Hemoglobin S and C heterozygosity enhances neither the magnitude nor breadth of antibody responses to a diverse array of Plasmodium falciparum antigens.

BACKGROUND: Heterozygous states of hemoglobin (Hb) A and HbS (HbAS, sickle-cell trait) or HbC (HbAC) protect against Plasmodium falciparum malaria by unclear mechanisms. Several studies suggest that HbAS and HbAC accelerate the acquisition of immunity to malaria, possibly by enhancing P. falciparum-specific antibody responses. METHODS: We used a protein microarray representing 491 P. falciparum proteins expressed during exoerythrocytic and erythrocytic stages of the life cycle to test the hypothesis that HbAS and HbAC enhance the P. falciparum-specific IgG response compared with normal HbAA. Plasma samples were collected from Malian children aged 2-10 years before and after a 6-month malaria season and were probed against the microarray. Immunoglobulin G (IgG) profiles of children with HbAA (n = 106), HbAS (n = 15), and HbAC (n = 20) were compared. RESULTS: Although the magnitude and breadth of P. falciparum-specific IgG responses increased with age and from before to after the malaria season in each antigen category, Hb type did not independently predict significant differences in P. falciparum-specific IgG profiles. CONCLUSIONS: These data do not support the hypothesis that HbAS and HbAC protect against malaria by enhancing P. falciparum-specific antibody responses. It remains possible that HbAS and HbAC protect against malaria by enhancing antibody responses to antigens not studied here or through other immune mechanisms.

Structural basis of peroxide-mediated changes in human hemoglobin: a novel oxidative pathway.

Hydrogen peroxide (H(2)O(2)) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H(2)O(2) to highly purified human hemoglobin (HbA(0)) induced structural changes that primarily resided within beta subunits followed by the internalization of the heme moiety within alpha subunits. These modifications were observed when an equal molar concentration of H(2)O(2) was added to HbA(0) yet became more abundant with greater concentrations of H(2)O(2). Mass spectrometric and amino acid analysis revealed for the first time that betaCys-93 and betaCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA(0) was treated with H(2)O(2). Oxidation of further amino acids in HbA(0) exclusive to the beta-globin chain included modification of betaTrp-15 to oxyindolyl and kynureninyl products as well as betaMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the beta subunits as a result of the H(2)O(2) attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two alpha-peptide fragments (alpha128-alpha139) and a heme moiety with the loss of iron, cross-linked between alphaSer-138 and the porphyrin ring. The novel oxidative pathway of HbA(0) modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

Development and validation of an ELISA for hemoglobin-A2: a novel method for beta-thalassemia screening in developing countries.

Hemoglobin-A2 (HbA2) measurement in human hemolysates has great significance, since its level can indicate beta-thalassemia carrier status in otherwise healthy individuals. An ELISA for HbA2 using antiserum monospecific to the delta chain of HbA2 and affinity purified antirabbit gamma globulins (ARGG) conjugated to horseradish peroxidase (HRP) have been developed. The monospecific antiserum used does not cross react with other hemoglobins. Hemolysates from volunteers are used for measurement of HbA2. In a limited trial for beta-thalassemia carrier screening (n = 350), the results obtained with the developed ELISA are comparable with those obtained with a micro-column chromatography method (r > or = 0.89). The developed ELISA is simple, accurate, precise, inexpensive, and several samples can be processed simultaneously with ease, making this system a suitable candidate for transforming into a user friendly kit.

Directing phage selections towards specific epitopes.

It is possible to direct selections from antibody repertoires displayed on filamentous phage towards unique epitopes on protein antigens by competing with related molecules. A phage display repertoire of human single chain Fvs (scFvs) was panned three times against foetal haemoglobin (HbF). The selection was dominated by one clone with a Kd of 10 nM but yielded at least 17 others, all of which bound HbF but crossreacted with adult haemoglobin (HbA). To direct selection towards HbF-specific epitopes, the repertoire was preincubated with HbA in solution before each panning. Crossreactive scFvs can form complexes with the soluble HbA and thereby be prevented from binding the immobilized HbF. Four clones with preferential binding to HbF emerged under these conditions. One of these (Hb-1), with a Kd of 6 microM, had exquisite specificity for HbF and could distinguish cells expressing HbF from those expressing HbA by immunocytochemistry and flow cytometry. This antibody has an affinity that is 600-fold lower than the dominant crossreactive clone, and so only emerged under conditions of 'competitive deselection'. Thus, competitive deselection is a viable means for directing selections towards useful epitopes. It permits a more effective 'search' of phage display repertoires and allows the emergence of lower affinity clones with useful specificities. These clones may be useful in themselves or may serve as leads for in vitro affinity maturation.

Screening for colorectal cancer by immunochemical fecal occult blood testing.

Screening for colorectal cancer using the conventional Hemoccult test has been shown to reduce mortality associated with cancer by 33% through a randomized controlled trial. However, the magnitude of effectiveness is small in terms of cost-effectiveness. The recently developed immunochemical fecal occult blood test (IFOBT) provides a potential replacement for the Hemoccult test as a screening test, due to its superior performance characteristics such as higher sensitivity shown in preliminary studies and the fact that it does not require any dietary restriction. The IFOBT method is reviewed, especially in relation to its specificity. In known colorectal cancer subjects, IFOBTs have shown both higher sensitivity and specificity than the Hemoccult test. Similarly, IFOBT has demonstrated a higher sensitivity than Hemoccult for colorectal cancer in an asymptomatic population. A nationwide screening program in Japan has demonstrated the feasibility of this approach for large population screening. However, the positivity rate varied according to the conditions at each screening facility. Therefore, technical factors that influence the positivity rate of IFOBTs in the screening program are discussed. Case-control studies have strongly suggested that screening using IFOBT would reduce mortality from colorectal cancer by 60% or more. Several observational studies have provided support for this estimate. The feasibility and effectiveness of population-based screening by IFOBT are discussed.

Binding specificity of a monoclonal antibody to human Hb A.

HemoCardTM kits use monoclonal antibody-conjugated metal sol particles in an immuno-concentration assay. The HemoCard Hbs S, C, and E kits detect the three most common hemoglobin variants in both adult and newborn samples. The HemoCard Hb A kit can be used in conjunction with HemoCard Hbs S and C (but not HemoCard Hb E) to distinguish heterozygotes from homozygotes. We examined the antigen-binding domain of a monoclonal antibody to Hb A by testing samples from human and animal sources. We confirmed that the Hb A conjugate recognizes an epitope in which glutamic acid at beta 6 is necessary to provide the proper conformation for binding. This binding is not affected by substitutions at positions directly adjacent to beta 6. However, extensive substitutions in the primary sequence (e.g. in chicken, beta 6: glutamic acid, substitutions at beta 3, beta 5, beta 9, beta 10, beta 11) may produce conformational changes, resulting in diminished binding.

Structural analysis of peptide-acetaldehyde adducts by mass spectrometry and production of antibodies directed against nonreduced protein-acetaldehyde adducts.

Acetaldehyde can form protein-acetaldehyde adducts (AAs) in vivo and may play a role in the genesis of alcoholic liver disease. The nature of the chemical modification of proteins by acetaldehyde in vivo has not been elucidated. In vitro, acetaldehyde can form reversible adducts including a Schiff's base with lysine (K) and imidazolidinone with terminal amino groups of proteins such as human hemoglobin (Hb). In this study, we used FAB/MS to analyze the products of peptide-AAs (pep-AAs) formed by incubating acetaldehyde with Hb peptides. We then used an octabranched multiple antigen peptide (MAP) system containing Hb peptide-AAs to raise antibodies. Three Hb peptides [i.e., 8-pep consisting of 8 residues (V1HLTPVEK8) at the N-terminus of beta-chain of human sickle-cell Hb, 11-pep-gly consisting of 11 residues (G56NPKVKAHGKK66) in a segment of beta-chain rich in lysine, and 11-pep-pro that consists of the same sequence as 11-pep-gly, except G56 was replaced by proline (P)] were incubated with 1 mM acetaldehyde at 4 degrees C for 7d without NaCNBH3 (nonreduced conditions). Analysis by FAB/MS showed that 8-pep formed an imidazolidinone at the N-terminal valine, 11-pep-gly formed a Schiff's base and imidazolidinone at the N-terminus, whereas 11-pep-pro that lacks a free alpha-amino group formed only a Schiff's base at K59. By contrast, incubation of these Hb peptides with 250 mM acetaldehyde and NaCNBH3 at 37 degrees C for 1 hr (reduced conditions) produced mono- and diethylated modifications of all available K residues, as well as the N-terminal amino group.(ABSTRACT TRUNCATED AT 250 WORDS)

A hemoglobin A1C immunoassay method not affected by carbamylated hemoglobin.

Hemoglobin A1C (HbA1C) methods based on charge separation of Hb species are subject to interference from carbamylated Hb (carb Hb). Carb Hb adducts are formed via interaction of terminal amino groups of HbA with isocyanic acid, after the spontaneous dissociation of urea to cyanate. It is hypothesized that a new immunoassay method, using a monoclonal antibody that recognizes the N-terminus of the Hb beta-chain and its sugar moiety, should be refractory to cross-reactive interference from carb Hb. To test this hypothesis, Hb was carbamylated in vitro and co-migration of carb Hb assessed with HbA1C using an electrophoretic method. Densitometric scans - post sodium cyanate incubation and electrophoretic separation - showed a 5 to 7 fold elevation of the HbA1C peak only, while HbA1C values obtained using immunoassay were unaffected. Also assessed was carbamylation interference in vivo, and a positive proportional bias with the electrophoretic system (Y) was observed compared to the immunoassay system (X) (y = 1.2x - 0.21 percent). Others have shown that carb Hb may cause a clinically significant false elevation in patient HbA1C values, when methods based on charge separation of Hb species are used. It is our conclusion, however, that while carb Hb may play a role, the differences observed in this study are largely due to calibration.

Utilization of monoclonal-antibody-based assay (HemoCard in screening for and differentiating between genotypes of sickle cell disease and other hemoglobinopathies.

Sickle cell disease covers a group of conditions in which pathology may be attributed to the presence of sickle hemoglobin (HbS). The identification of HbS and other variants including those in combination with HbS is commonly achieved by cellulose acetate electrophoresis at alkaline pH. Because many hemoglobin variants with similar charges have similar electrophoretic migration patterns, they are difficult to differentiate by electrophoresis. The HemoCard assays address this concern through the use of monoclonal antibodies capable of specifically recognizing the unique amino acid substitution in the variant hemoglobin. The panel of HemoCard monoclonal antibodies confirms the absence and presence of HbA, HbC, HbE, HbS, and other sickling hemoglobin variants. The combination of alkaline cellulose acetate electrophoresis and HemoCard assays allows the technologist to reach a final conformation of both common and much less common sickle cell disease genotypes, combinations of HbS with other hemoglobins that ordinarily do not produce sickle cell disease, and other clinically important hemoglobinopathies including HbE/beta-thalassemia and hemoglobin C disease.

Study of procainamide hapten-specific antibodies in rabbits and humans.

Procainamide (PA) is the drug most commonly associated with the induction of autoantibodies and drug-related lupus (DRL). While the majority of these patients express autoantibodies, antibodies to the parent drug and metabolites, PA-hydroxylamine (PAHA) or nitroso-PA (NOPA), have not been reported in humans. Hapten-carrier conjugates were prepared using human hemoglobin (HgB) or autologous rabbit erythrocytes with PAHA or NOPA. PA was conjugated to rabbit serum albumin (RSA) or egg albumin (OVA) via diazotization and condensation methods. Rabbits were immunized with hapten conjugates in Freund's adjuvant. These hapten-carrier compounds (5-10 micrograms/ml) were used as test antigens for antibodies in sera from the rabbits and 40 patients on chronic PA treatment. 10 SLE patients, 33 elderly and 20 young normal controls by ELISA. Type I and II collagens were also used as test antigens for human sera. Sera from rabbits immunized with the PA compounds had elevated IgG antibody values to PA, PAHA and NOPA, but no autoantibodies. Absorption of the rabbit sera with the PA compounds reduced the antibody levels; ssDNA and histones failed to inhibit the total binding values. Mean binding to PA-OVA was 0.95 +/- 0.41 for PA patients and 1.37 +/- 0.26 standard error of means (S.E.M.) in the SLE patients compared to 0.37 +/- 0.14 S.E.M. in the normal sera (P < or = 0.05); similar binding values to PAHA-HgB and NOPA-HgB were also observed. Sixty-eight percent of the PA patients had antibodies to type II collagen. Elevated binding values to PA compounds were inhibited by absorption of human sera with ssDNA or total histones; absorption with PA or PAHA had no significant effect. These findings suggest that sera from PA patients containing high titers of autoantibodies cross-react in vitro with unrelated antigens.

Production of antibodies that recognize the heterogeneity of immunoreactive sites in human hemoglobin chemically modified by acetaldehyde.

Human hemoglobin (Hgb) was incubated with acetaldehyde under two different conditions: (a) in the presence of 250 mM acetaldehyde for 1 hr then reduced with 100 mM NaCNBH3 for an additional 4 hr at room temperature; and (b) in the presence of 500 mM acetaldehyde for 10 days at room temperature and then reduced with 1 mM NaBH4 for 1 hr. It was found that 44% and 27% of free amino groups in Hgb-acetaldehyde adduct (AA) remained unmodified when Hgb was treated under conditions (a) and (b), respectively. SDS-PAGE analysis revealed that the molecular weight of Hgb-AA(a) [Hgb modified under condition (a)] was slightly greater than that of unmodified Hgb and extensive protein cross-linking had occurred in Hgb-AA(b) [Hgb modified under condition (b)]. Electrophoresis on agarose gel showed the order of negative charge was Hgb-AA(b) > Hgb-AA(a) > unmodified Hgb. Polyclonal antibody raised in rabbits using keyhole limpet hemocyanin as the carrier protein modified by acetaldehyde under condition (a) [i.e., KLH-AA(a)] preferentially recognized Hgb-AA(a), whereas antibody raised using KLH-AA(b) as the immunogen recognized only Hgb-AA(b). In conclusion, antibodies raised with protein-AA antigens produced under different conditions recognize different epitopes.

Serum immunoglobulin (G, A and M) classes and IgG subclasses in sickle cell anaemia.

Serum concentrations of immunoglobulins G, A, M and IgG subclasses were determined by single radial immunodiffusion assay in a population of sickle cell anaemia patients resident in the tropics. Fifty apparently healthy subjects of haemoglobin genotype AA, of comparable age, sex and socioeconomic status (SES), and in the same environment as the patients, were included as controls. Three indices of morbidity in SCA, namely frequency of crisis, degree of anaemia and the number of organ complications, were used to derive a severity score for each patient; and thus categorize the subjects into severity groups. Immunoglobulin levels were then correlated with the indices of morbidity as well as the derived severity score. IgG, IgA, IgM, IgG1 and IgG3 levels were significantly raised in the SCA subjects when they were compared as a group with the controls. When separated into disease severity groups, the mildly affected patients were found to have virtually normal levels of immunoglobulins. Total IgG concentration and level of the IgG3 subclass showed significant positive correlation with frequency of crisis and derived severity score. Markedly raised levels of IgG and IgG3 may be predictive of severity in sickle cell anaemia.

T cells specific for alpha-beta interface regions of hemoglobin recognize the isolated subunit but not the tetramer and indicate presentation without processing.

Processing of a protein antigen into fragments is believed to be a prerequisite for its presentation by the antigen-presenting cell to the T cell. This model would predict that, in oligomeric proteins, T cells prepared with specificity for regions that are buried within subunit association surfaces should recognize the respective regions in vitro equally well on the isolated subunit or on the oligomer. Three hemoglobin (Hb) alpha-chain synthetic peptides, corresponding to areas that are situated either completely [alpha-(31-45)] or partially [alpha-(41-45) and alpha-(81-95)] within the interface between the alpha and beta subunits of Hb, and a fourth peptide representing a completely exposed area in tetrameric Hb were used as immunogens in SJL/J (H-2s) mice. Peptide-primed T cells were passaged in vitro with the respective peptide to obtain peptide-specific T-lymphocyte lines. T-cell clones were isolated from these lines by limiting dilution. T-cell lines and clones that were specific for buried regions in the subunit association surfaces recognized the free peptide and the isolated subunit but not the Hb tetramer. On the other hand, T cells with specificity against regions that are not involved in subunit interaction and are completely exposed in the tetramer recognized the peptide, the isolated subunit, and the oligomeric protein equally well. The responses of the T-cell lines and clones were major histocompatibility complex-restricted. Since the same x-irradiated antigen-presenting cells were employed, the results could not be attributed to differences or defects in Hb processing. The findings indicate that in vitro the native (unprocessed and undissociated) oligomeric protein was the trigger of major histocompatibility complex-restricted T-cell responses.

Comparison of peptide-coating conditions in solid phase plate assays for detection of anti-peptide antibodies.

Mice were immunized with 14 free (i.e. not conjugated to any carrier) synthetic peptides representing the entire human hemoglobin alpha-chain. Antibodies against each peptide were determined using solid phase radioimmunoassay, both with free peptides and peptides coupled to a protein carrier as the coating antigen. It has been demonstrated that large improvements in the ability to detect anti-peptide antibodies were achieved in some cases by precoating the assay wells with free peptides and in other cases by precoating with peptide-protein conjugates. Sodium carbonate buffer, pH 9.6, had a favorable effect on the coating of two of the free peptides when compared with phosphate-buffered saline, pH 7.2. The assay with the plates coated with optimum peptide form (free peptide or peptide-protein conjugate) was superior in the detection of antibody binding to 9 of the peptides when compared with the assay using chemically activated plates. The results suggest that the appropriate form (conjugated or free) and conditions for immobilizing small peptides to plastic supports are not universal but will have to be determined for each test peptide.

The use of monoclonal antibodies UCH beta and UCH gamma for the antenatal diagnosis of beta-thalassaemia.

Monoclonal antibodies for the beta-globin chain of human HbA (UCH beta) and the gamma-globin chain of human HbF (UCH gamma) have been made. UCH beta indirectly labelled with rhodamine-labelled goat anti-mouse Ig and directly flouresceinated UCH gamma have been used, via double labelling immunofluorescence microscopy, to assay for the presence of beta-chains in fetal erythrocytes obtained at fetoscopy from fetuses at risk for beta-thalassaemia major. The results from 111 cases demonstrate that beta-chain synthesis of as low as 1.8% can be detected in fetal erythrocytes. The immunochemical labelling can be rendered more sensitive by the use of biotinylated UCH beta and avidin-FITC conjugates. This method is rapid and can be used for a sample that is highly contaminated with maternal erythrocytes.

Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin.

Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.

Antigenic structure of human haemoglobin. Localization of the antigenic sites of the beta-chain in three host species by synthetic overlapping peptides representing the entire chain.

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.

Antigenic structures of proteins. Their determination has revealed important aspects of immune recognition and generated strategies for synthetic mimicking of protein binding sites.

Studies in this laboratory have resulted in the delineation and synthetic verification of several complete protein antigenic structures that are recognized by antibodies. More recently, for the first time, the full profiles of the sites that are recognized by T cells have been localized and confirmed by synthesis for two proteins, myoglobin and lysozyme. These have thus far constituted the only complete antigenic structures to be determined. The availability of these antigenic structures has enabled us to investigate in detail the molecular and cellular parameters responsible for immune recognition, responses to, and control and regulation of these responses to protein antigens at the molecular and submolecular levels. Moreover, these investigations have afforded general strategies for the synthetic mimicking of not only antigenic sites, but also protein binding sites involved in other biological activities.