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S Avrameas

Publications and source records attributed to S Avrameas.

At least 91 records · Page 5Linked to original sources
PubMed1988

Development of a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for measuring venom antigens after an experimental snake bite.

We describe a new ELISA which allows the measurement of the concentration of venom antigens in whole blood. The assay can be performed in less than 20 min and requires a 200 microliters sample of blood. It allows the accurate evaluation of concentrations of Vipera ammodytes venom in quantities smaller than 1 ng/ml of blood. Using this ELISA, we were able to follow in rabbits the kinetics of experimental envenomation with non-lethal doses of venom. This ELISA was also used to measure post mortem the level of venom antigens in various tissues such as liver, kidney, muscles and abdominal serosity of a rabbit. The method, which might be adapted to measure envenomation by other snake species, seems to be sufficiently rapid and sensitive to allow routine evaluation of the gravity of a snake bite in humans and to estimate the efficacy of immunotherapy.

Animals↗
PubMed1988

[Immunoenzyme assay for histamine].

In order to produce monoclonal antibody to histamine, mice are immunized with histamine conjugated to bovine serum albumin (BSA) by the 1,4-benzoquinone method. After an initial screening using ovalbumin (OVA) and histamine-OVA conjugate as antigens to identify monoclonal antibody secreting clones, the hybridomas are isolated by limiting dilution cloning and grown in ascites. The specificity of selected monoclonal antibody (D22) is studied using a direct enzyme immunoassay and an ELISA-inhibition test. D22 antibody reacts with histamine-protein conjugates prepared by the 1,4-benzoquinone coupling procedure. On the contrary, this antibody is unreactive with native proteins, 1,4-benzoquinone treated proteins or various amine-protein conjugates. Free unconjugated histamine significantly inhibits antibody binding to histamine-OVA and 50% inhibition (IC50) is recorded at 5 X 10(-3) M. On a histamine molar concentration basis a much more lower inhibitory potency of free histamine is recorded, as compared to histamine-benzoquinone derivative (IC50 = 2 X 10(-8) M) and to histamine-OVA (IC50 = 7 X 10(-10) M). It is our interpretation that for the D22 antibody, the main epitope encompasses the 2-histaminyl-1,4-benzoquinone moiety and that this points to the importance of polyvalent ligands for efficient bivalent binding of the IgG antibodies. Using the D22 antibody we set up a competitive enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of monoclonal antibody which is used to coat the wells of a microtitration plate.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1988

Polyspecific natural antibodies and autoantibodies secreted by human lymphocytes immortalized with Epstein-Barr virus.

Recent studies have shown that autoreactive B cells and autoantibodies are present in pathological as well as in normal situations. In the present study, we immortalized human B cell lines from normal individuals and from patients with malignant or benign dysglobulinemia with Epstein-Barr virus and examined, after cloning, the autoantibody reactivities of the immunoglobulins secreted by these cells. Forty-two supernatants were analyzed by enzyme-immunoassay on a panel of 13 self and non-self antigens: trinitrobenzenesulfonic acid (TNP), DNA, L-glutamine, L-alanine, L-tyrosine (GAT), actin, myosin, tubulin, albumin, renin, spectrin, transferrin, thyroglobulin, myoglobin, peroxidase, and by immunofluorescence in tissue sections. Fourteen (33%) of the immunoglobulin-secreting cell lines were found to have an autoantibody function; seven secreted IgM, six IgA, and one IgG. The light chains were of the kappa type in 11 cases. The vast majority of these clones reacted with more than five antigens of the panel and all of them reacted with TNP. No correlation was found between a given isotype and an antibody specificity. More than half of these antibodies also reacted with cellular antigens present in tissue sections. None of the four cell lines secreting monoclonal antiviral antibodies reacted with any of the antigens of the panel. The results indicate that immunoglobulins secreted by human monoclonal lymphoid cell lines can have polyspecific autoantibody functions, similar to those found in normal human polyclonal antibodies, in human monoclonal paraproteins and in natural monoclonal antibodies synthesized by murine or rat clones obtained from physiologically normal animals.

Antibodies↗
PubMed1988

Specificity of natural serum antibodies present in phylogenetically distinct fish species.

The following fish orders, at different stages of phylogenetic development, were tested for serum antibody activity against actin, myosin, tubulin, thyroglobulin, haemocyanin, single-stranded DNA and trinitrophenyl (TNP): Elasmobranches (sharks), Chondrosteans (sturgeons), and Teleosts (bony fish). Significant antibody activity was found against all these self and non-self antigens but, in particular, high titres of natural anti-TNP antibodies were noted. Anti-TNP antibodies were then isolated by affinity chromatography from pooled sera of several representative fish species, and studied by a non-competitive and a competitive enzyme immunoassay. When the isolated antibodies were tested by the non-competitive assay, Teleost (tench) anti-TNP antibodies bound almost exclusively to TNP, whereas Chondrostean and Elasmobranche antibodies bound mainly to TNP but also to one or more other antigens. When the isolated antibodies were examined by the competitive assay, they could be separated into three groups: (i) anti-TNP antibodies whose binding could be inhibited by TNP only (Teleosts); (ii) anti-TNP antibodies mainly inhibited by TNP but also significantly inhibited by two or three other antigens (Chondrosteans); and (iii) anti-TNP antibodies binding to different antigens but only slightly inhibited by them (Elasmobranches). These antibodies were found by immunocytochemical staining to bind to normal cell constituents, and especially to cytoskeletal proteins. The results strongly suggest that natural antibodies against the seven antigens examined are present in normal fish serum, and that their specificities differ with the stage of phylogenetic development.

Animals↗
PubMed1987

A method for the quantification of a colored or fluorescent signal in enzyme immunoassays by photodensitometry.

A colored or fluorescent signal is generally evaluated with the naked eye, or by means of different more or less sophisticated and costly instruments. Photodensitometry is an additional technique which is both inexpensive and simple to perform. This technique can satisfactorily quantify a signal without the use of either a spectrophotometer or a fluorometer. In this study we compared readings obtained by spectrophotometry, fluorometry and photodensitometry in 96-well ELISA plates and in Terasaki plates. In ELISA plates, it is possible to detect 1220-300,000 femtograms (fg) of peroxidase by spectrophotometry and 4800-125,000 fg by photodensitometry. In Terasaki plates, we were able to measure between 3.8 and 8000 fg of beta-galactosidase per sample by spectrofluorometry, and from 30 to 8000 fg by photodensitometry. Using a sandwich procedure in Terasaki plates we were able to measure between 100 and 10,000 fg of IgE per sample by spectrofluorometry and from 500 to 10,000 fg by photodensitometry. Photodensitometry is the least expensive technique for the reliable detection of enzyme or enzymatic marker in small sample volumes treated with a fluorogenic substrate.

Chromogenic Compounds↗
PubMed1987

A highly sensitive microtitre plate enzyme immunoassay for oestradiol-17 beta.

A specific and sensitive solid-phase microtitre plate enzyme-linked immunosorbent assay for oestradiol-17 beta (E2) is described. After coating with an IgG anti-E2 fraction, we used E2-6-carboxymethyl-oxime-beta-galactosidase in a competitive binding assay and revealed the bound activity with a fluorogenic substrate. Two methods for the competitive binding assay were tested: (1) a classical one (method A) defined as a 'two-step competition' because the E2 sample was first incubated alone, and then E2-beta-galactosidase conjugate was added; (2) and a new one (method B) also performed in two steps but in which the E2 sample was evaporated to dryness. The detection limit of method A was 100 pg/ml (9 pg/well). Method B was more sensitive since 1 pg/ml (0.3 pg/well) was statistically different from 0 pg/ml. Specificity was equivalent with both methods while precision was better in B. Thus, this new method may be able to measure very low levels of oestradiol-17 beta in, for example, the blood of domestic mammals.

Binding, Competitive↗
PubMed1987

5-Bromodeoxyuridine in vivo labelling of M13 DNA, and its use as a non-radioactive probe for hybridization experiments.

We describe the in vivo production of 5-bromodeoxyuridine- (5-BUdR) labelled M13 DNA by a thymine-requiring Escherichia coli strain. We show that the 5-BUdR-labelled M13 single-stranded DNA is not extruded into the culture medium, but accumulates inside the bacterial cells. On the basis of this observation, a procedure involving FPLC gel filtration already reported and used for the isolation of plasmid DNA has been adapted for the isolation of at least 90% pure 5-BUdR-labelled single-stranded DNA. An M13 probe, containing part of the Hepatitis B Virus (HBV) genome was constructed, and the corresponding 5-BUdR-labelled single-stranded DNA was used in hybridization experiments to detect homologous HBV target DNA. Picogram amounts (10(-19) moles) of the probe itself or the target DNA could be detected, by monoclonal anti-5-BUdR antibodies in an immunoenzymatic assay.

Animals↗
PubMed1987

Single lipopolysaccharide-reactive B cells in the non-immune mouse spleen cell population secrete natural multispecific autoantibodies.

Starting from unimmunized BALB/c splenocytes, B-cell clones were obtained by lipopolysaccharide (LPS) stimulation, and the frequencies of their anti-TNP, anti-BALB/c actin, and anti-BALB/c tubulin secretion were determined. The culture conditions were then chosen so as to have one anti-TNP precursor per positive well. Out of the 41 wells containing one anti-TNP antibody-secreting cell, nine (22%) also reacted either with actin or with tubulin and five (12%) with both actin and tubulin. Using horse red blood cells to which trinitrophenyl (TNP) had been coupled, spleen cell rosettes were prepared, enriched, micromanipulated, and cultured individually. Of the 500 micromanipulated and cultured TNP antigen-binding cells, 28 were found to secrete antibodies directed against TNP. Eight of these 28 clones (28%) also reacted with either actin or tubulin, and five (17%) reacted with both actin and tubulin. The frequency of these multispecific clones is of the same order of magnitude as that obtained with the limiting dilution experiments. The results show that unimmunized BALB/c mouse spleen contains precursor cells that secrete natural multispecific autoantibodies.

Actins↗
PubMed1987

Detection of plasma cell immunoglobulins in tissue sections optimally fixed for ultrastructural immunocytochemistry.

We describe the ultrastructural localization of plasma cell immunoglobulins in vibratome sections of popliteal lymph nodes. Fixation with glutaraldehyde-paraformaldehyde gave better tissue and antigen preservation than paraformaldehyde or periodic acid lysine-paraformaldehyde; biotinylated Fab fragments of sheep anti-mouse IgG-streptavidin-biotinylated horseradish peroxidase (HRP) or Fab-HRP conjugates gave similar results. With both immunoreagents, excellent tissue preservation and antigen detection was observed in the first layer of cells sectioned with the vibratome. Conjugates of anti-mouse IgG with HRP did not show any staining. Peroxidase stain was observed in the nuclear envelope, cisternae of the rough endoplasmic reticulum, and the Golgi apparatus complex. In the Golgi apparatus, staining was seen consistently in cisternae of the cis face and in adjacent vesicles; the trans cisternae showed weak or no stain, and adjacent vesicles, "coated" vesicles, and granules were not stained. This study shows that high quality of tissue preservation and antigen detection, by both light and ultrastructural immunocytochemistry, is feasible in tissue fixed with glutaraldehyde-paraformaldehyde followed by vibratome sectioning and immunostaining with Fab-biotin-streptavidin-biotin-HRP, or Fab-HRP.

Animals↗
PubMed1987

In vitro proliferation of human lymphocytes measured by an enzyme immunoassay using an anti-5-bromo-2-deoxyuridine monoclonal antibody.

A previously described, non radioactive method for the measure of in vitro mouse lymphocyte proliferation was applied to human lymphocyte proliferation assays. It involved incorporation into DNA, during cell multiplication, of 5-bromo-2-deoxyuridine (BUdR), a thymidine analogue. BUdR-DNA was then assayed by a sandwich enzyme immunoassay (BUdR-EIA) using an anti-BUdR monoclonal antibody (McAb 76-7). BUdR-DNA from crude cell extracts was first immobilized on microtitration plates coated with McAb 76-7. In a second step BUdR-DNA was reacted again with McAb 76-7 conjugated to horse radish peroxydase. The quantity of peroxydase in microtitration wells was then measured by the coloration of o-phenylenediamine (492 nm). Titration curves obtained with dilutions of crude extracts were compared to the curve obtained with a purified BUdR-DNA reference solution. Results were expressed as equivalent ng BUdR-DNA/ml. BUdR-EIA was compared to 3H-thymidine incorporating assay for the measure of lymphocyte proliferation induced by PHA mitogen, candidine and tuberculine antigens and mixed lymphocyte culture. Excellent correlation between both assays was observed for each experiments (r = 0.953 to 0.999). Overall correlation coefficient for the 5 experiments was 0.785, indicating greater variation of BUdR than 3H-thymidine incorporation, according to the mitogen or antigen used and the culture conditions. This could be due to that fact that BUdR-EIA measured only BUdR incorporated into DNA, while 3H-thymidine incorporation assay measured 3H-thymidine both incorporated into DNA, and stocked into the cell before DNA incorporation. BUdR-EIA would thus reflect cell proliferation more exactly than 3H-thymidine incorporation assay. The sensitivities of both techniques were comparable.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1987

Natural autoantibodies in systemic lupus erythematosus.

We have tested the sera of 25 patients with systemic lupus erythematosus (SLE) for antibody activity against a panel of six antigens: DNA, TNP, actin, tubulin, myosin, albumin. Eluates from renal biopsy tissue were also tested. Sera from patients with lupus nephritis were found to contain high titres of IgA antibodies directed against the antigens of the panel, and marked IgG anti-DNA and anti-TNP antibody activity. The IgG anti-TNP antibodies isolated from SLE serum by affinity chromatography on a TNP-immunoadsorbent, were also found to possess anti-DNA activity. Kidney eluates obtained from biopsy specimens of SLE patients contained IgG antibodies strictly specific for DNA in three out of the nine patients tested, while three eluates from the remaining six patients reacted with DNA and TNP and three with DNA and all the other antigens of the panel. These results strongly suggest that in SLE sera there are at least three populations of circulating anti-DNA antibodies: those strictly specific for DNA, those recognizing DNA and TNP and those recognizing DNA and other macromolecules. Furthermore, because six out of nine of the eluates contained antibodies with an absolute or restricted specificity for DNA, this suggests that these antibodies are more often pathogenic than the polyspecific ones recognizing DNA and other macromolecules.

Actins↗
PubMed1987

Suppression of anti-DNA antibody production in MRL mice by treatment with anti-idiotypic antibodies.

Antibodies against idiotypic determinants carried by a monoclonal polyspecific natural autoantibody were raised in rabbits and in syngeneic BALB/c mice. These anti-idiotypic antibodies were administered to newborn and to pregnant BALB/c mice and to MRL-lpr/lpr mice. Serial measurements of the idiotypes, naturally occurring autoantibodies, and antibodies obtained after antigenic stimulation were performed in the sera of the injected mice and in the offspring of pregnant mice. No idiotypic suppression was noted in newborn injected mice. Transient suppression of idiotypes recognized by the syngeneic anti-idiotypic antibody was noted in the offspring of pregnant mice injected with the rabbit polyclonal anti-idiotypic antiserum. No changes in naturally occurring autoantibodies or in antibodies appearing after antigenic stimulation were noted in BALB/c mice. In contrast, a significant decrease of spontaneously occurring anti-DNA antibodies was found in MRL-lpr/lpr mice treated with rabbit polyclonal anti-idiotypic antiserum. Furthermore in these mice a slight decrease of anti-TNP antibodies was also observed. These results suggest that anti-idiotypic antibodies directed against natural autoantibodies may play a regulatory role in the immune system; this role is more easily appreciated in mice suffering from immune dysregulation.

Animals↗
PubMed1986

Enzyme immunoassay for the measurement of histamine.

This paper reports a competitive solid-phase enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone treatment and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of an anti-histamine monoclonal antibody which was used to coat the wells of a microtitration plate. After incubation and washing, peroxidase activity associated with the solid phase is measured. With this method the histamine concentration in blood or various tissues may be determined easily, safely and reproducibly. Histamine concentrations from 0.3 to 20 ng/ml may be measured with the procedure reported here.

Animals↗
PubMed1986

Autoimmunity induced by HgCl2 in Brown-Norway rats. II. Monoclonal antibodies sharing specificities and idiotypes with mouse natural monoclonal antibodies.

Spleen cells derived from BN rats receiving HgCl2 were fused with the nonsecreting rat myeloma cell line IR983F. We screened 59 supernatants from immunoglobulin-secreting hybrids for antibody activity against actin, tubulin, autologous and heterologous myosin, myoglobin, dsDNA, peroxidase, and the haptens TNP, NIP, NNP, and NBrP. Six monoclonal antibodies (mAb) were found to react with antigen(s) of the panel. At least three groups of antibody specificities were identified: clones reacting with TNP (1 IgM, 1 IgE); clones reacting with horseradish peroxidase (1 IgM); and clones possessing widespread reactivity for several antigens as found for mouse natural autoantibodies (2 IgM, 1 IgE). We also analyzed the idiotypic (Id) determinants of the 59 mAb by using anti-Id antibodies described elsewhere prepared in rabbits against the BALB/c D23 natural monoclonal autoantibody and recognizing a BALB/c recurrent Id (Id D23) of natural polyspecific autoantibodies. We found that all rat mAb that possessed widespread reactivities bore this Id. We performed similar studies in sera from normal and mercury-stimulated rats. The results indicate a role for HgCl2 in the stimulation of natural antibodies producing cells and the existence of interspecies cross-reactive Id among mouse and rat natural antibodies.

Animals↗
PubMed1986

Monoclonal anti-histamine antibody. Preparation, characterization and application to enzyme immunoassay of histamine.

An enzyme immunoassay to measure histamine has been developed. A histamine-bovine serum albumin conjugate was prepared using 1,4-benzoquinone as the coupling agent and was employed to immunize mice for the preparation of monoclonal antibodies against histamine. After an initial screening to identify antigen-binding monoclonal antibodies the clones were isolated by limiting dilution cloning, grown in ascites and antibodies which had been secreted into the ascitic fluid were precipitated by ammonium sulphate at 50% saturation. A systematic approach for the determination of epitope specificities of monoclonal antibodies was performed. It was found that for the most specific antibody the main epitope encompassed the 2-histaminyl-1,4-benzoquinone moiety and that the KD value determined by indirect ELISA was 1.5 X 10(-8) M for the hapten part of the immunogen and 4.6 X 10(-10) M for a histamine-Bq-ovalbumin conjugate. The selected monoclonal antibody could not recognize histidine or methyl-histamine. Using this antibody, we developed an enzyme immunoassay for histamine and pg amounts could be detected. The same assay was used to quantify the allergic release of histamine from guinea pig lung mast cells. Results obtained either by the present enzyme immunoassay or by a fluorometric assay were closely correlated (correlation coefficient r = 0.9702, n = 37).

Animals↗