Molecular Analysis of Common Types ofﾎｱ-Thalassemia Associated with(ﾎｲ-Thalassemia in Northern Thailand

We applied PCR strategies to detect the common types of ﾎｱ- thalassemia determinants which were associated with ﾎｲ- thalassemia in northern Thailand. Two types of deletions in the ﾎｱ-globin gene locus;the 18 kb deletion of Southeast Asian type(-ﾎｱSEA)and the 3.7 kb rightward deletion(-ﾎｱ3.7), and the most prevalent non-deletion mutation, Hb Constant Spring(ﾎｱC8, TAA to CAA at the codon 141)were investigated in 22 cases of ﾎｲ-thalassemia. Nine ﾎｲ-thalassemia patients were found to be associated with one or two of these defective ﾎｱ-globin gene determinants and the mean hemoglobin concentration in these patients was 6.3 ﾂｱ 1.1 g/dl whereas it was 5.6ﾂｱ.0.8 g/dl in 12 ﾎｲ-thalassemia patients without ﾎｱ- globin gene abnormalities ; the difference is statistically insignificant (p = 0.08). The level of anemia was severe in the ﾎｲ-thalassemia patients carrying a single ﾎｱ-globin gene abnormality in the heterozygous compounds ; whereas the (ﾎｲ-thalassemias withﾎｱ-globin gene defects in both alleles showed less severe anemia. A patient carrying the ﾎｱ ・｣8 determinant in homozygous compound showed the highest hemoglobin level among these ﾎｲ-thalassemia patients

Cloning and Expression of a Recombinant Single-Chain Variable Fragment Antibody Specific to Hemoglobin Bart’s

Hemoglobin Bart’s (γ4 ), an abnormal Hb, is a homotetramer of γ-globin chains . Τhe amount of this abnormal Hb in blood circulation can be used as an indicator for the presence of different genotypes of α-thalassemias. We successfully cloned and expressed a novel recombinant scFv antibody derived from mouse hybridoma producing monoclonal antibody highly specific to Hb Bart’s. The genes encoding variable regions of the heavy (VH) and light (VL) chains were cloned and identified by DNA sequencing. The VH and VL genes were connected via a short linker to form the full length VH-linker-VL construct and ligated into pET28a. The His tag-scFv fusion protein was expressed in E. coli and purified by affinity chromatography. The recombinant scFv antibody was mostly expressed as inclusion bodies with the predicted molecular weight of 28 kDa. This scFv antibody was very specific by reacting with Hb Bart’s (γ4) but not cross-react with HbA (α2β2), HbF (α2γ2), HbS (α2β2 S ), HbE (α2β2 E ), HbA2 (α2δ2 ), and HbH (β4 ), as determined by Western blot. The detection sensitivity of this scFv antibody was 5 µg/µl of Hb Bart’s by dot blot ELISA. The scFv antibody should be useful in development of an immunoassay with high sensitivity and specificity for the diagnosis of α-thalassemias

STAPHYLOCOCCAL DELTA HEMOLYSIN.

STAPHYLOCOCCAL DELTA HEMOLYSIN