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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↗

PubMed1975

A systematic approach to enzyme assay optimization illustrated by aminotransferase assays.

We have developed a systematic approach to optimization of reagent concentrations for assays of alanine aminotransferase and aspartate aminotransferase: (a) Michaelis constants describing the initial-velocity kinetics of the coupled enzyme reactions were evaluated by a nonlinear least-squares fit of the appropriate equation to measured enzyme activities. Activities of more than 50 normal and pathological sera were measured at 30 degrees C. (b) These kinetic equations are used to calculate the set of reagent amino- and keto-acid concentrations that all yield a selected fraction of the theoretical maximum enzyme velocity. An optimal pair is determined by defining an additional criterion, such as minimal reagent cost or minimal concentration to Km ratio. (c) The optimum amounts of reagent NADH and coupling enzyme, being a function of desired pre-incubation and measurement intervals, maximum aminotransferase activity to be measured, and endogenous keto-acid concentration, are determined by computer simulation. An approximate relationship and an exact method for computing assay lag time are presented, along with experimentally measured endogenous keto-acid concentrations in serum. All procedures may be applied to other enzyme assays if appropriately modified.

Alanine Transaminase↗

PubMed1975

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. I. Phagocytic and non-phagocytic effector cell activity against erythrocyte and tumour target cells in a 51Cr release cytotoxicity assay and [125I]IUdR growth inhibition assay.

Both phagocytic and non-phagocytic effector cells were able to kill rabbit antibody-coated chicken erythrocytes (CRBC) while only non-phagocytic effector cells were active against alloantibody-coated SL2 lymphoma. In addition to the variation in susceptibility of erythrocyte and tumour target cells to various effector cell populations, it was found that different tumour cells can vary markedly in their ability to be killed by non-immune spleen cells in the presence of antibody. It is postulated that both the type of antibody and certain characteristics of the cell membrane are important in determining whether target cells are susceptible to antibody-dependent cell-mediated cytotoxicity detected by the 51Cr release assay. It was also demonstrated that alloantibody-coated P-815-Y mastocytoma, which showed very little evidence of cytotoxicity in the 51Cr release assay, was markedly inhibited in its ability to incorporate [125I]IUdR after incubation with antiserum and non-immune spleen cells. This growth inhibition in the absence of cytotoxicity, or cytostasis, is discussed in relation to the potential mechanisms of target cell damage, and in the light of recent observations (Plata, Gomard, LeClerc and Levy, 1974; Newlands and Roitt, 1975) that cytotoxicity and growth inhibition assays detect different effector cell populations in tumour-bearing animals.

Animals↗

PubMed1975

A single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen. Proposals for an assay method for the haemagglutinin content of influenza vaccines.

Single-radial-diffusion techniques are proposed as possible alternatives to tests based on agglutination of erythrocytes for the assay of the haemagglutinin content of influenza vaccines. Two test procedures (microtest and macrotest) and the use of reference reagents to assay vaccines using these tests are described. The two tests are of similar reproducibility and accuracy but the macrotest is technically simpler to perform and results of assays may be obtained more rapidly.

Antigens, Viral↗

PubMed1976

Modified assay medium for the turbidimetric assay of chlortetracycline in feeds.

In previous experiments, the turbidimetric method for determining chlortetracycline-HCI (CTC-HCI) in feeds showed lower recovery than the AOAC plate method. Although the addition of vitamins to the turbidimetric medium improved results, values by the turbidimetric method remained about 10% lower than by the plate method. A modified (1.7X the weight recommended by the manufacturer) turbidimetric assay medium decreased assay sensitivity but did not significantly change the slope of S. aureus response to CTC-HCI. There was no evidence that vitamin fortification of the modified medium had any significant effect on the growth rate of test organism. Examination of about 100 samples of commercial feeds containing CTC-HCI showed excellent agreement in results between the turbidimetric and plate methods.

Animal Feed↗

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↗

PubMed1976

Fluorometric assay of vasopressin and oxytocin: a general approach to the assay of peptides in tissues.

A fluorometric method for the quantitative assay of vasopressin and oxytocin in individual rat pituitaries has been developed. Acid extracts of pituitaries are freed of amino acids and polyamines by passage over a copper-Sephadex column, and the peptides fraction is then labeled by reaction with fluorescamine. The resulting peptide fluorophors are separated by chromatography on a reverse-phase bonded column. Specificity of the procedure was ascertained by several criteria, including bioassay and amino-acid analysis of the eluted peptide fluorophors. The procedure serves as a model system for the assay of tissue peptides in the picomole range.

Amino Acids↗

PubMed1976

Quality control of serum alkaline phosphatase assays: project report and discussion of some factors affecting the assay.

The factors which affect the standardisation and quality control of serum alkaline phosphatase assays are discussed. A quality control project was designed to test the performance of seven Birmingham laboratories in the assay of sera with alkaline phosphatase activities outside the range normally tested by the National Quality Control and Wellcome schemes. The results showed that the precision of individual laboratories was satisfactory. Differences between the results of the laboratories were considerable and could be accounted for by differences in methodology. Auto-Analyzer methods employing phenyl phosphate as substrate would best be standardised by adopting the optimised reaction conditions of Buch and Buch (1939); but the borate buffer of these authors should be replaced by the carbonate-bicarbonate buffer of Moss et al. (1971). To avoid the confusion which may arise in future if alkaline phosphatases are reported in U/l irrespective of the substrate, it is suggested that some substrate--indicative nomenclature may be advisable.

Alkaline Phosphatase↗

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↗

PubMed1979

Assay of Fletcher factor (plasma prekallikrein) using an artificial clotting reagent and a modified chromogenic assay.

An artificial clotting reagent lacking in Fletcher factor (plasma prekallikrein, PPK) was made by mixing human plasma, activated by 5 mg/ml of celite, then kept 16 hours at 37 degrees to destroy most of the plasma kallikrein, plus rabbit plasma (which is devoid of XII and Fletcher activity). Chromogenic assay using a tripeptide substrate was also modified to exclude the interference of the endogenous contact factors. Celite eluate was used instead of kaolin or dextran sulphate for the activation. Using both these methods, it is possible to distinguish between Fletcher trait (PPK deficiency) and other contact factors such as factor XII and HMWK deficiencies, which do not activate with kaolin or dextran sulphate. These simple clotting and enzymatic assays give specific and well correlated results for PPK estimation.

Animals↗

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↗

PubMed1978

Radioaffinity assay, a new approach to binding assays applied to the measurement of serum albumin.

The affinity adsorbent Cibacron Blue F3GA-Sepharose 4-B has been used to develop a binding assay for human serum albumin. The method is simple, accurate and precise. It agrees well with the 'rocket' immunoelectrophoretic method and is proposed as an alternative technique for the re-estimation of albumin levels in the low range (less than 30 g/l) where the bromocresol green dye-binding method overestimates. Bilirubin, several drugs, gammaglobulin, haemoglobin and heparin do not interfere with the estimations.

Bromcresol Green↗

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↗