Application of a monoclonal antibody specific for the δ chain of hemoglobin A₂ in the diagnosis of β thalassemia

Abstract

We have developed a murine monoclonal antibody (mAb) specific for the delta chain of hemoglobin (Hb) A2 that does not cross-react with alpha, beta, or gamma chains. The mAb reacted with Hb P-Nilotic (beta delta hybrid), but not with Hb Lepore-Boston (delta beta hybrid), indicating an epitope consisting of positions 116 (Arg) and 117 (Asn) or 125 (Gln) and 126 (Met) of the delta chain. By using this antibody, we have established a simple and rapid enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of Hb A2 in adult, cord, and fetal hemolysates. We analyzed 70 adult, 8 newborn, and 19 fetal hemolysates from normal subjects and those with various hemoglobinopathies. The mean percentage of Hb A2 was 2.5 for normal adults, 5.4 for beta thalassemic (beta thal) heterozygotes, and less than 0.1% in beta thal fetal samples. We were able to distinguish and characterize certain phenotypes of beta thal patients such as beta thal heterozygotes, beta 0 thal homozygotes, and C beta 0 thal, and C beta + thal double heterozygotes with the aid of this and other mAbs we have generated. This technique is a valuable addition to current methods for the diagnosis of beta thal based on quantification of Hb A2.

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

“…Sandwich ELISA is an appropriate assay for determination of a target molecule present in the mixtures, which is in accordance with our aim of determining HbA 2 in the hemolysate containing various other proteins. The sandwich ELISA using anti-HbA 2 mAbs has more benefits compared to the immunological methods for HbA 2 quantification described previously [15][16][17][18]. HbA 2 monospecific polyclonal antibodies had been used to develop ELISA for determination of HbA 2 [15][16][17].…”

“…Using polyclonal antibodies for routine purposes is, therefore, inappropriate. MAbs specific to HbA 2 have previously been reported [18,27], and an ELISA was developed for HbA 2 determination using an anti-HbA 2 mAb [18]. However, the developed ELISA was based on the direct coating of hemolysate samples onto the ELISA plate.…”

“…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]. However, most of the described methods employ polyclonal antibodies in the assay [15][16][17].…”

“…This mAb specifically reacts with HbA 2 and was applied for an ELISA for HbA 2 quantification. However, the developed method was the direct ELISA by which the hemolysate samples were directly coated on ELISA plate and the HbA 2 was determined using the HbA 2 mAb [18]. This type of ELISA may produce erroneous results, as other proteins present in the sample may compete with HbA 2 in binding to the plate.…”

“…Therefore, using polyclonal antibodies for establishment of immunological methods is impractical. Monoclonal antibodies (mAbs) specific for the d-globin chain have been described [18]. This mAb specifically reacts with HbA 2 and was applied for an ELISA for HbA 2 quantification.…”