A sensitive radioimmunoassay for the detection of monoclonal anti-idiotope antibodies

A radioimmunoassay was developed in order to detect anti-idiotypic antibodies in the supernatants of hybrid cells. This assay is both sensitive and specific for anti-idiotypic (but not anti-allotypic) antibodies. Monoclonal antibodies present in test supernatants are bound by an anti-immunoglobulin coated solid phase. Subsequent incubation with a source of mouse immunoglobulin 'blocks' unreacted anti-immunoglobulin antibodies on the solid phase. Anti-idiotypic antibodies are then detected by their ability to bind 125I-labelled idiotype-bearing antibody. This paper describes the use of this assay to detect monoclonal anti-idiotypic antibodies in 2 systems: the cross-reactive idiotype of A/J anti-ABA antibodies, and the idiotype expressed by the myeloma protein HOPC 8. Similarly, 125I-labelled anti-idiotype antibodies may be used in this assay to detect monoclonal idiotype-bearing antibodies. Further modifications are described which would allow the detection of monoclonal anti-allotype antibodies.     

Control of idiotope expression by monoclonal anti-idiotope and idiotope-bearing antibody

Preinjection of C57BL/6 mice with nano-to microgram amounts of a monoclonal IgG1 antibody directed against a binding site-related idiotope of the anti-NP [(4-hydroxy-3-nitro-phenyl)acetyl] antibody B1-8 results in enhancement or suppression of the corresponding and of another B1-8 idiotope in a subsequent anti-NP response, depending on the dose of the injected anti-idiotope antibody. The enhancing and suppressive effects appear two weeks after anti-idiotope administration and are maximal after 6-8 weeks. They are predominantly expressed at the level of IgG, not IgM, antibodies. Enhancement of idiotype expression, i.e. idiotypic memory, can also be induced by the injection of idiotypic antibody of the IgM class, namely antibody B1-8. This effect might represent one of the general mechanisms by which immunological memory is established.     

Improved methodology for the production of monoclonal antibodies against parasites

A modification of the standard fusion methodology is described which results in greatly increased yields of monoclonal antibodies against certain organ-specific parasites. Several fusions were carried out using mice infected with Schistosoma mansoni or Nematospiroides dubius, using B lymphocytes harvested from either the spleen or the mesenteric lymph nodes. Results indicated a greatly improved yield of positive clones using the lymph nodes as a source of B cells for fusion. A 7-fold increase in the number of positive clones was seen with N. dubius injections, while S. mansoni fusions showed a 2-fold increase.     

Specificity, duration and mechanism of idiotype suppression induced by neonatal injection of monoclonal anti-idiotope antibodies into mice

Monoclonal antibodies detecting idiotopes on the germ line-encoded anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody B1-8 were injected at various doses into newborn mice and the expression of B1-8 idiotopes was measured in anti-NP responses in later life. Suppression was long lasting, and a 100-fold increase in the dose of anti-idiotope delayed recovery from suppression by 5-6 weeks. Upon injection of a single anti-idiotope, suppression was observed for all B1-8 idiotopes to various degrees. Certain idiotopically defined antibody phenotypes were much more efficiently suppressed, and later recovered from suppression, than others. This specificity pattern was observed at the level of both B and T cells from the manipulated animals, as demonstrated in cell transfer experiments in which such cells were mixed with normal T and B cells. In these experiments, there was evidence for suppression mediated by regulatory T (and possibly also B) cells. Whereas the B cells from the manipulated animals were idiotypically unresponsive in a T cell-dependent adoptive primary response, the frequency of lipopolysaccharide-reactive B cells expressing the target idiotype was only slightly reduced in these animals as compared to control mice. Together with data on the elimination of anti-idiotope antibody from the neonatally injected animals these results are interpreted in the following way: idiotype suppression is induced through the reaction of anti-idiotope with idiotopes expressed on the surface of newly generated B cells, at microgram concentrations of anti-idiotope. When the concentration of anti-idiotope fall below that level, recovery from suppression sets in. Two types of suppression are induced. The first, namely, direct blockade of B cell maturation, is short-lived. The second involves the induction of regulatory cells, perhaps through idiotope-bearing antibody V regions complexed by anti-idiotope. This type of suppression is long-lived and its specificity depends upon the distribution of the target idiotope in the antibody repertoire and/or peculiarities of the T cell receptor repertoire. It impinges on the selection of the B cell repertoire in the animal as expressed in T cell-dependent (and possibly other) responses and is thus hardly seen at the level of lipopolysaccharide-reactive (immature) cells. Idiotype suppression by regulatory cells may be perpetuated by antigen interacting with idiotypic antibodies on the B cell surface and may therefore play a role in establishing tolerance not only for the expressed antibody repertoire, but for self antigens in general.     

Hybrid hybridomas and the production of bi-specific monoclonal antibodies

Cell fusion techniques can now be used to generate antibodies with two different specificities. Here Cesar Milstein and A. C. Cuello discuss the theoretical basis of the method they have devised and its first applications in histochemistry and immunoassay.     

Monoclonal antibodies to rat sarcomata. I. Immunization procedures and source of lymphoid cells for hybridoma production.

Hybridomas secreting monoclonal antibodies with specificity for Hooded rat fibrosarcomata have been obtained by fusion of the rat myeloma Y3 Ag 1.2.3. (Galfre, Milstein & Wright, 1979) with cells taken from spleens or lymph nodes of immunized syngeneic and allogeneic donors. Two of the monoclonal antibodies, both derived from he spleens of tumour bearers, showed specificity for individual tumours one for MC24 (M10/76) and the other for HSNTC (11/160). These two antibodies had a long half-life in the blood when injected intravenously showing that they had a low affinity for normal tissue antigens. Monoclonal antibodies exhibiting broad tumour specificity or extensive cross-reactivity with normal cells were secreted by many of the hybridomas derived from both syngeneic and allogeneic rats that had been hyperimmunized with tumour cells. These results are discussed in relation to the production of monoclonal anti-tumour antibodies for use in experimental therapy.     

New heteromyeloma cell lines for the production of human monoclonal antibodies.

The aim of this study was to establish hybridomas capable of long-term production of human monoclonal antibodies (mAbs). Heterohybridization was performed between the mouse myeloma cell line P3X63Ag8.653 and activated human peripheral blood lymphocytes (PBL). In order to achieve better retention of human chromosomes, as well as to improve the stability of the heterohybrids, one HAT-sensitive immunoglobulin (Ig)-non-secreting human x mouse (h x m) heteromyeloma was fused for a second time with activated human PBL. In this way, a panel of HAT-sensitive Ig-non-secreting h x h x m heteromyelomas was obtained and tested for its ability to generate stable human Ig-secreting heterohybrids with activated human PBL. Six lines were selected on the basis of their enhanced characteristics of fusion efficiency and genetic stability. When fused with in vitro immunized human PBL, they generated several h x h x h x m hybridomas stably secreting high yields (10-23 micrograms/ml/24 h) of human mAbs reactive with recombinant HBV core antigen (rHBcAg). Moreover, a continuous production of human Ig was observed when two h x h x m heteromyelomas, previously made ouabaine-resistant, were hybridized with EBV-transformed lymphoblastoid cell lines. These h x h x m heteromyelomas are ideal fusion partners for the production of human mAbs.     

In vitro stimulation of nonimmunized, naturally infected rat spleen cells for the production of Mycoplasma pulmonis-specific monoclonal antibodies

Spleen cells derived from rats naturally-infected with Mycoplasma pulmonis were stimulated in vitro, and then fused with a mouse myeloma cell line. The resulting hybridomas were screened for mycoplasma-specific Mabs by ELISA and for hemolysis-blocking activities. Fusions performed with in vitro-treated spleen cells yielded larger numbers of growth-positive wells and antibody secreting cells than untreated spleen cells from the same animals. Hybridomas derived from naturally-infected animals gave a higher percentage of hemolysin-specific monoclonal antibodies than did hyperimmunized animals. This indicated that B cell priming during mucosal infections can produce antigen-primed spleen cells. Stimulation of these cells in vitro can result in monoclonal antibodies against antigens not normally recognized during immunization with in vitro grown pathogenic bacteria.     

Large-scale production of monoclonal antibodies in dialysis tubing

A simple procedure for the large-scale production of monoclonal antibodies has been developed. Hybridomas were grown in dialysis tubing containing medium with a low concentration of proteins. The dialysis tubing was inserted into a flask with medium containing 10% or 30% foetal calf serum. The flask was placed on a roller and medium was changed every other day. Monoclonal antibodies were harvested after about 10 days in culture. Immunoglobulin concentrations up to 5.4 mg/ml and cell yields of 85 X 10(6) cells/ml have been obtained. The low concentration of contaminating low molecular weight proteins in the supernatant from cells grown in dialysis tubing facilitated purification of the monoclonal antibodies.     

Immunization strategies for the prevention of pneumovirus infections

Pneumoviruses, which are viruses of the family Paramyxoviridae, subfamily Pneumovirinae, are pathogens that infect the respiratory tract of their host species. The human pneumovirus pathogen, human respiratory syncytial virus (RSV), has counterparts that infect cows (bovine RSV), sheep (ovine RSV), goats (caprine RSV) and rodents (pneumonia virus of mice). Each pneumovirus is host specific and results in a spectrum of disease, ranging from mild upper-respiratory illness to severe bronchiolitis and pneumonia with significant morbidity and mortality. Given the public health burden caused by human RSV and the concomitant agricultural impact of bovine RSV, these two viruses are considered as prime targets for the development of safe and effective vaccines. In this review, we describe the strategies used to develop vaccines against human and bovine RSV and introduce the pneumonia virus mouse model as a novel and invaluable tool for preclinical studies and new vaccine strategies.     

Immunization with immune complexes: Characterization of monoclonal antibodies against a TSH-antibody complex

Monoclonal antibodies (MAb) to human thyrotropin (hTSH) were prepared by immunization of mice and rats according to different procedures. We have previously demonstrated that a specific antigenic region on the surface of the hTSH molecule was highly immunogenic; in order to produce specific MAb to weakly immunogenic regions of hTSH, we immunized mice and rats with a complex composed of hTSH and an anti-β hTSH MAb 27 directed against the highly immunogenic region. Monoclonal antibodies elicited by this immunization procedure were highly specific and a high percentage was found complementary to the MAb 27 used in the immunogen. We did not search for anti-MAb 27 antibodies, however one hybridoma produced antibody that preferentially reacted with the immune complex. This antibody, called 515, is an IgG₁ that binds the complex with 100-fold greater affinity than it does to the anti-β hTSH MAb 27 alone. This enhancement was also observed with the Fab fragment of the MAb suggesting that the epitope recognized by this anti-complex MAb is displayed in a very different way when hTSH is bound to the first MAb.     

B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage-associated antigens

Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double-) immunofluorescence staining of single cell suspensions from lymphoid organs. HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compartment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes. HIS22 and HIS24 detected B lineage-associated antigens expressed by major subpopulations of B cells. HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone. In accordance with this, substantial proportions of sIg+ cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively). In bone marrow the vast majority of cytomplasmic mu+ pre-B cells were HIS14+ and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis. The antigens recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: none of the mAb bound to plasma cells. As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22, or HIS24. In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig- cells. HIS22 did not bind to Ig- peripheral lymphocytes. Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches. As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen. In addition, the antigens recognized by HIS14, HIS22 and HIS24 co-capped with the leukocyte common antigen. This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen.     

The production and characterization of two monoclonal antibodies to rat kidney L-arginine: glycine amidinotransferase

Rat kidney L-arginine:glycine amidinotransferase (transamidinase) has been purified previously to homogeneity as two fractions, designated alpha and beta. No differences in the properties of these two fractions could be found. Two monoclonal antibodies (Tran/NS-1/1 and Tran/NS-1/3) to the purified alpha fraction of rat kidney transamidinase were produced, purified, and characterized. The results of competitive binding studies of the two monoclonal antibodies to alpha transamidinase were as follows: 1) Tran/NS-1/3 had no effect on 125I-Tran/NS-1/1 binding while Tran/NS-1/1 inhibited 125I-Tran/NS-1/1 binding; 2) Tran/NS-1/3 inhibited 125I-Tran/NS-1/3 binding while Tran/NS-1/1 had no effect on 125I-Tran/NS-1/3 binding. Therefore, Tran/NS-1/1 and Tran/NS-1/3 bound to different antigenic determinants on alpha transamidinase. 125I-Tran/NS-1/1 and 125I-Tran/NS-1/3 each had high avidity constants (approximately 10(7)-10(9)) for both alpha and beta rat kidney transamidinase. Tran/NS-1/1 and Tran/NS-1/3 bound to human kidney transamidinase in ELISA assays. A quantitative immunosorbent inhibition assay for rat kidney transamidinase was developed with 125I-Tran/NS-1/3. Approximately 30 ng of immunoreactive transamidinase could be detected by this immunosorbent inhibition assay. The amount of Tran/NS-1/3 immunoreactive species in rat lung and testicular tissue by the immunosorbent inhibition assay correlated well with the amount of transamidinase activity found in those tissues. The availability of the monoclonal antibodies, Tran/NS-1/1 and Tran/NS-1/3, should facilitate studies of rat and human transamidinase structure and regulation.     

Analysis of rat hemopoietic cells using monoclonal antibodies

Rat hemopoietic cells were analyzed with immunohistochemical technique, binding inhibition assay and flow cytometer using a monoclonal antibody (UB-12) to rat fetal liver hemopoietic cells. UB-12 positive cells were recognized in only red pulp but not in white pulp of spleen. The number and fluorescence intensity of UB-12 positive cells in spleen appeared to reach to peak at 6 weeks old occupying about 60 to 70% of total cells in red pulp. On the other hand, OX-7 (anti-Thy-1) positive and W3/13 (anti-leuko-sialoglycoprotein) positive cells were found in both red and white pulp, but not in marginal zone of spleen. UB-12 antigen was found on the surface of the cells only in the early stages of hemopoiesis: relatively large nuclei of UB-12 positive cells were rich in heterochromatin. There were a large number of free-ribosomes and some mitochondria in cytoplasm, and a centriole was observed in cytoplasm at some sections of UB-12 positive cells. From the EPICS analysis of adult rat bone marrow cells using UB-12, OX-7 and W3/13 monoclonal antibodies, the percent of UB-12, OX-7 and W3/13 positive cells was 82%, i.e., 18% was negative from these monoclonal antibodies. UB-12 single positive, OX-7 single positive and W3/13 single positive cells were 7%, 7% and 47%, respectively. The percent of triple positive cells with these antibodies was about 2%.