Testing of a Chemiluminescence Enzyme Immunoassay for selective detection of E. Coli O157 from ground beef samples.

The aim of this study was to evaluate a Chemiluminescence Enzyme Immunoassay (CLIA) developed for the detection of E. coli O157:H7, using different E. coli O157 serotypes. The sensitivity and specificity of the kit were determined from the tenfold dilutions of the 24-hour broth cultures of the test strains. According to the results obtained in this trial, the sensitivity of the kit is 10(3)-10(4) cells ml-1, and it is specific for E. coli O157. Twenty-five g ground raw beef samples were prepared and inoculated with E. coli O157:H7 at different CFU g-1. The samples were incubated in 225 ml of modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 4 h and the immunoassays were performed following the instructions of the manufacturer. According to the results obtained by the CLIA test 10(1)-10(2) E. coli O157 g-1 can be detected from the sample. So this kit seems to be suitable for screening the samples before selective cultivation of E. coli O157:H7.

Comparison of a tissue transglutaminase ELISA with the endomysium antibody test in the diagnosis of gluten-sensitive enteropathy.

BACKGROUND: Tissue transglutaminase (tTG) has recently been found to be the major if not the only autoantigen of gluten-sensitive enteropathy (GSE). OBJECTIVES: To further determine the significance of this finding for diagnostic (screening) and follow-up purposes, we performed tTG-based ELISAs, and compared the results to the endomysium antibody test (EMA). PATIENTS: We examined 120 serum samples from patients with celiac disease (CD) including 72 on a gluten-free diet (GFD) and eleven on a gluten challenge, 47 with dermatitis herpetiformis (DH) including 16 on a GFD and one on a gluten challenge, 96 with non-CD gastrointestinal diseases, and 117 with others; i.e. 380 serum samples altogether. Follow-up sera from 13 patients were included. METHODS: Results of an ELISA with guinea pig liver tTG were compared with the EMA test using monkey esophagus. Inhibition of endomysial staining was performed with sera positive on the EMA test but negative with the guinea pig tTG ELISA. RESULTS: The specificity and sensitivity of the tTG ELISA are high (98.6% and 92.5%). The serum IgA antibody titers against tTG decrease after introduction of a GFD. In one case, endomysial staining could not be inhibited. CONCLUSIONS: Our results show that the guinea pig tTG ELISA is suitable for use as a simple diagnostic screening and follow-up method for GSE. Further studies are necessary to identify possible additional minor antigens in GSE.

Chromogen-substrate assay--as a tool for monitoring a new thrombin inhibitor.

The N-methyl-D-phenylalanyl-L-prolyl-arginine-aldehyde sulfate tripeptide-aldehyde (GYKI-14766) is an anticoagulant with specific thrombin inhibitor action. The molecule proved to be effective in rabbits, rats and dogs upon i.v. administration. Chromogen-substrate assay was developed for monitoring of biologically active tripeptide-inhibitor GYKI-14766 in plasma. The assay based on the inhibition of the active center of the thrombin enzyme, so it is suitable also for the assay of all those active metabolites which inhibit thrombin by a mechanism similar to the active parent compound. The chromogen substrate assay was performed in a range of 0.625-10 micrograms/ml GYKI-14766 in dog plasma. The assay was employed in pharmacokinetic study in dogs after i.v. administration. The data obtained in the chromogen-substrate assay were analyzed according to a one-compartment model. The major parameters of the plasma level studies were: D/V = 8.6 microEqv/ml t1/2 = 30.8 min AUC = 380 min microEqv/ml.

Radioimmunoassay for a new phenothiazine derivative and its application.

Fluphenazine-4-chlorophenoxy-isobutyrate ester, a new phenothiazine derivative was synthesized in the Institute for Drug Research Budapest. Radioimmunoassay was developed for the therapeutic monitoring of the drug level after intramuscular depot injection. The fluphenazine hapten was coupled to BSA by mixed-anhydride method. Antisera were produced to this conjugation in New-Zealand white rabbits and were tested for the antibody-titer. The specificity was tested by the cross-reaction with phenothiazine-analogues and other psychotropics. Strong cross-reaction was found with compounds possessing piperazine in side chain (trifluoperazine, perphenazine), but other psychotropic drugs did not react. Tritium-labelled trifluoperazine (spec. activity: 3.5 TBq/mmol) was used as a tracer in the radioimmunoassay. The detection limit was 75 pg with a CV of < 5% in 50 microliters plasma sample (equivalent to 1.5 ng/ml concentration) and a standard curve in the 3 ng/ml-50 ng/ml GYKI-22441 concentration range showed a CV of < 10%. Preliminary pharmacokinetic study was performed in Beagle dogs after intramuscular depot injection with GYKI-22441 in sesame oil in a dose of 0.1 mg/kg. The GYKI-22441 concentration of the plasma samples were measured by the RIA method during a 28-day interval after the treatment and was evaluated by the MultiCalc Immunoassay Data Management program (Pharmacia).