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. Monoclonals 16-2 and 37-8 are beta-chain specific. Antibody 31-2 recognizes an antigenic determinant common to the alpha and beta subunits. Monoclonal 30-3 recognizes determinants best expressed in the alpha 2 beta 2 tetramer. Antibody 45-1 recognizes a determinant common to beta and gamma subunits, while antibody 51-7 is gamma-chain specific. None of the monoclonal antibodies recognizes mouse hemoglobin, and they display significant differences in binding to hemoglobins of various species. The species-specific reactions and the knowledge of the primary structures of globins allowed deductions about the antigenic sites recognized by two of the monoclonals (16-2 and 45-1). These antihemoglobin monoclonal antibodies will provide useful probes for studying hemoglobin expression in vivo and in vitro.

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 7.4 X 10(-9) per nucleotide per generation; that derived using de novo alpha-gene mutants is 10.0 X 10(-9). Subsequent calculations of mutation rates per alpha- and beta-chain gene and extrapolation of these rates to a hypothetical gene of 1000 nucleotides yield an estimated mutation rate of 8.6 X 10(-6) per 1000 nucleotides per generation. Even though some instances of false paternity may have biased these estimates in an upward direction, under-reporting of Hb M cases, and particularly of unstable hemoglobins, makes it likely that the cited values are minimal estimates of mutation rates at the molecular level.

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. The primary structural site recognised by monoclonal antibody HuHb beta 3-2(an antibody that reacts with the adult haemoglobins from man and macaque monkey, but not with those from baboon and mouse) includes the aspartic acid residue at position 52 of the beta-globin subunit.

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 mamalian 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 structural site recognised by monoclonal antibody HuHb á 3-2 (an antibody that reacts with the adult haemoglobins from man and macaque monkey, but not with those from baboon and mouse) includes the aspartic acid residue at position 52 of the á-globin subunit.(AU)

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.

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. Additional pedigree data on several published cases were collected, and a depository containing all available information rekated to de novo Hb mutants was established. The 50 probands were born in 14 countries between 1922 and 1976. Paternity was tested in 36% of the cases, and no instance of false paternity was noted.The data were used to test for an association of advanced parental age with the appearance of de novo mutants. Paternal ages at the probands' births ranged from 20 to 50 years, with a mean of 32.7 years. Maternal ages ranged from 18 to 43 years, with a mean of 28.5 years. For each year and country (or, where necessary, for the nearest possible year and/or a demographically similar country), the cumulative frequency distributions of the ages of parents who had a child in that country and year were computed; the ages of each proband's father and mother were then expressed as percentiles on these distributions. The distribution of paternal age percentiles was shifted toward the upper end of the range, with 11 of the 50 paternal ages falling between the 90th and 100th percentiles. The distribution of maternal age percentiles was more complex, with one peak (10 of 50 ages) falling between the 30th and 40th percentiles and a second peak (10 of 50 ages), between the 90th and 100th percentiles. These distributions, though suggestive of an association of advanced parental age and the appearance of de novo mutations that cause unstable Hb disease or methemoglobinemic cyanosis, were not significantly different from those uniform distributions expected in the absence of a parental age effect.

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). On these grounds, we assume that this minor component contains, like Hb FI, gamma chains that differ from those of the major component by virtue of acetylation of their amino-terminal glycyl residues. Although the gamma chains of most antropoid primates examined to date are structurally heterogeneous and, hence, appear to be encoded by nonallelic genes, no sign of structural heterogeneity was detected at any position in the major gamma chain from M. mulatta. Thus, if nonallelic gamma-chain genes exist in this species, the chains encoded by them may be identical in sequence. The gamma chain from M. mulatta is but the sixth primate gamma chain whose primary structure has been fully characteerized. The slight extent of structural divergence among these chains (the four chains from various species of Old World monkeys differ from one another by no more than two substitutions, while the human and cercopithecoid gamma chains differ at no more than five sites) attests to the conservative nature of gamma-chain evolution among the higher primates.

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. On the basis of the structures and the molecular lesions underlying production of the two abnormal hemoglobins, we predict that the anti-Hb Wayne antibody will detect several frameshift mutants resulting from deletion of 3n + 1 nucleotides or insertion of 3n + 2 nucleotides at the 5' side of the codon normally specifying residue 139 of the alpha chain. The anti-Hb Cranston antibody should be capable of detecting beta chains, the corresponding genes of which have sustained insertions of 3n + 2 nucleotides or deletions of 3n + 1 nucleotides on the 5' side of the codon normally specifying residue 144. The two antibodies may, therefore, prove to be valuable in the development of a system aimed at detecting rare erythrocytes that express mutations which arise in the hemopoietic stem cells of normal individuals and subjects exposed to mutagens.

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. Of the cercopithecoid beta chains whose complete sequences have been determined or inferred from compositions of small peptides, that of P. cynocephalus is most like the beta chain of the gelada baboon, an observation in accord with assessment of a close phylogenetic relationship between the genera Papio and Theropithecus.

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. This gamma chain, the first from a nonhuman primate to be sequenced, differes from the human G gamma and A gamma chains at but 4 and 5 positions, respectively. All substitutions are conservative and unlikely to produce alterations in the oxygen-binding properties of tetrameric fetal hemoglobin. Consideration of the data presented herein, together with published observations made on portions of other primate gamma chains, provides some insight into the evolutionary history of the multiple gamma-globin chains observed in several anthropoid primates.

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. cynocephalus that have been well characterized attests to the conservative nature of gamma-chain evolution among the Anthropoidea, the differences in sequence between any two of these chains ranging from none (between the A gamma and G gamma chains of P. troglodytes and H. sapiens) to no more than five (between the V gamma chains of P. cynocephalus and the A gamma chains of H. sapiens).

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. Cultured cells from subjects without a hemoglobinopathy produced an average of 8.2% gamma chains (range 3.1%-20.3%), while cultured cells from subjects homozygous for HbS or HbC produced an average of 16.6% gamma chains (range 12.2%-20.4%). These findings indicate that fetal Hb production was regularly enhanced in adult bone marrow cells triggered in vitro to clonal growth in the plasma clot culture system.

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.