Immunoelectrophoretic methods can be employed to examine lipid haptens if these are present in the form of negatively charged micelles. Lipid haptens may be carriers of charges like cardiolipin or, as in the case of the neutral hapten galactocerebroside, charged groups are introduced into the micelles by incorporation of anionic amphiphilics.
The new serological group ofStreptococcus sanguis provisionally ealled “group 10043” has been examined The group specific polysaccharide antigen was isolated and compared serologically and chemically with group H antigen fromS. sanguis strain 13843 and strain NCTC 7868 Challis. The results confirm thatS. sangruis strain 10043 (NCTC 11085, ATCC 29667) contains a new antigen which does not react in anti-group H serum, nor in other grouping sera against groups A through V. The group specific antigen isolated from strain 10043 does not contain rhamnose in contrast to the group H antigen. The new antigen appears to be analogous to the other streptococcal group antigens and to deserve recognition as such. The designation W is suggested for the new group antigen. All three antigens examined contain glucose.
The enhancement of guinea-pig complement by car☐ypeptidase A is due primarily to the effect of the enzyme on the activity of the fourth component (C4). The result is an increase in the number of SAC14 capable of reacting with terminal components to produce lysis without any detectable change in the ability of SAC14 to react with C2. as shown by the rate of generation of SAC142 and the amount of C2 consumed. Experiments utilizing guinea-pig C4 deficient scrum indicate that the enzyme acts on C4. either cell-bound or in the fluid phase, and not on an inhibitor associated with this component.
Repeated injections in rabbits of pneumococcal vacxine strains R 36 A induce antibodies of restricted heterogeneity as evidences by electrophoresis and isoelectric focusing. An immunoadsorbent containing purified C-polysaccharide was used to isolate and characterize the induced antibodies. Specificity for phosphorylcholine was demonstrated by hapten elution and by inhibition of precipitation. Addition of the ligand caused an enhancement of the intrinsic fluorescence of the anti-phosphorylcholine antibodies. The association constants determined by fluorometric titration were of the order of 103M−1 and were only slightly influenced by temperature.
Antibodies against methylated bases were highly specific. In the case of dimethylated bases, antibodies reacted only with the homologous hapten, slightly with the monomethylated bases at the same position and not at all with the original non-methylated base, or with different methylated bases, Antibodies against monomethylated bases are also very specific. They reacted only with homologous hapten and slightly with the original non-methylated base. Antibodies against inosine distinguished it from 6-mercaptopurine riboside which differs from inosine only in that it has a sulfur atom instead of an oxygen atom. 6-Azauridine, which has a different conformation from uridine, reacted very poorly with anti-uridine antibodies. Antibodies against pseudouridine did not react at all with uridine. Antibodies are very powerful tools to determine conformation of coenzymes containing nucleosides or nuclcotides. Using this approach we were able to suggest that ATP, ADP, CoA, UDP, UTP, CDP and CTP are in a folded configuration.
Pre-incubation of BSA-anti-BSA precipitates with Clq or bivalentStaphylococcus protein A inhibits the solubilization of the precipitates due to the complement dependent complex release activity (CRA) of fresh sera. The inhibition was dose dependent. The results support the intercalation hypothesis of CRA.
The affinity values of antibodies to the random synthetic antigen poly-(Tyr,Glu)-poly(dl Ala)-poly(Lys) [known as (T,G)-A-L] and to the ordered (Tyr-Tyr-Glu-Glu)-poly(dlAla)-poly(Lys) [known as (T-T-G-G)-A-L] were measured. Determinations of the association constants were performed by antigen binding capacity assay, ABC, using the whole antigen, and by equilibrium dialysis, using a radioactive conjugate of the ordered peptide T-T-G-G which was found to represent the major determinant of the random (T,G)-A-L. The affinity values of antibodies elicited by high responder mice to (T,G)-A-L and to (T-T-G-G)-A-L were found to be similar. However, a difference of two orders of magnitude was found between the values obtained by the two methods. This difference is partially explained by probability analysis.
Purified di- and hexamannoside components ofMycobacterium 607 (M. 607) were analysed for their immunochemical relationships in agglutination and precipitation tests, using antisera against the MBSA complexes of total mannosides, M2-3F† and M2-4F components of dimannosides. The agglutinating antibody to total mannosides seemed to be different from that given by the precipitation test. There was no cross-reactivity between PI and the mannoside antigens, which suggested the association of antigenicity with the mannose units of the mannosides. Dimannosides with 2, 3 and 4 esters showed complete cross-reactivity. However, different antigenic specificities were found to be associated with di- and hexamannoside antigens in the immunochemical reactions. The deacylated mannoside lacked serological reactivity with antiserum to the mannosides. However, the additional fatty acids in the inositol-oligomannosyl region of the dimannosides did not seem necessary for reactivity or specificity.
Both lipopolysaccharides and lipid A moiety prepared fromVibrio el-tor exhibited almost equal endotoxic activities in eliciting the local Shwartzman reaction, in Limulus-lysate gelation, in chick-embryo lethality and pyrogenicity. The structural part responsible for these activities of lipopolysaccharides is the lipid A moiety. The high anti-complementary activities present in lipopolysaccharides and lipid A moiety was destroyed by alkaline digestion of the samples. Lipid A antiserum can protect against some of the toxic activities of the endotoxin in animals.
A glycopeptide which has inhibitory activity against guinea pig lymphotoxin (GLT) was isolated from the cell surfaces of L cells. It has an approximate mol. wt of 1600 (gel filtration) and contains galactose, mannose. fucose and N-acetylglucosamine in a molar ratio of 2:2:1:3. β-Galactosidase treatment of the glycopeptide significantly diminished its inhibitory activity against GLT. Furthermore. GLT was found to be inhibited by the lectins (Ricinus communis haemagglutinin. Concanavalin A. andPhaseolus vulgaris haemagglutinin) which bind preferentially to sugar chains like those of serum glycoproteins (serum glycoprotein-type sugar chains) on the cell surface. These data indicate that serum glycoprotein-type sugar chains on the cell surface of target cells serve as receptors for GLT and that the β-galactosyl residue at the non-reducing terminal of the sugar chain is an important part of the receptor.
The complement activating capacity and complement dependent complex release activity (CRA) of immune complexes (IC) formed at different antigen:antibody ratios were investigated. The correlation between antibody avidity, precipitating capacity and the effect of these parameters on Fcdependent biological functions was studied in BSA-anti-BSA and OA-anti-OA systems. The results suggest that antibody excess favours the complement activating effect of ICs. No correlations between antibody precipitating capacity and avidity was found. However, antibody avidity and complement activating capacity were related: the higher the avidity index of antibodies, the higher the complement activation. The kinetics of CRA are not directly influenced by these characteristics of antibodies. The intercalation of C3b into the lattice interferes not only with the primary antigen—antibody bonds, it may result in the rearrangement of the lattice through the disruption of the non-specific intermolecular interactions.
In the present work, we have studied the antigenically reactive regions of S.C. with immune sera of well-defined specificity. Immunochemical analyses conducted with an I.S. containing only precipitating antibodies against a hidden S.C. group of determinants, unravels the existence of a new antigenic specificity that we chose to call provisionally “R” (from mercapto-resistant). R was shown to be completely independent from Brandtzaeg's I and A1 by its resistance to strong reduction and to be distinct from A2 by the fact that anti-R antibodies do not precipitate sIgA or tryptic S.C.
The content and the avidity curves of IgM-positive spleen B lymphocytes forming rosettes with TNP-SRBCI were compared in C57BL/6 and BALB/c mice (C57BL/6 × BALB/c)F1 hybrids, as well as F1 × C57BL/6 and F1 × BALB/c backcross hybrids. Conjugates of bovine serum albumin with trinitrophenyl. dinitrophenylsulfonic and sulfanilic acids were used as inhibitors (TNP24BSA, DNP23BSA, Sulf17BSA).
It was recently shown that complement can dissociate BSA-anti-BSA immune complexes (Miller, 1977). In the present study we have formed specific complexes of idiotype and anti-idiotype. The idiotype used was the phosphorylchotine-binding Balb/c myeloma protein, HOPC-8, and the anti-idiotype was raised against purified HOPC-8 protein in A/J mice. Idiotypic complexes were prepared in either of 2 ways: (1) HOPC-8 protein was immobilized in test tubes and allowed to bind purified radiolabeled anti-idiotype. (2) Anti-idiotype was immobilized in test tubes and allowed to bind purified labeled HOPC-8 protein. Then both types of immobilized idiotypic complex were incubated with fresh or heat-inactivated rabbit serum at 37°C. The dissociation of either labeled idiotype or anti-idiotype was measured as a function of time. Active complement increased the rate und extent of dissociation of the idiotypic complex regardless of whether the idiotype or the anti-idiotype was immobilized.
Affinity chromatography with AMP-Sepharose allows us to purify malate dehydrogenase ofSchistosoma mansoni and to perform the studies about the immunochemical and biological properties of this enzyme. MDH has a mol. wt 60,000 and has two isoenzymes (pI 7.1 and 8.5), which are antigenically different. Isoenzyme (pI 7.1) is the antigen called 4, specific of the genusSchistosoma and is highly immunogenic. By immunofluorescence, MDH was localized in the cellular layer of the coecum. Mice immunized with purified MDH exhibited a significant decrease of worm burden.
The isolation of human alpha-1-antitrypsin has been realized by a new method: the immunoelectroelution. The principle of the method is the dissociation of some antigen-antibody complexes in an electric field when the antibodies are attached to an agarose matrix. The apparatus built for this purpose is described. The purity of the isolated alpha-1-antitrypsin has been shown by immunological means. The integrality of the isolated protein has been demonstrated by the fact that it has the same mol. wt, electric charges, antigenic properties and antitrypsic activity as the native one. Although the yield of the isolation process is only 5%, it is concluded that the immuno-electroelution may be used to isolate non-denatured human alpha-1-antitrypsin.
