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Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The new technology of non-radioactive probes (cold probes) allows the routine use of this method outside of the specialized molecular biology laboratory. Hybridization makes use of a nucleic acid probe to detect a complementary nucleic acid target present in biological fluid or in a biopsy tissue. Hybridization leads to the formation of a double-stranded molecule called hybrid or duplex, which can be detected with a great sensitivity by using high-energy radioisotope. However, the use of radioisotope labeled probes is limited by the short half-life of the isotope, radiolysis of the probe and the radioactive hazards. In order to overcome autoradiography delay and the drawbacks of techniques employing radioisotopes, various non-radioactive labels have been proposed. Labeling and detection systems are currently designed in two ways: The label molecule can be attached directly to the DNA probe (direct labelling) or it can be attached to a molecule which binds either to a modified probe or specifically to the duplex (indirect labeling). Various substances has been used to label directly a nucleic acid probe. The assay detection limit depends largely on the detection limit of the label, therefore, probe assay based on label which provides signal amplification (e.g., enzymes) is likely to be more sensitive than an assay using a label which provides only a single signal per molecule (e.g., fluorochromes). In the indirect labeling, the probe cannot be detected alone; rather, it requires the addition of a detection system.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The possibility of using them in clinical biology has been considered for several years. The transfer from basic to applied research can now take place due to the new technology of non-radioactive probes (cold probes). Hybridization features the use of a probe nucleic acid molecule and of a target nucleic acid molecule. It leads to the formation of a double-stranded molecule, also called duplex, which can be detected with great sensitivity. For this to be done, the probe can be tagged with a variety of labels such as a radio-isotope or a non-isotopic marker which can be detected for its fluorescence, luminescence or enzyme activity or by immuno-reaction(s). Hybridization can be performed either in a liquid solution or on a solid support. In the first case, hybridization is generally followed by a separation step which makes use of solid phase to isolate the hybrid (duplex) formed. This separation can be achieved by biochemical means (adsorption chromatography, differential precipitation, electrophoresis, etc.) or by immunological means (affinity chromatography, immuno-capture). In the second case, the support can be used in different ways according to the assay format employed to perform hybridization. The target DNA can be immobilized by contacting the sample to be tested with the support: simple specimen deposit with a pipette (for low volumes), or by aspiration under vacuum or by (capillary or electrophoretic) transfer from an agarose gel. In the sandwich assay format, probe DNA is immobilized onto the support to specifically extract the target DNA from the medium under assay. Support to be employed for hybridization purpose can be selected from the group consisting of: nylon (N), nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes; N+NC mixed membranes; cellulose and its derivatives; polyoside supports (Sephacryl, Sepharose, etc.), latex particles, polystyrene microplates. The coupling procedures and capacity vary according to the type of support used.

The principle of this thin-layer immunoassay (vapour condensation technique or TVAP) is based on the ability of antibodies to absorb firmly to polystyrene surfaces and to retain their reactivity. A condensation pattern consisting of large confluent water drops is noticable when an antibody-antigen reaction takes place. We used this technique to detect and assay the Escherichia coli heat-labile enterotoxin (ETEC LT+) and compared the results of 53 strains (40 positives and 13 negatives) with single radial immune haemolysis, Gm1-ELISA and Vero cell culture tests. With the reagents used, this reaction was specific for a toxin dilution up to 1/14. As little as 0.025 micrograms/ml of cholera toxin could be detected. The TVAP-test is simple, rapid and cost-effective. It is thus quite suitable for use in diarrhoeal endemic areas.

A GM1 erythroassay (GERYDO) for heat-labile Escherichia coli enterotoxin (LT) and cholera toxin (CT) is described. This assay was developed for use in poorly equipped laboratories in developing countries. It uses GM1-coated polystyrene plates and is based on the competition between the toxin to be assayed and CT covalently bound to sheep red blood cells. GERYDO can detect 0.9 or 0.5 ng of CT per ml depending on the method of sensitization of erythrocytes. Good quantitative and qualitative correlation with the enzyme-linked immunosorbent assay and the Vero cell test was observed.

An ELISA method on microtitration plates to detect and assay Escherichia coli heat-labile enterotoxin (LT) is described. This technique is rapid and simple to perform in any laboratory. It allows detection of the presence of LT with the naked eye within 10 h in a 12-h E. coli culture supernatant. The reaction is based on immunological cross-reaction between LT and the Vibrio cholerae toxin (CT). In place of traditional microtitration plates coated with ganglioside Gm1, we propose a new polystyrene support coated with purified anti-CT antibodies. This coated support has been conditioned in a kit to be used in laboratories in bush dispensaries of endemic areas. It was tested with 40 enterotoxigenic (LT+) strains isolated from stools of diarrhoeal children and with 14 LT- strains. All supernatants LT+ and LT- were found positive and negative, respectively, with the ELISA method and with the new polystyrene support. Field tests (in Wallis, Futuna et Vanuatu) with the new kit and standard method were satisfactory.

The titration of rabbit anti-Escherichia coli heat-labile enterotoxin antibodies by an immunosorbent assay (ELISA) generally uses a GM1 coating step to lend specificity to the ELISA reaction at a time when the method is tempered by a lack of purified E. coli toxin. We have developed an adsorption method of the toxin to the polystyrene in order to simplify the technique. Three different immunosorbent preparations were tested to determine which of them yielded the most sensitive results.

A sensitive solid-phase enzyme-immunoassay for the detection of rubella virus specific IgG and IgM antibodies has been developed. Wells of polystyrene trays were sensitized with purified rubella antigen. A substrate employed in enzyme-histochemistry, which gives a stable staining, was used to allow direct determination of titre values. One hundred and thirty-five human sera were tested. Antibodies titres, detected using this glucose oxidase immunoassay, correlated well with those obtained using a virus haemagglutination inhibition test. With this procedure sera can be tested without any pretreatment and evaluated within one day. Moreover, TIE is more sensitive and specific antibodies are detected earlier.

Complex antigens were coupled to polyacrylamide agarose beads which had been made magnetic with iron oxide. Those beads were then used in an enzyme-immunoassay for quantitation of specific antibodies to grass-pollen and mites in rabbit antisera. The results obtained by a direct or an inhibition technique proved the specificity of this assay towards homologous antigens. Moreover, cross-reactions between the various species of grass-pollen could be shown. One advantage of this simple method is that it may avoid use of radioactive compounds.

Two simple two-step competitive enzyme-linked immunoassays for human E. coli heat-labile enterotoxin (LTh) employing microtitration plates coated with rabbit anti-LTh antibody (ELISA) or GM1 ganglioside (GM1-ELISA) are described. LTh of the test sample competed with the same toxin coupled with horse-radish peroxidase. ELISA and GM1-ELISA were able to detect, respectively, as low as 5 ng and 6.5 ng TLh/ml and up to 9 and 11 micrograms TLh/ml. Both techniques were applied to the study of 167 infant diarrhoeas; ETEC producing LTh were identified in 17 diarrhoeal stools. When the faeces were diluted in phosphate buffer, only 17.6% (3 stools) and 29% (5 stools) of LTh-positive faeces were identified in ELISA and GM1-ELISA. When the stools were diluted with phenylmethylsulphonyl fluoride (PMSF), a synthetic protease inhibitor, 82% (14 stools) and 88% (15 stools) of LTh-positive stool supernatants were detected. Aprotinin, another protease inhibitor, was without effect and foetal calf serum, horse serum and bovine serum albumin enabled detection of only a low percentage of LTh-positive stools.

In order to produce monoclonal antibody to histamine, mice are immunized with histamine conjugated to bovine serum albumin (BSA) by the 1,4-benzoquinone method. After an initial screening using ovalbumin (OVA) and histamine-OVA conjugate as antigens to identify monoclonal antibody secreting clones, the hybridomas are isolated by limiting dilution cloning and grown in ascites. The specificity of selected monoclonal antibody (D22) is studied using a direct enzyme immunoassay and an ELISA-inhibition test. D22 antibody reacts with histamine-protein conjugates prepared by the 1,4-benzoquinone coupling procedure. On the contrary, this antibody is unreactive with native proteins, 1,4-benzoquinone treated proteins or various amine-protein conjugates. Free unconjugated histamine significantly inhibits antibody binding to histamine-OVA and 50% inhibition (IC50) is recorded at 5 X 10(-3) M. On a histamine molar concentration basis a much more lower inhibitory potency of free histamine is recorded, as compared to histamine-benzoquinone derivative (IC50 = 2 X 10(-8) M) and to histamine-OVA (IC50 = 7 X 10(-10) M). It is our interpretation that for the D22 antibody, the main epitope encompasses the 2-histaminyl-1,4-benzoquinone moiety and that this points to the importance of polyvalent ligands for efficient bivalent binding of the IgG antibodies. Using the D22 antibody we set up a competitive enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of monoclonal antibody which is used to coat the wells of a microtitration plate.(ABSTRACT TRUNCATED AT 250 WORDS)

An India ink immune reaction was used for the direct detection, in diarrhoeal stools, of Escherichia coli possessing the CFA/I, CFA/II and E8775 fimbriae. With this method, a presumptive diagnosis of enterotoxigenic E. coli (ETEC) can rapidly be made. Staining required the bivalency of rabbit anti-fimbriae IgG; the F(ab')2 fragment was necessary. The reaction was impossible with purified Fab fragment. A comparative study of this technique and detection by culture of ETEC strains showed good correlation when the India ink immune reaction was performed at the beginning of infection.