HEMOGLOBIN, 14(4), 389-398 (1990)

ENZYME IMMUNOASSAY FOR THE I D E N T I F I C A T I O N OF HEMOGLOBIN VARIANTS

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H. MOSCOSO, C.R. Kiefer, M. Shyamala and F.A. Garver Department of Cell and Molecular Biology Medical College of Georgia, Augusta, Georgia 30912-2100, U.S.A. ABSTRACT

We have prepared monospecific antibodies to Hbs D-Los Angeles, J-:altimore, O-Arab and J-Paris-I and developed an enzyme immunoassay (ELISA) for their identification in hemolysates. Hbs in adult or cord blood hemolysates were coated to the wells of microtiter plates and reacted with the appropriate antisera followed by the detection system which contains antirabbit IgG/peroxidase conjugate and the substrate tetramethylbenzidine. Sixty-nine samples were tentatively considered to contain the above hemoglobin variants by isoeleutrofocusing and Some o f the the identity of 83% of them was confirmed by ELISA non-reacting hemolysates were shown by amino acid sequence analysis to contain Hbs Korle-Buy D-Ibadan, G-Copenhagen and the new variant Chandigarh. This ELISA offers specificity and simplicity for tile confirmatory identification of hemoglobin variants. INTRODUCTION

The presumptive identification of hemoglobin (Hb) variants i s usually carried out by electrophoresis on cellulose acetate (1). However, a significant number of variants have similar substitutions that do not alter the overall charge of the protein (2). To identify these variants conclusively other techniques __-___--.

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Received : Januaruj 70, 1 9 9 0 .

Accepted : June 13, 7 9 9 0 . 389

Copyright 0 1990 by Marcel Dekker, Inc.

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such as electrophoresis on citrate agar at pH 6-6.5 (3), electrophoresis of globin chains (4), isoelectrofocusing (IEF) ( 5 ) , high performance liquid chromatography (HPLC) ( 6 ) or even amino acid sequence analysis (1) must be employed. Hb 0-Los Angeles [ a2B2121(GH4)Glu+Gln] is the most commonly encountered " 0 " variant ( 7 ) and the fourth most frequent B chain variant worldwide ( 8 ) . Under standard electrophoresis conditions, " 0 " and "GI' variants comigrate with Hb S, and citrate agar electrophoresis or a solubility test are required for a tentative identification of Hb S (8). In addition, " 0 " and "G" variants cannot be resolved under alkaline or acid conditions and their identifications are based upon globin chain studies ( 8 ) . Although homozygotes for Hb D-Los Angeles have no clinical significance, the double heterozygotes, Hb S/Hb 0-Los Angeles and Hb 0-Los Angeles/B"-thalassemia (thal), produce adverse clinical effects (8). Similarly, Hb O-Arab [a2B2121(GH4)Glu+Lys] comigrates with Hbs Ap, C and E on cellulose acetate (9) and further studies on citrate agar (10) or HPLC (11) are necessary for a tentative identification, especially in the case of the double heterozygote Hb S/O-Arab, which is a severe sickling disorder (8). The identification of "J" variants is even more difficult since 38 "J" variants have been described (2). Among these are Hb J-Baltimore [a2B216(A13)Gly+Asp], the most common "J" variant (12) , and Hb J-Paris-I [a212(AlO)Ala+AspB2], one of the four most common " J " variants of the a. chain (13). Immunological approaches for identification of Hb variants provide specificity, sensitivity, simplicity and rapidity. We have utilized these qualities by incorporating monospecific antibodies in RIA (14,15), ELISA (16,17,18), Western blot (19,20) and magnetic affinity immunoassays (21), and more recently by the development of monoclonal antibody-based immunoassays (22,23,24). Here we describe the development of an ELISA for the detection of four relatively common Hb variants, 0-Los Angeles, J-Baltimore, O-Arab and J-Paris-I.

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ENZYME IMMUNOASSAY

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MATERIALS AND METHODS Blood Samples and Hb Purification. Hemolysates of blood samples from normal adult individuals or from adults heterozygous for the variants were prepared as described elsewhere (14). Normal and variant Hbs were purified by column chromatography on DEAE-cel lulose (Whatman DE-52) (25). Antiserum Preparations. Antisera were generated by injecting the purified Hb variants into rabbits ( 1 4 , 1 5 ) . Monospecificity for the variants was obtained by imrnunoaffinity absorptions with Hb A. Antiserum to Hb O-Arab was also absorbed with Hb A2. For immunoabsorptions, Hbs A or A2 were conjugated to AH-sepharose 46 (Sigma Chemical Co., St. Louis, MO) (17). Imnunoassavs. The specificity and titers of these antisera were evaluated by RIA, and the identification of Hb variants utilized inhibition-RIA, as previously described (14,15). The basic principles of the ELISA for Hb have been reported (22). Specificities and titers of the antisera were determined with 5 pg of Hb variants or normal Hbs attached to wells of microtiter plates. Unoccupied binding sites were blocked and the wells were incubated with various dilutions of the antisera. The detection system utilized goat anti-rabbit IgG/peroxidase conjugate (Bio-Rad Laboratories, Richmond, CA) and the substrate tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO). Sensitivity of the assays was determined for cord or adult hemolysates which contained the appropriate variants by coupling the Hbs to the wells at 10-fold dilutions from 500 pg to 0.5 ng/well and reacted with a constant dilution of the corresponding antisera. We analyzed 98 hemolysates (79 adults, 19 cords) which had been previously identified by IEF (5) and/or HPLC ( 6 , l l ) . These hemolysates included 36 D-Los Angeles, 26 J-Baltimore, four O-Arab, three J-Paris-I, 20 normal adults (AA) , and nine normal cords (FA).

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RESULTS

The rabbit anti-human Hb variant sera were absorbed with Hb A to achieve specificity for the variants. Fig. 1 shows the RIA titrations of the absorbed antisera. Although significant cross-reactivities with Hb A were observed at serum dilutions

Enzyme immunoassay for the identification of hemoglobin variants.

We have prepared monospecific antibodies to Hbs D-Los Angeles, J-Baltimore, O-Arab and J-Paris-I and developed an enzyme immunoassay (ELISA) for their...
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