Monoclonal antibodies specific for globin chains.

Six monoclonal antibodies specific for human globin chains are described. They are produced by stable clones obtained by raising hybridomas using cells of mice immunized with either adult or fetal hemoglobin. Characterization of the antibodies included testing against tetrameric human and other animal hemoglobins, isolated hemoglobin chains, and when indicated, cyanogen bromide fragments.

De novo mutations producing unstable Hbs or Hbs M. II. Direct estimates of minimum nucleotide mutation rates in man.

Cases of unstable hemoglobin and hemoglobin M disease that have appeared as de novo mutants over a span of approximately 50 years were used to deriving minimal, direct estimates of mutation rates per nucleotide per generation in man. The estimates are based upon analysis of data related to 40 cases of unstable Hbs and 15 of Hbs M that arose in 13 countries. The estimated rate calculated using all de novo beta-gene mutants is 7.

Mapping of antigenic sites on human haemoglobin by means of monoclonal antibodies and haemoglobin variants.

Monoclonal antibodies specific for human globin chains have been prepared and the following strategy has been applied in delimiting the antigenic sites involved in antibody binding. The structural sites of the human globin subunit that might be recognised by the monoclonal antibody were deduced from comparisons of the primary structures of mammalian globin chains that did or did not react with the antibody. The involvement of individual residues at these specific sites was subsequently tested by reacting the antibody with abnormal human haemoglobins in which there was either a substitution or a deletion of one of the residues in question.

De novo mutations producing unstable hemoglobins or hemoglobins M : I. Establishment of a Depository and use of data to test for an association of de novo mutation with advanced parental age.

Hemoglobins M and unstable hemoglobins cause clinical syndromes that are transmitted in autosomal dominant fashion. Pedigrees of 50 probands with de novo mutations producing unstable Hb disease or Hb M disease were compiled. Cases were ascertained (1) by screening the relevant literature published from 1950 through 1980 and (2) through personal communication.

Toward a system for detecting somatic-cell mutations. V. Preparation of fluorescent antibodies to hemoglobin hasharon, a human alpha-chain variant.

Antibodies against the abnormal human hemoglobin, Hb Hasharon (alpha 47 Asp leads to His), were raised in horse and purified by absorption against Sepharose 4B to which normal hemoglobins or Hb Hasharon were bound. The purified, non-precipitating antibodies were tested for specificity against normal hemoglobins and Hb Hasharon by immunodiffusion in the presence of anti-horse IgG, and by exposing mixtures of normal and Hb Hasharon-containing red cells to the antibodies after conjugation of the latter with fluorescein isothiocyanate. The ease with which antibodies specific for different variant hemoglobins have been prepared, and their potential for identifying individual erythrocytes that contain these hemoglobins by virtue of somatic mutation, underscore their value as aids to detection and analysis of mutational events in human subjects.

Fetal hemoglobin of the rhesus monkey, Macaca mulatta: complete primary structure of the gamma chain.

The complete amino acid sequence of the gamma chain from the major one of two fetal hemoglobins from the rhesus monkey, Macaca mulatta, was determined by automated, stepwise degradation of selected fragments produced by cleavage at methionyl and tryptophanyl residues and at the single aspartylprolyl bond. The minor fetal hemoglobin is single aspartylprolyl bond. The minor fetal hemoglobin is similar to human Hb F1 in relative electrophoretic and chromatographic properties and in the level at which it is found (about 12% of the total Hb F).

Development of a somatic mutation screening system using Hb mutants. IV. Successful detection of red cells containing the human frameshift mutants Hb Wayne and Hb Cranston using monospecific fluorescent antibodies.

The production and purification of antibodies detecting Hb Wayne, an alpha-globin frameshift mutant, and Hb Cranston, a beta-globin frameshift mutant, are described. The antibodies are of a nonprecipitating nature, and they permit strong fluorescent labeling of erythrocytes containing Hb Wayne or Hb Cranston. Studies using artificial mixtures containing cells with either of the two mutants in frequencies ranging from 1 in 10(2) to 1 in 10(5) showed that fluorescent antibodies can detect rare mutant red cells in the presence of vast excesses of normal erythrocytes.

Complete primary structure of the beta chain from the hemoglobin of a baboon, Papio cynocephalus.

The complete primary structure of the beta chain from the adult hemoglobin of a baboon, Papio cynocephalus, has been determined by automated, Edman degradation of the intact chain and four fragments derived therefrom by specific cleavage reactions. The analysis was facilitated by application of a modified solvent system that permits unambiguous identification, by high-performance liquid chromatography, of the 17 amino acids whose phenylthiohydantoin derivatives are soluble in ethyl acetate. The sequence obtained differs from that of the human beta chain at eight sites, a degree of divergence similar to that observed when human and macaque beta chains are compared.

Hb F production of endogenous colonies of polycythemia vera.

Fetal hemoglobin was studied in endogenous colonies produced in plasma clot and methylcellulose cultures of circulating progenitors from patients with polycythemia vera (PV). Analysis of globin chain synthesis showed that gamma chains constituted from 13% to 42% of the non-alpha chains produced in cultured cells, whereas from 27% to over 50% of the endogenous colonies contained Hb F, as indicated by the fluorescent antibody probe. Since the endogenous colonies in PV cultures originate from the abnormal PV clone, the findings provide direct evidence that a single pluripotent stem cell can have committed progeny that differ in their expressions of the Hb F production program.

Complete amino acid sequence of the gamma chain from the major fetal hemoglobin of the pig-tailed macaque, Macaca nemestrina.

The complete primary structure of the gamma chain of the major fetal hemoglobin from the pig-tailed macaque, Macaca nemestrina, was obtained by the automated sequencing of fragments produced by three nonenzymatic cleavage reactions. About two-thirds of the sequence was established from the amino terminus of the intact chain and two of the three fragments produced by cleavage at methionyl residues by cyanogen bromide. Acid clevage at the single aspartyl-prolyl linkage and cleavage at tryptophanyl residues in intact chains yielded the two fragments necessary to complete the sequence.

Complete sequence of the gamma chain from the fetal hemoglobin of the baboon, Papio cynocephalus.

The amino acid sequence of the hemoglobin gamma chain from a baboon, Papio cynocephalus, was determined by automated sequencing of the intact chain and six fragments generated by specific clevage reactions. The existence of structural heterogeneity at position 75, where both valyl and isoleucyl residues were found, is suggestive of the presence of nonallelic V gamma- and I gamma-chain genes in this species, and further emphasizes the extent to which the genetic basis of hemoglobin production among many higher primates is similar. Comparison of the sequences of those gamma chains from Homo sapiens, Pan troglodytes, Macaca nemestrina and P.

Consistent activation of fetal hemoglobin synthesis in cultured adult bone marrow cells.

The production of fetal hemoglobin was investigated in plasma clot cultures of adult bone marrow cells from normal donors and from individuals with homozygous HbS or HbC disease. Synthesis of gamma and beta chains was assessed either by 35S-methionine labeling of cultures and measurement of the radioactivity incorporated into the methionine-containing tryptic peptides of the gamma and beta subunits or by 3H-leucine labeling and measurement of the radioactivity incorporated into the globin chains of Hbs F0 and A0 isolated by ion-exchange chromatography. The cultures from all individuals responded with increased production of HbF.

Erythroid progenitors circulating in the blood of adult individuals produce fetal hemoglobin in culture.

Erythroid colonies, raised from erythroid stem cells circulating in the peripheral blood of normal adult individuals, synthesize considerable amounts of fetal hemoglobin. In cultures from persons with sickling disorders, amounts of hemoglobin F that are known to inhibit sickling in vivo are produced. The results provide evidence that primitive erythroid progenitors are able to express the hemoglobin F production program and that cultures of mononuclear cells of the adult blood can be used to investigate the mechanisms involved in regulation of gamma-globin gene switching.

Hemoglobin ontogenesis: test of the gene excision hypothesis.

The gene excision hypothesis of hemoglobin ontogenesis was tested in persons with HbSC disease, with the use of monospecific fluorescent antibodies for the identification of hemoglobins S, C, and F in individual erythrocytes. The results are incompatible with the prediction that only one gamma- or beta-globin gene may be active in any single chromosome and provide further evidence for incomplete repression of gamma-globin genes lying cis to active beta-globin genes.

Use of specific fluorescent antibodies for the identification of hemoglobin C in erythrocytes.

Antibodies against hemoglobulin C (alpha2beta2 6Glu leads to Lys) were produced by immunizing horses and were purified by affinity chromatography. As expected from the bivalency of both the antibody and the antigen, the purified antibodies failed to produce immunoprecipitates upon reaction with the corresponding antigens. Identification of hemoglobin C in individual erythrocytes was achieved by reacting the fluorescein isothiocyanate-conjugated antibodies with the hemoglobin antigen in fixed smears of peripheral blood.

Identification of Haemoglobin S in red cells and normoblasts, using fluorescent anti-Hb S antibodies.

Anti-haemoglobin S antibodies were raised in horses and purified by affinity chromatography. These antibodies recognize beta6 valine, while they fail to bind to haemoglobins with a glutamy1 (Hb A) or a lysy1 (Hb C) residue in this position. The purified anti-Hb S antibodies were composed of equine IgG(a, b) and IgG(T) subclasses and failed to cause precipitation with Hb S, evidently because of the bivalencey of both antibodies and antigen.

Haemoglobin M Hyde Park occurring as a fresh mutation: diagnostic, structural, and genetic considerations.

Hb M Hyde Park disease was detected in a girl who for several years was thought to have cyanotic heart disease. The problems of recognizing the condition are outlined and clues to diagnosis are discussed. Evidence for heme loss from the aberrant beta chains of Hb M Hyde Park and production of an unstable molecule is presented.

The Kenya form of hereditary persistence of fetal haemoglobin: structural studies and evidence for homogeneous distribution of haemoglobin F using fluorescent anti-haemoglobin F antibodies.

Several members of a Ugandan family were heterozygous for the gamma beta fusion gene of Haemoglobin Kenya. Levels of Hb Kenya were significantly higher than those in subjects of previous reports, ranging from 20.68 to 23.