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G. Norheim,
A. Aseffa,
M. A. Yassin,
G. Mengistu,
A. Kassu,
D. Fikremariam,
W. Tamire,
Y. Merid,
E. A. Hoiby,
D. A. Caugant,
E. Fritzsonn,
T. Tangen,
B. Melak,
D. Berhanu,
M. Harboe, J. Kolberg,
E. Rosenqvist
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ABSTRACT: Dissecting the specificities of human antibody responses following disease caused by serogroup A meningococci may be important for the development of improved vaccines. We performed a study of Ethiopian patients during outbreaks in 2002 and 2003. Sera were obtained from 71 patients with meningitis caused by bacteria of sequence type 7, as confirmed by PCR or culture, and from 113 Ethiopian controls. Antibody specificities were analyzed by immunoblotting (IB) against outer membrane antigen extracts of a reference strain and of the patients' own isolates and by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) levels against lipooligosaccharide (LOS) L11 and the proteins NadA and NspA. IB revealed that the main antigens targeted were the proteins PorA, PorB, RmpM, and Opa/OpcA, as well as LOS. MenA disease induced significant increases in IgG against LOS L11 and NadA. The IgG levels against LOS remained elevated following disease, whereas the IgG anti-NadA levels returned to acute-phase levels in the late convalescent phase. Among adults, the anti-LOS IgG levels were similar in acute-phase patient sera as in control sera, whereas anti-NadA IgG levels were significantly higher in acute-phase sera than in controls. The IgG antibody levels against LOS and NadA correlated moderately but significantly with serum bactericidal activity against MenA strains. Future studies on immune response during MenA disease should take into account the high levels of anti-MenA polysaccharide IgG commonly found in the population and seek to clarify the role of antibodies against subcapsular antigens in protection against MenA disease.
Clin Vaccine Immunol. 01/2008; 15(5):863-71.
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ABSTRACT: Mouse monoclonal antibodies (MoAbs) of the four IgG isotypes, all specific for the P1.16 epitope on the meningcoccal PorA protein, were tested for functional activities. The avidities of the antibodies, measured by NH4SCN elution in enzyme-linked immunosorbent assay, showed similar values for all the MoAbs. The serum bactericidal activity (SBA) defined as the lowest concentration of antibodies giving 50% reduction in the number of meningococcal colony-forming units using human serum as complement, showed a hierarchy of IgG3 > IgG2b > IgG2a > IgG1. For the opsonophagocytosis (OP), the hierarchy was IgG3 > IgG2b = IgG2a > IgG1. OP was measured in flow cytometry using log-phase live meningococci as target cells, normal human peripheral blood polymorphonuclear cells (PMNs) as effector cells and human serum as a complement source. The mouse MoAbs were negative in OP when using human PMNs in the absence of complement. The results demonstrate the importance of choosing the right isotype of mouse MoAbs when using them to judge the potential vaccine importance of their corresponding antigen. If such MoAbs should be used for passive vaccination against infectious diseases, the isotype would presumably play an important role for their anticipated clinical effects.
Scandinavian Journal of Immunology 01/2004; 59(1):34-9. · 2.23 Impact Factor
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ABSTRACT: We have constructed chimaeric (ch) mouse/human antibodies with identical binding regions isolated from the V-genes of two mouse parent hybridoma cell lines, with specificity against the P1.7 and P1.16 epitopes on the outer-membrane protein PorA on meningococci. The chimaeric antibodies can be used to analyse relationships between specificity, binding activity (avidity and kinetics), isotype (antibody class and antibody subclass) and in vitro anti-bacterial activity of meningococcal antibodies. The antibody sets represented the human isotypes IgG1, IgG3 and IgM, which dominate during immune response against protein antigens. The binding activities were quite similar for all these isotypes, surprisingly also for the pentameric IgM. Interestingly, monomeric IgM, prepared from pentameric IgM by partially reduction and alkylation, had similar binding activities as the original pentameric IgM. Regarding in vitro anti-bacterial activity, chIgG1 was superior in SBA (serum bactericidal activity) compared with chIgG3, while chIgG3 was more efficient in OP (opsonophagocytosis; measured by flow cytometry) than chIgG1. ChIgM showed slightly higher SBA than chIgG1 on molar basis, and much higher OP than chIgG3 and chIgG1. A lower concentration of antibodies was needed against the P1.16 than against the P1.7 epitope to induce SBA, but this was not the case for OP.
Biochemical Society Transactions 11/2003; 31(Pt 5):1032-5. · 3.71 Impact Factor
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ABSTRACT: Immunisation of BALB/c mice with seven heat-treated Norwegian clinical isolates of Streptococcus pneumoniae of different serotypes elicited mainly monoclonal antibodies (mAbs) to pneumococcal surface protein A (PspA). It was remarkable that the fusions resulted only in a few mAbs directed against other protein antigens. Dot blot analysis with 16 mAbs using clinical isolates representing 23 different capsular types and the uncapsulated reference strain R36A showed that some of the mAbs bound to PspA epitopes expressed by a low number of strains whereas others bound to broadly distributed epitopes. On the basis of their reactivities, seven of these mAbs could be divided into two groups recognising different subsets of pneumococci. The three mAbs in the narrow reacting group bound to epitopes found in 21-25% of the strains whereas the four mAbs in the broad reacting group detected more than 57% of the analysed strains. The epitopes for these seven antibodies were surface exposed on live exponential phase grown pneumococci as shown by flow cytometry. The finding that a combination of mAb 180,C-1 (IgG2a) from the first group and mAb 170,E-11 (IgG2a) from the second group detected 94% of the examined strains is interesting because PspA has been reported by others to be a serological highly variable protein.
FEMS Immunology & Medical Microbiology 11/2001; 31(3):175-80. · 2.44 Impact Factor
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ABSTRACT: The RmpM protein has been reported to be present only in pathogenic Neisseria species. In the present study we demonstrate that this protein is also present at least in N. lactamica and N. sicca strains. The N. lactamica protein reacts with a RmpM-specific monoclonal antibody (185,H-8), having a molecular mass ( approximately 31 kDa) slightly lower than that of the meningococcal RmpM, and mouse antibodies from sera against outer membrane vesicles from both N. lactamica and N. sicca strains cross-react with the meningococcal RmpM. PCR and hybridization experiments with a complete rmpM probe agree with the immunodetection experiments. Our results strongly suggest that the meningococcal RmpM should not be considered a virulence marker, and the presence of this protein in the commensal species agrees with its role as a structural protein, proposed for the RmpM, which should be considerably conserved in the Neisseria species.
FEMS Microbiology Letters 06/2001; 199(2):171-6. · 2.04 Impact Factor
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ABSTRACT: It is reported here that the PorB3 porin proteins of serotype 4 and 15 are poorly accessible for antibody binding on live Neisseria meningitidis bacteria, whereas the allelic PorB2 and the PorA outer membrane protein appear to be highly accessible. This was revealed by flow cytometry analysis using several mouse monoclonal antibodies (mAbs) as well as PorB3 specific antibodies isolated from post vaccination and patient sera. However, strong antibody binding to the PorB3 protein was observed after killing the bacteria with ethanol. The reason for the lack of epitope exposure could be a shielding effect of the carbohydrate chains of lipopolysaccharides (LPS) possibly combined with short extra-cellular loops in the PorB3 protein. The findings indicate that the PorB3 protein is not an optimal target for protective antibodies induced by vaccination.
Vaccine 02/2001; 19(11-12):1526-33. · 3.77 Impact Factor
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ABSTRACT: Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.
FEMS Immunology & Medical Microbiology 01/2001; 29(4):289-94. · 2.44 Impact Factor
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E Rosenqvist,
A Musacchio,
A Aase,
E A Høiby,
E Namork, J Kolberg,
E Wedege,
A Delvig,
R Dalseg,
T E Michaelsen,
J Tommassen
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ABSTRACT: Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.
Infection and Immunity 04/1999; 67(3):1267-76. · 4.16 Impact Factor
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ABSTRACT: Streptococcus pneumoniae group 9 includes four capsular polysaccharide types: 9A, 9L, 9N and 9V. We have generated four mouse monoclonal antibodies against group 9 polysaccharide using heat-treated S. pneumoniae strains of different capsular polysaccharides types as immunogens. The specificities of the monoclonal antibodies were determined by ELISA using capsular polysaccharide directly coated to the wells as antigens and by dot blotting with heat-treated bacteria. Two groups of monoclonal antibodies were found. The first group included two monoclonal antibodies which were found to be capsular type specific. The second group was monoclonal antibodies that bound to epitopes shared by two or three pneumococcal group 9 types. The monoclonal antibody 204,A-4 (IgM) was found to be specific for S. pneumoniae type 9N. The binding of the type 9V specific monoclonal antibody 206,F-5 (IgG1) was found to be dependent upon O-acetyl groups. Monoclonal antibody 205,F-3 (IgM) reacted also with type 9V, but was found to cross-react with types 9A and 9L. The binding of this monoclonal antibody to polysaccharide 9V was not dependent upon O-acetyl moieties. The fourth monoclonal antibody (214,G-5, isotype IgM) did not show any correlation between reactivity with isolated polysaccharides and dot blotting with relevant bacteria. The monoclonal antibody reacted with polysaccharides 9A and 9L in ELISA, but not with the homologous bacteria.
FEMS Immunology & Medical Microbiology 05/1998; 20(4):249-55. · 2.44 Impact Factor
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ABSTRACT: The sequence diversity of 45 Opa outer membrane proteins from Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria sicca, and Neisseria flava indicates that horizontal genetic exchange of opa alleles has been rare between these species. A two-dimensional structural model containing four surface-exposed loops was constructed based on rules derived from porin crystal structure and on conservation of sequence homology within transmembrane beta-strands. The minimal continuous epitopes recognized by 23 monoclonal antibodies were mapped to loops 2 and 3. Some of these epitopes are localized on the bacterial cell surface, in support of the model.
Journal of Bacteriology 04/1998; 180(5):1323-30. · 3.83 Impact Factor
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ABSTRACT: We wanted to compare the potential protective capacity of antibodies to meningococcal lipopolysaccharides (LPS). The frequency of occurrence and degree of expression of the epitopes recognized by murine monoclonal antibodies (MAbs) to immunotypes L3,7,9 (9-2-L379) and L8 (2-1-L8) and to the LPS inner core (216-Lc and 217-Lc), were determined among 77 consecutive Norwegian meningococcal patient isolates from 1995. The immunotype L3,7,9 was strongly expressed by 95% of the isolates, whereas L8 was weakly to moderately expressed by 9%. The inner core epitopes, were widely distributed among the serogroup B organisms, but were proved weakly expressed. The bactericidal activity of the four MAbs to various selected strains, was found to correlate positively with the quantity of the LPS epitopes recognized by these four MAbs in the bacteria. When tested in the serum bactericidal assay (SBA), often a few percent of the colonies of the inocula survived high concentrations of the MAbs. The results indicate that escape from the bactericidal action could be achieved through: (i) selection of variants not expressing the LPS-epitope of the actual MAb, (ii) a relative reduction in the density of the LPS-epitope achieved by dilution with another LPS structure or (iii) other factors, not yet understood. In conclusion, antibodies to the L3,7,9 epitope seem to be of importance for protection, whereas antibodies to the epitopes of the LPS inner core or immunotype L8, are not likely to offer protection alone. However, in order to prevent escape through alteration of the LPS pattern of the microbes, various LPS structures should probably be present in the OMV vaccine.
Microbial Pathogenesis 10/1997; 23(3):139-55. · 1.94 Impact Factor
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ABSTRACT: The phosphorylcholine (PC) determinant in Streptococcus pneumoniae is known to be linked to the cell wall polysaccharides (C-Ps) and to the lipoteichoic acid (LTA) (Forssman antigen) of the plasma membrane. Western blotting with two PC specific murine monoclonal antibodies (MAbs) designated 145,F-2 (IgM) and 147,A-1 (IgA) showed a similar ladder-like pattern for all examined strains of S. pneumoniae and Streptococcus mitis. Purified antigens run in parallel indicated that this ladder pattern is due to the PC of LTA. Unlike other techniques, Western blotting thus enables the identification of only one of the streptococcal structures carrying the PC epitope. Gram-negative organisms were also examined, and six of 11 Haemophilus influenzae strains reacted with the MAbs. For this species, unlike the streptococci, only one fast moving band was detected. Analyses by thin-layer chromatography (TLC) detected the PC epitope in lipopolysaccharide (LPS) fraction from H. influenzae. Some strains of the Neisseriaceae family were also positive by Western blotting, but TLC and immunostaining did not detect the PC determinant in LPS.
Microbial Pathogenesis 07/1997; 22(6):321-9. · 1.94 Impact Factor
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ABSTRACT: Two monoclonal antibodies (mAbs) designated 144,H-3 (IgG2a) and 218,C-5 (IgM) were produced after immunization of mice with two different heat-treated and sonicated pneumococcal strains. Western blotting, with solubilized proteins from different bacterial genera and from mammalian lymphocytes, showed that both mAbs reacted with a protein of approximately 12 kDa in all 66 strains of eubacteria examined, representing 27 different species. The 12 kDa protein was isolated by immunoaffinity chromatography. Subsequent preparative Western blotting enabled N-terminal amino acid sequence analysis by microsequencing. A high degree of amino acid sequence similarity with eubacterial ribosomal proteins L7/L12 was demonstrated. One of the mAbs (144,H-3) also cross-reacted in Western blotting with a 43 kDa protein, but only from streptococci. The 43 kDa protein carrying the common streptococcal epitope was isolated and sequenced in the N-terminal region. A high degree of amino acid sequence identity was found to elongation factor Ts from Escherichia coli.
Microbiology 02/1997; 143 ( Pt 1):55-61. · 3.06 Impact Factor
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ABSTRACT: Monoclonal antibodies (MAbs) against clinical isolates of Streptococcus pneumoniae were produced in a search for common pneumococcal proteins. One of the fusions generated two MAbs, 174,B-8 (immunoglobulin G2a) and 177,D-8 (immunoglobulin G1), which by Western blotting (immunoblotting) stained with a main band of 40 kDa found in all isolates of S. pneumoniae examined. Cross-reactivity studies with streptococci other than pneumococci revealed very weak or moderate reactions with the MAbs. The 40-kDa protein was isolated by immunoaffinity chromatography and subsequent preparative Western blotting. N-terminal amino acid sequencing showed 90% amino acid sequence homology with a surface-located glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes. This protein has also been reported to exhibit binding to mammalian proteins such as fibronectin, which may serve as host receptors. The epitopes for MAbs 174,B-8 and 177,D-8 reacting with the pneumococcal analog were not accessible to antibody binding in live bacteria but were exposed after heat killing. The MAbs showed negligible cross-reactions with S. pyogenes.
Infection and Immunity 10/1996; 64(9):3544-7. · 4.16 Impact Factor
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ABSTRACT: The antibody kinetics in sera from 27 adults after three doses of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine was studied. The vaccinees received the third dose 4 to 5 years after the first two. Antibody responses against outer membrane proteins (OMPs) and lipopolysaccharides were studied by enzyme-linked immunosorbent assay and immunoblotting and with serum bactericidal assays (SBA) with three variants of the vaccine strain, 44/76. Six weeks after the second injection, the geometric mean (GM) of the levels of immunoglobulin G (IgG) against OMVs was about sevenfold higher than that of prevaccination levels, and 74% of the vaccinees developed a greater-than-twofold rise in SBA titer. After 6 months, the GM of IgG levels declined to about threefold higher, and after 4 to 5 years it declined to about twofold higher, than that before vaccination. The third dose induced a rapid increase in SBA titers in 96% of the vaccinees, and the GM of levels of IgG against OMVs rose to about 14-fold the prevaccination level. One year later, the IgG antibody levels had dropped to 4.6-fold the prevaccination level, but 88% of the vaccinees still showed bactericidal activity. The response after the two first doses was higher in individuals with prevaccination antibodies, but no such effect was found after three doses. The use of defined mutants in SBA and linear multiple regression analyses indicated that among the major OMPs, antibodies to the Opc and class 1 proteins made the most important single contributions to the bactericidal activity against the vaccine strain, but it also demonstrated the importance of antibodies against other antigens. After three doses, 68% of the vaccinees showed a significant SBA response against a strain lacking both the Opc and the class 1 proteins. Three doses converted almost all subjects to SBA responders and gave higher antibody levels and relatively less serosubtype-specific bactericidal activity than did two doses, probably indicating a broader cross-protection against heterologous strains.
Infection and Immunity 01/1996; 63(12):4642-52. · 4.16 Impact Factor
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ABSTRACT: The class 3 outer-membrane protein (OMP) of Neisseria meningitidis is a potential target for bactericidal and opsonic antibodies in humans. Synthetic peptides spanning the class 3 OMP from the vaccine strain 44/76 (B:15:P1.7,16:L3,7) were synthesized on pins and screened with serum obtained from Norwegian adolescents immunized with a meningococcal serogroup B outer-membrane vesicle (OMV) vaccine. A strong IgG response to a single peptide (19FHQNGQVTEVTT30) located within loop 1 (VR1) was stimulated after three doses of OMV vaccine in three vaccinees selected on the basis of their antibody response to class 3 OMP. No clear linear B-cell epitopes were recognized by four different murine serotype 15-specific mAbs. A 23mer peptide (D63b2) containing loop 1 of the class 3 OMP was synthesized, and the IgG responses were measured in pre- and post-vaccination serum from 27 vaccinees. Specific IgG rose significantly in 37% of vaccinees 6 weeks after the second dose and in 74% of the vaccinees 6 weeks after the third dose of the OMV vaccine. Most immune sera reacted distinctly on immunoblots with denatured class 3 OMP, and the immunoblotting reactivity correlated strongly with concentration of the IgG antibodies specific for peptide D63b2. When added to a post-vaccination serum from one vaccinee, peptide D63b2 competed efficiently with the class 3 OMP for specific antibody binding on immunoblots and in pin ELISA. The results show that the significant part of the humoral response to the meningococcal class 3 OMP elicited by vaccination with the Norwegian OMV vaccine was directed against a single continuous epitope.
Microbiology 08/1995; 141 ( Pt 7):1593-600. · 3.06 Impact Factor
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ABSTRACT: An increase in B:15:P1.12 meningococci among isolates from patients with Neisseria meningitidis infection in Norway in recent years led to further characterization of such strains. Between 1987 and 1992, B:15:P1.12 strains constituted 9.8% (24 strains) of B:15 isolates. The B:15:P1.12 strains belonged to the electrophoretic type 5 (ET-5) complex, but 17 (71%) strains were a new clone (ET-5c) not found elsewhere in the world. All but one strain of ET-5c were responsible for a localized outbreak of systemic meningococcal disease in western Norway. A novel monoclonal antibody (202,G-12), developed against the unknown variable region 2 on the class 1 protein of one of these strains, bound to 19 of the 15:P1.12 strains, 4 strains bound the subtype P1.13 reference monoclonal antibody MN24H10.75, and the remaining strain showed no reaction. Sequencing of porA genes demonstrated a series of nine threonine residues in the deduced variable region 2 of the latter strain, while four and five threonine residues were found in the corresponding regions of strains reacting with the monoclonal antibodies 202,G-12 and MN24H10.75, respectively. Epitope mapping with synthetic peptides showed that 202,G-12 bound to a sequence of 11 amino acids which included the four threonine residues specific for subtype P1.13a. Immunoglobulin G antibodies against the P1.7,16 subtype protein, induced in volunteers after vaccination with the Norwegian meningococcal vaccine, did not cross-react on immunoblots with the subtype protein of clone ET-5c. Thus, postvaccination class 1 protein antibodies, assumed to be protective, may not be effective against infection with the new clone.
Clinical and Diagnostic Laboratory Immunology 06/1995; 2(3):314-21. · 2.51 Impact Factor
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ABSTRACT: Two sets of monoclonal antibodies (mAbs) probably reacting with two different epitopes in the CH3 domain of the human IgG4 molecule were studied. We observed that the commercially available mAb HP 6011 inhibited the antigen binding of the three mutually inhibitable mAbs, 40-A2, 41-E8 and 43-F11 (40-series), made by us. However, the 40-series mAbs, including those with similar affinity such as mAb HP6011, were not able to inhibit mAb HP 6011. When the 40-series mAbs were preincubated with IgG4, the mAb HP 6011 could partially displace these antibodies. This one-way inhibition indicates that upon binding mAb HP 6011 changes the antigenic structure of the IgG4 molecule by disrupting the epitope for the 40-series mAbs. A steric hindrance of this epitope by mAb HP 6011 is more unlikely, since the small Fab fragment of mAb HP 6011 also inhibited the reaction of the 40-series mAbs.
Apmis 08/1992; 100(7):615-22. · 1.99 Impact Factor
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ABSTRACT: Binding patterns of mouse monoclonal antibodies (mAb) to P1, Pk, N, I, H, Y or A antigens were visualized in the backscatter electron imaging mode of a scanning electron microscope by indirect immunogold labelling. Experiments were performed at room temperature (RT) and at 4 degrees C. In experiments with anti-P1 and anti-Pk, clusters of immunolabelling particles dominated the immunolabelling pattern much more at RT than at 4 degrees C. By contrast, no clustering was seen with anti-N, even at RT. Clustering was also observed at RT with anti-I, anti-H and anti-Y, and on some Ax and A3 cells with anti-A, but was much reduced at 4 degrees C. Immunolabelling was stronger at 4 degrees C than at RT with all mAb except anti-N and anti-A. The results indicate that glycolipid blood group antigens are more mobile in the membrane of intact erythrocytes at RT than at 4 degrees C, and that the cells bind more antibodies to such antigens at 4 degrees C than at RT. We suggest that antigen immobilization in the cold will reduce cross-linking of antigens and hence increase the number of antibody molecules needed for epitope saturation, leading to increased binding of antibody in the cold. This may be the main reason for cold-enhanced agglutination with human blood group antibodies.
Transfusion Medicine 04/1992; 2(1):7-15. · 1.14 Impact Factor
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J Kolberg
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ABSTRACT: Two mouse monoclonal antibodies (IgM) were produced against the N-acetylglucosamine-specific rice bran lectin. It was difficult to establish antibody-producing hybrids when soluble rice lectin was used for immunization. Therefore a complex of rice lectin and chitin, a water-insoluble polymer of N-acetylglucosamine, was used as immunogen. Antibody reactivity against Gramineae lectins from barley, rice, rye and wheat (wheat germ agglutinin) was tested in ELISA and two (137,E-1 and 140,B-3) were found to be specific for rice lectin. Gramineae lectins were separated by sodium dodecyl-sulphate-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose papers. Analyses showed that the antibodies reacted strongly with non-reduced rice lectin, whereas only weak staining was seen with the other lectins. The binding was abolished after treatment with 2-mercaptoethanol suggesting that disulphide bond tertiary structures were necessary for epitope integrity.
Biological chemistry Hoppe-Seyler 03/1992; 373(2):77-80.
