Production and characterization of a rat monoclonal antibody against leu5 enkephalin.

A rat X mouse hybridoma line producing a monoclonal antibody against leu5 enkephalin has been obtained. The monoclonal antibody belongs to the IgG2b class and has an affinity constant of 8.0 X 10(8) M-1 at 4 degrees C. This antibody exhibits approximately 40% cross-reactivity with 1-6 dynorphin but very weak cross-reactivities with met5 enkephalin (1.4%), 1-13 dynorphin (1.3%) and beta-endorphin (0.0045%). The detailed study, by competitive assay, of the interaction between this antibody and various enkephalin derivatives shows that the carboxy-terminal part of the molecule and, particularly, the leu side chain constitutes the immunodominant group. Nevertheless, the tyrosyl residue also contribute considerably to the binding, probably via a peculiar conformation effect, although we can not exclude the tyrosyl residue acting as a secondary contact residue. This monoclonal antibody has been used in a radioimmunoassay to determine leu5 enkephalin like immunoreactive material present in rat brain and hypothalamus.

Isolation and structure-functional characterization of phage display library-derived mimotopes of noxiustoxin, a neurotoxin of the scorpion Centruroides noxius Hoffmann.

Noxiustoxin (NTX) is a short-chain toxin from the venom of the scorpion Centruroides noxius Hoffmann, whose molecular structure and physiological effects have been characterized in detail, whereas the antigenic properties of this and other K(+) channel-blocking toxins are poorly studied. A monoclonal antibody against NTX, BNTX18, able to inhibit the binding of NTX to rat brain synaptosomes, was used in the present study for selecting immunoreactive peptides, mimotopes, from a 12mer and a 7mer phage library. The peptides were characterized immunologically and used for mapping the epitope on NTX. In total, 75 phage clones carrying 43 different peptides were analyzed of which 42 clones carrying 17 different peptides, twelve 12mer and five 7mer peptides, presented a single consensus motif: Leu(Ile, Val)-Tyr(Phe, Trp, Leu)-Gly-Met(Ala). All but three of the peptides containing this motif were reactive with selected mAb BNTX18 in a dot-blot assay of which eight were clearly positive in ELISA and exhibited in competition-inhibition assay the antibody binding specificity of the NTX epitope recognized by BNTX18. The two most reactive mimotopes injected into mice showed the ability to induce antibodies reacting with NTX, thus, to mimic the epitope of NTX antigenically. Sequence comparison and the analysis of the three-dimensional structure of NTX led to the proposal that residues Glu19-Leu20-Tyr21-Gly22 and the hydrophobic part of the side chain of Lys18 form the C-terminal part of the epitope. Due to the frequent presence of residues Pro, Leu, Thr, Arg, and Gln in the N-terminal part of the mimotopes, corresponding homologous residues in the N-terminal proximity of the partial epitope may be part of an additional more hydrophilic epitope element.

Idiotypic studies of monoclonal anti-tobacco mosaic virus antibodies from one mouse.

Monoclonal antibodies have been prepared from one BALB/c mouse immunized with tobacco mosaic virus. The monoclonal antibodies are distributed into three subgroups recognizing different epitopes on tobacco mosaic virus subunits. The idiotypic specificities of these monoclonal antibodies have been studied using syngeneic antiidiotypic sera. A sharing of idiotypic specificities has been observed between members of each subset. These idiotypes are not recurrent in BALB/c mice immunized with tobacco mosaic virus.

Expression of human alpha 1-antitrypsin in Escherichia coli.

Complementary DNA coding for human alpha 1-antitrypsin has been placed under the control of the lambda PR promotor carrier by the expression vector pCQV2 [1]. In conditions which allow transcription from this promotor (thermoinactivation of the repressor), Escherichia coli cells harbouring the recombinant plasmid pULB1114 express human alpha 1-antitrypsin (+/- 9000 molecules/cell). The product has a Mr of 44000, corresponding to mature unglycosylated alpha 1-antitrypsin.

Cloning, characterization and functional expression of a cyclophilin of Entamoeba histolytica.

Full-length Entamoeba histolytica cyclophilin gene (EhCyp) was isolated, characterized and recombinantly expressed in bacterial cells. The deduced amino acid sequence of EhCyp shows 60-70% identity with cyclophilins from other organisms and has conserved the cyclophilin signature motifs and residues involved in cyclosporin A binding. Upstream of the 501 bp open reading frame of EhCyp, sequences resembling the putative consensus E. histolytica CE1, CE2 and CE3 regulatory elements were found. Northern blot assays revealed a single transcript of 0.63 kb. The transcription start was determined by primer extension at position -13 relative to the initial ATG codon. Cyclosporin A binding and peptidyl-proplyl cis-trans isomerase activities characteristic of cyclophilin were detected in soluble extracts of E. histolytica trophozoites and in the recombinant protein. In both cases, the isomerase activity was inhibited by nanomolar concentrations of cyclosporin A. Treatment of cultured trophozoites with cyclosporin A decreased their proliferation with a 50% inhibition value of 1 microg/ml and was lethal in doses over 50 microg/ml.

Purification of urokinase by monoclonal antibody affinity chromatography.

Matrix-bound monoclonal antibodies against urokinase have been used to purify this enzyme by affinity chromatography. In a single-step procedure, urokinase can be isolated from crude preparations with high yield and high purity, and without loss of enzymatic activity.

Monoclonal antibodies against plasma protease inhibitors: II. Production and characterization of 25 monoclonal antibodies against human alpha 1-antitrypsin. Correlation between antigenic structure and functional sites.

Twenty-five hybridomas secreting monoclonal antibodies against human alpha 1-antitrypsin have been produced by the cell-fusion technique (Köhler and Milstein, 1976). All antibodies are specific for alpha 1-antitrypsin and carry gamma 1 heavy chains and kappa light chains. Inhibition experiments showed that these monoclonal antibodies define three independent antigenic regions on the alpha 1-antitrypsin molecule; one of these domains appears to be involved in the interaction between alpha 1-antitrypsin and trypsin. In addition, one monoclonal antibody, AATY39, was used to develop an enzyme-linked immunosorbent assay capable of detecting low levels of alpha 1-antitrypsin in the range of 1 to 2 ng/ml.

Monoclonal antibodies against urokinase.

Hybridomas secreting antibodies against human urokinase have been produced by the cell-fusion technique (Köhler & Milstein, 1976). They belong to the IgG1 and IgG2 classes. Fixation and inhibition of the binding of 125 I-labelled urokinase, in radioimmunoassay, show that two of the monoclonal antibodies exhibit a high titer in ascitic fluids, a good sensitivity, and no cross reaction with other enzymes showing partial sequence homology with urokinase. Moreover, one of the monoclonal antibodies is able to inhibit the enzymatic activity of urokinase using a chromogenic substrate.

Solid-phase enzyme immunoassay of urokinase using monoclonal antibodies.

Using two monoclonal antibodies directed against urokinase, we have developed a micro enzyme-linked immunosorbant assay (ELISA) to detect and measure urokinase in biological fluids. The system presents the following characteristics: simple and rapid procedure, reproducibility, sensitivity (urokinase levels down to 1 ng/ml) and evaluation of the enzyme in biological fluids such as urine, pleural effusions, and ascitic fluids without preliminary purification.

Monoclonal antibodies against plasma protease inhibitors: I. Production and characterization of 23 monoclonal antibodies against human alpha 2-antiplasmin.

23 hybridomas secreting monoclonal antibodies against human alpha 2-antiplasmin, the fast-acting inhibitor of plasmin present in plasma, have been produced by the cell-fusion technique. Isotyping of the monoclonal antibodies has revealed that 14 monoclonal antibodies belong to the class IgG1, 6 to the class IgG2a, and 3 to the class IgG2b. All light chains belong to the kappa group. The specificity and relative avidity of these monoclonals have been determined using an indirect enzyme-linked immunosorbent assay. 13 monoclonals exhibit a relatively high avidity for alpha 2-antiplasmin, 5 are of intermediate avidity, and 5 of low avidity. The epitope specificity of these 23 monoclonal antibodies, originating from a single mouse, have been examined in inhibition experiments. A group of 10 monoclonal antibodies exhibit a very similar inhibition pattern. Partial inhibition effects displayed by 10 other antibodies define partially overlapping antigenic regions. The binding of these antibodies seems to produce a conformational change in the alpha 2-antiplasmin molecule, reducing the binding of two other antibodies. The last antibody defines an independent epitope.

Subcellular localization of the 54-kDa antigen of Toxoplasma gondii.

A 54-kDa protein antigen of Toxoplasma gondii recently was cloned and expressed in Escherichia coli and shown to display immunoprotective properties. To determine the subcellular localization of this antigen, a fusion protein containing the 330 carboxy-terminal residues of the sequence coded by cDNA clone Tg34 was expressed in E. coli, purified, and used to raise antibodies in mice. Western blot analysis confirmed that the resulting antibody reacted with the 54-kDa antigen of T. gondii. Immunofluorescence and immunoelectron-microscopy showed that the antibody reacted with an antigen localized in the rhoptries, 1 of the organelles of the apical complex of the zoites involved in host cell invasion. Western blot studies using a recombinant fusion protein containing the full amino acid sequence encoded by the cDNA clone Tg34 and rhoptry protein-specific monoclonal antibodies (mAbs) previously described showed a reactivity of the recombinant antigen with 2 mAbs (T4 2F8 and T5 2D1) specific for the ROP2 protein, and with a mAb (T3 4A7) directed against an epitope shared by ROP2 and ROP4, but not with a mAb (T2 2H3) specific for the ROP4 protein. We thus conclude that the 54-kDa T. gondii antigen encoded by cDNA clone Tg34 is the previously described rhoptry protein ROP2.

Monoclonal antibodies identify new Toxoplasma gondii soluble antigens.

In order to characterize Toxoplasma gondii antigens, we have produced a panel of monoclonal antibodies specific for the parasite. A total of 22 hybridomas were derived from the spleen cells of mice immunized either with a 100,000 g supernatant of a sonicate from the RH strain (called F3), or chronically infected with the Wiktor or the 76K strain. Except for one hybridoma producing an IgM, all the hybridomas derived from mice immunized with F3 produced IgG1 antibodies while those obtained from chronically infected mice produced antibodies belonging to the IgG2b, IgG2a and IgM subclasses. Western-blot analysis showed that the panel of monoclonal antibodies defines at least 7 distinct antigens or antigen families. An antigen of apparent Mw 25 kD present exclusively in the 100,000 g supernatant of the T. gondii sonicate was recognized by the majority of monoclonal antibodies derived from mice immunized with the F3 fraction. Two other antigens of apparent Mw 27 kD and 29 kD present in the soluble and insoluble fractions of the sonicate were also identified. Monoclonal antibodies against the previously described 21 kD and 31 kD surface antigens and belonging to the IgG2a but also to the IgG1 subclasses were able to mediate lysis of the parasite in the presence of human non immune serum. The 22 monoclonal antibodies did not identify antigenic differences between the two independently isolated RH and Wiktor strains.

Characterization of high affinity monoclonal antibodies against the luteinizing hormone-releasing hormone.

Four hybridoma clones (BKL1, BKL2, BKL5 and BKL6), secreting monoclonal antibodies against the decapeptide luteinizing hormone releasing hormone (LHRH), were obtained by the fusion of Sp 2/0-Ag14 myeloma cells with spleen cells of a Balb/k mouse immunized with a conjugate of thyroglobulin-LHRH. All four monoclonal antibodies belong to the IgG1 subclass. The antibodies cross-reacted in RIA from 9.3 to 21% with 4-10 LHRH and less than 1% with 7-10 LHRH, 1-3 LHRH and LHRH-OH; 4-6 LHRH showed no cross-reaction. A RIA based on the BKL2 antibody and able to detect 4 pg LHRH per tube was developed. An extract of rat hypothalamus was submitted to Sephadex G50 separation and a single peak of LHRH immunoreactivity corresponding to synthetic LHRH, was detected with the antibody BKL2. When this material was further analyzed by HPLC, we found that the major peak of immunoreactivity co-migrated with synthetic LHRH. The data shows that the antibodies should be useful tools for biochemical and physiological studies on LHRH.

Monoclonal antibodies against noxiustoxin.

Noxiustoxin, a 39-amino acid residue peptide isolated from the venom of the Mexican scorpion Centruroides noxius, has previously been shown to affect voltage-dependent K+ channels. Here we describe the isolation and characterization of monoclonal antibodies (MAbs) against this toxin and their use in structure-function relationship studies. Six hybridoma clones (BNTX4, -12, -14, -16, -18, and -21) producing MAbs against noxiustoxin were isolated. The epitopes defined by the MAbs are overlapping or in close proximity because no MAb pair could bind simultaneously to the toxin. All the MAbs inhibited to various degrees the binding of the toxin to its receptor sites on rat brain synaptosomal membranes. The venom from other Centruroides species was shown to contain components cross-reacting with the MAbs, suggesting the existence of other NTX-like toxins.

Human T-cell clones against Toxoplasma gondii: production of interferon-gamma, interleukin-2, and strain cross-reactivity.

The soluble fraction from a sonicate of Toxoplasma gondii tachyzoites (termed F3) was shown to induce dose-dependent blastic transformation of peripheral-blood mononuclear cells (PBMC) from seropositive individuals only and was used to isolate a panel of T-cell clones from the PBMC of an immune donor. Proliferation assays using F3 showed that 15 (14 CD4+ and 1 CD8+) of the 18 isolated clones were specific for T. gondii. In response to antigen stimulation, 5 of the 15 clones produced detectable levels of interleukin-2 (IL-2, 0.2-15 u/ml) and 9 clones produced significant levels of interferon-gamma (IFN-gamma, 17.5-1400 IU/ml). Seven of the 7 T-cell clones tested reacted with two different Toxoplasma strains (RH and Wiktor). When used as antigen-presenting cells, an autologous B-lymphoblastoid cell line could efficiently present the antigen to only three of the six T-cell clones tested. This study identifies and characterizes cellular probes that could be useful for future vaccine design.

Genetic immunization with plasmid DNA coding for the ROP2 protein of Toxoplasma gondii.

The ROP2 protein of Toxoplasma gondii has previously been proposed as a vaccine candidate against toxoplasmosis. In this work we characterize the immune response induced by injection of plasmid DNA coding for this protein in three strains of mice (BALB/c, C57BL/6, and CBA/J) displaying different levels of susceptibility to toxoplasmosis and compare it with that obtained by vaccination with the live attenuated ts-4 strain of T. gondii. The ROP2 gene was cloned in the eukaryotic expression vector pcDNA3 and the resulting plasmid, named pcDNA3/ROP2, was used to immunize mice. After three immunizations with the plasmid, mice developed antibodies that could be detected by ELISA using a recombinant truncated form of ROP2; and these antibodies also recognized the natural protein by Western blot. Plasmid immunization generated antibodies against the ROP2 of both of the IgG1 and IgG2a isotypes in CBA/J and BALB/c mice and both of the IgG1 and IgG2c isotypes in C57BL/6 mice. However, animals vaccinated with the ts-4 strain generated only IgG2a (in CBA/J and BALB/c mice) or IgG2c (in C57BL/6 mice) against ROP2. Kinetic studies of the generation of isotypes indicated that both isotypes were generated at the same time. Mice immunized with the plasmid DNA did not resist a challenge with the virulent RH strain of T. gondii, while mice vaccinated with the ts-4 strain resisted the same challenge. However, in pcDNA3/ROP2-immunized BALB/c mice, death was significantly delayed with respect to the pcDNA3-immunized control group. These results suggest that plasmid immunization using the ROP2 gene generates a mixed T(H1)/T(H2) response against ROP2, which is different from that obtained by vaccination with live tachyzoites of the ts-4 strain (T(H1) response) and is not protective against the highly virulent RH strain of the parasite.

MVA ROP2 vaccinia virus recombinant as a vaccine candidate for toxoplasmosis.

Toxoplasma gondii is the aetiological agent of toxoplasmosis and is the most frequent and best known of the parasitic diseases. In the United States, a serological survey from the Third National Health and Nutrition Examination Survey found that an estimated 23% of adolescents and adults have laboratory evidence of infection with T. gondii. Although toxoplasmosis is asymptomatic or shows self-limited symptoms in adults, in pregnant women infections can cause severe health problems to the fetus if the parasites are transmitted. Also, in immunodeficient patients, chronic infection with T. gondii can reactivate and produce encephalitis, which is frequently lethal. In addition, in veterinary medicine, T. gondii infection is of economic importance due to abortion and neonatal loss in sheep and goats. Recently, the development of vaccines against toxoplasmosis has progressed considerably. The live attenuated S48 strain of Toxoplasma has been broadly used for veterinary purposes. DNA vaccines containing the full-length of SAG1/P30, ROP2 or ROP1 genes have proved to be a promising candidate to induce protection against toxoplasmosis. Viral vectors have proved to be the best candidates for vaccination in different diseases. A recombinant Herpes virus carrying the ROP2 gene is able to induce protective immunity in cats. In the present work we describe the potential of the MVA ROP2 recombinant vaccinia virus as a vaccine against toxoplasmosis. MVA ROP2 induces antibodies against the ROP2 protein in similar amount and types as the thermo-sensible strain ts-4 of T. gondii, which is able to fully protect mice against challenge with the virulent RH strain of T. gondii. Also, the life-span of mice is increased in MVA ROP2 vaccinated animals. We conclude that MVA ROP2 vaccine can possibly generate an immune response, which could be useful in protection against toxoplasmosis.

Monoclonal antibodies against plasma protease inhibitors: production and characterization of 15 monoclonal antibodies against human antithrombin III. Relation between antigenic determinants and functional sites of antithrombin III.

Fifteen hybridomas secreting monoclonal antibodies against human antithrombin III, originating from two mouse strains, have been produced by the cell fusion technique. Eight monoclonal antibodies belong to the class IgG1, five to the class IgG2a, and two to the class IgG2b. All light chains belong to the kappa group. No cross-reaction of the monoclonal antibodies have been observed with a crude preparation of albumin nor with alpha 1-antitrypsin and alpha 2-antiplasmin. Five of these monoclonal antibodies exhibit a relatively high avidity for antithrombin III. Inhibition experiments showed that the 15 monoclonal antibodies define seven more or less independent antigenic regions on the antithrombin III molecule. Examination of the effects of these antibodies on the inhibitory capacity of antithrombin III toward thrombin activity, either in the presence or in the absence of heparin, showed that several monoclonal antibodies inhibit the antithrombin III activity and allowed to relate some of the antigenic determinants to functional sites on the antithrombin III molecule.

Human T cell clone identifies a potentially protective 54-kDa protein antigen of Toxoplasma gondii cloned and expressed in Escherichia coli.

Protective immunity against Toxoplasma gondii is recognized to be cell mediated and IFN-gamma is considered to be the major mediator of resistance. Thus, protective Ag of the parasite must induce IFN-gamma-producing T cells. In order to identify such Ag, we have constructed a T. gondii cDNA library in the cloning/expression vector lambda gt11, screened this library with a pool of sera of immune donors, and further screened the set of selected recombinant Ag using, as probe, a T. gondii-reactive T-cell clone (TCC) derived from an infected/immune individual and producing a high level of IFN-gamma. One recombinant Ag was shown to induce TCC proliferation and was characterized. The corresponding mature T. gondii Ag has an apparent molecular mass of 54 kDa and the sequence of the cDNA clone suggests that it is membrane associated. The epitope defined by the TCC on this Ag was found to be present in three Toxoplasma strains independently of their phenotype (virulent or cyst forming). Recognition of this Ag by the TCC was shown to be restricted by HLA-DPw4, the most frequent allele in the Caucasian population (approximately 40%). The use of this Ag as a vaccine component is proposed.