skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Monoclonal antibodies specific for sickle cell hemoglobin

Abstract

Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Lawerence Livermore National Lab., CA
OSTI Identifier:
6105446
DOE Contract Number:
W-7405-ENG-48
Resource Type:
Journal Article
Resource Relation:
Journal Name: Hemoglobin; (United States); Journal Volume: 9:4
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; HEMOGLOBIN; IMMUNE REACTIONS; SCREENING; MONOCLONAL ANTIBODIES; BIOCHEMICAL REACTION KINETICS; AMINO ACIDS; CELL FLOW SYSTEMS; ELECTROPHORESIS; HYBRIDOMAS; MICE; PEPTIDES; SICKLE CELL ANEMIA; ANEMIAS; ANIMAL CELLS; ANIMALS; ANTIBODIES; CARBOXYLIC ACIDS; DISEASES; GLOBIN; HEMIC DISEASES; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; KINETICS; MAMMALS; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PIGMENTS; PORPHYRINS; PROTEINS; REACTION KINETICS; RODENTS; SYMPTOMS; VERTEBRATES; 550200* - Biochemistry

Citation Formats

Jensen, R.H., Vanderlaan, M., Grabske, R.J., Branscomb, E.W., Bigbee, W.L., and Stanker, L.H. Monoclonal antibodies specific for sickle cell hemoglobin. United States: N. p., 1985. Web. doi:10.3109/03630268508997010.
Jensen, R.H., Vanderlaan, M., Grabske, R.J., Branscomb, E.W., Bigbee, W.L., & Stanker, L.H. Monoclonal antibodies specific for sickle cell hemoglobin. United States. doi:10.3109/03630268508997010.
Jensen, R.H., Vanderlaan, M., Grabske, R.J., Branscomb, E.W., Bigbee, W.L., and Stanker, L.H. Tue . "Monoclonal antibodies specific for sickle cell hemoglobin". United States. doi:10.3109/03630268508997010.
@article{osti_6105446,
title = {Monoclonal antibodies specific for sickle cell hemoglobin},
author = {Jensen, R.H. and Vanderlaan, M. and Grabske, R.J. and Branscomb, E.W. and Bigbee, W.L. and Stanker, L.H.},
abstractNote = {Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.},
doi = {10.3109/03630268508997010},
journal = {Hemoglobin; (United States)},
number = ,
volume = 9:4,
place = {United States},
year = {Tue Jan 01 00:00:00 EST 1985},
month = {Tue Jan 01 00:00:00 EST 1985}
}
  • Antibodies to hemoglobin have been studied with a radioimmunoassay which employs [$sup 14$C]carbamylated (= carbamoylated) hemoglobin S. An antiserum raised against hemoglobin S, which initially discriminated poorly between hemoglobins S and A, was fractionated by absorption to a column of Sepharose to which a synthetic peptide corresponding to the first 13 amino-acid residues of the $beta$ chain of sickle hemoglobin had been covalently bound. A subpopulation of the antiserum was eluted from this column with 4 M guanidine . HCl. These antibodies showed binding to hemoglobin S but not to hemoglobin A and this interaction could be inhibited by themore » synthetic peptide. These antibodies, of demonstrated fine structural specificity, may be useful in the detection of sickle hemoglobin and in the study of its structure in solution. (auth)« less
  • The kinetics of deoxyhemoglobin S gelation have been investigated using photolytic dissociation of the carbon monoxide complex to initiate the process. Measurements over a wide range of times, 10/sup -3/ -10/sup 4/s, show that both the concentration dependence of the tenth-time (i.e., the time required to complete one-tenth the reaction) and the time dependence of the process decrease as gelation speeds up. In slowly gelling samples, where single domains of polymers are formed in the small sample volumes employed with this technique (1 to 2 x 10/sup -9/ cm/sup 3/), there is a marked increase in the variability of themore » tenth-times. These results are explained by a mechanism in which gelation is initiated by homogeneous nucleation of polymers in the bulk solution phase, followed by heterogeneous nucleation on the surface of existing polymers. At the lowest concentrations, homogeneous nucleation is so improbable that stochastic behavior is observed in the small sample volumes, and heterogeneous nucleation is the dominant pathway for polymer formation, thereby accounting for the high time dependence. At the highest concentrations homogeneous nucleation becomes much more probable, and the time dependence decreases. The decrease in concentration dependence of the tenth-time with increasing concentration results from a decrease in size of both the homogeneous and heterogeneous critical nuclei. The model rationalizes the major observations on the kinetics of gelation of deoxyhemoglobin S, and is readily testable by further experiments.« less
  • The four recognized levels of organization of protein structure (primary through quaternary) are extended to add the designation quinary structure for the interactions within helical arrays, such as found for sickle cell hemoglobin fibers of tubulin units in microtubules. For sickle cell hemoglobin the main quinary structure is a 14-filament fiber, with a number of other minor forms also encountered. Degenerate forms of the 14-filament fibers can be characterized that lack specific pairs of filaments; evidence is presented which suggests an overall organization of the 14 filaments in pairs, with particular pairs aligned in an antiparallel orientation. For tubulin, amore » range of quinary structures can be detected depending on the number of protofilaments and whether adjacent protofilaments composed of alternating ..cap alpha..- and ..beta..-subunits are aligned with contacts between like or unlike subunits and with parallel or antiparallel polarity. Thus, in contrast to quarternary structure, which generally involves a fixed number of subunits, the quinary structures of proteins can exhibit marked plasticity and inequivalence in the juxtaposition of constituent molecules.« less
  • The locus control region (LCR), composed of four hypersensitive sites (HS1-4) 5{prime} of the {epsilon} globin gene, confers strong, copy-number dependent expression on globin genes in transgenic mice. Several {beta}-globin gene cluster haplotypes carry the sickle cell gene, and show variable levels of fetal hemoglobin (Hb F) expression in association with DNA sequence differences in HS2, {gamma} and {beta} globin promoters, and {gamma}IVSII: The Senegal (SEN or No. 3) haplotype generally has high (>10%) Hb F, Benin (BEN or No. 19) has intermediate Hb F (but some low and some high), and Banu (BAN or No. 20) generally has lowmore » Hb F. Huisman and colleagues have proposed that `factors produced under conditions of hematopoietic stress, together with genetic determinants on the haplotype-3 like LCR sequences, allow for high level expression of {gamma} globin genes`. We have now used slot blot to screen high Hb F (>9.5%) and low Hb F cases for two of the three HS2 point mutations described by Oener et al. Comparing eight high Hb F BEN/BEN with two low Hb F BEN/BEN, all ten had the BEN mutations considered by Oener et al. to be associated with low Hb F. Comparing three high Hb F BEN/BAN with two low Hb F BEN/BAN, all five were heterozygous at three positions; this is consistent with BEN having G and T and BAN having A at both positions. DNA sequencing of HS2 for BAN, which is generally associated with low HB F, showed that the point mutations at all three positions were those seen in SEN (generally high Hb F); only the AT repeat region showed major differences, confirming results of Huisman and colleagues. Hence, if there is any effect of HS2 of the Senegal sickle cell haplotype in causing elevated Hb F under hematopoietic stress, it must be due to specific variation in the AT repeat region, which Oener et al. have suggested may bind a silencer.« less