Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: Specificities and reactivities of the antibodies formed in rabbits to stearyl-isomaltosyl oligosaccharides

The specificities and the sizes and shapes of the antibody combining sites of the 15 antisera raised against various stearyl-isomaltosyl oligosaccharides were studied by quantitative precipitin and precipitin inhibition. The antibodies precipitated well with dextrans B512 and B1424 but less well with B1299S and B1355S. Only 3 of the 15 antisera reacted with linear dextrans; however, with about 50% of the added antibodies being precipitated, showing that most of the antibodies cannot bind to internal determinants along the dextran molecules and are similar to myeloma protein W3129 in having cavity-type sites which bind only to terminal nonreducing ends of α1 → 6 dextran. Antibodies differing in the sizes of their antibody combining sites were elicited in different rabbits by the same antigen. Of the 15 antisera studied, four have antibody combining sites as large as IM3, five as large as IM4, three as large as IM5 and three as large as IM6. The association constants for various isomaltose oligosaccharides of an antiserum (R-862) showing fewest bands in isoelectric focusing gel were determined by affinity electrophoresis and were comparable to W3129.     

Specificities of human heterophile Hanganutziu and Deicher (HD) antibodies to glycosphingolipids and a glycoprotein

The specificities of five heterophile Hanganutziu and Deicher (HD) antibody-containing sera from four different cancer patients and one other diseased patients were compared. Three glycosphingolipids and one glycoprotein antigens and their chemically modified derivatives were used. The antibodies of all whole sera showed similar specificities. IgG and IgM antibody fractions of each serum were separated. Although antibodies of the same class showed similar specificities, differences were detected between the specificities of IgG and IgM. IgG antibody specificities were dependent on the hydrophobic (ceramide) group while IgM antibodies were directed more to the terminal sialic acid moiety of the glycosphingolipid antigens. The results suggested that a similar population of IgG-producing lymphocytes is stimulated in patients. Due to the similarities in specificities of HD antibodies, the results of this study will facilitate the future isolation of either IgG or IgM antibody-producing lymphocyte(s) from a patient with HD antibodies and the establishment of a monoclonal antibody through hybridization with a human myeloma cell line.     

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.     

Tumor cell reactivity mediated by IgM antibodies in sera from melanoma patients vaccinated with GM2 ganglioside covalently linked to KLH is increased by IgG antibodies

 Natural IgM antibodies against the melanoma cell-surface ganglioside GM2, and IgM antibodies induced by vaccination with GM2 adherent to bacillus Calmette-Guerin, have been correlated with increased disease-free and overall survival in melanoma patients in previous phase I and II clinical trials. A vaccine containing GM2 covalently attached to keyhole limpet hemocyanin (KLH) plus the immunological adjuvant QS-21 now induces higher-titer, longer-lasting IgM antibodies against GM2 and has recently entered phase III clinical trials. For the first time this new vaccine also induces IgG antibodies against GM2 in the majority of immunized patients. With regard to immunity against bacteria, IgM antibodies have been described to be 1000-fold more effective than IgG antibodies at opsonification, complement-mediated cytotoxicity and protection from bacterial challenge. Though IgG antibodies have the theoretical advantage of being able to mediate antibody-directed cell-mediated cytotoxicity (ADCC), they may inhibit complement mediated IgM effector mechanisms against melanoma cells. Our goal was to confirm the functional characteristics of the anti-GM2 IgM and IgG antibodies induced by vaccination and to determine the impact that IgG antibodies might have on IgM antibody reactivity with GM2-positive tumor cells. Post-immunization sera from seven immunized patients were separated by size-exclusion chromatography into IgM and IgG fractions and a variety of serological assays were performed with the individual fractions and their combinations. Assays identifying specific IgM or IgG reactivity demonstrated partial inhibition by the opposite fraction. However, when the endpoint was complement-mediated lysis or overall antibody binding, which may more faithfully predict in vivo complement-mediated opsonification and lysis, the combinations of IgM and IgG fractions consistently demonstrated higher reactivity than either fraction alone. In addition, ADCC was induced in all seven patients. The results were the same whether the sera were obtained after 2 months or 2 years of immunizations. These findings suggest that IgG antibodies induced by the GM2-KLH plus QS-21 vaccine will not inhibit and should further augment the clinical impact of induced IgM antibodies. Received: 25 April 1996 / Accepted: 21 October 1996     

Immunoglobulin classes of anti-Sendai virus antibody detected by ELISA in infected nude mouse serum

Sendai virus-infected nude mouse sera obtained on the seventh day after infection or later, in which anti-Sendai virus antibodies were undetectable by hemagglutination-inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase-conjugated anti-mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti-Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti-Sendai virus antibody activity of fractions from Sephadex G-200 gel filtration was detected in the IgM fraction when anti-mouse mu chain-specific antiserum was used and in both IgG and IgM fractions when heavy and light chain-specific anti-mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A-Sepharose affinity chromatography of Sendai virus-infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti-Sendai virus anti-bodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.     

The intrinsic affinity constant (K) of anticapsular antibody to oligosaccharides of Haemophilus influenzae type b

Antibody to the polyribosylribitol phosphate (PRP) capsular polysaccharide of Haemophilus influenzae type b is crucial to host defense. Affinities of antibody elicited by vaccination with PRP and PRP-diphtheria toxoid conjugate were determined using oligosaccharides (OS) from PRP. The affinities of antibody induced by vaccination with PRP to OS of three and four repeat units were similar but greater than the affinity to the two-unit OS. NaBH4 reduction of the three-unit OS did not alter the binding affinity, indicating that the reducing end of the OS did not participate in antibody binding. Over the range of OS concentrations tested, antibody affinity appeared to be homogeneous. Antibody concentration could be determined from binding experiments independently from affinity. Whole serum had 8- to 40-fold less antibody detected by binding analysis than by RIA, but the antibody concentration of an IgG fraction measured by the two methods agreed within a factor of two. We could not account for the discrepancy in concentrations found with whole serum by the presence of IgM or IgA antibody. The average affinity of antibodies of 10 adults vaccinated with PRP was similar to that of antibodies elicited in 14 adults vaccinated with a PRP-diphtheria toxoid conjugate (10.7 x 10(5) vs 7.6 x 10(5) liter/mol, respectively, p greater than 0.05). We conclude that the intrinsic affinity of antibody after vaccination with PRP is low and is not different from that of antibody elicited by PRP diphtheria toxoid conjugate.     

Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L-Asp-L-Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo- and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. (1998). Clin. Pharmac. Ther. 63, 580-593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid-phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl(2) to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp-Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG(1) antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp-Phe and Phe-Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer-assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG(4) antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints.     

Comparison of the C-mediating killing activity and C-activating properties of mouse monoclonal and polyclonal antibodies against Trypanosoma cruzi

A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG(1) and one IgG(2a)) recognize and combine with membrane components of trypomastigote forms of the parasite as revealed by immunofluorescence. Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C) system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabmu fragments were able to induce trypanosome lysis by the alternative C pathway.     

Subpopulations of antibodies to phosphocholine in human serum

We investigated the heterogeneity of anti-phosphocholine (PC) antibodies present in human serum taken from individuals before and after immunization with a multivalent pneumococcal vaccine. The fine specificity of IgM, IgG, and IgA anti-PC antibodies was determined in an ELISA by using phosphocholine or p-nitrophenyl phosphocholine (NPPC) to inhibit binding of antibody to PC-histone. We identified two populations specific for PC that differed in their binding properties. One population is inhibited by NPPC much better than by PC and is most evident in IgG antibodies. The second population has similar avidity for PC and NPPC and is consistently associated with the IgM and IgA isotypes as well as with IgG. The IgG antibodies in both populations were predominantly of the IgG2 subclass. Both populations were found in serum samples taken before immunization with pneumococcal vaccine, suggesting that they had been stimulated through prior environmental contacts with PC-containing antigens. Previously, we found populations with similar fine specificity patterns in the murine response to PC. The two murine antibody populations have been shown to derive from different immunoglobulin variable region genes. The presence of comparable antibody populations in the human suggests the possibility that these two fine specificity families have been conserved in evolution.     

Specific and shared idiotypes found on hybridoma anti-DNA autoantibodies derived from rheumatoid arthritis and systemic lupus erythematosus patients

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.     

Amino Acid Sequence of αkSubstance,a Mating Pheromone of Saccharomyces kluyveri

A fraction containing IgA (IgA-rich fraction) was prepared from bovine colostrum by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200. A large amount of IgG1-dimer was found in this fraction, which could not be separated from IgA by repeated gel filtration.

The Fc fragment of bovine colostral IgG (IgG-Fc) was prepared from papain digestion mixtures. IgG-Fc was found to be heterogeneous on DEAE-cellulose column chromatography. Two IgG-Fc fractions were obtained, but no antigenic difference was found between them. Anti-IgG-Fc antibodies raised in rabbits by injection of these Fc preparations reacted only with IgG1 and IgG2. An immunoadsorbent (anti-IgG-Fc-Sepharose) was prepared by coupling these anti-IgG-Fc antibodies to CNBr-activated Sepharose 4B.

IgA was purified from the IgA-rich fraction by affinity chromatography on anti-IgG-Fc-Sepharose adsorbent. IgG1-dimer was effectively removed by this treatment. The purified sample gave only one precipitin arc characteristic of IgA on immunoelectrophoresis with multiple anti-bovine colostral whey antiserum. A small amount of IgA was found to be adsorbed to the affinity column nonspecifically.

When a rabbit was immunized with the purified IgA, besides anti-IgA antibodies, antibodies against the secretory component (SC) were found in the antiserum. This finding leads us to expect that the purified IgA is secretory IgA containing SC.     

The presence of antibody in mice chronically infected with Schistosoma mansoni which blocks in vitro killing of schistosomula

We have previously reported that IgM monoclonal antibodies (mAb) that recognize surface carbohydrate determinants shared between schistosomula, cercariae, and miracidia block antibody/complement dependent killing of schistosomula in vitro. Binding assays that make use of one of the IgM mAb labeled with 125I demonstrated that serum from chronically infected mice (CMS) contained high levels of competing antibody, whereas serum from mice vaccinated with irradiated cercariae (VMS) contained little antibody of this specificity. Absorption of CMS with cercariae that removed antibodies to schistosomulum surface carbohydrate determinants increased its ability to kill schistosomula in vitro; absorption of VMS with cercariae failed to alter the lethal activity of the serum. Furthermore, fractionation of CMS by protein A Sepharose chromatography demonstrated that the IgG fraction had an increased lethal activity compared with unfractionated serum; this result was not seen with VMS. Finally, the IgM fraction of CMS was shown to block in vitro killing of the IgG fractions of both CMS and VMS. These data suggest that the blocking activities observed with the IgM mAb are contained within the serum of chronically infected mice but not in the serum of mice vaccinated with irradiated cercariae.     

Immunochemical studies with a specific antiserum to rabbit fibroblast collagenase.

1. Antisera were raised against the collagenase from rabbit synovial fibroblasts and characterized by immunoprecipitation and immunoinhibition reactions. 2. Immunoglobulins from the antisera were potent inhibitors of the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. 3. The antibody-binding fragment, Fab', produced by digesting the IgG (immunoglobulin G) with pepsin, inhibited collagenase activity just as well as whole IgG. 4. A specific antiserum to the rabbit collagenase was raised by a multi-step procedure. An initial antiserum was made by injecting partially purified collagenase as a complex with sheep alpha2-macroglobulin into a sheep. The non-specific antiserum so obtained was used to produce precipitin lines with the purified enzyme, and these lines were used as antigen for the production of the specific antiserum. 5. An IgG preparation from the specific antiserum was a specific and potent inhibitor of the rabbit synovial fibroblast collagenase. Neutral metallo-proteinase activity secreted by the rabbit fibroblasts was not inhibited by the antibody to the rabbit collagenase. 6. Criteria for determination of the specificity of antisera are discussed.     

Heterogeneity of antibody response to Salmonella lipopolysaccharide measured by passive hemagglutination and hemolysis in mice

The complement-requiring passive hemolysis test with Salmonella typhimurium lipopolysaccharide-coated sheep erythrocytes is more sensitive for antibodies directed against the lipopolysaccharide than is the passive hemagglutination test. The hemagglutinating and hemolyzing antibodies produced in Swiss mice by hyperimmunization, either with or without Freund's adjuvant, were distributed in both the light and heavy fractions isolated by sucrose density gradient fractionation and gel filtration. IgM fractions, whether tested by hemagglutination or hemolysis, were sensitive to 2-mercaptoethanol (0.15 m). On the other hand, IgG hemolytic antibodies were more sensitive to 2-mercaptoethanol than were IgG hemagglutinating antibodies. The resistance of IgG hemagglutinating activity amounted to about 72 to 95% of the total IgG recovered, whereas the resistant portion of the IgG hemolytic activity was approximately 40 to 53%. It is suggested that, although mercaptoethanol sensitivity is not a definitive test for IgM antibody, its use in connection with the hemagglutination test gives at least an approximation of the IgG antibody, whereas the hemolysis test gives a better approximation of maximal measurable antibody against Salmonella lipopolysaccharides.     

Interference of rheumatoid factor activity by aspartame,a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L ‐Asp‐L ‐Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo‐ and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. ( 1998 ). Clin. Pharmac. Ther. 63 , 580–593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid‐phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl₂ to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp–Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG₁ antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp–Phe and Phe–Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer‐assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG₄ antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints. Copyright © 1999 John Wiley & Sons, Ltd.     

Immunohistochemical observation on the antigens inducing IgG and IgM antibodies against sparganum]

Localization and characterization of the antigenic components of sparganum which induced IgG and IgM antibodies in the host were studied by immunohistochemical techniques and SDS-PAGE and Western blotting. The antigen recognized by IgG antibody of rats or mice which were immunized by infection or injection of crude extracts of metacestodes of Spirometra erinacei, was located in the parenchyma of sparganum, especially at the cortex and around the calcareous corpuscles. The immunoreaction was demonstrated not only in the encysted fibrous wall of host but around the arterioles or venules in the connective tissue of host. The antigen recognized by IgM antibody of rats or mice was also observed in the parenchyma of sparganum and in the connective tissue of host. By 5-20% gradient SDS-PAGE and EIBT, we detected antigenic components by IgG and IgM antibodies of the rat or mouse immunized by infection or injection of crude extract of spargana. Twenty-three antigenic bands from crude extracts of spargana were recognized by IgG antibody and 15 components by IgM antibody of immunized rats. Out of the bands recognized by IgG and IgM antibodies, 15 were cross-reacted each other. Twenty components of excretory-secretory proteins from spargana were recognized by IgG, and 5 components by IgM antibody of immunized rats. By IgG and IgM antibodies of immunized mice, 16 components of crude extracts were recognized by IgG antibody and 9 components by IgM antibody. Twenty components of excretory-secretory preparation were recognized by IgG antibody and 5 components by IgM antibody. Thirteen components of crude extracts were cross-reacted by IgG antibody of rats and mice.     

Response and Specificity of Antibodies for Candida albicans

Rabbit antibodies for Candida albicans, reacting in agglutination and fluorescent-antibody reactions, were present in both IgM and IgG protein fractions. The two types of immune globulins were separated from ammonium sulfate-precipitated gamma-globulin either by filtration through a column of Sephadex G-200 or by diethylaminoethyl column chromatography performed by stepwise elution with various concentrations of sodium chloride. In the fluorescent-antibody test, initial separation of the IgG fraction, prior to its conjugation with dye, proved to be essential for the high specificity of this reaction. Investigation of the specificities of the two types of antibodies revealed that the IgG was highly specific, whereas the IgM was not very specific. Each antigen fraction, extracted by various methods, demonstrated its own characteristic antibody response. Only the IgG fraction yielded serotype-specific antibody useful for detection of a serotype of C. albicans in agglutination and fluorescent-antibody tests. The results indicate the importance of IgG for specific serological reactions with the Candida species.     

Study of interaction between IgG and IgM antibodies against rubella virus by the immunofluorescence method.

In immunoglobulin fractions or after elimination of IgG by absorption the immunofluorescence test for rubella IgM antibodies is more sensitive than in whole serum. Blocking of IgM activity by IgG antibodies was eliminated when the time of incubation of the serum with virus antigen was prolonged. After prolonged incubation higher titres of rubella antibodies were also obtained in the IgM immunoglobulin fractions. Protein A in Staphylococcus aureus suspension effectively absorbs antibodies of IgG class. The IgM antibody titres in absorbed sera of patients infected with rubella were in some cases 2 to 4 times higher than in unabsorbed sera.     

Determination of binding constants of antibodies fractionated according to their affinity to insulin

The number of populations of antibodies differing in their affinity for insulin was determined in guinea pig antiserum. The antibody fractions differing in their affinity were obtained by affinity chromatography on an antigenic sorbent. Elution with a stepwise pH gradient from 4 to 2 differing in the number and form of the steps resulted in 10 fractions of antibodies obtained from individual animal sera. The binding constants of fractionated antibodies were determined by a bioluminescent immunocofactor method. With a change in elution pH from 4 to 2, the equilibrious binding constant of antibodies increased from 10(6) to 5 X 10(9) M-1.     

Thymus-dependent network responses to a monoclonal cross-reactive antiidiotypic antibody.

BALB/c mice were inoculated i.p. with a cross-reactive anti-Idiotypic mAb (designated FD5-1) in the absence of Ag or adjuvants. Injection with unmodified FD5-1 resulted in the induction of serum antibodies reactive with both FD5-1 (Ab3) and the hapten DNP (Ab1'). Endpoint titers of the Ab3 response showed an increase in serum IgM, which was dose-responsive to both the number of injections and the amount of FD5-1 antibody injected. The serum IgM Ab3 response was found to be thymus dependent and idiotypically specific for FD5-1. Athymic mice injected with FD5-1 were unable to produce a serum IgM Ab3 response, whereas their euthymic littermates produced strong Ab3 responses. Serum Ab3 responses and Ab1' were detectable only in the IgM isotype; no specific IgG responses were observed. Indeed, IgG recognized by FD5-1 appeared to be suppressed by FD5-1. Injection of mice with FD5-1 modulated serum IgM responses to DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one (OX), phosphorylcholine (PC), and alpha 1,3-dextran (DEX) in a thymus-dependent manner. FD5-1 injection induced IgM responses against DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one, and DEX but decreased IgM binding to PC. No detectable Ab1' responses to any of the aforementioned molecules were found when the same sera were probed for IgG. The specificity of serum Ab1' from FD5-1-injected mice was evaluated by antigenic inhibition. Binding of serum Ab1' to DNP-BSA was inhibitable by DNP-lysine, whereas equivalent concentrations of lysine alone had no inhibitory effect. The antigenic specificity of IgM from normal serum binding to PC-BSA was demonstrated by inhibition with free PC, and the binding of Ab1' from FD5-1-injected mice to DEX-coated plates was shown to be inhibitable by DEX. We have described in vivo network perturbation in adult BALB/c mice injected with anti-Id antibody in the absence of Ag or adjuvants. Our findings show that injection of the cross-reactive anti-Id FD5-1 can induce thymus-dependent Ag-specific responses. In two systems where FD5-1 functions as an anti-anti-anti-Id antibody (PC and DEX), thymus-dependent responses were also observed. FD5-1 injection suppressed antibodies binding to PC, whereas DEX-specific responses were induced.