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Development of a rapid and accurate immunological diagnostic method for alpha-thalassaemia screening
Author
Lai, Choon Ming
Supervisor
Gan-Yap, Yik Yuen
George, Elizabeth
Abstract
Alpha thalassaemia is a major genetic disorder that affects the production of a-globin chain of the haemaglobin commonly found in Southeast Asia. As a result of defective α-globin chain synthesis, individuals with this disorder show varying degree of anaemia due to ineffective erythropoiesis and haemolysis.
The percentage of α-thalassaemia occurrence in this region is thought to be increasing if no proper preventive programmes are taken place. To date, rapid detection and prenatal diagnosis followed by genetic counseling are the effective means to prevent the elevated valency of a-thalassaemia spectrum in Southeast Asia.
The presence of Hb H inclusion bodies in a-thalassaemia subjects has enabled the detection of Hb H using immunologic methods. The objective of this study is to develop a simple, rapid and accurate immunological diagnostic kit for the detection of α-thalassaemia by means of an Enzyme-linked Immunosorbent Assay (ELISA).
A murine monoclonal antibody specific to Hb H (В4) was developed by immunising mice with reconstituted В4. The synthetic В4 was generated by separating the α and В chains from normal Hb A and reconstituted the В chains to form В4. A few hybridoma clones showed specificity to Hb H but three clones with the highest specificity were evaluated (2C65B/9E, 5E5 and 8E5). Meanwhile, polyclonal antibodies specific to Hb H were developed by immunising sheep with FPLC-purified Hb H.
These antibodies were employed in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect and quantify Hb H in haemolysates from subjects with a-thalassaemia. Various designs of ELISA were tried but a three-layer sandwich-ELISA was found to be most suitable. The sandwich design consisted of sheep polyclonal antibodies that were specific to Hb H immobilized on microtiter plate as the first layer, followed by haemolysates as second layer; the third layer comprised of the murine monoclonal antibody specific to В4 and conjugated to horseradish peroxidase; and lastly tetramethyl benzidine was used as substrate.
Using the third sandwich ELISA method , a total; of 768 blood samples, consisting of 511 anaemic samples with low haemoglobin count and low mean corpuscular volume, 223 samples from normal subjects, 34 samples with abnormal haemoglobins, in which 18 samples were confirmed to have Hb H disease and 16 samples with α-thalassaemia trait were screened. An OD value of 0.2 was chosen as the cut-off value for normal subjects. Samples with OD values between 0.2 and 0.4 would be diagnosed as positive for α-thalassaemia trait, and those with OD values above 0.4 would be diagnosed as Hb H disease. By using these criteria, the sensitivities for detecting the Hb H disease and α-thalassaemia trait were 100% and 68.8% respectively. Using the same criteria, the specificity of the assay was tested and found to be 98.9%.
A second group of samples were screened using the second sandwich ELISA design. This group of samples consisted of 28 Hb H disease, 36 α-thalassaemia traits and 31 normal subjects. An OD value of 0.14 was chosen as the cut-off for normal subjects. Samples with OD values between 0.14 and 0.2 would be diagnosed as positive α-thalassaemia trait, and those with OD values above 0.2 would be diagnosed as Hb H disease. By using these criteria, the sensitivities for detecting Hb H disease and α-thalassaemia trait were 89.3% and 61.1% respectively.
During the analysis, 8 samples which were diagnosed as normal by the conventional methods in the hospital were shown to have abnormally high OD values by the second sandwich ELISA design. These samples were further analyzed and confirmed as Hb Constant Spring carriers.
There was one sample which was diagnosed as Hb Constant Spring (αα/ααcs) showed unexpected low OD value in the range of normal samples. This sample was further investigated by DNA analysis and confirmed to have concomitant inheritance of В-thalassaemia. Thus, the reason for the low sensitivity in detecting α-thalassaemia trait may be due to concomitant inheritance of В-thalassaemia
This method is very suitable for the large scale screening of Hb H disease and α-thalassaemia trait due to its simplicity and feasibility. In addition, this method may be useful for detecting carriers of (--SEA/ ) deletion and deletions involving the complete ζ-α-globin gene cluster, such as (--Fil/ ), (--Thai/ ) and (--HW/ ), which are the common deletional α-thalassaemias in Southeast Asians.
The percentage of α-thalassaemia occurrence in this region is thought to be increasing if no proper preventive programmes are taken place. To date, rapid detection and prenatal diagnosis followed by genetic counseling are the effective means to prevent the elevated valency of a-thalassaemia spectrum in Southeast Asia.
The presence of Hb H inclusion bodies in a-thalassaemia subjects has enabled the detection of Hb H using immunologic methods. The objective of this study is to develop a simple, rapid and accurate immunological diagnostic kit for the detection of α-thalassaemia by means of an Enzyme-linked Immunosorbent Assay (ELISA).
A murine monoclonal antibody specific to Hb H (В4) was developed by immunising mice with reconstituted В4. The synthetic В4 was generated by separating the α and В chains from normal Hb A and reconstituted the В chains to form В4. A few hybridoma clones showed specificity to Hb H but three clones with the highest specificity were evaluated (2C65B/9E, 5E5 and 8E5). Meanwhile, polyclonal antibodies specific to Hb H were developed by immunising sheep with FPLC-purified Hb H.
These antibodies were employed in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect and quantify Hb H in haemolysates from subjects with a-thalassaemia. Various designs of ELISA were tried but a three-layer sandwich-ELISA was found to be most suitable. The sandwich design consisted of sheep polyclonal antibodies that were specific to Hb H immobilized on microtiter plate as the first layer, followed by haemolysates as second layer; the third layer comprised of the murine monoclonal antibody specific to В4 and conjugated to horseradish peroxidase; and lastly tetramethyl benzidine was used as substrate.
Using the third sandwich ELISA method , a total; of 768 blood samples, consisting of 511 anaemic samples with low haemoglobin count and low mean corpuscular volume, 223 samples from normal subjects, 34 samples with abnormal haemoglobins, in which 18 samples were confirmed to have Hb H disease and 16 samples with α-thalassaemia trait were screened. An OD value of 0.2 was chosen as the cut-off value for normal subjects. Samples with OD values between 0.2 and 0.4 would be diagnosed as positive for α-thalassaemia trait, and those with OD values above 0.4 would be diagnosed as Hb H disease. By using these criteria, the sensitivities for detecting the Hb H disease and α-thalassaemia trait were 100% and 68.8% respectively. Using the same criteria, the specificity of the assay was tested and found to be 98.9%.
A second group of samples were screened using the second sandwich ELISA design. This group of samples consisted of 28 Hb H disease, 36 α-thalassaemia traits and 31 normal subjects. An OD value of 0.14 was chosen as the cut-off for normal subjects. Samples with OD values between 0.14 and 0.2 would be diagnosed as positive α-thalassaemia trait, and those with OD values above 0.2 would be diagnosed as Hb H disease. By using these criteria, the sensitivities for detecting Hb H disease and α-thalassaemia trait were 89.3% and 61.1% respectively.
During the analysis, 8 samples which were diagnosed as normal by the conventional methods in the hospital were shown to have abnormally high OD values by the second sandwich ELISA design. These samples were further analyzed and confirmed as Hb Constant Spring carriers.
There was one sample which was diagnosed as Hb Constant Spring (αα/ααcs) showed unexpected low OD value in the range of normal samples. This sample was further investigated by DNA analysis and confirmed to have concomitant inheritance of В-thalassaemia. Thus, the reason for the low sensitivity in detecting α-thalassaemia trait may be due to concomitant inheritance of В-thalassaemia
This method is very suitable for the large scale screening of Hb H disease and α-thalassaemia trait due to its simplicity and feasibility. In addition, this method may be useful for detecting carriers of (--SEA/ ) deletion and deletions involving the complete ζ-α-globin gene cluster, such as (--Fil/ ), (--Thai/ ) and (--HW/ ), which are the common deletional α-thalassaemias in Southeast Asians.
Date Issued
1995
Call Number
RC641.7.T5 Lai
Date Submitted
1995