection of botulinum neurotoxin serotype A is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant protein fragment of the toxin heavy chain ( approximately 50 kDa) as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate non-overlapping epitopes of the full botulinum holotoxin ( approximately 150 kDa). Both of the targeted epitopes are located on the recombinant fragment. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by a sequential injection flow system, creating a 3.6 microL column. After perfusing the bead column with sample and washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degrees angle to one another delivered excitation light from a HeNe laser (633 nm) using one fiber and collected fluorescent emission light for detection with the other. After each measurement, the used Sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes using this system.
Assuntos
Toxinas Botulínicas/química , Imunoensaio/instrumentação , Imunoensaio/métodos , Anticorpos , Técnicas Biossensoriais , Fluorescência , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Receptores FcRESUMO
These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.
Assuntos
Antígenos de Superfície/imunologia , Clonagem Molecular/métodos , Fragmentos de Imunoglobulinas/metabolismo , Biblioteca de Peptídeos , Leveduras/imunologia , Algoritmos , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/fisiologia , Reações Antígeno-Anticorpo/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Linfócitos B/imunologia , Separação Celular/métodos , Humanos , Fragmentos de Imunoglobulinas/genética , Magnetismo , Leveduras/genéticaRESUMO
Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.
Assuntos
Anticorpos/química , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Proteômica/métodos , Animais , Separação Celular , Fator de Crescimento Epidérmico/química , Epitopos/química , Humanos , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Região Variável de Imunoglobulina/química , Camundongos , Análise Serial de Proteínas/métodos , Proteínas/química , Tiorredoxinas/químicaRESUMO
Although anti-CD25 antibodies exist for clinical use in patients, there is a need for the development of a human Treg antibody that will abrogate the immunosuppressive function of this small but critical T cell subtype. Based upon mounting evidence that the level of Treg cells in the tumor microenvironment correlates with clinical prognosis and stage in man, it appears that Treg cells play an important role in the tumor's ability to overcome host immune responses. In mice, the rat anti-mouse CD25 antibody PC61 causes depletion of CD25-bearing Treg cells both peripherally in lymphatic tissues and in the tumor microenvironment, without inducing symptoms of autoimmunity. A similar antibody, though with the ability to delete Treg cells specifically, would be an important new tool for reversing tumor escape associated with Treg immunosuppression in man. To begin to generate such a reagent, we now describe the development of a human anti-CD25 antibody using a novel yeast display library. The target antigen CD25-Fc was constructed and used for five rounds of selection using a non-immune yeast display library that contained as many as 10(9) single chain variable fragments (scFv). Two unique clones with low K(D) values (RA4 and RA8) were then selected to construct fully human anti-CD25 antibodies (IgG1/kappa) for stable expression. One antibody, RA8, showed excellent binding to human CD25(+) cell lines and to human Treg cells and appears to be an excellent candidate for the generation of a human reagent that may be used in man for the immunotherapy of cancer.
Assuntos
Anticorpos Monoclonais/biossíntese , Subunidade alfa de Receptor de Interleucina-2/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Sequência de Bases , DNA Complementar/genética , Humanos , Hibridomas/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Técnicas In Vitro , Camundongos , Biblioteca de Peptídeos , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Saccharomyces cerevisiae/genéticaRESUMO
Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca(2+) binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca(2+)-bound (Ca(2+)-CaM) or Ca(2+)-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (K(d) = 0.8 nM) and specificity (>1000-fold) for Ca(2+)-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (K(d) = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.
Assuntos
Afinidade de Anticorpos/genética , Evolução Molecular Direcionada/métodos , Conformação Proteica , Saccharomyces cerevisiae/genética , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/genética , Calmodulina/imunologia , Calmodulina/metabolismo , Clonagem Molecular , Citometria de Fluxo , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas S100/imunologia , Proteínas S100/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Troponina C/imunologia , Troponina C/metabolismoRESUMO
Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mgL(-1) culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships.
Assuntos
Clonagem Molecular , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Escherichia coli/genética , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genéticaRESUMO
The ability to rapidly and consistently measure aqueous solubility in a preclinical environment is critical to the successful identification of promising discovery compounds. The advantage of an early solubility screen is timely attrition of compounds likely to fail due to poor absorption or low bioavailability before more costly screens are performed. However, due to the large number of compounds and limited sample amounts, thermodynamic solubility measurements are not feasible at this stage. A kinetic solubility measurement is an alternative to thermodynamic measurements at the discovery stage that provides a rank listing of solubility values with minimal sample requirements. A kinetic solubility measurement is attractive from an automation vantage because it features rapid data acquisition and is amenable to multi-well formats. We describe the use of a robotic liquid/plate handler coupled to nephelometry detection for the measurement of kinetic solubility. We highlight the liquid handling validation, serial dilution parameters, and a comparison to the previous method. Experiments to further enhance throughput, or increase confidence in the automation steps, are described and the effects of these experiments are presented. In our integrated nephelometry method, we observe rapid liquid handling with an error of less than 10%, after a series of validation studies, and a sample throughput up to 1800 compounds per week. We compare the nephelometry method with our semi-thermodynamic flow-injection analysis (FIA) method, and find a 75% bin agreement between the methods.
Assuntos
Lasers , Preparações Farmacêuticas/análise , Robótica/métodos , Cinética , Nefelometria e Turbidimetria/instrumentação , Nefelometria e Turbidimetria/métodos , Preparações Farmacêuticas/metabolismo , Robótica/instrumentação , Solubilidade , Água/análise , Água/metabolismoRESUMO
In order to more productively utilize the rich source of antigen-specific reagents present in the previously described non-immune single chain fragment variable (scFv) yeast display library, one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi Macs system to recover greater than 90% of the antigen-specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen-specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope-specific scFv. Combining these two improvements in library screening allowed isolation and characterization of three epitope-specific scFv, including a previously uncharacterized epitope to a 6-kDa protein, epidermal growth factor.