Generation of a mouse monoclonal TSH receptor antibody with stimulating activity.

A Balb/c mouse was subjected to genetic immunization with a cDNA construct encoding the human thyrotropin receptor (TSHr). The immune response of the mouse resulted in the production of immunoglobulins recognizing the TSHr in three different assays: (1) flow immunocytometry (FACS) with CHO cells expressing the receptor; (2) receptor-dependent stimulation of cAMP production in the same cell line; and (3) competition with labeled TSH for binding to the receptor. One thousand hybridomas were generated from the spleen of the mouse and their supernatants were screened. A single monoclonal, IRI-SAb1, scored positive in all three assays and was studied further. It stimulated 13-fold cAMP production in TSHr-expressing CHO cells, with an EC50 in the low nanomolar range. When compared with bovine TSH, IRI-SAb1 behaved as a partial agonist. Contrary to the expectation from the characteristic of autoantibodies of Graves' patients, IRI-SAb1 recognized a linear epitope, which was localized in a segment encompassing the first 281 residues of the receptor.

Cytokine production and T-cell activation by macrophage-dendritic cells generated for therapeutic use.

Clinical grade ex vivo-generated antigen-presenting cells, macrophage-dendritic cells (MAC-DCs) or macrophage-activated killers (MAKs) were derived from peripheral blood mononuclear cells (PBMCs). Cultures (7 d) were performed in non-adherent conditions in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and either interleukin 13 (IL-13) or dihydroxy-vitamin D3 respectively. MAKs were activated during the last 24 h with interferon gamma (IFNgamma). Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses indicated that IL-1beta and tumour necrosis factor alpha (TNFalpha) were produced by both cells. Higher pro-inflammatory cytokine (IL-1beta and TNFalpha) amounts were detected on average in MAK supernatants. In contrast, IL-12 p40 was found only in MAC-DC supernatants, but the biologically active IL-12 form (p70) was never detected. T-cell cytokines (IL-2, IL-4, IL-10) were not produced in culture conditions in which T cells were nevertheless present. At d 7, TNFalpha or lipopolysaccharide (LPS) upregulated IL-12 p40 production by MAC-DCs, while IL-12 p70 remained undetectable. LPS stimulation also increased TNFalpha production by these cells. Allogeneic mixed lymphocyte reactions (MLR) showed that MAKs are poor stimulatory cells compared with MAC-DCs. The MAC-DC stimulatory capacity was enhanced by LPS, although the expression of HLA class II, CD83, CD80 and CD86 was unmodified. Thus, MAC-DCs represent a tool for triggering adaptative immunity, while MAK should be primarily used as effector killer cells.

Characterisation of monoclonal antibodies against human interleukin-10 and their use in an ELISA for the measurement of this cytokine.

Biological and biochemical characteristics of monoclonal antibodies (MABs) raised against human interleukin-10 (IL-10) are described as well as their use in the design of a specific ELISA for the measurement of the cytokine. 21 murine anti-human interleukin-10 (IL-10) MABs were obtained by fusion of splenocytes from mice immunized against human recombinant IL-10 with SP2/0 myelomatous cells. These antibodies define three major antigenic areas on the IL-10 molecule, one of which comprises epitopes involved in receptor binding and induction of biological activity. They recognize recombinant human IL-10 with affinities ranging from 1.3 x 10(-7) to 3 x 10(-11), as well as natural IL-10. Most of them also recognize viral IL-10 (vIL-10) encoded by the Epstein-Barr virus (EBV). A specific human-IL-10 ELISA has been developed using two MABs (18 and 19) as capture antibody and one MAB (17) as detector. The sensitivity (3 pg/ml), precision (intra-assays < 4%), reproducibility (interassay < 3%), and accuracy (recoveries, ranging between 84 and 107%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances, permits accurate cytokine measurement in biological fluids such as serum, plasma, bronchoalveolar lavage, urine and culture supernatants. Using the assay, IL-10 was measurable in the plasma of patients with septic shock (range 11-2740 pg/ml) whereas IL-10 plasma levels were < 7.8 pg/ml in healthy volunteers.

An ELISA for the measurement of human leukemia inhibitory factor in biological fluids and culture supernatants.

A new monoclonal antibody-based ELISA for leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) measurements is described. The sensitivity (56 pg/ml after 4 h incubation, 14 pg/ml after 24 h incubation), precision (intra-assays < 5%), reproducibility (interassay < 10%), and accuracy (recoveries, ranging between 98 and 119%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances guarantee accurate cytokine measurement in biological fluids such as serum, plasma, synovial fluid, follicular fluid, urine and culture supernatants. Using the assay, LIF/HILDA was measurable in supernatants after in vitro whole blood stimulation with phytohemagglutinin (PHA), OKT3, and phorbol myristate acetate (PMA) but not with lipopolysaccharide (LPS) or Ca ionophore. LIF/HILDA production was not measurable until after 24 h of culture, when cytokine levels were seen to increase linearly in the supernatant to reach values of up to 40 ng/ml after 96 h of culture. Finally, a good correlation was found (r = 0.96; p < 0.0001; y = 23.1x + 233) between the LIF/HILDA values obtained using the ELISA and DA-1a bioassay.

Detection of rotavirus in faecal specimens with a monoclonal antibody enzyme-linked immunosorbent assay: comparison with polyclonal antibody enzyme immuno-assays and a latex agglutination test.

Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen. A sandwich ELISA procedure using the chosen monoclonal as "capture and detecting" antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results.

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.

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 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.

Idiotypic regulation of the immune system. Common idiotypic specificities between idiotypes and antibodies raised against anti-idiotypic antibodies in rabbits.

Anti-idiotypic antibodies (Ab2) were raised in allotype-matched rabbits against anti-carbohydrate or anti-tobacco mosaic virus antibodies (Ab1). Several Ab2 were purified and injected into a third series of rabbits III which synthesized antiantiidiotypic antibodies (Ab3). Antigen was then given for the first time in those rabbits who had synthesized Ab3. The specific antibody synthesized in rabbits III was called Ab1'. Anti-idiotypic antibodies were raised against purified Ab3 antibodies (Ab4). In most cases, Ab1' antibodies are sharing idiotypic specificities with Ab1. Ab3 did not react with antigen but shared idiotopes with Ab1 and Ab1' because Ab4 antibodies, which are anti-idiotypes to Ab3 do recognize specifically Ab1 and Ab1' antibodies belonging to the same chain of immunization. It seems therefore that Ab3 looks idiotypically like Ab1 and Ab4 displays the same behaviour as Ab2. A general view of the functioning of the immune system is presented.

Idiotypic regulation of the immune system by the induction of antibodies against anti-idiotypic antibodies.

Anticarbohydrate antibodies (Ab1) were isolated from a rabbit hyperimmunized with Micrococcus lysodeikticus and injected into allotype-matched rabbits in order to obtain specific anti-iodiotypic antibodies (Ab2). Ab2 was isolated by means of a Sepharose column coupled to the anticarbohydrate antibodies and was injected into two allotype-matched rabbits. These latter rabbits produced specific anti-anti-idiotypic antibodies (Ab3) probably sharing idiotypic specificities with Ab1. However, these Ab3 did not react with the antigenic carbohydrate moiety of bacteria. The two rabbits that had produced Ab3 were then immunized with M. lysodeikticus and synthesized anticarbohydrate antibodies (Ab1') bearing idiotypic specificities similar to those of Ab1. The immune repertoire which is effectively expressed in one individual depends not only on the antigenic stimulation but also on the previous idiotypic history of the individual. These data support the concept that the immune system is a functional idiotypic network.