Individual Antigenic Determinants Research Articles

Clonal nature of the immune response to phosphorylcholine (PC). V. Cross-idiotypic specificity among heavy chains of murine anti-PC antibodies and PC-binding myeloma proteins.

Seven mouse myeloma proteins with specificity for phosphorylcholine (PC) were found to share a common antigenic determinant. This group of proteins contained members which differed in genetic origin, heavy chain class, kappa-chain subgroup, individual antigenic determinants and specificity for choline analogues. The cross-idiotypic determinant, VH-PC, was antigenically similar in each of the proteins and was associated with the variable portion of the heavy chain in the region of the antibody combining site. Further studies showed that an indistinguishable determinant was present on IgM anti-PC antibodies isolated from all strains of mice tested regardless of histocompatibility or heavy chain allotype. In view of the finding that this cross-idiotypic determinant was not found on antibodies or myeloma proteins which lacked specificity for PC, the data strongly suggest that a particular heavy chain variable region has been preserved in all mouse antibodies with specificity for PC.

The Role of Lipid in the Induction of Hapten-Specific Delayed Hypersensitivity and Contact Sensitivity

Abstract Guinea pigs immunized with bovine serum albumin conjugated with dinitrophenol (BSA-DNP), in Freund's complete adjuvant, produce antibody to the DNP and delayed-type hypersensitivity (DTH) to the BSA. We report that immunization with BSA-DNP doubly conjugated with medium chain fatty acids (L-BSA-DNP) stimulates the production of DTH specific for the hapten DNP with minimal antibody to DNP and no detectable immune response to the carrier BSA. The production of hapten-specific DTH reactions was assessed by two methods. First, animals immunized with L-BSA-DNP produced typical contact sensitivity reactions to epidermal challenge with dinitrofluorobenzene. Second, they produced grossly and microscopically typical delayed hypersensitivity reactions to the intradermal injection of DNP conjugated either to BSA or to a non-cross-reacting carrier, hen egg albumin. In further experiments, DNP-specific DTH was produced by animals immunized with BSA-DNP to which lipid groups had been attached by electrostatic rather than covalent bonds. Evidence is discussed which indicates that the production of DTH to haptens and proteins is largely a function of the lipophilic nature of the entire immunogen and is not dependent upon properties of individual antigenic determinants. Autoradiographic studies with 125I-labeled material demonstrated that the lipid-rich antigens are localized in the paracortical area of lymph nodes, in close proximity to the thymus-derived cells which are stimulated in the induction of DTH.

Studies on IgA and IgG Monoclonal Proteins Derived from a Single Patient

Abstract The serum from a patient (GR) with multiple myeloma was shown to contain two unique homogenous bands by cellulose acetate electrophoresis. These components were further identified as a monoclonal IgG2k and a monoclonal IgAk. Rabbit antisera directed against IgG (GR) and IgA (GR) monomer were absorbed to render them specific for individual antigenic (ind) determinants. Both proteins were capable of inhibiting the reaction between labeled protein and its homologous ind antiserum in the radioimmunoassay indicating that both immunoglobulins shared ind determinants. In addition, the determinants shared between the two proteins appear to be identical. The potential sharing of any two heavy chain constant genes among all heavy chain constant genes is discussed.

Fab-related fragments of human IgG produced by partial tryptic proteolysis

A 2·2−2·5 S, Fab-related fragment (tFab′) was isolated from trypic digests of normal human IgG and of IgG1 myeloma proteins. Reductive cleavage of this gragment led to dissociation into polypeptide constituents of a mol. wt corresponding to one half of a light chain or of an Fd piece. Indirect evidence is presented that the disulphide bridge linking a κ to a γ 1 heavy chain is contained by tFab′. Heterologous anti-Fab immune sera precipitated fragment tFab′ as well as Fab (produced by papain) or the 3·9−4·4 S tryptic Fab fragment (tFab). Fragment tFab′ from a myeloma protein was found to be deficient in individual specific antigenic determinants of the parent protein. ‘Trypsin-site’ (determinant of IgG revealed by tryptic proteolysis) was found to be fully retained by tFab′, while the Gm(f) allotype-determinant was not detectable on it serologically.

Immunochemical analysis of the basic immunoglobulin in papular mucinosis

The basic immunoglobulin (PM protein) isolated from the serum of a patient with papular mucinosis is unique in that it appears to be a homogeneous immunoglobulin distinct from myeloma paraproteins. The PM protein is electrophoretically homogeneous, always has λ light chains, and in most cases has γG 1 heavy chains. Individual antigenic determinants of the PM protein were detected with rabbit anti-PM protein serum. The diagnostic PM protein seems to be a γG 1 immunoglobulin whose only unusual feature is its marked basicity. The PM protein has a net positive charge much greater than that of normal IgG fractions. This difference in net charge is associated with the light chains of the PM protein.

Sharing of individual antigenic determinants between a gamma G and a gamma M protein in the same myeloma serum.

SummaryTwo lambda proteins, one a γM and the other a γG3, were isolated from the same human myeloma serum and were shown to share individual antigenic (idiotypic) specificity when tested with several antisera prepared against the intact proteins and pepsin digested fragments. These shared determinants were localized to the heavy chains and the combinational structure of the heavy plus light chains. Shared individual determinants of the light chains were also demonstrated utilizing antisera prepared against the isolated light chains. These results indicate that the light chains and the variable regions of the heavy chains of the two proteins are extremely similar. The peptide map and mobility differences in the light chains indicate that they are not identical.

Immunochemical Studies of Foot-and-Mouth Disease

Abstract Immunodiffusion and inhibition studies indicated that 19S class guinea pig antibodies to foot-and-mouth disease virus were specific for distinctive antigenic determinants on the virus particles, whereas 7S antibodies had a single specificity for the virus particles and were unable to discriminate between the different individual antigenic determinants. This was interpreted as meaning that the antigenic determinant site encompassed by 7S antibodies consists of several smaller regions (subdeterminants) each of which may serve as an antigenic determinant site for 19S antibodies. It was also proposed that the combinding sites of 19S antibodies are smaller than those of 7S antibodies.

Studies on the Immunogenicity of Antigen-Antibody Precipitates

Summary Guinea pigs immunized with antibody excess immune precipitates of human γG globulin (HGG) and rabbit or guinea pig anti-Fab or anti-Fc antibody make little antibody to HGG. The small amount of anti-HGG antibody produced in each case was directed against antigenic determinants present in both the Fab and Fc portions of the HGG molecule, and thus bore no reflection on the specificity of the antibody in the complexes. This finding suggests that the suppression is to the whole antigen molecule rather than its individual antigenic determinants. The mechanism of the suppression of antibody formation to complexes comprised of immunogenic components cannot be explained in terms of antigenic competition for antibody-producing cells, nor in terms of “blocking” of antigenic determinants. It was shown that the combination of HGG with specific anti-Fab antibody at the equivalence point hinders only 21% of the Fc antigenic determinant, while in 4-fold antibody excess precipitates 66% of the Fc antigenic determinants are still capable of reacting with homologous antibody. Similarly, equivalence precipitates of HGG with anti-Fc antibody “block” only 16% of Fab determinants, whereas in 4-fold antibody excess precipitates 43% of the Fab determinants are “hidden.” Quantitatively, HGG bound in a 4-fold antibody excess precipitate with either anti-Fab or anti-Fc antibody has approximately 0.5% of the immunogenicity of the uncombined antigen. It thus appears that antigen in antibody excess immune precipitates is non-immunogenic, and that the antibody produced against HGG can be explained in terms of a 0.5% dissociation of the complex in vivo.

Specificity of the Anamnestic Response to a Double Hapten Conjugate in Guinea Pigs Primed with a Single Hapten

Summary Genetic responder guinea pigs primed with either benzylpenicilloyl-poly-l-lysine (BPO13-PLL287) or dinitrophenyl-polylysine (DNP13-PLL287) and boosted with the double hapten conjugate BPO13-DNP14-PLL287 gave an anamnestic response to the priming hapten and a primary response to the new hapten. Primary and anamnestic responses were clearly differentiated on the basis of antibody titers, time of attainment of maximal titers, and mercaptoethanol sensitivity of the antibodies. Both haptens were shown to be present on the double conjugate molecule. The absence of a booster response to the new hapten did not appear to be due to antigenic competition. These findings indicate for the present system, that the specificity of the anamnestic response relates only to individual antigenic determinants of a complex antigen, and not to the entire antigen molecule. These results would tend to exclude an enhanced processing of the antigen molecule as the crucial mechanism of the anamnestic response. The relationship between these results and the apparently discordant results of others is discussed.

The effect of passively administered antibody on antibody synthesis.

Suppression of the primary response of rabbits to intravenously administered KLH can be achieved with very small amounts of hyperimmune anti-KLH administered a day later since the rabbit apparently rapidly eliminates most of the KLH by nonimmunologic means. The amount of passive anti-KLH needed to achieve immunosuppression was directly proportional to the dose of injected antigen. Antibody passively administered as much as 6-8 days after antigen still can be strongly immunosuppressive, which suggests that the antibody must be reacting with immunogen in or on responding cells or perhaps in the process of transfer between cells. There was no evidence that the presence of passively administered hyperimmune anti-KLH prior to the injection of antigen had any immunosuppressive action beyond the direct neutralization of the injected antigen. When KLH was injected in Freund adjuvant, anti-KLH incorporated with the KLH in the adjuvant was much more efficient in causing immunosuppression than anti-KLH given intravenously. The primary responses to 2 mg KLH given intravenously and 2 microg given in adjuvant reached approximately equal peaks and were suppressible by comparable amounts of intravenously administered anti-KLH. Two observations suggest that passive antibody neutralizes the immunogenic stimulus at the level of individual antigenic determinants and not merely by aggregating or precipitating entire antigenic molecules. First, anti-abalone hemocyanin (AH) which cross-reacts approximately 50% with KLH was only partially immunosuppressive even in extremely large amounts, i.e., amounts which could react with and precipitate much more KLH than could the smaller but more suppressive doses of anti-KLH. Second, when KLH and anti-KLH were given together in adjuvant, effective immunosuppression was achieved only with amounts of anti-KLH sufficient to saturate or cover virtually all available antigenic determinants. The immunosuppressive quality of passive antibody increases with time after immunization and with repeated immunization of the donor. In view of their relatively weak immunosuppressive properties, antibodies formed in the first weeks of a primary response may not contribute significantly to the turning off of the antibody response. In any event, results obtained by passive transfer of hyperimmune antibody to animals early in a primary response cannot be applied to the natural events in a primary response.

Tolerance Specificity and the Immune Response to Lactic Dehydrogenase Isoenzymes

Whereas it seems well established that antibody production is due to monospecific effector cells, the transmission mechanism from individual antigenic determinants to individual antibody producing cells and their production units is not clearly understood. Immunochemical work on the specificity of antigen in the course of the immune response is difficult to reconcile with a simple linear transmission. Phenomena like the hapten-carrier effect (see below), antigenic competition (Adler, 1964), original antigenic sin (cf. Fazekas de St. Groth and Webster, 1966) and tolerance specificity as distinct from antibody specificity (Austin and Nossal, 1966), all suggest that the process of immune initiation is a complex one.