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OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Sensibilidad y Especificidad , Prueba de COVID-19 , InmunoensayoRESUMEN
Early detection of lung cancer allows for earlier stage treatment initiation and improved patient prognosis. This report focuses on utilization of combining patient demographic information with non-invasive biomarkers and their potential ability to predict risk of malignancy of nodules. A pilot study cohort of 141 subjects with IPNs (105 stage I cancer and 36 benign nodules) were collected by RUMC. The demographic variables of gender, age, sex, race, ethnicity, nodule size (mm), and smoking pack years, as well as the plasma levels of CA-125, SCC, CEA, HE4, ProGRP, NSE, Cyfra 21-1, hs-CRP, Ferritin, IgG, IgG1, IgG2, IgG3, IgG4, IgE, IgM, IgA, KFLC, and LFLC, were assessed for this cohort. Multivariable analyses of the previously aforementioned biomarkers and demographic variables yielded a reduced algorithm consisting of CA-125, total IgG, IgA, IgM, IgE, LFLC, nodule size, and smoking pack years with improved performance (AUC 0.82, 95 %CI 0.74-0.90) over the same analysis of the demographic variables (age, nodule size, and smoking pack years) alone (AUC 0.70, 95 %CI 0.61-0.78). This reduced algorithm of biomarkers and demographic variables may aid in assessing the risk of IPN malignancy which could be a useful stratification tool in early detection of lung cancer in high-risk subjects.
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Neoplasias Pulmonares , Nódulos Pulmonares Múltiples , Humanos , Proyectos Piloto , Nódulos Pulmonares Múltiples/diagnóstico , Algoritmos , Biomarcadores , Inmunoglobulina G , Inmunoglobulina A , Inmunoglobulina E , Inmunoglobulina MRESUMEN
B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high-affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5-13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three-dimensional structure by X-ray diffraction to 2.1 A resolution. The structure reveals the detailed interactions that five of the complementarity-determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, DeltaG = -54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion-limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine-scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides.
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Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Cristalografía por Rayos X , Péptido Natriurético Encefálico/química , Animales , Línea Celular , Epítopos/química , Ratones , Conformación Proteica , TermodinámicaRESUMEN
The T cell stimulatory activity of peptides is known to be associated with the cell surface stability and lifetime of the peptide-MHC (pepMHC) complex. In this report, soluble high-affinity T cell receptors (TCRs) that are specific for pepMHC complexes recognized by the mouse CD8+ clone 2C were used to monitor the cell surface lifetimes of synthetic agonist peptides. In the 2C system, L(d)-binding peptide p2Ca (LSPFPFDL) has up to 10,000-fold lower activity than peptide QL9 (QLSPFPFDL) even though the 2C TCR binds to p2Ca-L(d) and QL9-L(d) complexes with similar affinities. Unexpectedly, p2Ca-L(d) complexes were found to have a longer cell surface lifetime than QL9-L(d) complexes. However, the strong agonist activity of QL9 correlated with its ability to participate in efficient intracellular delivery followed by cell surface expression of the peptide, resulting in high and persistent surface levels of QL9-L(d). The ability of target cells to take up and present QL9 was observed with TAP-deficient cells and TAP-positive cells, including dendritic cells. The process was brefeldin A-sensitive, indicating a requirement for transport of the pepMHC through the ER and/or golgi. Thus, strong T cell stimulatory activity of some pepMHC complexes can be accomplished not only through long cell surface lifetimes of the ligand, but through a mechanism that leads to delayed presentation of the exogenous antigen after intracellular uptake.
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Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos/inmunología , Péptidos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Brefeldino A/farmacología , Línea Celular Tumoral , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Péptidos/antagonistas & inhibidores , Péptidos/química , Linfocitos T/efectos de los fármacos , Factores de TiempoRESUMEN
BACKGROUND: Periostin is being investigated as a potential biomarker for T-helper-2 (Th2)-driven asthma or eosinophilic inflammation and may help to identify patients more likely to benefit from interleukin-13-targeted treatments. We report the development and analytic performance of the investigational use only ARCHITECT Periostin Immunoassay, a new automated assay developed to detect serum periostin concentrations. METHODS: We assessed assay performance in terms of precision, sensitivity, linearity, interference from classical immunoassay interferents and representatives of common asthma medications, specimen handling, and isoform reactivity. The assay was also used to assess the biological variability of serum periostin concentrations in samples from healthy volunteers and from subjects with uncontrolled asthma (the intended use population). RESULTS: The percentage CVs for 5-day total precision, assessed using two instruments, was <6% across 2 controls and one serum-based panel. Limit of quantitation was 4ng/mL (dilution adjusted concentration), suiting the needs for this application. Dilution analysis yielded linear results and no endogenous sample or drug interferences were observed. All known periostin isoforms expressed in the mature human lung were detected by the assay. CONCLUSION: Our studies provide support that the ARCHITECT Periostin Immunoassay is a reliable and robust test for measuring serum periostin concentrations.
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Análisis Químico de la Sangre/métodos , Moléculas de Adhesión Celular/sangre , Inmunoensayo/métodos , Adolescente , Asma/sangre , Automatización , Biomarcadores/sangre , Recolección de Muestras de Sangre , Estudios de Casos y Controles , Femenino , Humanos , Límite de Detección , Modelos Lineales , Masculino , TemperaturaRESUMEN
BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) may be a suitable biomarker to identify people with severe asthma who have greater activation of the interleukin-13 (IL-13) pathway and may therefore benefit from IL-13-targeted treatments. We report the analytical performance of an Investigational Use Only immunoassay and provide data on the biological range of DPP-4 concentrations. METHODS: We assessed assay performance, utilising analyses of precision, linearity and sensitivity; interference from common endogenous assay interferents, and from asthma and anti-diabetic medications, were also assessed. The assay was used to measure the range of serum DPP-4 concentrations in healthy volunteers and subjects with diabetes and severe, uncontrolled asthma. RESULTS: The total precision of DPP-4 concentration measurement (determined using percentage coefficient of variation) was ≤5% over 20 days. Dilution analysis yielded linear results from 30 to 1305 ng/mL; the limit of quantitation was 19.2 ng/mL. No notable endogenous or drug interferences were observed at the expected therapeutic concentration. Median DPP-4 concentrations in healthy volunteers and subjects with asthma or Type 1 diabetes were assessed, with concentrations remaining similar in subjects with diabetes and asthma across different demographics. CONCLUSION: These analyses indicate that the ARCHITECT DPP-4 Immunoassay is a reliable and robust method for measuring serum DPP-4 concentration.
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In a cellular immune response, antigenic peptides derived by intracellular processing of foreign pathogens are bound to the class I major histocompatability complex (MHC I) and presented to CD8(+) cytotoxic T cells. Although the crystal structures of several different MHC products have been solved, many MHC molecules, including some associated with diseases, have not been amenable to biochemical and structural studies. The variability in this success is based largely on the fact that peptide-MHC complexes vary extensively in their stability. These properties also are intimately tied to the biological activity of the complexes. The ability to apply the techniques of directed evolution to this system in order to engineer stable complexes has been complicated by the trimeric structure of peptide-MHC complexes, requiring association of three polypeptides: the heavy chain, beta2-microglubulin (beta2m), and a short peptide. We show here that single-chain forms of peptide-MHC complexes can be expressed as Aga-2 fusions on the surface of yeast. Three different complexes, SIYRYYGL-K(b)-beta2m (SIYR-K(b)), EQYKFYSV-K(b)-beta2m (dEV8-K(b)), and SIINFEKL-K(b)-beta2m (OVA-K(b)), were expressed on yeast and detected by flow cytometry with a conformation-specific anti-K(b) antibody (B.8.24.3). In addition, yeast displaying K(b) loaded with exogenous SIYR and OVA peptides were recognized by a high-affinity T cell receptor that is specific for SIYR-K(b) and by an antibody (25.D1-16) that is specific for OVA-K(b), respectively. Finally, yeast that display the SIYRYYGL-K(b) also directly stimulated CD69 up-regulation on naive 2C T cells. Hence, yeast display represents a technology that can be used for directed evolution of any of the components of the trimeric pep-MHC complex.
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Antígenos de Histocompatibilidad Clase I/química , Técnicas Inmunológicas , Oligopéptidos/química , Ingeniería de Proteínas , Saccharomyces cerevisiae/genética , Animales , Anticuerpos , Evolución Molecular Dirigida , Vectores Genéticos , Antígenos H-2/química , Antígenos H-2/genética , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Técnicas In Vitro , Ratones , Oligopéptidos/genética , Oligopéptidos/metabolismo , Biblioteca de Péptidos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunologíaRESUMEN
Studies of the broader autism phenotype, and of subtle changes in autism symptoms over time, have been compromised by a lack of established quantitative assessment tools. The Social Responsiveness Scale (SRS-formerly known as the Social Reciprocity Scale) is a new instrument that can be completed by parents and/or teachers in 15-20 minutes. We compared the SRS with the Autism Diagnostic Interview-Revised (ADI-R) in 61 child psychiatric patients. Correlations between SRS scores and ADI-R algorithm scores for DSM-IV criterion sets were on the order of 0.7. SRS scores were unrelated to I.Q. and exhibited inter-rater reliability on the order of 0.8. The SRS is a valid quantitative measure of autistic traits, feasible for use in clinical settings and for large-scale research studies of autism spectrum conditions.
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Síndrome de Asperger/diagnóstico , Trastorno Autístico/diagnóstico , Trastornos Generalizados del Desarrollo Infantil/diagnóstico , Inventario de Personalidad/estadística & datos numéricos , Conducta Social , Síndrome de Asperger/psicología , Trastorno Autístico/psicología , Niño , Trastornos Generalizados del Desarrollo Infantil/psicología , Preescolar , Femenino , Humanos , Masculino , Psicometría , Reproducibilidad de los ResultadosRESUMEN
Development of a robust immunoassay requires the selection of monoclonal antibodies with desired properties. These properties generally include kinetics parameters such as on-rate and off-rate (i.e., binding affinity), and, often times, the ability to form a sandwich with the analyte of interest. We sought to obtain antibodies suitable for development of an immunoassay capable of detecting human neutrophil gelatinase-associated lipocalin (NGAL), a glycosylated lipocalin of 25 kDa expressed in kidney tubules in response to injury that has been shown to be a urinary biomarker capable of diagnosing acute kidney injury. We immunized CAF1/J and RBF/DnJ mouse strains with recombinant NGAL, and a robust immune response, as measured by serum antibody titer, was observed among all CAF1/J mice. Antibodies secreted from mouse B cell-myeloma hybridomas were screened by enzyme immunoassay (EIA) and by surface plasmon resonance using a method we termed hybrid supernatant kinetic screening. Approximately 300 hybrid clones were evaluated by this technique to identify antibodies with the kinetic binding parameters meeting criteria required for further assay development (i.e., rapid association and slow dissociation). This data, along with epitope grouping, cell growth, cell viability, and antibody secretion, were used to identify antibodies for testing in the ARCHITECT assay.
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Proteínas de Fase Aguda/inmunología , Anticuerpos Monoclonales de Origen Murino/química , Lipocalinas/inmunología , Proteínas Proto-Oncogénicas/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/biosíntesis , Anticuerpos Monoclonales de Origen Murino/inmunología , Afinidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos/inmunología , Humanos , Hibridomas , Cinética , Lipocalina 2 , Ratones , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Mouse-human chimeric antibodies (cAbs) against hepatitis C virus (HCV) core, NS3 (nonstructural), NS4, and NS5 antigens were developed as quality control (QC) reagents to replace the use of human sera/plasma for Abbott HCV immunoassays. The cAb retains the mouse monoclonal antibody (MAb) specificity and affinity but still reacts in the existing HCV assay format, which measures human anti-HCV immunoglobulin. Mouse heavy-chain (V(H)) and light-chain (V(L)) variable regions of anti-HCV core, NS3, NS4, and NS5 antigens were PCR amplified from hybridoma lines and then cloned with human IgG1 heavy-chain (C(H)) and light-chain (C(L)) constant regions, respectively. A single mammalian expression plasmid containing both heavy-chain and light-chain immunoglobulin genes was constructed and transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells. The transfected CHO cells were selected using hypoxanthine- and thymidine-free medium and screened by an enzyme immunoassay (EIA). The clone secreting the highest level of antibody was isolated from the CHO transfectants and further subcloned. Each cAb-expressing CHO cell line was weaned into serum-free medium, and the cAb was purified by protein A affinity chromatography. The levels of cAb production for the various CHO cell lines varied from 10 to 20 mg/liter. Purified anti-HCV cAbs were tested with Abbott HCV immunoassays and showed reactivity. Moreover, yeast surface display combined with alanine-scanning mutagenesis was used to map the epitope at the individual amino acid level. Our results suggest that these HCV cAbs are ideal controls, calibrators, and/or QC reagents for HCV assay standardization.
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Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Antígenos de la Hepatitis C/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas del Núcleo Viral/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/biosíntesis , Anticuerpos Monoclonales de Origen Murino/genética , Células CHO , Cricetinae , Cricetulus , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/biosíntesis , Anticuerpos contra la Hepatitis C/genética , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.
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Técnicas Biosensibles/instrumentación , Calorimetría/instrumentación , Calorimetría/normas , Inhibidores de Anhidrasa Carbónica/clasificación , Inhibidores de Anhidrasa Carbónica/metabolismo , Sulfonamidas/antagonistas & inhibidores , Benchmarking , Investigación Biomédica , Técnicas Biosensibles/normas , Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/metabolismo , Variaciones Dependientes del Observador , Unión Proteica , Sulfonamidas/clasificación , Resonancia por Plasmón de Superficie/instrumentación , Resonancia por Plasmón de Superficie/normas , TermodinámicaRESUMEN
Insecure mental representations of attachment, a nearly invariant feature of cluster B personality disorders, have never previously been studied in twins. We conducted the Adult Attachment Interview (AAI) on 33 pairs of monozygotic (MZ) female twins reared together as an initial exploration of causal influences on mental representations of attachment. As predicted by attachment theory, we observed substantial twin-twin concordance for attachment security (odds ratio 13.8; P = 0.001), a similar level of concordance between twins and their non-twin siblings, and an inverse relationship between attachment security and current level of aggression (P = 0.01). These data indicate that there are minimal effects of non-shared environmental influences (or measurement error) on attachment classifications derived from the AAI. In this sample of twins with and without histories of Conduct Disorder, mental representations of attachment appear to be highly familial, i.e., strongly influenced by either shared environmental factors, genetic factors, or both.
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Cognición , Trastorno de la Conducta/epidemiología , Trastorno de la Conducta/psicología , Apego a Objetos , Gemelos Monocigóticos/psicología , Adolescente , Adulto , Demografía , Femenino , Humanos , Variaciones Dependientes del Observador , PrevalenciaRESUMEN
The major histocompatibility complex (MHC) is the most polymorphic locus known, with thousands of allelic variants. There is considerable interest in understanding the diversity of structures and peptide-binding features represented by this class of proteins. Although many MHC proteins have been crystallized, others have not been amenable to structural or biochemical studies due to problems with expression or stability. In the present study, yeast display was used to engineer stabilizing mutations into the class I MHC molecule, Ld. The approach was based on previous studies that showed surface levels of yeast-displayed fusion proteins are directly correlated with protein stability. To engineer a more stable Ld, we selected Ld mutants with increased surface expression from randomly mutated yeast display libraries using anti-Ld antibodies or high affinity, soluble T-cell receptors (TCRs). The most stable Ld mutant, Ld-m31, consisted of a single-chain MHC module containing only the alpha1 and alpha2 domains. The enhanced stability was in part due to a single mutation (Trp-97 --> Arg), shown previously to be present in the allele Lq. Mutant Ld-m31 could bind to Ld peptides, and the specific peptide.Ld-m31 complex (QL9.Ld-m31) was recognized by alloreactive TCR 2C. A soluble form of the Ld-m31 protein was expressed in Escherichia coli and refolded from inclusion bodies at high yields. Surface plasmon resonance showed that TCRs bound to peptide.Ld-m31 complexes with affinities similar to those of native full-length Ld. The TCR and QL9.Ld-m31 formed complexes that could be resolved by native gel electrophoresis, suggesting that stabilized alpha1/alpha2 class I platforms may enable various structural studies.