Determination of antibodies and non-specific immunoglobulins produced by single cells

To determine whether one cell simultaneously produces antibodies and non-specific immunoglobulins (nIg), spleen cells from mice immunized twice with SRBC were cultivated on membrane filters, firstly on the layer of native SRBC in agarose and then on the layer of SRBC sensitized with anti-mouse IgG antibodies. A specific immunoadsorbent, eliminating all anti-SRBC antibodies, was placed between the filters with spleen cells and sensitized SRBC. By comparing the location of indirect and reverse plaques (i.e. the location of cells producing antibodies to SRBC and nIg) it was shown that antibody-forming cells do not synthesize nIg.     

Effect of antibodies against immunoglobulins and the theta antigen on the specific and non-specific stimulation of mouse spleen cells in vitro

Normal mouse spleen cells were stimulated in culture by phytohaemagglutinin (PHA), pokeweed mitogen (PWM), allogeneic cells; keyhole limpet haemocyanin (KLH) sensitized cells by the specific antigen. The stimulation of the cells was measured by [³H]thymidine incorporation into the TCA precipitable fraction of the cultures. In this system, the effect of treating the cells with an antibody against the theta antigen and an antibody against immunoglobulin, with or without complement, was investigated. Treatment of the cells with antisera and complement or without complement gave similar results. The secondary in vitro stimulation with soluble antigen KLH could be markedly reduced with both anti-θ and anti-immunoglobulin serum. The response to allogeneic cells in the mixed lymphocyte reaction was only reduced by anti-θ serum and not by anti-immunoglobulin serum. No definite effect could be demonstrated by either antibody on the non-specific stimulation by PHA or PWM.     

Changes in the synthesis of antibodies and non-specific immunoglobulins in the spleen perfused in vitro

The synthesis of antibodies and non-specific immunoglobulins by the rabbit spleen perfused with culture medium in vitro has been studied using the incorporation of [¹⁴C]glycine. Separate perfusion of the two halves of the same spleen provided an opportunity to determine the influence of several factors on the synthesis of these proteins.

It was shown that changes in the rate of synthesis of antibodies and non-specific immunoglobulins in the perfused spleen are in some cases similar to the changes observed in the whole organism, and in some cases they are not. Thus, the synthesis of antibodies in the spleen withdrawn 2 days after secondary immunization increased during the 2 days of perfusion and in the spleen taken 4 days after immunization it decreased in the same way as in the whole organism. However, in the spleen taken 3 days after secondary immunization, the synthesis of antibodies during the perfusion period remained unchanged. Another distinction from the processes occurring in the living body was that the increased rate of antibody synthesis in the perfused spleen was not associated with an increase in nonspecific immunoglobulins synthesis.

     

Possible use of similar framework region amino acid sequences between human and mouse immunoglobulins for humanizing mouse antibodies.

We have previously noted that a specific amino acid sequence could form the second framework region of human, mouse and rabbit immunoglobulin light chains, suggesting that this sequence has been preserved for 80 million years. Through divergent evolution, each species has acquired a different set of framework region sequences; however, these sets still share a few similar or identical amino acid sequences. In the present study, we have identified such sequences for all four framework regions between human and mouse immunoglobulin light and heavy chains. They may be useful in humanizing or reshaping mouse or rat antibodies for therapeutic applications in human patients.     

Biological mimicry of antigenic stimulation: analysis of the in vivo antibody responses induced by monoclonal anti-idiotypic antibodies

In this study, the induction of protective antibodies against a bacterial pathogen in mice was used as a model for idiotype vaccine development. The antibody responses induced in different strains of mice by the hapten phosphorylcholine (PC) coupled to ovalbumin, PC-OVA, were compared with the responses induced by carrier conjugates of two different anti-idiotopic antibodies. One anti-idiotope, 4C11, exhibits the characteristics of an internal image of phosphorylcholine, and therefore is classified as an Ab2 beta; the other, F6, does not mimic antigen, and therefore is classified as an Ab2 alpha. The analysis of the temporal kinetics of the IgM and IgG1 anti-PC responses induced by nominal and idiotope antigens revealed dynamic responses characterized by changes in the quality and quantity of the antibody populations during the course of the immune response. All three antigens could stimulate antibodies that were PC-specific and T15 idiotope-positive in BALB/c and A/St mice. The highest titre of T15+ anti-PC antibodies was achieved with an immunization protocol which involved priming with Ab2 alpha followed by challenge with PC-OVA. Antibodies specific for the extended hapten, diazophenylphosphorylcholine, and hapten-carrier bridge determinants were being stimulated late in the responses to PC-OVA. BALB-c, A/St and CBA/N (Xid) mice all produced, late in the response to Ab2 alpha, high T15+ antibody titres which do not bind PC. The induction of T15+, non-PC binding, antibody suggests that T15 is a regulatory idiotope, expressed on antibodies having differing antigenic specificities. With regard to vaccine development, these results support the contention that effective induction of antibodies does not depend on stimulating a unique idiotope but can be achieved by anti-idiotypes reacting with different idiotopes. In addition, these results suggest that the combined use of idiotope and nominal antigens in an immunization protocol may provide the maximal protective immunity.     

Immunogenic and antigenic epitopes of immunoglobulins I. Cross-reactivity of murine monoclonal antibodies to human IgG with the immunoglobulins of certain animal species

Antibody-producing hybridoma clones have been isolated following immunization of mice with human IgG. Twenty-five monoclonal antibodies (nine anti-C gamma 3, fourteen anti-C gamma 2, one anit-kappa and one anti-lambda) were selected for study of their cross-reactivity with the IgG of fifteen mammalian species and chicken immunoglobulin. Each antibody exhibited a unique reaction profile suggesting that human IgG expresses a very large repertoire of immunogenic epitopes. Whilst some antibodies showed a very restricted cross-reactivity profile for others a very wide reactivity profile was observed-including two clones producing autoantibodies. Antibodies demonstrating cross-reactivity between human Fc gamma and 7S chicken immunoglobulin allow its definitive assignment as a homologue of human IgG. Four clones demonstrated specificity for bovine IgG subclass gamma 1 and gamma 2 and the degree of reactivity allows their application to qualitative and quantitative assay systems. These studies suggest new perspectives for the characterization of immunoglobulins and the standardization of anti-immunoglobulin reagents.     

Role of different lymphocyte subpopulations in the formation of non-specific immunoglobulins induced by antigen injection

The formation of antibody and non-specific immunoglobulin under the influence of T-dependent (TD) and type 2 T-independent (TI-2) antigens in mice of two congenic strains CBA (Lyb5-, Lyb5+) and CBA/N (Lyb5-) was studied. TD antigens induced in mice of both strains not only the appearance of antibody-forming cells (AFC), but also a great increase in the number of cells producing non-specific immunoglobulins (nIFC). TI-2 antigens induced the AFC and antigen-dependent nIFC formation in CBA mice only. It is concluded that during immune response to TI-2 antigens not only the AFC appearance but the increase in nIFC formation (polyclonal activation) is due mainly to the mature Lyb5+ B cells.     

Grass pollen allergens: antigenic relationships detected using monoclonal antibodies and dot blotting immunoassay

Monoclonal antibodies against major rye-grass pollen allergens have been used to detect cross-reactive determinants in other grass pollen extracts. Antibody binding was detected by the dot blotting immunoassay in which alkaline phosphatase-conjugated anti-mouse IgG was used as secondary antibody. Taxonomically ordered variations were found between pollen of 22 grass species representing all major natural groups. One of the antibodies, that bound to the 28 to 30Kd allergen, showed high species specificity; immunoblotting showed binding to similar polypeptides in Festuca elatior but to none of the other grasses tested. This simple assay has applications in standardizing grass pollen extracts.     

Possible relationship between antigenic properties and isolation history of HSV-1 strains

29 monoclonal anti-herpes simplex virus (HSV)-1 antibodies were produced and characterized with regard to virus neutralizing activity, intracellular or cell-surface location of viral antigens and, where possible, molecular weight of the viral protein recognized. 13 antibodies recognized viral antigens expressed on the surface of infected cells and 16 were directed to intracellular viral components. Only two antibodies exhibited virus neutralizing activity. Application of these antibodies to an antigenic comparison of standard laboratory HSV-1 strains F, HFEM, mP, Glasgow-17 and MAC revealed unique antigenic differences among these strains. The antibodies were further used in an antigenic comparison of 45 human HSV-1 isolates with defined isolation history. Except for two paired isolates from left and right trigeminal ganglia of two human cadavers, the antibody panel revealed antigenic differences among all isolates, including paired isolates from three additional cadavers. Overall, isolates from different human donors showed greater antigenic dissimilarity from each other in cell-surface associated than in intracellular antigens. The data suggest the possibility of a correlation between antigenic and biologic properties of HSV-1.     

Possible antigenic relationship between varicella/ zoster virus and herpes simplex virus

Summary Cultures of the Gabi strain of diploid cells derived from human fetal lung fibroblasts were used for isolation of virus from typical cases of varicella and herpes zoster. CF antigens were prepared from 2 varicella and 2 zoster strains. Similar antibody responses to these antigens were demonstrated in paired sera from the four patients who provided the virus strains and one other case of herpes zoster. None of these sera reacted with herpes simplex antigen.Out of 49 patients with a significant rise of CF antibody titer against herpes simplex virus, 12 had a similar response against varicella antigen. These findings suggest a minor antigenic relationship between both viruses.     

T cell dependence of cells synthesizing immunoglobulin without detectable antibody function induced after an antigenic stimulation.

A previous study of primary and secondary immune responses of rats and mice immunized against various protein antigens allowed us to describe a population of immunocytes containing, synthesizing and secreting immunoglobulins without detectable antibody function against the antigen injected (IFC). We here report their T cell-dependence. Mice, thymectomized, irradiated and reconstituted with bone marrow or fetal liver cells, generally show neither antibody-forming cells (AFC) nor cells synthesizing immunoglobulin without detectable antibody function. In some mice, probably only partly T cell-deprived, antibody-containing cells were seen, and at that time they were associated with cells synthesizing immunoglobulin without detectable antibody function. For most of the animals studied in primary response, however, the IFC population remained higher than the AFC population throughout the immune response. In normal animals immunoglobulin-synthesizing cells were predominant at the beginning of the immune response, then decreased and progressively replaced by antibody-synthesizing cells. After the first injection or after two stimuli, the number of responders among T cell-deprived mice increased progressively. Finally, these experiments indicate that both cells synthesizing immunoglobulins without detectable antibody function and specific AFC are thymus-dependent and rule out the hypothesis according to which horseradish peroxidase is a polyclonal B cell activator.     

Idiotypic relationships between allotypically different rabbit antibodies

Homozygous rabbits genotypically different for the allotypy of the VH region and the C kappa region of their immunoglobulins (al/al, b4/b4 and a3/a3, b5/b5, respectively) were transferred together at the blastocyst stage to be born from the same foster mother. At the time of transfer the foster mothers were active anti-anti-idiotypic (Ab3) antibody producers and, according to their own genotype, the idiotypes (Ab1) which initiated the immunization cascade ending with their Ab3 were allotypically either a1,b4 or a3,b5. This experimental scheme was carried out, at the level of the Ab1 and of the Ab'1 (antibodies produced by the transferred rabbits subjected to the Ab3 action), with three different exogenous antigens, namely a protein, a polysaccharide and a hapten which elicited in normal rabbits the production of antibodies with private idiotypic specificities. Two of them, the protein and the polysaccharide enabled us to find idiotypic relationships between antibodies totally different for their allotypy and particularly for the allotypy of the variable region of their heavy chains. In these examples, the structural coercions implied by the different allotypic markers of the VH did not seem to prevent the association of at least related genetic elements responsible for the observed idiotypic community. In these idiotypic relationships, only subpopulations of the Ab1 and of the Ab'1 were implicated and they could be different for the diverse littermates.     

Lysosomal enzyme changes in mouse spleen cells after antigenic stimulation

Changes in the lysosomal enzyme acid phosphatase have been assayed in mouse spleen cells after injection of the animal with various antigens. Disruption by freezing and thawing or sonication of spleen cells or the large granular fraction showed, 48 hours after injection of antigen, an increase in enzyme level irrespective of the antigen used. In contrast, if non-disrupted lysosomes were examined only cells from animals treated with aggregate-free antigen (BSA) showed an increase in level. Spleen cells from animals treated with the other antigens, heat-aggregated BSA, haemocyanin (KLH) and sheep red cells, showed that the enzyme was less accessible to its substrate, reflected by lower levels of enzyme. The results are discussed in relation to possible changes in the stability of the lysosome membrane and the involvement of lymphocytes.     

Calculation of secretory antibodies and immunoglobulins

There is increasing evidence for the secretion of IgG and IgE, as well as IgA, antibodies by the nasal mucosa. Because the former immunoglobulins lack an identifiable secretory piece, estimates of their secretion have been based on their ratio to albumin or to total immunoglobulin of the same type in the nasal secretion as compared with serum. It is shown, however, that use of the ratio to albumin will result in large errors if the amount of antibody secreted by the nose is relatively small, and ratios based on total secretory Ig will, at least hypothetically, lead to error if antibodies with multiple specificities are being secreted. It is suggested that a more valid quantitation will be obtained from the expression

$INS=\frac{{Ab}_N}{{Alb}_N}-\frac{{Al}_S}{{Alb}_S}$

where INS is an index of nasal secretion, Ab = antibody, Alb = albumin, N = nasal secretion, and S = serum.     

Observations on the antigenic relationships between Epstein-Barr virus and herpesvirus saimiri.

The antigenic relationships between Epstein-Barr (EB) virus and herpesvirus saimiri (HVS) have been investigated in comparative immunofluorescence, microimmunodiffusion and serum neutralization tests. No similarity was detected between the structural antigens of the two herpesviruses but the results of microimmunodiffusion tests showed that they shared a non-structural, virus-determined antigen. The implications of this finding are discussed in relation to the importance of HVS and its monkey lymphoma as a model system of herpesvirus oncogenesis.     

Monoclonal antibodies to human embryonal carcinoma cells: antigenic relationships of germ cell tumors

Fifteen monoclonal antibodies (mAb) that show specificity for human embryonal carcinoma cells are described. C57BL/6 mice were immunized with Tera-2 embryonal carcinoma cells, and hybridomas were isolated and tested versus a set of human developmental tumor cell lines. The antigens exhibit relatively restricted and unique distributions on normal tissues as shown by immunohistochemistry. The mAbs recognize a series of differentiation antigens since their expression changes when teratocarcinoma stem cells are induced to differentiate in vitro. The expression of these molecules in germ cell and related tumors is consistent with the data obtained from in vitro cell studies. Seven of these mAbs immunoprecipitate molecules from surface labeled Tera-2 cells that show distinct molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenic phenotypes defined by the set of mAbs can be used to investigate possible pathogenetic relationships of the various testicular tumors.     

Immunogenic and antigenic epitopes of immunoglobulins. XIV. Antigenic variants of IgG4 proteins revealed with monoclonal antibodies

Two IgG4 paraproteins having heavy chains of normal molecular weight are shown to be antigenically distinct in their reactivity profiles with 18 monoclonal antibodies having specificity for the IgG2 or IgG4 subclasses. One protein expresses IgG2 and IgG4 epitopes within the C gamma 2 domain. A second protein is deficient in the expression of IgG4 Fc-specific epitopes. These proteins will be of value in defining the structural basis of Fc effector functions and individual epitope expression.     

Structural relationships between carrier epitopes and antigenic epitopes on hen egg-white lysozyme.

The spatial relationship for T-B cell cooperation between antigenic epitopes and carrier epitopes on the antigen molecule was studied. Two mono-DNP substituted derivatives of hen egg-white lysozyme (HEL), DNP1-33HEL and DNP1-96HEL were used as antigens; the former is dinitrophenylated only at lysine-33 and the latter only at lysine-96. Fragment peptides of HEL were used to induce specific T cells to the respective sites of the antigen. Adoptive cell-transfer experiments of site-specific T cells and DNP-primed B cells directly showed that multiple distinct carrier epitopes for T cells could help the antibody responses to the single antigenic epitope for B cells and that a single carrier epitope could help antibody responses to multiple antigenic epitopes. T cells primed with a synthetic peptide SP34-54 (corresponding to sequence 34-54 of HEL) co-operated with DNP-primed B cells on challenge with DNP1-96HEL, but not with DNP1-33HEL.     

Possible linkage relationships between certain blood groups and schizophrenia or other psychoses

A reanalysis of a body of data on dizygotic twins may suggest a genetic relationship between the Gc locus and psychosis in general and the possibility of linkages between schizophrenia and theGm and the Rhesus systems.This investigation was supported by a Public Health Service Research Career Development Award (1-K3-GM-31, 732) and a research grant (GM HD 16697) from the National Institute of General Medical Sciences.