Comparison of time-resolved fluoroimmunoassay and immunoenzymometric assay for clenbuterol.

A time-resolved fluoroimmunoassay (TR-FIA) for the direct determination of clenbuterol residues in horse urine using a highly specific monoclonal antibody has been compared with an immunoenzymometric assay (IEMA). The sensitivity of both methods was 10 pg; the calibration curve was linear between 10 and 10(5) pg for the TR-FIA and between 10 and 10(4) pg for the IEMA.

Determination of aflatoxin B1 in agricultural commodities by time-resolved fluoroimmunoassay and immunoenzymometric assay.

A time-resolved fluoroimmunoassay (TR-FIA) and an immunoenzymometric assay (IEMA) were applied for the measurement of aflatoxin B1 in soya seeds, dried figs and raisins. The extraction procedure was simple and no clean-up was found to be necessary. Limits of detection were 0.5 microgram kg-1 for TR-FIA (range of linearity of calibration graph 2.5-5 x 10(4) pg; inter-assay relative standard deviation Sr < 5%) and 0.2 microgram kg-1 for IEMA (linear range 1.0-5 x 10(3) pg; Sr < 11%).

Relationship between the intrahepatic expression of 'e' and 'c' epitopes of the nucleocapsid protein of hepatitis B virus and viraemia.

The relationship between hepatitis B viraemia and intrahepatic HBV nucleocapsid proteins (HBcAg and HBeAg) was studied in 18 patients with chronic hepatitis B. Monoclonal antibodies (MoABs) were obtained in BALB/c mice primed with recombinant HBV nucleocapsid proteins. Four MoABs reacting with recombinant proteins gave positive results in competitive assays. Two reacted as anti-HBc and two as anti-HBe. One of them showed a strong affinity for the cytoplasmic, membrane-bound antigen (P23e) of infected hepatocytes while the latter showed a higher specificity for serum HBeAg than for the intrahepatic antigen. Anti-HBc MoABs had a staining capacity for liver cell nuclei comparable with that of polyclonal antibodies. Overall the anti-HBc MoABs stained the liver cell nuclei in 86% of cases, while anti-HBe MoABs stained in 58% of cases. The hepatocyte cytoplasm was stained by anti-HBc MoABs and anti-HBe MoABs in 64% and 72% of cases respectively. Not one of 12 control liver biopsies was stained. Viraemia (HBV-DNA) was measured by dot blot hybridization and was correlated with the number of hepatocytes containing the nucleocapsid antigen. The highest levels of HBV-DNA (greater than 10(8) genomes/ml) were detected in patients with prevalent nuclear staining while the lowest ones were observed in those with prevalent cytoplasmic expression of this antigen. The application of anti-HBV-nucleocapsid MoABs in diagnostics requires careful scrutiny since some are specific for the circulating antigen while others show a higher affinity for the intrahepatic antigen.

Synergistic neutralization of rubella virus by monoclonal antibodies to viral haemagglutinin.

Using murine monoclonal antibodies (MAbs) to rubella virus haemagglutinin, five epitopes were identified in competitive ELISA binding assays: A, B, D and E by haemagglutination-inhibiting (HI) MAbs with no neutralizing (Nt) activity, and C by a MAb with neither activity. However, when HI and Nt activities were determined in the presence of anti-mouse immunoglobulins, epitopes A, B and D were defined by both HI and Nt MAbs, whereas epitopes C and E were identified by HI MAbs without Nt activity. A synergistic Nt activity, in the absence of anti-mouse immunoglobulins, was displayed by mixtures of antibodies of different epitope groups. Analysis of mixtures of MAb pairs each belonging to a different epitope class, showed that synergistic Nt activity was elicited primarily by the group A epitope, secondarily by groups B and D and only minimally by groups C and E.

Development and evaluation of a capture enzyme-linked immunosorbent assay for determination of rubella immunoglobulin M using monoclonal antibodies.

A capture enzyme-linked immunosorbent assay (ELISA) for detection of virus-specific immunoglobulin M (IgM) antibody was developed which used a panel of labeled monoclonal antibodies to rubella virus hemagglutinin. The rapidity of the test system was increased by using, after 1-h incubation of the test serum, a second 1-h incubation of the serum with a mixture of viral antigen and labeled monoclonal antibody. The new assay was tested for specificity on 371 human sera from people without any recent contact with rubella virus; of these, 66 were sera selected from people with rheumatoid factor or IgM antibody to human cytomegalovirus, Epstein-Barr virus, or other viruses. In parallel, the new assay was performed on 191 sera from patients having recent contact with rubella virus. Results were compared with those obtained by an indirect ELISA method on IgM serum fractions, using purified rubella virus as a solid phase. Of the 371 sera tested for specificity, 5 (1.3%) gave false-positive results with indirect ELISA (1 rheumatoid factor, 2 heterophil antibody, and 2 human cytomegalovirus sera positive for IgM), and none were false-positive with the capture assay. Two sera from a patient with primary cytomegalovirus infection, which were positive for rubella IgM antibody with both methods and were initially interpreted as false-positive, were finally considered to be true-positive, since they were reactive only in the presence of IgM antibody and viral antigen. Of the 191 sera from 92 patients (84 patients with acute rubella, four newborns from mothers with rubella during pregnancy, and four vaccinees), 136 (71.2%) were found to be positive for IgM by direct ELISA, and 128 (67.0%) were positive by capture ELISA; 12 sera drawn during the first 2 days of disease, or at least 40 days after onset (or after vaccination), were detected only by indirect ELISA, and 4 sera were detected only by capture ELISA. Thus, specificity and sensitivity, respectively, were 100 and 91.4% for capture ELISA and 98.6 and 97.1% for indirect ELISA. However, when the number of patients was considered, 86 were detected as IgM positive by indirect ELISA, and 87 were detected positive by capture ELISA. The overall agreement between the two assays was 96.2%. Capture ELISA using monoclonal antibody appears preferable over indirect ELISA on IgM serum fractions because of its higher specificity and shorter time for test performance; furthermore, there is no need for serum fractionation or virus purification for the capture ELISA.

Rapid detection of antibodies to infectious bovine rhinotracheitis by 'macro' and 'micro' ELISA.

Two kinds of enzyme-linked immunosorbent assay were evaluated in their ability to detect specific antibodies against Bovine Rhinotracheitis Virus (IBR-IPV). The tests were called MACROELISA and MICROELISA, according to the kind of the solid support used for antigen insolubilization, polystyrene beads and microtitration plates respectively. Partially purified virus was used to coat both beads and plates; a single dilution of examples was tested and protein A linked to peroxidase was employed as enzyme tracer. Quantitative instrumental results from MACROELISA and qualitative visual results from MICROELISA were compared with serum neutralization titers. The results clearly show that ELISA tests are suitable for IBR serologic detection, being sensitive, specific and accurate over serum neutralization method.

ELISA for toxoplasma antibody detection: a comparison with other serodiagnostic tests.

An ELISA method was developed for the measurement of toxoplasma IgG antibodies in human serum using antigen-coated polystyrene beads as a solid phase and anti human IgG-horse radish peroxidase conjugate as an enzymatic tracer. In order to assess ELISA sensitivity and specificity, a between methods comparison was made using 'conventional' serological tests as reference (dye-test, crossover-linked immunoassay, passive haemagglutination, indirect immunofluorescence). From an analysis of the group classifications obtained some considerations emerged: the ELISA specificity looks comparable with that of the 'reference' tests, as no sample classified as negative by all these tests was ELISA-positive, and vice versa; ELISA appears to correlate better with haemagglutination and immunofluorescence, on the basis of the respective class frequencies; in particular, the number of positives, which is much lower for the dye-test and crossover-linked immunoassay, suggests that a higher sensitivity is reached in the former cases.

[The immune response of the fetus in the diagnosis of intrauterine infections: IgM and IgA concentrations in the umbilical cord blood and titers of rubella and cytomegalic virus antibodies]. / La risposta immunitaria del feto nella diagnosi delle infezioni intrauterine: concentrazione delle IgM e delle IgA nel sangue funicolare e titolo degli anticorpi anti rosolia ed anti virus citomegalico.

Two serological methods, hemagglutination and complement fixation, have been used respectively to determine HAI titers to rubella virus and C fixing antibodies to cytomegalovirus in cord blood of newborn infants delivered in Turin from August 1973 to April 1974. Sera have been divided according to antibody titer and aspecific IgM and IgA concentrations. By statistical analysis we observed a highly significant association between both IgM and IgA concentration and antibody titers to rubella virus.

[Antibodies against the cytomegalic virus and Toxoplasma gondii in a sample of the Monrovia population].

Cytomegalovirus and Toxoplasma gondii antibodies were titered in 133 serum specimens from students, physicians and technical staff of Monrovia hospital. The findings obtained show that both infections are widely spread: Cytomegalovirus antibody was detected in 81%, and Toxoplasma antibody in 70% of tested sera. No significative differences were observed among sera from different tribes and birth places.