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

Abstract

Hemoglobin (Hb) A2 (alpha2delta2) is a minor hemoglobin in human red blood cells. An abnormal increase in the level of HbA2 is the most significant parameter in the diagnosis of beta-thalassemia carriers. In this study, we produced two monoclonal antibodies (mAbs) that specifically react to the delta-globin chain of HbA2. A sandwich type ELISA was developed employing the produced anti-HbA2 mAbs. HbA2 levels quantified by the developed sandwich ELISA were highly correlated with those obtained from the standard HPLC method (r = 0.934, p < 0.001). HbA2 levels determined by the ELISA were 4.4 +/- 1.9% in beta-thalassemia heterozygotes compared to 1.4 +/- 0.8, 1.9 +/- 0.8, 1.5 +/- 0.8 and 1.5 +/- 0.6% in normal subjects, HbE heterozygotes, suspected alpha-thalassemia heterozygotes and HbE homozygotes, respectively. Using a cut-off value of 2.5%, beta-thalassemia heterozygotes could be separated from non-beta-thalassemia heterozygotes with the same accuracy as obtained using the standard HPLC method. More importantly, the developed ELISA was able to determine HbA2 levels in HbE-bearing individuals which could not be done by the HPLC method. Our results suggest that this sandwich ELISA can be applied for mass screening for beta-thalassemia heterozygotes, especially in resource-limited countries, where beta-thalassemia is highly prevalent.

Production of monoclonal antibodies to P-glycoprotein: its application in detection of soluble and surface P-glycoprotein of leukemia patients

“…In order to determine soluble P-gp, along with that 2 mAbs to P-gp were generated in our laboratory, a sandwich-type ELISA was chosen to be the assay system [22]. Employing the produced anti-P-gp mAbs, an efficient sandwich ELISA was successfully developed.…”

Laboratory Diagnosis of β-Thalassemia and HbE

“…Using this ELISA, Shyamala found mean value of HbA 2 in normal and β-thalassemia carrier to be 2.5 and 5.4%, respectively. The mAb against HbA 2 was also produced and sandwich ELISA developed in the author's laboratory [30]. Under this developed sandwich ELISA, Kuntaruk found that the levels of HbA 2 between normal and β-thalassemia carrier were also significantly different (Figure 21).…”

“…• Hemoglobin studies: Tests for hemoglobin studies include cellulose acetate electrophoresis, microcolumn chromatography, alkaline denaturation test, cation-exchange high performance liquid chromatography (HPLC) [17][18][19], cation-exchange low pressure liquid chromatography (LPLC) [20][21][22], capillary zone electrophoresis (CZE) [23][24][25][26][27], sandwich enzyme linked immunosorbent assay (ELISA) for Hb F [28], Hb Bart's [29] and Hb A 2 [30], flow cytometric analysis of F cells [31], and immunochromatographic strip (IC strip) test for Hb Bart's [32].…”

“…In author's laboratory, this PCR protocol was modified and adapted to identify HbA 2 levels determined by sandwich ELISA set up in the author's laboratory. Note that HbA 2 levels in β-thalassemia trait are higher than those in normal, HbE trait, homozygous HbE, and suspected α-thalassemia trait (modified from [30]).…”

Les β-thalassémies de l'enfant camerounais : étude de la symptomatologie en fonction des différentes formes biologiques

“…La β-thalassémie est caractérisée par le déficit de synthèse de chaînes globiniques β [2]. Le manque d'hémoglobine normale A 1 , engendré par ce déficit, est compensé par une ou plusieurs hémoglobines compatibles avec la vie (HbF, HbA 2 ) [1,3,7,11,14]. L'électrophorèse de l'hémoglobine (Hb) est une technique qui permet de mettre en évidence ses différentes fractions et ainsi d'identifier plusieurs types d'hémoglobinopathies [4,10].…”