ABSTRACT
Couples in which both partners carry the α-thalassemia-1 trait have a 25% risk of hemoglobin Bart's hydrops fetalis in each pregnancy. Identification of α-thalassemia-1 trait is, therefore, necessary in order to control this severe form of α-thalassemia. We have generated monoclonal antibodies specific to the ζ-globin chain without cross reaction with other globin chains. A simple and sensitive ELISA was developed by using poly-l-lysine to increase the protein binding to the ELISA plate. The developed poly-l-lysine pre-coated ELISA has a very high sensitivity (100%) and specificity (97%) for detection of carriers of α-thalassemia-1 with Southeast Asian-type deletion.
Subject(s)
Asian People/genetics , Enzyme-Linked Immunosorbent Assay/methods , Gene Deletion , alpha-Thalassemia/diagnosis , alpha-Thalassemia/genetics , zeta-Globins/analysis , zeta-Globins/genetics , Animals , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Asia, Southeastern , Humans , Mice , Mice, Inbred BALB C , Sensitivity and Specificity , zeta-Globins/immunologyABSTRACT
AIMS: The Southeast Asian (SEA) deletion (--(SEA)) represents the most common determinant causing alpha thalassaemia in Southeast Asian countries. The embryonic zeta-globin chain has been defined as a marker for the detection of this deletion in adults. The aim of this study was to develop an appropriate low-cost ELISA for zeta-globin chain detection as a routine screening test for (--(SEA)) alpha thalassaemia deletion. METHODS: A sandwich ELISA system for zeta-globin chains was established with a pair of zeta-globin-specific monoclonal antibodies prepared in-house, and locally made products. Against a gap-PCR method that was taken as the standard, this assay was validated in a cohort study testing a total of 526 individuals comprising patients scheduled for haemoglobinopathy diagnostic analysis and normal individuals. Routine screening of the (--(SEA)) deletion in 300 random student volunteers was conducted using the assay. RESULTS: While the cut-off point was set at a percentage positive value of 30, the sensitivity and specificity of this ELISA method were 100% and 99.24%, respectively. The mean intra-assay and inter-assay coefficients of variation among the different concentrations in the optimised ELISA conditions were 2.1-11.4% and 4.3-13.2%, respectively. Seventeen of the 300 volunteers sampled were determined by the ELISA to have the (--(SEA)) deletion; these results were in 100% agreement with the gap-PCR results. CONCLUSIONS: This study validates the ELISA method described here as a simple, rapid and cost-effective assay that is potentially adaptable for application in large-scale population screening for this prevalent disorder in SEA areas such as southern China.
