Specific radioimmunochemical identification and quantitation of hemoglobins a2 and f

Abstract

Hyperimmune antisera to chromatographically purified hemoglobins F and A2 were produced in rabbits and made specific for the immunogen by adsorption with normal human hemoglobin A conjugated to cyanogen bromide-activated agarose. A radioimmunoassay was established that permitted identification and quantitation of each of these two minor hemoglobins in hemolysates containing other hemoglobin components. The quantities of hemoglobins A2 and/or F present in hemolysates of individuals with beta-thalassemia, sickle cell anemia, Hb-C disease, and other hematological disorders were determined immunochemically, and the results were commpared to values obtained by microcolumn chromatography for the measurement of Hb-A2 or with the alkali denaturation technique in quantitating Hb-F. The immunoassay procedure has a greater sensitivity than other commonly employed techniques and can detect as little as 0.05 mug of these hemoglobins.

Detection of proteins like human gamma and beta globins in Escherichia coli carrying recombinant DNA plasmids.

“…Preparation and Absorption of Antisera. New Zealand White rabbits were immunized with the purified hemoglobins according to the procedure of Garver et al (16).…”

“…Hemoglobins were labeled with 125I by the chloramine-T procedure (20). Unbound 125I was eliminated by gel filtration and dialysis (16), and the preparations were stored at 40C.…”

“…A standard radioimmunochemical inhibition test (16,20) was used to determine the presence of immunoactive proteins from crude lysates of bacterial cultures. In these tests, proteins obtained from bacterial cultures were assayed for their ability to inhibit an 125I-labeled hemoglobin-antihemoglobin precipitation reaction.…”

Comparison of radial immunodiffusion and alkaline cellulose acetate electrophoresis for quantitating elevated levels of fetal hemoglobin (HbF): Application to evaluating patients with sickle cell disease treated with hydroxyurea

“…Monospecific polyclonal antibodies and monoclonal antibodies specific for HbF are available and have been incorporated into enzyme-linked immunosorbent assays (ELISA) (21,22), radioimmunoassays (RIA) (23,24), and magnetic affinity immunoassay (25), but their usage has been limited to a small number of investigators. Introduction by Helena Laboratories of a ready-to-use monospecific antibody-(antiserum) based immunodiffusion assay (HbF QUIPlate) into the clinical laboratory has made immunologic testing easier and available to a greater number of laboratories (26).…”

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

“…At birth, Hb A, is present in low amounts (0.05-0.45%) and then gradually increases to reach adult levels of 2.5-3% by the 40th week [2]. A large proportion of adult individuals heterozygous for p thal have elevated levels (3.5-7%) of Hb A, [3], and its quantitation in blood has become useful for diagnosis [4,5]. Hb A, is elevated in individuals heterozygous for p thal, S/p thal, and some acquired disorders such as megaloblastic anemia and hyperthyroidism.…”