Decarboxylases

The IC and commercial i+LAB THAL IC strip tests were validated simultaneously using samples from various thalassemia and non-thalassemia subjects

The IC and commercial i+LAB THAL IC strip tests were validated simultaneously using samples from various thalassemia and non-thalassemia subjects. IC strip tests. These results led to a 0-thalassemia screening being proposed in which blood samples are first evaluated by MCV, MCH and Hb typing. Samples with high MCV and MCH values are excluded for the presence of the 0-thalassemia gene. Samples with low MCV or MCH values are assayed using the developed IC strip assessments, where only samples screening positive are further assayed for 0-thalassemia by PCR. Patients with Hb H, EA Barts or EF Barts diseases do not need to use this IC strip assay. Thus, in this study, a simple and cost effective 0-thalassemia point of care test was developed. Introduction -Thalassemia is usually a genetic disorder caused by a defect in the -globin gene [1, 2], the severe form of which (0-thalassemia) is usually characterized by the deletion of both pairs of linked -globin genes, whereas a single -gene deletion is present in individuals with +-thalassemia. Accordingly, Tropanserin couples who carry the 0-thalassemia trait have a 25% risk of hemoglobin (Hb) Barts hydrops fetalis in each pregnancy Tropanserin due to the absence of -globin genes [3C5]. Hb Barts hydrops fetalis is the most severe type of thalassemia and causes fetuses pass away in utero. Their mothers also often suffer from several obstetric complications and must cope with the psychological burden of transporting a nonviable fetus to term [6, 7]. Currently, new cases of Hb Barts disease still occur and need to be prevented [2, 8]. Providing appropriate genetics counselling to individuals recognized -thalassemia can prevent severe thalassemia disease and reduce the spread of the -thalassemia gene [9C12]. Polymerase chain reaction (PCR) is currently the most commonly used technique to diagnose 0-thalassemia [13C16]. However, this technique is not widely employed in routine laboratories of rural hospitals in resource-limited countries. Thus, the development of more cost effective and simplified techniques for identifying 0-thalassemia service providers are greatly needed for incorporation into the routine thalassemia screening programs of health promotion guidelines. In Southeast Asian countries, the Southeast Asian (SEA) deletion (–SEA) is the most common 0-thalassemia genotype [2, 8, 11, 17, 18]. The minute amounts of Hb Barts and -globin chains in red blood cells (RBCs) are especially observable in 0-thalassemia subjects, including those with 0-thalassemia (–SEA) [19C24]. Using a monoclonal antibody (mAb) generated in our lab, we previously developed an immunochromatographic (IC) strip test for detecting Hb Barts in RBC hemolysates [21, 25C27]. In this study, using a panel of our generated anti–globin chain mAbs [28], we established another IC strip test that can detect -globin chains in RBC lysates. The IC strips for Hb Barts and -globin chain detection were affirmed for their potential use in 0-thalassemia differentiation, especially in 0-thalassemia (–SEA) service providers. The clinical sensitivity, clinical specificity, positive predictive value (PPV) and unfavorable predictive value (NPV) of both IC strip tests were validated, and a new 0-thalassemia screening strategy was also proposed. Tropanserin Materials and methods Antibodies and reagents The anti–globin chain mAbs PL2 (IgG1 isotype) and PL3 (IgG1) [28] and the mouse anti-Ag85B mAb clone AM85B-8B (IgG1) [29] were generated in our laboratory. Goat anti-mouse IgG antibody was obtained from Jackson ImmunoResearch (West Grove, PA, USA). EZ-Link? Sulfo-NHS-LC-Biotin was purchased from Pierce (Rockford, IL, USA). Horseradish peroxidase (HRP)-labeled streptavidin and 3,3,5,5-tetramethylbenzidine (TMB) substrate were purchased from Invitrogen (Camarillo, CA, USA). Goat anti-mouse immunoglobulins antibody was obtained from KPL (Gaithersburg, MD, USA). The IC strip test for Tropanserin the determination of Hb Barts in RBC hemolysates was purchased from i+Med Laboratories Co., Ltd. (Bangkok, Thailand). Identification of Tropanserin an anti–globin chain mAb pair for use in an immunochromatographic strip test To identify an anti–globin chain mAb pair suitable for use in an IC strip test, a sandwich ELISA was employed. The anti–globin chain mAbs PL2 and PL3 or the isotype-matched control mAb were first biotinylated using EZ-Link? Sulfo-NHS-LC-Biotin according to manufacturer instructions. For the sandwich ELISA, the anti–globin chain mAbs PL2 and PL3 or the isotype-matched control (10 g/ml) were coated on 96-well ELISA plates (Costar, Corning, NY, USA) in carbonate/bicarbonate covering buffer CD350 pH 9.6 overnight at 4C. After washing, the plate was blocked with 2% skim milk in PBS at 37C for 1 hour. Hemolysates of Hb Barts hydrops fetalis were added and incubated at.