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At least 19 recordsLinked to original sources

PubMed1977

Biological activities of tritiated endotoxins: correlation of the Limulus lysate assay with rabbit pyrogen and complement-activation assays for endotoxin.

Tritiated endotoxins were prepared by three different methods. The biological activities of the tritiated endotoxins were determined by the Limulus amebocyte lysate assay, a rabbit pyrogen assay, and a complement-activation assay and were compared to native, unlabeled endotoxin. All three tritiated endotoxin preparations manifested adequate biological activity in each of the three assay systems, and all three assays ranked the biological activity of the different endotoxin preparations in the same order. Endotoxin tritiated by the Wilzbach procedure retained most of its biological activity and also had the highest specific radioactivity. The good correlation between the Limulus lysate, rabbit pyrogen, and complement-activation assays suggests that the same active site of the endotoxin molecule is identified by the three different assays.

Animals↗

PubMed1975

Evaluation of the cell-mediated immune response to murine sarcoma virus by (125I)iododeoxyuridine assay and comparison with chromium 51 and microcytotoxicity assays.

The cell-mediated immune response of C57BL/6 mice to murine sarcoma virus (MSV) was examined by the [125I]-iododeoxyuridine release cytotoxicity assay using MSV-induced sarcoma tissue culture cell lines as target cells. Cellular cytotoxicity was detected as early as 3 days after virus inoculation. Most mice assayed between 12 and 17 days after MSV inoculation gave positive results with maximum levels of activity present on Days 13 and 14. Reactivity was frequently detected for up to 100 days after MSV inoculation, although at low levels (5 to 10%). Additional experiments comparing the kinetics of the cellular response as measured by different in vitro cytotoxicity assays were performed. The results showed a good direct correlation between the [125I]iododeoxyuridine release assay and a 51Cr release assay. A similar pattern of reactivity was also observed when the cellular response was measured by a visual microcytotoxicity assay, although reactivity dropped off more rapidly and became undetectable in most instances by 20 days after injection of MSV. Studies on effector cell type revealed that cytotoxicity in all three assays was T-cell dependent, being eliminated by treatment with anti-theta plus complement. Macrophages did not appear to play a role, since treatment with carbonyl iron and magnet had no effect.

Animals↗

PubMed1977

Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones.

Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique.

Adrenocorticotropic Hormone↗

PubMed1978

Isotope-release cytotoxicity assay with the use of indium-111: advantage over chromium-51 in long-term assays.

The adaptation of indium-111-oxine (also known as 8-hydroxyquinoline) (111In Ox) chelate for long-term (18-48 hr) isotope-release assays of cell-mediated cytotoxicity (CMC) and its advantages over the use of 51 Cr are described. Labeling of DBA/2 P815 mastocytoma cells with 111InOx resulted in the incorporation of as many as a million counts per minute in 10(5) cells with no reduction in cell viability. 111InOx labeled both mouse and human tumor cells. 111InOx, like 51Cr, primarily labeled cytoplasmic constituents; up to 80% of the label existed in a releasable form. 111InOx was quantitatively released from labeled P815 in response to specifically sensitized C57BL/6 lymphocytes. The high labelling efficiency of 111InOx offered a significant advantage over 51Cr in 18- to 48-hour assays for CMC by reducing the counting error and thus making the assay more precise. Because of its higher labeling efficiency, 111InOx can be used in microcytotoxicity assays. 111InOx has the added advantage of a lower spontaneous release in culture than 51Cr. This feature of 111InOx also makes the calculation of specific isotope release more accurate than that achieved with 51Cr in long-term cytotoxic assays.

Animals↗

PubMed1977

Rapid fluorimetric assay of minocycline in plasma or serum: comparison with microbiological assay.

1. Plasma concentrations of minocycline were assayed by measuring the fluorescence emission of an aluminum-minocycline chelate in a spectrofluorimeter. 2. The assay gives reproducible results over the concentration range 0.05-5.0 mug ml-1, and compared favourably with results achieved using a conventional large plate agar diffusion microbiological assay in a double-blind comparison of the two methods. 3. Fluorimetric assay of minocycline in plasma was found to correlate closely with antibiotic activity (r = 0.95). The method is easy and economical to perform, results being obtained in 1 h.

Bacillus cereus↗

PubMed1979

Enzyme-linked fluorescence assay: Ultrasensitive solid-phase assay for detection of human rotavirus.

Enzyme-linked immunosorbent assay (ELISA) has proven to be a useful assay system for the direct detection of infectious agents. However, when the usual color-producing substrates are employed, relatively large amounts of substrate must be hydrolyzed by the bound enzyme before detection can be achieved. We attempted to improve the sensitivity of ELISA by utilizing a substrate that yields a fluorescent product on enzyme action. The enzyme-linked fluorescence assay (ELFA) based on this principle was approximately 100 times more sensitive than the corresponding ELISA or radioimmunoassay for the detection of human rotavirus in a standard stool suspension. In addition, the ELFA for human rotavirus was capable of detecting antigen in six specimens that were negative by ELISA. Five of these specimens were obtained late in the course of confirmed rotavirus infections. ELFA provides a simple, reliable, ultrasensitive method for the rapid detection of viral antigen.

Antigens, Viral↗

PubMed1978

Measurement of rotavirus antibody by an enzyme-linked immunosorbent assay blocking assay.

A new method for the measurement of rotavirus antibody is described, utilizing the system of enzyme-linked immunosorbent assay (ELISA). In this method, serum is incubated with a fixed amount of rotavirus antigen, and the amount of antibody is determined by measuring the amount of unneutralized antigen. Such an assay system proved to be as efficient as the other available rotaviral antibody systems. The ELISA blocking assay also has the advantages of not requiring purified or gnotobiotic antigen and of being able to measure rotaviral antibody in all animal species.

Adult↗

PubMed1978

An isotope-release assay and terminal-labeling assay for measuring cell-mediated allograft and tumor immunity to small numbers of adherent target cells.

A 51Cr-release assay and terminal 51Cr-labeling assay for measuring cell-mediated immunity to adherent target cells is described. Both techniques utilize small 10 microliter-per well microtiter plates, require low numbers of target cells (50-500 per well), and consequently, relatively small numbers of effector cells per well (3x10(3) -1x10(5)). Both assays are objective, quantitative, and simple to perform. The suitability of these techniques for monitoring immunologically specific, cellmediated, cytotoxic response to syngeneic and allogeneic tumor cells and normal skin fibroblasts is demonstrated. Lymph node cells, spleen cells and peritoneal exudate cells serve as effectors.

Animals↗

PubMed1977

Assay of proteolytic activity by gelatin liquefaction (trypsin assay by gelatin liquefaction).

A new assay for proteolytic enzymes and their inhibitors based on the liquefaction of gelatin gels has been developed. The assay is more sensitive than colorimetric tests, can be carried out upon colored or turbid samples and does not require the use of a spectrophotometer. The procedure consists of incubating the test sample with a fluid gelatin solution, cooling the solution so that it sets to a firm gel and then incubating at a warmer temperature until the gel iquefies. The time taken for liquefaction is several days for a sample of pure buffer, about one minute for a sample containing 0.5 microgram of trypsin per ml and longer for samples containing less trypsin, following an empirical calibration. An appreciable decrease in tryptic activity can be detected in the presence of only 0.1 KIU of Trasylol (bovine pancreatic trypsin inhibitor) by this method.

Aprotinin↗

PubMed1976

An assay for total hexosamine and a differential assay for glucosamine and galactosamine.

Two new procedures are presented for quantitative determination of glucosamine and galactosamine. One, which is proposed for total hexosamine, yields chromogens of equal intensity with equal concentration of glucosamine and galactosamine. There is addition of the correspondent chromogens when they are present in mixtures. The procedure is presented as a manual as well as an automated assay. The other procedure is a differential assay which allows the detection of galactosamine without interference by glucosamine. By the two procedures, the hexosamines present in acid mucopolysaccharides and/or glycoproteins can be determined.

Acetylgalactosamine↗

PubMed1977

[Fast immunochemical assay of proteins with Gemsaec centrifugal analyser. Application to serum transferrin and IgA assay].

An automated method for measurement of specific proteins on a centrifugal analyser is reported. Based on the immunoprecipitin turbidimetric reaction enhanced by polyethylene-glycol, the technique, simple and fast, gives precise and accurate results with following cautions: the antigen and antibody concentrations must be carefully chosen according to defined specific antiserum, and two dilutions of each serum sample must be assayed to control quite satisfactory determination and exhibit any antigen excess error. Large series of serum transferrin and IgA assays were studied and compared with radial immunodiffusion and continuous-flow immunonephelometric method: good correlations prove the value of the reported method.

Autoanalysis↗

PubMed1978

[Precipitating immunoenzyme revealed tests on cellulose acetate. Use of ELIEDA (enzyme-linked-immuno-electro-diffusion assay) and ELIDEPA (enzyme-linked-immuno-double-electrophoresis-assay) in parasitology].

Precipitating tests carried out on cellulose acetate membrane were revealed by treating the immune complexes with enzyme linked anti-immunoglobulin antibodies. We insist on the importance of ELIEDA (enzyme-linked-immuno-electro-diffusion-assay) and ELIDEPA (enzyme-linked-immuno-double-electro-phoresis-assay) for the study of antibody classes involved in the immune response.

Antibodies, Anti-Idiotypic↗

PubMed1978

Enzyme-linked immunosorbent assay for detection of hepatitis A antigen in stool and antibody to hepatitis A antigen in sera: comparison with solid-phase radioimmunoassay, immune electron microscopy, and immune adherence hemagglutination assay.

Previously described techniques for detection of hepatitis A antigen (HA Ag) and antibody (anti-HA) have required purified HA Ag and expensive equipment. Herein is described an enzyme-linked immunosorbent assay (ELISA) for specific detection of HA Ag in human stool filtrates and of anti-HA in sera by using selected HA Ag-containing human stool filtrates as the antigen source. Because human stools often react nonspecifically in serological tests for HA Ag, blocking with preexposure and hyperimmune anti-HA sera from a chimpanzee inoculated with hepatitis A virus was used to confirm specific detection of HA Ag. The sensitivity of ELISA was found to be comparable to that of solid-phase radioimmunoassay (SPRIA) and immune electron microscopy (IEM). Of 37 acute-phase stools collected from nine patients, 16 were positive for HA Ag by ELISA. In 13 of these, HA Ag particles were found by IEM, and an additional 3 stools negative by ELISA contained HA Ag particles by IEM. Eight control stools were negative by both ELISA and IEM. Anti-HA was measured in sera by demonstrating its ability to block binding of the enzyme conjugate to HA Ag in a stool without detectable nonspecificity. This test (blocking ELISA) was as sensitive and specific as blocking SPIRA, IEM, and immune adherence hemagglutination and, like SPRIA and IEM, detected early-developing antibody. The ELISA is simple to perform and requires only a minimum of equipment. It is useful for screening stools for HA Ag and for monitoring HA Ag during purification, as well as for detecting early and late anti-HA in sera.

Acute Disease↗

PubMed1978

Simple assay for staphylococcal enterotoxins A, B, and C: modification of enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay (ELISA) introduced for the detection of staphylococcal enterotoxins by Saunders et al., Simon and Terplan, and ourselves has proved to be a simple, reliable, and sensitive test. A new modification is described that uses polystyrene balls (diameter, 6 mm) coated individually with antibody against one of the toxins A, B, or C. In a single tube, 20 ml of the food extract was incubated with the three balls differently stained, which were then each tested for the uptake of enterotoxin by a competitive ELISA. A concentration of 0.1 ng or less of enterotoxin per ml can be measured, making tedious concentration procedures of the extracts superfluous. Culture supernatants and extracts from foods artificially or naturally contaminated with toxin were successfully examined. Cross-reactions did not occur, and nonspecific interfering substances did not create serious problems.

Enterotoxins↗

PubMed1977

Detection of mitogen-activated T and non-T lymphocytes by virus plaque assay. Virus plaque assay on the cells fractionated by unit gravity sedimentation.

Virus plaque assay (VPA) was utilized for the quantitative evaluation of activated lymphocytes. We examined what types of cells, especially which of activated T and non-T lymphocytes, were detected as infective centres after infection with vesicular stomatitis virus. Marked increases in DNA synthesis and in virus-plaque forming cells (V-PFC) were observed not only during the activation of T lymphocytes with Con A, but also, though to a lesser extent, during the activation with lipopolysaccharide (LPS) of non-T lymphocyte preparations of nude spleen from which theta-positive lymphocytes and macrophages were completely depleted. The latter observation was further confirmed by the VPA on the populations enriched in LPS-activated non-T lymphocytes fractionated by the unit gravity sedimentation method. Fast sedimenting cells were found to be more active in DNA synthesis and contained more infective centres after infection than those sedimenting slowly and original unfractionated cells. Both the capacity for DNA synthesis and virus-replication were considered to be general properties accompanying lymphocyte activation.

Animals↗

PubMed1979

Immunoglobulins G, A and M in normal and pathologic human sera determined with the spot immunoprecipitate assay (SIA) in multi-assay plates.

A simple rapid spot immunoprecipitate assay (SIA) is presented. The antigen--antibody reaction takes place in a field of alternating current; small amounts of specific antisera are used, and the influence of diffusion or electromobility of the antigen is avoided. Determinations of IgG, IgA and IgM in sera from healthy individuals correlated highly significantly with radial immunodiffusion (RID) results. The reaction conditions in SIA, particularly the availability of a sufficiency of specific antibodies, may permit more accurate estimates of monoclonal forms of Ig than in RID, where the combination depends on diffusion of one reactant. The precision of SIA quantitation of IgG and IgA was within +/- 10% and +/- 13% for IgM. Working ranges of the standard curves were from about 25--150 ng for G and A and 75--300 ng for IgM; least squares regression coefficients for four sets of standards on three SIA plates were 0.995 for IgG, 0.969 for IgA, and 0.986 for IgM. IgA at 7.5 ng in a 2.5 mg/l dilution was at the lower limit of demonstrability. Photometric registrations and estimates made by visual inspection compared highly significantly (P less than 0.001), differing by +/- 3%.

Antigen-Antibody Reactions↗

PubMed1979

Effects of platinum antitumor agents on in vitro assays of human antitumor immunity. I. Effects of cis-[pt (NH3)2Cl2] on the mixed lymphocyte tumor assay.

Human lymphoblast cell lines (LCL) were treated with the antitumor complex Cis-[pt(NH3)2Cl2] (CPDC). Direct effects of CPDC treatment on LCL growth, as well as on LCL recognition by human lymphocytes in vitro are reported. Treatment of LCL with 10 micro m CPDC strongly inhibits DNA and protein synthesis, but has little effect on LCL viability. At this concentration, CPDC also inhibits the proliferation of human peripheral blood lymphocytes (PBL) in response to mitogens or allogeneic cells. Therefore, studies of LCL recognition by human PBL were conducted using CPDC pretreated and extensively washed LCL. Recognition of CPDC pretreated LCL in the in vitro mixed lymphocyte tumor (MLT) cell assay for the activation phase of cellular immunity is reduced. The decrease in MLT response is not due to direct effects of the complex on the responding T lymphocytes, but may be due to lowered antigen expression of treated LCL or other mechanisms. If CPDC does enhance tumor rejection in vivo, it does not appear to be due to enhanced recognition by T cells.

Cell Division↗

PubMed1976

The "echis carinatus venom" prothrombin assay in coumarin treated patients. A comparison with one-stage and immunological assays.

Prothrombin (factor II) was assayed in a group of coumarin treated patients using the Echis carinatus venom as thromboplastin. The levels obtained were comparable to those observed using the classical one-stage method. A good correlation was in fact observed between the two methods. The levels observed by the Echis carinatus method were definitely lower than those obtained using two immunological methods indicating that Echis carinatus venom activated, in our system, only normal prothrombin. However, even the levels obtained immunologically were slightly decreased, regardless of the method used, as compared to pooled normal plasma. In congenital prothrombin deficiency (homozygotes and heterozygotes) the level obtained by the Echis carinatus method was comparable to that observed by the one-stage method. On the contrary, in a congenital dysprothrombinemia (prothrombin Padua) a normal level was observed whereas the one-stage and two-stage methods yielded constantly levels of about 50% of normal.

Animals↗