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T Porstmann

Publications and source records attributed to T Porstmann.

At least 37 records · Page 2Linked to original sources
PubMed1992

Human-immunodeficiency-virus-type-1-encoded Vpu protein is phosphorylated by casein kinase II.

Vpu as a human-immunodeficiency-virus-type-1-encoded 81-amino-acid integral-membrane protein was expressed in Escherichia coli using the inducible ptrc promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of E. coli and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII-related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with E. coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled gamma ATP and gamma GTP were used as phosphate donors in vitro phosphorylation of recombinant Vpu. In vivo phosphorylation of Vpu in HIV-1-infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV-1-infected human host cells. Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus S/TXXD/E for CKII. These potential phosphorylation sites are located within a well-conserved dodecapeptide of Vpu (residues 47-58), which is found in different HIV-1 strains as well as in a Vpu-like protein of SIVCPZ. Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV-1-infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV-1-infected cells as well as recombinant Vpu expressed in E. coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.

Adenosine Triphosphate↗
PubMed1992

Inhibition of HIV-1 infection in vitro by murine monoclonal anti-p24 antibodies.

Murine monoclonal antibodies (Mabs) to the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were tested for their ability to inhibit the replication and spread of the virus in permanent cell cultures (Molt4/8, K37, H9) and in the culture of II-2 stimulated T cells of healthy donors. After addition of ascitic fluid containing monoclonal anti-p24 antibodies or purified anti-p24 antibodies or the respective control to co-cultures of infected and non-infected cells, HIV-1 replication was evaluated by determining the percentage of infected cells and the activity of reverse transcriptase (RT) in cell-free supernatant. In addition, the supernatant's infectivity was determined. FACS analysis demonstrated p24 antigen in about 40% of unfixed HIV-1 infected cells at the cell membrane. Monoclonal anti-p24 antibodies of different epitope specificity added to the cells but not to the virus delayed the spread of HIV-1 infection in permanent cell culture. Furthermore, anti-p24 Mabs inhibited the release of RT-active virus particles by HIV-1 infected cell lines or II-2 stimulated T-lymphocytes, respectively, up to 60%. The mode of action of anti-p24 antibodies after HIV-1 infection is discussed on the basis of the data obtained.

Animals↗
PubMed1991

A solid-phase enzyme immunoassay for the detection of tetanus toxin using human and murine monoclonal antibodies.

Three human and three murine monoclonal antibodies were tested for their reactivity to tetanus toxin and toxoid and used to establish an enzyme immunoassay specific for tetanus toxin. The dissociation constants of the monoclonal antibodies were between 3.91 x 10(-9) and 8.48 x 10(-12). Two human monoclonal antibodies recognized conformation determinants on the toxin, whereas the others reacted to the heavy chain. Only a combination of antibodies of the two species allowed the development of an enzyme immunoassay for the detection of tetanus toxin with a lower detection limit of 1.2 micrograms/l.

Animals↗
PubMed1991

Cu/Zn superoxide dismutase quantification from fetal erythrocytes--an efficient confirmatory test for Down's syndrome after maternal serum screening and sonographic investigations.

An enzyme immunoassay especially designed for the quantification of Cu/Zn superoxide dismutase (SOD) in erythrocytes has been applied to measure the SOD of outcomes with high risk for Down's syndrome. From 148 fetuses SOD was quantified from erythrocytes of umbilical vein blood and related to the number of cells, the content of haemoglobin (Hb), and to the haematocrit (Hc). Comparative studies between the SOD content of erythrocytes from the fetuses and their mothers resulted in similar SOD levels (14.09 +/- 1.20 for fetal and 14.48 +/- 1.63 for maternal cells) with a 1.84-fold smaller variance for fetal cells. The best differentiation between normal fetuses and fetuses with Down's syndrome resulted from the SOD/cell ratio followed by the SOD/Hb ratio. Fixing a cut-off value from the probability density functions that the method results in a specificity of 99.99 per cent, the sensitivity to detect cases of Down's syndrome was 99.71 per cent for the SOD/cell ratio, 70.92 per cent for the SOD/Hb ratio, and 60.21 per cent for the SOD/He ratio. Nine cases with Down's syndrome were correctly diagnosed by the SOD/cell ratio determination. Eight of these were confirmed as free trisomy 21 by karyotype analysis and one was found to be a triploidy. The latter was not detected by the SOD/Hb and SOD/Hc ratios because of the one-third higher content of haemoglobin and the larger volume of the erythrocytes which resulted in ratios within the normal range.

Adult↗
PubMed1991

Comparison of four anti-HIV screening assays which belong to different test generations.

There are three different test generations of enzyme-linked immunosorbent assays (ELISA) for the detection of human immunodeficiency virus (HIV) infection, depending on whether virus lysate, recombinant proteins or synthetic peptides are used as solid phase antigen. Four different assays, i.e., three sandwich ELISAs and one competitive test, were used to demonstrate differences between the three systems with regard to the content of different diagnostically relevant virus proteins. The sensitivities and specificities of these assays were compared by using 312 anti-HIV positive sera and 500 sera of healthy blood donors. The highest sensitivity and specificity were achieved by the competitive ELISA based on recombinant proteins, and by the sandwich ELISA based on synthetic peptides.

Blotting, Western↗
PubMed1990

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA).

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.2 X 10(10) l/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, and additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 micrograms SOD/l.

Antibodies, Monoclonal↗
PubMed1990

Immunochemical quantification of Cu/Zn superoxide dismutase in prenatal diagnosis of Down's syndrome.

Cu/Zn superoxide dismutase (SOD) was quantified by enzyme immunoassay for prenatal diagnosis of Down's syndrome. Overall, 154 samples of amniotic fluid, 72 samples of amniotic cells and 31 samples of chorionic tissue were investigated. Due to the large biological variance of the SOD concentrations in normal pregnancies (range for amniotic fluid 10.5-154.9, for amniotic cells 40.0-338.8, and for chorionic tissue 132.2-649.5 g SOD/g protein) the cases of Down's syndrome detected by karyotype analysis were not reliably identified by Cu/Zn SOD quantification. As in erythrocytes obtained from patients with Down's syndrome, a trisomy 21 was easily and accurately detected in the erythrocytes from very small quantities (about 50 microliters) of umbilical blood. The SOD concentrations in normal cases (n = 40) varied between 11.4 and 17.3 and in the cases of trisomy 21, as confirmed by karyotyping (n = 4), between 22.5 and 23.2 ng/one million cells. SOD quantification in fetal erythrocyte is a helpful additional method in prenatal Down syndrome diagnosis under certain conditions, which are discussed.

Amniotic Fluid↗
PubMed1990

Binding behaviour of antibodies reacting specifically with an immunodominant region of the transmembrane protein gp41 of HIV-1.

A highly conserved region of the transmembrane protein gp41 of the HIV-1 was found in which the amino acid sequence 606 to 620 was characterized as an immunodominant region. The binding behaviours of a human monoclonal antibody and of polyclonal specific antibodies in sera of HIV-infected persons to this particular sequence have been studied. In two sandwich-type ELISAs with synthetic peptides as antigen representing this region, sera of 312 HIV-infected persons at all stages could be clearly discriminated against 500 anti-HIV antibody negative sera of healthy blood donors.

Amino Acid Sequence↗
PubMed1990

Development of an immunoenzymometric assay for alpha 1-microglobulin and measurement of its serum concentration in normal and HIV-infected persons.

alpha 1-Microglobulin was purified from urine to a purity of 97.7% in a yield of 25.8%, and was used to produce antibodies in sheep. These antibodies, purified by affinity chromatography, were used to develop a rapid one-step and a two-step immunoenzymometric assay (IEMA). The equilibrium in the reaction between solid phase-adsorbed antibodies and antigen and between the antigen and enzyme-labelled antibodies was attained within 30 and 100 min, respectively. The one-step IEMA permits a good differentiation of low alpha 1-microglobulin concentrations after 30 min reaction time. Its detection limit is 0.35 micrograms/l, and its measurement range is between 0.5 and 100 micrograms/l. The IEMA correlates well with radial immunodiffusion (r = 0.973). The mean alpha 1-microglobulin serum concentration in women is insignificantly lower (33.2 mg/l) than in men (36.1 mg/l). In both sexes the alpha 1-microglobulin concentration increases with age. HIV-infected symptomless men have a significantly lower (15.9 mg/l) alpha 1-microglobulin concentration in serum than normal persons, whereas in AIDS patients it is significantly higher (45.5 mg/l).

Acquired Immunodeficiency Syndrome↗
PubMed1990

[Enzyme immunoassays for the quantification of human gamma-gamma-enolase (NSE)].

A direct two-site binding assay on the basis of antibodies from sheep for the quantification of human gamma-gamma enolase is described. The antibody was produced by immunization with human NSE coupled to horse spleen ferritin. The assay shows two feature: a decreased reactivity with NSE from rat and NSE from human serum in spite of 100% recovery of purified human brain NSE. The sheep antibody seems to react with epitopes less accessible on the rat NSE and on the NSE of human serum. The assay is characterized by gamma-gamma enolase specificity, a high sensitivity (2 pg) and a precision of CV = 3-7%.

Animals↗
PubMed1990

Multireactive human monoclonal antibodies.

Human monoclonal antibodies were tested using different immunochemical procedures for their reactivity with various antigens. The great majority of human monoclonal IgM antibodies (15 out of 24) turned out to bind to a whole series of recognized antigens (DNA, keratin, tetanus toxin, ricin etc.). The specificity of these reactions was detected by competitive assays. For some antibodies a simultaneous reaction with two antigens could be demonstrated by capture bridge technique. IgG antibodies also turned out to be multireactive, but not to the same extent (range of antigens much smaller, only 5-6 out of 53).

Antibodies, Antinuclear↗
PubMed1989

Application of a human monoclonal antibody in a rapid competitive anti-HIV ELISA.

The ELISA is the established screening technique for the detection of antibodies directed against HIV. The first generation assays, mostly based on the sandwich principle, employed purified virus from cell culture and gave both false-positive and false-negative results. Sandwich-type assays preferentially detect IgG antibodies, require a high serum dilution and are two-step procedures. In order to detect an immune response as early as possible after infection anti-HIV antibodies of the IgM class should also be measured. To this end a competitive ELISA has been developed using a solid phase-adsorbed recombinant HIV envelope protein and an enzyme-labelled human monoclonal antibody. This detects both IgM and IgG antibodies, the results are available within 1 h and a serum predilution is not necessary.

Antibodies, Monoclonal↗
PubMed1989

Fine mapping of an immunodominant region of the transmembrane protein of the human immunodeficiency virus (HIV-1).

Peptides of HIV sequences are significant for antibody screening systems, and because of the limited number of amino acids they have to represent immunodominant regions of the virus proteins in order to maintain sensitivity. We detected, in a region of the outside loop of the transmembrane protein gp41 of the human immunodeficiency virus HIV-1 (amino acid 586-620), two immunodominant sequences which are distinct from each other. Whereas in the first immunodominant region the sensitivity and specificity of ELISA were inadequate, a neighbouring region is well suited for use as antigen for an anti-HIV screening ELISA.

Amino Acid Sequence↗
PubMed1989

Measurement of lysozyme in human body fluids: comparison of various enzyme immunoassay techniques and their diagnostic application.

Three variants of the immunoenzymometric assay of human lysozyme with HRP-labeled antibodies were compared. The highest sensitivity (with a detection limit of 0.2 micrograms lysozyme/L) was achieved by a one-step assay lasting 2 h. Between-batch precision for the techniques was 6-11%. Lysozyme reference values were determined in serum, cerebrospinal fluid and urine. In serum they are age-dependent and in urine sex-dependent when related to creatinine excretion. Serum lysozyme is increased in only 57% of the patients with active rheumatoid arthritis and is also unreliable for indicating remission. In Crohn's disease the serum lysozyme reflects activity better, but it does not exceed the diagnostic value of alpha-1-acidic glycoprotein (orosomucoid). The lysozyme quantification in cerebrospinal fluid is useful in distinguishing between viral or bacterial meningitis.

Acute Disease↗