Simple method for screening of α-thalassaemia 1 carriers

Abstract

Alpha-thalassaemia 1 genetic disorder occurs when there is a deletion of two linked alpha-globin genes. The interaction between these abnormal genes leads to the most severe type of thalassaemia disease, haemoglobin (Hb) Bart's hydrops fetalis. The identification of alpha-thalassaemia 1 carriers and genetic counselling are essential for the prevention and control of severe thalassaemia diseases. In this study, we have developed a rapid screening method for identifying alpha-thalassaemia 1. A sandwich-type immunochromatographic (IC) strip test was developed, using the generated monoclonal anti-Hb Bart's antibody, to trace the Hb Bart's in haemolysates. When assayed by our IC strip test, all alpha-thalassaemia 1, HbH disease, HbH-Constant Spring (H-CS) disease, HbH-CS and heterozygous HbE (CSEA) Bart's disease, and homozygous alpha-thalassaemia 2 showed positive results. No false negative results were observed in these blood samples. In alpha-thalassaemia 2 heterozygotes, 83% of them showed positive reactivity. Among HbE (both homozygotes and heterozygotes), beta-thalassaemia (heterozygotes, homozygotes and beta-thalassaemia/HbE) and normal subjects, the IC strip test revealed negative reactivity of 100, 85 and 97%, respectively. These results indicate that this novel immunodiagnostic kit, in combination with red blood cell indices, is suitable for screening and ruling out mass populations for the presence of alpha-thalassaemia 1.

Routine screening for α-thalassaemia using an immunochromatographic strip assay for haemoglobin Bart's

“…13,14 Polymerase chain reaction (PCR) and related methods are routinely utilized to identify b-thalassaemia mutations and six a-thalassaemia alleles common in Thailand (ie. a 0 -thalassaemia SEA & THAI deletions, a þ -thalassaemia 3.7 & 4.2 kb deletions, Hb Constant Spring and Hb Pakse´1 [5][6][7][8][9][10][11][12][13][14][15][16][17] ). Thalassaemia genotypes were defined.…”

A modified hybridoma technique for production of monoclonal antibodies having desired isotypes

“…Hemoglobin F (HbF) was purified from hemolysates of normal umbilical cord blood by DEAE Sepharose chromatography (Tayapiwatana et al 2009). Briefly, the hemolysates were dialyzed against the binding buffer (Tris-HCl-KCN (THK) pH 9.0) for overnight.…”

Sandwich ELISA for hemoglobin A2 quantification and identification of β-thalassemia carriers

“…Hbs A 2 , A, F, E were isolated from hemolysates via anionexchange liquid chromatography, whereas Hb Portland and Hb Bart's were purified by cellulose acetate electrophoresis with elution as described elsewhere [3,19]. The purified HbA 2 was used as an immunogen for antibody production and the rest used as antigens for determining specificity of the produced mAbs.…”

“…This genetic disorder leads to a reduction or total absence of one or more globin chains of Hb in affected individuals. The imbalance in globin chain synthesis leads to homotetramer formation of excess c-and b-globin chains in a-thalassemia, and to excess a-globin in b-thalassemia [1][2][3]. Currently, both a and b thalassemias have become global concerns, not only as health problems but also as a socioeconomic burden.…”

“…Furthermore, a large number of samples can be analyzed in a short time. Therefore, the immunological technique has been suggested to be an appropriate method for screening and diagnosis of thalassemia in resource-limited countries [3,15]. Several immunological techniques have been developed for HbA 2 quantification [15][16][17][18].…”