Serodiagnostic potential of immuno-PCR using a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor in tuberculosis patients.

A novel indirect immuno-polymerase chain reaction (I-PCR) assay was developed for the detection of circulating anti-Ag85B (antigen 85B, Rv1886c), anti-ESAT-6 (early secretory antigenic target-6, Rv3875) and anti-cord factor (trehalose 6,6'-dimycolate) antibodies from the sera samples of pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients and the results were compared with an analogous enzyme-linked immunosorbent assay (ELISA). We covalently attached the amino-modified reporter DNA to the dithiothreitol (DTT)-reduced anti-human IgG antibody through a chemical linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The detection of cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies was found to be superior to the detection of individual antibodies. The sensitivities of 89.5% and 77.5% with I-PCR and 70.8% and 65% with ELISA were observed in smear-positive and smear-negative PTB cases, respectively with high specificity (90.9%). On the other hand, a sensitivity of 77.5% with I-PCR and 65% with ELISA was observed in EBTB cases. The detection of cocktail of antibodies by I-PCR is likely to improve the utility of existing algorithms for TB diagnosis.

Validation of Total and Specific IgE Measurements in Induced Sputum

Durante los últimos años, se están analizando de forma exhaustiva los componentes solubles del esputo inducido, no obstante pocos estudios analizan la IgE total y específica y ninguno de ellos ha intentado validar la técnica. El objetivo de este estudio fue validar la cuantificación de IgE total y específica en el sobrenadante del esputo inducido y evaluar la influencia del procesamiento del mismo con dithiothreitol (DTT). La IgE total y específica fueron determinadas mediante el fluoroenzymoinmunoensayo ImmunoCAP (Phadia ThermoFisher Scientific). Para el proceso de validación utilizamos experimentos de adición de células del esputo con IgE total y específica (frente a Dermatophagoides pteronyssinus y Phleum pratense) en un rango de concentraciones de acuerdo a las recomendaciones de la ATS/ERS. Para las comparaciones intragrupo se utilizó el test de Wilcoxon y el coeficiente de correlación intraclase para evaluar el grado de acuerdo entre ambas mediciones. Fueron considerados como significativos valores de p <0.05. En cuanto a los resultados obtenidos en las muestras de esputo procedentes de 18 pacientes (13 con enfermedad pulmonar intersticial, 2 con asma alérgica y 3 controles sanos), 12 varones, con una edad media de 45.6 ± SD 15.8 años, se obtuvo una media de IgE total de 5.4 (P25-754.0-6.0) kU/L. La IgE específica frente a Dermatophagoides pteronyssinus y Phleum pratense fueron inferiores a 0.35 kUA/L en todas las muestras. Las cifras de recuperación de la IgE total y específica estaba por encima del 80% con un amplio rango de valores. No se hallaron diferencias significativas entre la cuantificación de IgE total y específica con PBS o con DTT, con un buen coeficiente de correlación intraclase (0.81; p=0.01). En conclusión, la cuantificación de IgE total y específica en esputo inducido utilizando un imnunoensayo comercializado es válido y presenta una alta dispersión de rangos de niveles de IgE (AU)

Background and objectives: Soluble components are increasingly analyzed in induced sputum supernatant. However, only a few studies have measured total or specific immunoglobulin (Ig) E in sputum and none have attempted to validate it. We aim to validate laboratory measurements of total and specific IgE in induced sputum supernatant and to evaluate the influence of sputum processing with dithiothreitol (DTT) on IgE measurements. Methods: Total and specific IgE were measured by ImmunoCAP and the process was validated using sputum spiking experiments with total and specific IgE (to Dermatophagoides pteronyssinus and Phleum pratense) over a range of concentrations according to international recommendations. The Wilcoxon signed-ranks test was used for within-group comparisons and intraclass correlation coefficients were used to evaluate agreement between measurements. Two-tailed P values lower than .05 were considered significant. Results: Samples from 18 patients (13 with interstitial lung disease, 2 with allergic asthma, and 3 healthy controls; 12 men; mean [SD] age, 45.6 [15.8] years) were evaluated. Median total IgE was 5.4 kU/L (interquartile range, 4.0-6.0 kU/L). Specific IgE levels to D pteronyssinus and P pratense were below 0.35 kUA/L in all samples. Recovery rates were above 80% for total and specific IgE over a wide range of values. No differences were found in total IgE measurements of sputum dispersed with DTT or phosphate-buffered saline, with a good intraclass correlation coefficient between both measurements (0.81, P=.01). Conclusions: Total and specific IgE measurements performed in induced sputum with a commercially available immunoassay are valid over a wide range of IgE levels (AU)

Preliminary characterization of a structural defect in homozygous sickled cell alpha spectrin demonstrated by a rabbit autoantibody.

We have identified a rabbit autoantibody that strongly reacts with the core membrane skeleton of control red blood cells, and does not react with low- or high-density sickle cell core skeletons upon indirect immunofluorescence. Western blot analysis of red blood cell membrane proteins, utilizing this autoantibody, indicated no reactivity to any protein when SDS-PAGE was conducted in the presence of the reducing agent, dithiothreitol. However when SDS-PAGE was performed on control red blood cell membrane proteins separated in the absence of dithiothreitol, the autoantibody specifically reacted with a high molecular weight polypeptide (apparent Mr approximately equal to 310 kD) representing a DTT sensitive form of control alpha spectrin, which we refer to as alpha' spectrin. There was no staining of high density or low density sickle cell alpha or alpha' spectrin. This autoantibody should be an excellent tool for the fine mapping of structural change(s) in control vs. sickle cell alpha spectrin, and determination of whether the structural alteration effects spectrin dimer-tetramer interconversion and/or the spectrin-actin interaction. The modification in alpha spectrin, detected by this antibody, is very specific for homozygous SS alpha spectrin because sickle cell beta+ thalassemic alpha spectrin and sickle cell trait alpha spectrin react intensely with the autoantibody.

[Detection of rubella specific IgM on gel filtration through Sephadex G 200: use of dithiothreitol and limits with MnCl2-heparine pretreatment (author's transl)]. / Détermination des anticorps rubéoliques de classe IgM par gel-filtration sur Sephadex G 200. Utilisation du dithiothréitol et limites du traitement à l'héparine-chlorure de manganèse.

The detection of rubella haemagglutination inhibiting antibody, in the IgM fraction of the serum, on gel filtration through Sephadex G 200, needs precautions to exclude false results. Treatment with dithiothreitol is a satisfactory method for confirming the content of rubella IgM antibody. The failure of MnCl2-heparin pretreatment to remove non specific inhibitors of rubella hemagglutinin is unfrequent (7/108) and so do be repeated. Rarely (1/108) aggregated IgG fractionates with IgM and yield false positive results.