T cell allotypic determinants encoded by genes linked to the immunoglobulin heavy chain locus. I. Establishment of monoclonal antibodies against allotypic determinants

Monoclonal alloantibodies for T cell allotypic determinants were obtained by hybridizing SP-2 tumor cells with BALB/c (H-2d, Igh-1a) spleen cells, which had been repeatedly immunized with Con A-stimulated CB-20 (H-2d, Igh-1b) spleen cells. It was found that these monoclonal anti-CB-20 antibodies detect the new allotypic determinants (distinct from the B cell Igh-C region determinant) expressed only on the augmenting or suppressor T cells. Genetic analysis of these antigenic determinants revealed these antibodies react with the gene products located on the telomeric side chromosome of the Igh variable region gene (Igh-V) cluster. These antibodies when given in vivo caused a modification of antibody production. The antibody activity was absorbed by Con A-stimulated B10.BR (H-2b, Igh-1b), C57BL/6 (H-2b, Igh-1b), CWB (H-2b, Igh-1b), CB-20 (H-2d, Igh-1b), and BAB-14 (H-2d, Igh-1b) spleen cells, but not by Con A-stimulated C3H.SW (H-2b, Igh-1j), BALB/c (H-2d, Igh-1a), A/Sn (H-2a, Igh-1e), and C.AL-20 (H-2d, Igh-1d) spleen cells. In addition, in vivo these monoclonal antibodies modified anti-SRBC antibody production only in Igh-1b allotype-bearing mice. One monoclonal antibody reacted with 4-hydroxy-3-nitrophenyl acetyl- (NP) hapten-specific augmenting T cells, and the other two batches of monoclonal antibodies reacted with NP-specific suppressor T cells of NP-mediated cutaneous responses. A mapping study with these recombinants limits the gene coding for the T cell-specific determinants to a gene within the variable region to the telomeric side of NP-VH and to the centromeric side of prealbumin. This segment is inclusive of all immunoglobulin genes, the region Owen named IgT-C, and a histocompatibility gene (H-Ig).     

Further studies of specificity of antibodies contained in antiserum against poliovirus.

Antigenic components of Mahoney strain (poliovirus type 1) involved in virus neutralization reaction were analyzed with mutant Mahoney strains resistant to inhibitors in equine serum (inhibitor-resistant mutants) by means of the kinetic neutralization test. It was shown that absorption of anti-Mahoney serum with five inhibitor-resistant mutants yielded sera with different antibodies, of which three had distinct specificities and two specificities possibly partly related to one of those three sera. Further, it was found that step wise selection of Mahoney variants resistant to one, two, three and four different inhibitors resulted in gradual deviation of its antigenic composition from that of the original strain. From these results, the possible presence of three or more distinct antigenic determinant sites on the surface of Mahoney strain was indicated.     

ELISA for the detection of specific IgM and IgG in human leptospirosis

ELISA was used to detect specific IgM and IgG in sera from humans with current or past leptospirosis. A serological pattern of a high IgM titre (greater than or equal to 1280), or moderately increased IgM (160-640) in conjunction with a low IgG titre (less than or equal to 20), with serovar copenhageni antigen was characteristic for approximately two-thirds of the sera from serovar icterohaemorrhagiae patients obtained in the first two months of the disease. The antigen was the supernatant of a heated and centrifuged culture of leptospires. Antigens were prepared from serovars copenhageni, grippotyphosa, hardjo and patoc. Sera from patients with icterohaemorrhagiae, grippotyphosa and hardjo infections showed cross-reactivity when different antigens were used. In past infections the IgG titres were clearly higher with the homologous antigen. ELISA for IgM and IgG allows the rapid diagnosis of acute leptospirosis.     

Age of appearance of IgG, IgM, and IgE antibodies specific for Loa loa in Gabonese children

IgG, IgM, and IgE antibodies against the filaria Loa loa were measured in umbilical cord blood and in blood from young Gabonese children by an ELISA technique using a homologous metabolic antigen. For children in eight consecutive age groups and adults the percentage of the population positive for each of the antibody classes was determined. The number of children with maternal IgG decreased until one year of age when new synthesis began to become apparent. IgM antibodies were detected only after six months, probably indicating an early infancy as opposed to a fetal infection. The percentage of individuals positive for IgM or IgE reached a peak between two and three years old, followed by a slight decline. Over half of the individuals over one year of age had IgM antibody against L. loa, indicating long-term synthesis of this class of immunoglobulin in many people. In the first two years of life, IgE antibodies were usually accompanied by L. loa-specific IgM. This specific IgE did not appear to trigger the synthesis of nonspecific IgE. By the age of two, 95% of the population had some antibodies against L. loa and by five the percentage of individuals positive for each antibody class had reached adult levels.     

Use of near-infrared raman spectroscopy to detect IgG and IgM antibodies against Toxoplasma gondii in serum samples of domestic cats.

Near-infrared Raman spectroscopy (NIRS) is a particularly promising technique that is being used in recent years formany biomedical applications. Optical spectroscopy has gained increasing prominence as a tool for quantitative analysis of biological samples, clinical diagnostic, concentration measurements of blood metabolites and therapeutic drugs, and analysis of the chemical composition of human tissues. Toxoplasmosis is an important zoonosis in public health, and domestic cats are the most important transmitters of the disease. This disease can be detected by several serological tests, which usually have a high cost and require a long time. The goal of this work was to investigate a new method to diagnosis Toxoplasma gondii infections using NIRS. In order to confirm antibody detection, 24 cat blood serum samples were analyzed by the Raman spectra, from which 23 presented positive serology to toxoplasmosis and one was a reference negative serum. Characteristic Raman peaks allowed differentiation between negative and positive sera, confirming the possibility of antibody detection by Raman spectroscopy. These results give the first evidence that this technique can be useful to quantify antibodies in cat sera.     

Characterization of murine monoclonal antibodies against Helicobacter pylori lipopolysaccharide specific for Lex and Ley blood group determinants.

The cell envelope of Helicobacter pylori contains lipopolysaccharide (LPS), the O-chain of which expresses type 2 Lex and Ley blood group antigens, which mimic human gastric mucosal cell-surface glycoconjugates and may contribute to the survival of H. pylori in gastric mucosa. Here we describe the generation of monoclonal antibodies specific for Lex and Ley blood group determinants and the characterization of their binding properties using purified, structurally defined H. pylori LPS, synthetic glycoconjugates, and H. pylori cells. Analysis of oligosaccharide binding by SPR provided a rapid and reliable means for characterization of antibody affinities. One of the antibodies, anti-Lex, was of IgG3 subclass and had superior binding characteristics as compared with the commercially available anti-Lex IgM. These antibodies could have potential in the immunodiagnosis of certain types of cancer, in serotyping of H. pylori isolates, and in structure-function studies.     

Comparative evaluation of three recombinant antigen-based enzyme immunoassays for detection of IgM and IgG antibodies to human parvovirus B19

Background: Diagnosis of acute and past infection with parvovirus B19 is based on detection of IgM and IgG antibodies.Objectives: To evaluate two commercial recombinant antigen-based enzyme immunoassay (EIA) test kits for detection of IgM and IgG antibodies to parvovirus B19 and to compare the commercial EIAs to in-house EIA test procedures.Study design: A panel of 121 sera was used to compare the three IgM EIAs. The panel included 84 sera submitted for parvovirus B19 testing and 37 sera that were IgM positive for other viral pathogens. The same serum panel plus an additional 14 sera submitted for B19 testing was used to compare the three IgG EIAs. The commercial EIAs were performed according to manufacturers' instructions. Using the in-house EIA test procedures as the reference, sensitivity and specificity for each of the commercial EIAs was determined.Results: The commercial B19 IgM EIAs showed agreements of 95.0 and 93.4% to the in-house IgM EIA. Compared to the in-house B19 IgM EIA, the commercial B19 IgM EIAs, were 97.4 and 97.5% sensitive, respectively. Specificities were 93.5 and 91.4%, respectively. Sensitivities for the commercial IgG EIAs, compared to in-house IgG EIA, were 88.0 and 85.2%, respectively, and specificities were 94.1 and 98.0%.Conclusion: We found that the commercial parvovirus B19 IgM and IgG EIAs are comparable to standard in-house EIAs and are suitable for testing for B19 antibodies in human sera.     

Development of monoclonal IgA and an apparent IgG in a patient with macroglobulinemia: sharing of individually specific antigenic determinants among IgM, IgA, and IgG.

Patient CM, who initially was diagnosed as having macroglobulinemia (IgM, kappa) was subsequently found to develop a monoclonal IgA(kappa) protein. Rabbit antisera directed against the patient's IgAm and IgM were rendered specific for individual antigenic (ind) determinants. The anti-IgAm and IgM ind sera reacted with both 131I labeled monoclonal proteins in a double antibody radioimmunoassay (RIA). In addition, both monoclonal immunoglobulins inhibited the reaction between labeled immunoglobulin and both ind antisera, and statistical analysis of the data suggested that the shared ind determinants were identical. The IgG fraction of patient CM's serum also contained a component which competed with both monoclonal IgA (CM) and IgM (CM) in the RIA specific for ind determinants. Analysis of serum samples taken over a 2-year period revealed that, in addition to IgM, both the IgA and IgG components possessing the shared ind determinant(s) were present in low concentrations in the earliest sample, although not detected by conventional techniques. The monoclonal IgA and the IgG component were found to increase in concentration over this time interval with a concomitant decrease in IgM. The regulation of immunoglobulin expression with respect to the proposed models of gene organization in antibody-producing cells was discussed.     

Comparative study of cell-immunoenzymatic methods for the estimation of IgG and IgM anti-brucella antibodies in the diagnosis of human brucellosis

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11 % positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM anti-brucella antibodies.     

Xenogeneic antibodies with apparent public idiotypic specificity for anti-Ia.7 antibodies are directed in part against V kappa 21D and E subgroup marker

Public idiotypes (IdX) expressed on monoclonal antibodies (mAb) against a monomorphic alpha-chain determinant of the I-E molecule (Ia.7 epitope cluster I) have been studied by using xenogeneic anti-Id reagents derived from pig, rabbit, and rat. IdX+ anti-Ia.7 mAb were recently demonstrated to be structurally related by a high frequency expression of the V kappa 21E light chain subgroup. This raised the question of whether V region determinants of the IdX were related to V kappa 21E sequences or whether they were unique to hypervariable regions of Ia.7 binding antibodies. To clarify this question, the possible association between the expression of the public Id (IdX(s)Ia.7) and the presence of V kappa sequences (V kappa 21E and/or J kappa segment) was examined. The reactivity of the anti-Id reagents with a random panel of 28 myeloma products (each containing a light chain from one of the different V kappa 21 subgroups) was studied by assaying the ability of these mAb to inhibit the binding between the anti-Id and anti-Ia.7 mAb. This analysis demonstrates that what has previously been defined as IdX Ia.7 includes determinants shared by V kappa 21E and V kappa 21D light chain V regions. The structures recognized are expressed irrespective of the J kappa segment. In addition, this study demonstrates interspecies variation in immune responses to such V kappa 21E antigenic determinants. Additional IdX components are found on anti-Ia.7 mAb but not on other V kappa 21E or D proteins. Thus V region subgroup considerations have crucial implications for Id characterization. In addition, this work describes the first division of the V kappa 21 subgroup into component parts by a mAb.     

Comparative study of cell-immunoenzymatic methods for the estimation of IgG and IgM anti-Brucella antibodies in the diagnosis of human brucellosis

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11% positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM antibrucella antibodies.     

Immunity in human schistosomiasis mansoni: cross-reactive IgM and IgG2 anti-carbohydrate antibodies block the expression of immunity

We have previously reported that the slow development of immunity to reinfection after treatment of Schistosoma mansoni infections is partly attributable to the continued presence of 'blocking' antibodies in young, susceptible children. A further analysis of this phenomenon supports the hypothesis that such blocking antibodies can be of the IgG2 as well as the IgM isotype, and that they react with carbohydrate epitopes expressed both on egg polysaccharides and on schistosomulum surface antigens, of particular importance being those antigens that are shed from the schistosomulum surface during the early stages of maturation in vitro. Evidence is also presented that, in those patients lacking high levels of IgG2 blocking antibodies, resistance to reinfection after treatment is associated with the presence of other IgG isotypes against the same shed antigens.