Boundary condition of grounding lines prior to collapse, Larsen-B Ice Shelf, Antarctica.

Grounding zones, where ice sheets transition between resting on bedrock to full floatation, help regulate ice flow. Exposure of the sea floor by the 2002 Larsen-B Ice Shelf collapse allowed detailed morphologic mapping and sampling of the embayment sea floor. Marine geophysical data collected in 2006 reveal a large, arcuate, complex grounding zone sediment system at the front of Crane Fjord. Radiocarbon-constrained chronologies from marine sediment cores indicate loss of ice contact with the bed at this site about 12,000 years ago. Previous studies and morphologic mapping of the fjord suggest that the Crane Glacier grounding zone was well within the fjord before 2002 and did not retreat further until after the ice shelf collapse. This implies that the 2002 Larsen-B Ice Shelf collapse likely was a response to surface warming rather than to grounding zone instability, strengthening the idea that surface processes controlled the disintegration of the Larsen Ice Shelf.

The production, serologic evaluation, and epitope mapping of ten murine monoclonal Dombrock antibodies.

The Dombrock (Do) glycoprotein is a glycosylphosphatidylinositol(GPI)-linked membrane protein carrying Dombrock blood group antigens. There are no standardized typing reagents for Do(a) or Do(b). We have developed ten different monoclonal antibodies(MoAbs) that are specific for Dombrock. The objectives of this study were to characterize these MoAbs serologically and determine the epitopes they recognize. MoAbs were generated by standard fusion methods. Mice were immunized with transfected human embryonic kidney 293T cells expressing high levels Do(a) or Do(b). The MoAbs were tested serologically with untreated and enzymatically or chemically modified red blood cells (RBCs).Serologic inhibition studies were performed with synthetic peptides corresponding to Do(a) and Do(b) amino acid sequences.Pepscan epitope analysis was done on an array of immobilized tridecapeptides corresponding to the full-length polypeptide. All ten antibodies were serologically specific for Dombrock. Eight of the antibodies recognized epitopes that were resistant to treatment with ficin, pronase, a-chymotrypsin, and neuraminidase,but sensitive to trypsin and 0.2 M dithiothreitol (DTT). Five have anti-Do(b)-like specificity. The epitope recognized by MIMA-52 was neuraminidase sensitive, and MIMA-127 epitope recognized a DTT-resistant, linear epitope (90)QKNYFRMWQK(99) of the Dombrock polypeptide. MIMA-127 was the only one of the ten Dombrock MoAbs mapped to a specific sequence of the Dombrock glycoprotein; the other nine MoAbs did not provide aspecific peptide binding pattern. The other MoAbs could not be mapped as they most likely recognize nonlinear, conformation-dependent epitopes, as is evident by their sensitivity to reduction of disulfide bonds by DTT. The dependence of some epitopes on antigen glycosylation is also a possibility.

Characterization of three novel monoclonal anti-OKa.

Anti-Ok(a) was first described by Morel and Hamilton in 1979. The Ok(a) antigen has a very high incidence, and only eight probands that are Ok(a-) have been found; all are of Japanese heritage. In this study,we describe the generation and characterization of three novel monoclonal antibodies (Mabs), MIMA-25, MIMA-144, and MIMA-149. The reactivity of these three Mabs was compared with the original human polyclonal anti-Ok(a). Mice were immunized with transfected HEK cells to induce an immune response, and the spleen B lymphocytes were fused with mouse myeloma X63-Ag8.653 cells to form antibody-secreting hybridomas. The resulting Mabs were tested serologically, by flow cytometry, and by immunoblotting. The specificity of each antibody was determined after excluding specificities to common antigens in the Rh, Kell, Duffy, Kidd, MNS, Lewis, Lutheran, P1, Colton, Diego, Xga, and Dombrock blood group systems. In each case only the Ok(a-)RBC sample was nonreactive. The Mabs and the original human anti-Ok(a) each have a unique pattern of reactivity when tested with enzyme-treated cells; however, none were reactive by immunoblotting. We have generated three novel anti-Ok(a) Mabs: MIMA-144 is an indirectly agglutinating IgG2b antibody, and MIMA-25 and MIMA-149 are directly agglutinating antibodies (IgM and IgA, respectively), underscoring their usefulness as typing reagents for the clinical laboratory.

Efficacy of murine monoclonal antibodies in RBC phenotyping of DAT-positive samples.

Determining the phenotype of patient RBCs that are positive by the DAT may prove problematic. Antigen typing of RBCs coated with IgG requires direct agglutinating reagents or chemical treatment (such as chloroquine diphosphate [CDP] or citric acid) to remove sufficient IgG to permit testing with IAT-reactive reagents. The citric acid elution method is commonly used in the United States; however, antigens in the Kell system are altered to the extent that they may appear to be absent by this method. There are a limited number of direct agglutinating monoclonal antibodies available. Murine monoclonal antibodies provide an additional tool for typing RBCs with a positive DAT. Five murine monoclonal IgG antibodies (anti-K: MIMA-22, MIMA-23; anti-Kpa: MIMA-21, MIMA-27; anti-Fya: MIMA-19) were used in this study. Donor RBCs with known phenotypes were sensitized in vitro with alloanti-D, alloanti-c, and alloanti-K and with 20 autoantibodies (autoanti-D [n=3], autoanti-e [n=5], autoanti-Ce/e [n=5], autoanti-e+D+E [n=1], autoanti-I [n=1], and nonspecific [n=5]) to simulate a positive in vivo DAT. The sensitized RBCs were treated with CDP to remove IgG. To determine the efficacy of the murine monoclonal antibodies when testing DAT-positive samples, both sensitized and CDP-treated RBCs were tested with these monoclonal antibodies by the IAT using anti-mouse IgG. No discrepancies were noted with the unsensitized, sensitized, or CDP-treated RBCs. An exception was noted with a potent autoanti-I, where direct agglutination of the sensitized RBCs was obtained. This study demonstrates the value of using murine monoclonal antibodies to determine the phenotype of RBCs with a positive DAT caused by autoantibodies (e.g., in autoimmune hemolytic anemia) and supports previous studies showing that RBCs sensitized in vivo can be typed without chemical manipulation.

Altered glycosylation leads to Tr polyagglutination.

BACKGROUND: Polyagglutination refers to red blood cells (RBCs) that are agglutinated by a high proportion of ABO-matched adult sera but not by cord sera. Polyagglutinable RBCs have been associated with microbial infection, myeloproliferative disorders, and myelodysplasia. Lectins aid in the identification of polyagglutination. CASE STUDY: A Hispanic male infant with mild hemolytic anemia, a "Bernard-Soulier-like" syndrome, intermittent neutropenia, mitral valve regurgitation, ligament hyperlaxity, and mild mental retardation was studied. The patient's Group O RBCs were polyagglutinable; they were agglutinated by normal human sera, several lectins [including Arachis hypogea, Salvia sclarea, Salvia horminum, Glycine max, Ulex europaeus, Griffonia simplicifolia I, and Gr. simplicifolia II], and some monoclonal antibodies. His RBCs were not agglutinated by cord sera, Dolichos biflorus, or Phaseolus lunatus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the RBC membranes followed by staining with periodic acid-Schiff stain showed markedly reduced staining of glycophorins A and B. Staining with Coomassie brilliant blue revealed that Band 3 has a faster mobility than normal. CONCLUSIONS: Collectively, the results suggest that the patient's RBCs have a reduction in N-acetylneuraminic acid on both N- and O-glycans, exposing, respectively, beta1,4-galactosidase and beta1,3-galactosidase. The patient likely has an altered glycosyltransferase that results in defective glycosylation in RBCs and other cell lineages. This type of polyagglutination was named Tr.

Applications of murine and humanized chimaeric monoclonal antibodies for red cell phenotyping.

The limited supply of reagent human polyclonal antibodies to high prevalence antigens, like Js(b), is driving the search for alternative reagents. Murine immunoglobulin G (IgG) monoclonal antibodies (Mabs) and their humanized chimaeric IgM isoforms can now be used for typing patients and screening donors. Antigen typing of red blood cells (RBC) with a positive direct antiglobulin test (DAT) is also possible using these antibodies. Blood from patients with sickle cell disease and African donors were tested with reagent anti-Js(b), murine Mab IgG anti-Js(b) [murine immunochemistry monoclonal antibody-8 (MIMA-8)], and humanized chimaeric IgM anti-Js(b) [human immunochemistry monoclonal antibody-8 (HIMA-8)] by haemagglutination and gel cards. RBC samples that were DAT positive were used to evaluate the humanized chimaeric IgM monoclonal anti-Fy(a) (HIMA-19). RBC samples (n = 243) of known Js(b) type were tested in parallel with MIMA-8 and reagent anti-Js(b), and 132 samples were tested with MIMA-8 in gel cards and HIMA-8 by direct tube testing. No discrepant results were obtained. DAT-positive RBC samples (n = 27) were correctly phenotyped using HIMA-19. We conclude that MIMA-8 is suitable for screening donors and typing patient RBCs. Testing MIMA-8 with gel cards containing anti-mouse IgG enables the screening of donors by automated methods. Humanized chimaeric IgM anti-Js(b) and anti-Fy(a) are suitable as typing reagents by direct agglutination methods.

Production of recombinant murine-human chimeric IgM and IgG anti-Js(b) for use in the clinical laboratory.

BACKGROUND: Directly agglutinating MoAbs are more useful than IgG MoAbs of murine origin for typing RBCs from donors and patients. The molecular manipulation and conversion of a murine IgG MoAb into mouse- human chimeric IgM and IgG antibodies are described. STUDY DESIGN AND METHODS: cDNA encoding the variable heavy- and light-chain genes of a murine hybridoma anti-Jsb cell line (MIMA-8) were cloned into human IgM or IgG expression vectors, which were then separately stably transfected into SP2/0-Ag14 B-cells. The secreted antibodies were screened by ELISA and analyzed by flow cytometry and hemagglutination. RESULTS: Forty percent (16 of 40) of the stable clones secreted IgM and 66 percent (12 of 18) of the stable clones secreted IgG. The chimeric IgM from the highest expressing clone reacted 4+ in LISS at room temperature. The chimeric IgG from one clone reacted 4+ by the IAT, resembling the specificity of the original murine antibody. Both manipulated MoAbs reacted specifically with RBCs as assessed by flow cytometry. CONCLUSION: Human-mouse chimeric IgM and IgG from a murine IgG MoAb anti-Jsb has been successfully engineered for use in the clinical laboratory. This approach can potentially be used to manipulate other murine MoAbs to blood group antigens into more clinically useful human isotypes.

Studies on the Dombrock blood group system in non-human primates.

The Dombrock blood group system consists of five distinct antigens: two antithetical antigens, Doa and Dob, and three high-frequency antigens:Gya,Hy, and Joa. Although the prevalence of Doa and Dob in different populations makes them useful as genetic markers, the scarcity of reliable antibodies to these antigens has prevented this potential from being realized. The gene (DO;ART4) encoding the Dombrock glycoprotein has been cloned and sequenced, and the molecular bases of the various Dombrock phenotypes have been determined. The purpose of this study was to perform DNA-based assays on the DO homolog in non-human primates to determine the degree of conservation in the DO gene. Murine MoAbs to Dombrock protein were developed by standard hybridoma technologies and used to test RBCs from non-human primates by hemagglutination. PCR-RFLP analysis for the six single-nucleotide polymorphisms (SNPs) that have been defined in human alleles were performed on DNA extracted from fresh or frozen blood samples from numerous non-human primates. Hemagglutination tests with six MoAbs to the Dombrock glycoprotein revealed distinct epitopes on RBCs from the non-human primates. The gorillas and orangutans had the same PCR-RFLP digestion pattern for the six SNPs studied as chimpanzees. Old world monkeys (macaques) were identical at nucleotides (nt) 323, 350, 624, and 793 with the chimpanzees, and at nt 898 the digestion pattern was the same as for the HY1 allele in humans. For the new world monkeys (tamarins and squirrel monkeys) the digestion pattern was conserved for nt 793 but different for nt 624; the other SNPs could not be determined because there was no amplification. The presence of epitopes recognized by the MoAbs and PCR-RFLP results among the non-human primates shows considerable conservation of the DO gene. The difficulties we encountered with the amplification of DNA from the non-human primates lower in the phylogenetic tree are probably due to divergence in sequence.

Murine monoclonal antibodies can be used to type RBCs with a positive DAT.

RBCs with a positive DAT due to IgG coating require the use of directly agglutinating reagents or treatment with chemicals to remove sufficient IgG to permit typing of the RBCs with antisera that require use of the IAT. In this study we demonstrate that murine IgG MoAbs to human RBC antigens can be used as an alternative if the anti-mouse IgG is neutralized or affinity purified to prevent cross-reaction with cell-bound IgG. We performed DATs on RBC samples coated with IgG in vivo and in vitro, comparing two anti-human IgG reagents (Organon Teknika, Durham, NC, and Ortho-Clinical Diagnostics, Raritan, NJ) with two affinity-purified anti-mouse IgG reagents (The Binding Site, San Diego, CA, and Sigma, St. Louis, MO), and one non-purified anti-mouse IgG reagent. The affinity-purified anti-mouse IgG reagents were nonreactive with the four in vitro sensitized RBC samples and were nonreactive with 8 of 11 in vivo sensitized RBC samples. Non-purified antimouse IgG and both anti-human IgG reagents reacted with every sample. Use of murine MoAbs to antigen type RBCs coated with human IgG is reliable only when the anti-mouse IgG reagents have been affinity purified or neutralized to prevent cross-reactivity. Our results also show the importance of including a saline/RBC control as well as an anti-mouse IgG/RBC control. Murine MoAbs are valuable reagents and we have applied them successfully in typing patients' RBCs that have a positive DAT.

MIMA-9, a valuable antibody for screening for rare donors.

Since monoclonal antibodies (Mabs) are potentially available in an unlimited volume, they can be used to screen numerous donor blood samples to identify antigen-negative donors. We have used a Mab (MIMA-9) with characteristics that allow for the simultaneous screening of RBCs of any ABO group for high-incidence antigen-negativity in the Kell and Gerbich blood group systems. MIMA-9, a murine IgG2a antibody, previously shown to facilitate the identification of K+k-, Kp(a+b-), K0, McLeod, or Ge:-3 red blood cells (RBCs), was used in MTS gel cards containing anti-mouse IgG as the second antibody to test 1134 K- donors. Among the 1134 donors tested, we found one Kp(a+b-) and one Ge:-2,-3,4 donor. If random donor samples had been used instead of preselecting for K-, we would have expected to identify two K+k- donors. One reagent (MIMA-9) can be used to simultaneously screen for K+k-, Kp(a+b-), K0, McLeod, and Ge:-3 RBCs and thereby conserve rare antisera. Inclusion of anti-mouse IgG in gel cards allowed for rapid screening. MIMA-9 is also a useful reagent to type RBCs with a positive direct antiglobulin test. This antibody is available to donor screening laboratories at no cost for this specific use.

Immunization of transgenic mice for production of MoAbs directed at polymorphic blood group antigens.

BACKGROUND: Antibodies of human origin for blood typing are increasingly difficult to obtain, and, despite aggressive efforts, MoAbs with specificities to several blood group polymorphisms have eluded production. As an approach for the generation of MoAbs with defined specificities, the feasibility of immunizing mice that are transgenic for the target polymorphism, Fy(a)/Fy(b) of the Duffy blood group system, was tested with a source of the antithetical antigen. STUDY DESIGN AND METHODS: Nontransgenic mice were immunized with recombinant Fy(b), and transgenic mice expressing human Fy(b) were immunized with recombinant Fy(a). RESULTS: Immunization of the nontransgenic mice resulted in the production of MoAbs to the Duffy protein, but not to the Fy(a)/Fy(b) blood group polymorphism. However, immunization of the transgenic mice resulted in production of the first example of murine Fy(a) MoAb (MIMA-19). This antibody is being used to screen for Fy(a-) blood donors and has been evaluated by many laboratories in an international workshop. CONCLUSION: This approach provides an effective method for producing MoAbs with specificities to polymorphic epitopes. These MoAbs are needed in transfusion medicine to identify antigen-negative donors and to alleviate the critical shortage of blood bank typing reagents, which currently are available only from human-derived sources.

Direct evidence for the existence of Miltenberger antigen.

The Miltenberger (Mi) subsystem, which originally consisted of four phenotypes, now has 11 phenotypes. The antigens of this subsystem belong to the MNS blood group system. The Mia antigen has been reported to be present on red blood cells with several Miltenberger phenotypes, namely: Mi.I, Mi.II, Mi.III, Mi.IV, Mi.VI and Mi.X. However, the existence of the Mia antigen as a separate entity has been in question and difficult to prove with polyclonal reagents. We report the first monoclonal anti-Mia (GAMA210), whose epitope is TNDKHKRD or QTNDMHKR, and thereby confirm the existence of the Mia antigen.

A DNA-based immunization protocol to produce monoclonal antibodies to blood group antigens.

A major challenge facing transfusion medicine is the establishment of immunological methods to produce specific and avid blood group typing reagents to the many polymorphic blood group antigens. This is especially true when sources of human antibody are limited. Based on the knowledge that inoculation with plasmid DNA can induce a humoral response in the host animal, we inoculated mice with plasmid DNA followed by a single boost injection with plasmid-transfected cells that have a high level of expression of the same target protein. Using this method, several hybridoma clones that produced strongly reactive antibodies specific for the Kell polymorphic antigens (anti-K, anti-k, anti-Kp(a)) were isolated. The monoclonal antibodies that were produced with this method have potential clinical utility for identifying a patient's blood type and for screening for antigen-negative donor blood.

Molecular basis for Rh(null) syndrome: identification of three new missense mutations in the Rh50 glycoprotein gene.

Rh(null) is a rare autosomal recessive disorder characterized by an absence of Rh antigens and a varying degree of hemolytic anemia and spherostomatocytosis. We report studies of two Japanese Rh(null) cases and describe three new missense mutations of RHAG, the locus that encodes Rh50 glycoprotein and modulates Rh antigen expression. In Rh(null)(HT), RHAG harbored in exon 6 two G-->A transitions, GTT-->ATT and GGA-->AGA, which cause Val(270)-->Ile and Gly(280)-->Arg substitutions, respectively. These missense mutations were cotransmitted from the propositus to the children and were predicted to reside in endoloop 5 and transmembrane (TM) segment 9, respectively. In Rh(null)(WO), RHAG contained in exon 9 a single G-->T transversion, GGT-->GTT, which caused a Gly(380)-->Val missense change in TM12 segment. The G-->T transversion, which is located at the +1 position of exon 9, had also affected pre-mRNA splicing and caused partial exon skipping. Although both Rh(null) cases had a structurally normal RH antigen locus, hemagglutination and immunoblotting showed no expression of Rh antigens or proteins. These results correlate each mutation with a structural defect in the respective TM domain of Rh50 glycoprotein.

Evaluation and comparison of three human monoclonal anti-S, two human polyclonal anti-S, and one murine anti-GPB.

Polyclonal anti-S react with Met29 of red blood cell (RBC)-bound glycophorin B (GPB) but may also require adjacent amino acids. Treatment of RBCs with certain enzymes and sodium hypochlorite-based bleach (NaClO) affect the interaction of GPB with anti-S. Some, but not all, anti-S react with hybrid glycophorin molecules associated with the TSEN antigen. The purpose of this study was to characterize monoclonal anti-S and to compare their reactivity to polyclonal anti-S in order to determine their potential as blood group reagents and research tools. Furthermore, through inhibition experiments, we attempted to define the epitope recognized by the antibodies. Three monoclonal (MS-93; MS-94; MS-95) and two polyclonal (A1958; X1960) anti-S and a monoclonal anti-GPB (Mab 148) were tested by standard hemagglutination with RBCs of known common and rare phenotype, with S+ RBCs treated with enzymes, with different concentrations of NaClO, and after incubation with synthetic peptides. The anti-S gave different patterns of reactivity. Reactivity with sialidase-treated RBCs showed that MS-93, MS-95, Mab 148, and X1960 recognize sialic acid independent epitopes, whereas MS-94 and A1958 require sialic acid for optimal reactivity. MS-95 and X1960 were strongly reactive with TSEN+ RBCs and only Mab 148 agglutinated S- Dantu+ and S- St(a+) RBCs. MS-94 and Mab-148 agglutinated S+ RBCs treated with NaClO. MS-93 was inhibited only by the 14-mer S-specific synthetic peptide whereas MS-95 was inhibited by all three synthetic peptides containing S-relevant residues. This study clearly demonstrates that different anti-S have different characteristics that should be analyzed before selecting monoclonal antibodies for the basis of reagents for use in the clinical laboratory. These anti-S, because of their varied characteristics, will be useful research tools.

Use of LOR-15C9 monoclonal anti-D to differentiate erythrocytes with the partial DvI antigen from those with either partial D antigens or weak D antigens.

Historically, red blood cells (RBCs) with partial D antigens have been defined serologically by their pattern of reactivity with polyclonal and monoclonal anti-D. Although numerous variants have been described in tests with well-characterized monoclonal anti-D, definition remains difficult to ascertain serologically. RBCs of known partial D type were tested with LOR-15C9 (a monoclonal anti-D) and commercial anti-D by the tube indirect antiglobulin test (IAT), by micro typing system IgG gel cards, and by immunoblotting. By IAT, LOR-15C9 reacted strongly with DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs in addition to RBCs with common D antigens; weakly with DII, DNU, and DIIIb RBCs; and not at all with DIVa, DIVb, DBT, or R0 Har RBCs. Reactivity was variable (1+ to 4+), with RBCs classified as weak D (Du). As expected, the commercial anti-D agglutinated all D variants and weak D RBC samples by the IAT and by using IgG gel cards; however, the reactivity with DVI RBCs was weaker than with LOR- 15C9. By immunoblotting, LOR-15C9 detected a band with an apparent molecular mass of approximate Mr 30,000-34,000 in membranes prepared from D-positive, DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs and an additional band of Mr 20,000-22,000 in membranes prepared from DVI RBCs. No band(s) was detected in membranes from DII, DNU, DIIIb, DIVa, DIVb, DBT, R0 Har, weak D, or D-negative samples. LOR-15C9 provides a useful tool to identify positively DVI samples and thereby differentiate this partial D from other D variants and from weak D samples.

A human monoclonal anti-D antibody which detects a nonconformation-dependent epitope on the RhD protein by immunoblot.

We describe the first human monoclonal anti-D (LOR-15C9) which reacts with a D-specific motif exposed either on a native form on intact D-positive red cells or on a denatured form of the RhD protein (33 kD), and detected by immunoblotting. LOR-15C9 was able to precipitate RhD but not RhcE proteins produced by in vitro transcription-translation assays. The reactivity of the antibody, using panels of red cells with various partial D phenotypes known to lack some D epitopes and corresponding in RHD gene variants, suggested that LOR-15C9 reactivity depends on the portion of the RhD polypeptide encoded by the exon 7 (amino acids 314-358). These findings correlate well with the reactivity of LOR-15C9 with erythrocytes of some nonhuman primates (D(gor)-positive gorillas), but not of chimpanzee and Old or New World monkeys. In membrane proteins from partial D(VI) red cells, LOR-15C9 detected two proteins of molecular weight 33 and 21 kD: the presence of the latter was specific for category D(VI) and presumably represented the product of an alternatively spliced RHD(VI) transcript in these cells. This is consistent with the finding that LOR-15C9 can precipitate a shortened D protein mutant resulting from in vitro transcription-translation and lacking amino-acids 163-313 encoded by exons 4-6. In addition, a 21 kD band polypeptide was detected by immunoblot in all red cell samples but D--, using a rabbit anti-Rh polypeptide antibody (MPC8) raised against the C-terminal domain of Rh proteins. This 21 kD polypeptide most probably results from the translation of an alternatively spliced RHCE gene transcript. This study demonstrates that LOR-15C9 detects an epitope on the RhD protein that is independent of the membrane environment, and therefore could be a useful tool for the study of RhD polypeptides.

Characterization of monoclonal antibodies in section 2B using enzymes and variant red blood cells.

Antibodies in Section 2B of the Third International Workshop on Monoclonal Antibodies were tested against human erythrocytes treated with different enzymes and against erythrocytes with well-defined rare phenotypes by immunoblotting and hemagglutination. In some instances, the epitope recognized by the Mab was precisely determined.