Production of human antibodies by in vitro immunization using a fusion protein containing the transcriptional transactivator of HIV-1.

Antigen-specific activation of human B cells represents a key step for the production of monoclonal antibodies. Several approaches have been developed over the last thirty years in order to improve the process of lymphocyte activation in vitro. In the present study, we investigated whether the transcriptional transactivator (Tat) of human immunodeficiency virus, which possesses numerous biological activities, is able to trigger antibody secretion when incubated with human peripheral blood mononuclear cells. No such effect was observed when using Tat as a free protein. However, we found a significant IgM antibody production when Tat was previously fused to a double domain, called ZZ, derived from protein A of Staphylococcus aureus. The effect was also observed when the fusion protein, called ZZTat101, was incubated with purified B cells, indicating that the phenomenon does not require T-cell help. Antibody secretion was observed in the absence of cytokines that are usually used during in vitro immunization experiments, indicating that ZZTat101 provides the signals required for the initiation of the immune response. Antibody secretion was observed using a ZZTat mutant, containing only the Tat residues 22 to 57, called ZZTat22-57, indicating that this region is sufficient to initiate the immune response. In contrast, the effect was not found with a ZZTat22-57 mutant devoid of the seven Tat cysteines located between residues 22 and 37, demonstrating that these residues play a crucial role in the phenomenon. Our results pave the way to the development of a new in vitro immunization method based on antigens associated with ZZTat.

Facile and rapid access to linear and truncated microcystin analogues for the implementation of immunoassays.

A series of simplified microcystin-LR analogues based on Adda [(2S,3S,8S,9S,4E,6E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldecadienoic acid] or its corresponding aldol precursor linked to a polypeptide moiety have been synthesised and assessed for their binding affinity by the monoclonal antibody mAb MC159, an anti-microcystin-LR mAb recently selected by us for the detection of microcystins through various immunoassay formats. Some modifications have been brought to the enantiospecific synthesis of N-Boc-Adda developed by Pearson et al. (Org. Lett., 2000, 2, 2901) which enabled us to access in an economical and time-saving manner a small library of MC-LR linear analogues. Among which Adda was chosen to synthesise, as an illustrative example, a fluorescent probe derived from this beta-amino acid. This probe was subsequently solid-phase immobilised by means of oxime ligation in order to lead to biochips suitable for microcystin detection through the SPIT-FRI method.

A novel fluorescence-based array biosensor: principle and application to DNA hybridization assays.

A novel fluorescence-based array biosensor targeted for field applications, such as environmental monitoring, has been developed, and successfully applied to DNA hybridization assays. The purpose was to meet the demand for automated, portable but easy-to-maintain systems allowing continuous flow monitoring of surface reactions. The biosensor presented here can be distinguished from the existing systems by the optical method used, which provides an enhanced simplicity and robustness, and enables a simple maintenance by potentially unskilled personnel. The system is based on a conventional microscope slide which acts both as transducer and biological array sensor. The excited fluorescence is guided by total internal reflection into the slide to the detector which is directly interfaced to the slide. Each region of the sensor array is successively optically interrogated, and the detection of the corresponding fluorescent emission synchronized. A real-time three-analyte analysis is thus feasible without any mechanical scanning movement or optical imaging systems as generally used in the existing instruments. The ability of the biosensor to operate in continuous flow for several tens of hours has been demonstrated. The biosensor has been assessed in terms of stability, and slide-to-slide reproducibility, which is found to be less than 3.7%, thus far below the standard biological reproducibility. DNA hybridization assays were performed to estimate a limit of detection, which was found to be 16 mol/microm(2), and to determine the reaction kinetics associated to the DNA model used. The developed biosensor is thus shown to be able to predict reaction kinetics, and to monitor in real time surface reactions between targets and probes.

Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry.

The toxin ricin is a biological weapon that may be used for bioterrorist purposes. As a member of the group of ribosome-inactivating proteins (RIPs), ricin has an A-chain possessing N-glycosidase activity which irreversibly inhibits protein synthesis. In this paper, we demonstrate that provided appropriate sample preparation is used, this enzymatic activity can be exploited for functional ricin detection with sensitivity similar to the best ELISA and specificity allowing application to environmental samples. Ricin is first captured by a monoclonal antibody directed against the B chain and immobilized on magnetic beads. Detection is then realized by determination of the adenine released by the A chain from an RNA template using liquid chromatography coupled to tandem mass spectrometry. The immunoaffinity step combined with the enzymatic activity detection leads to a specific assay for the entire functional ricin with a lower limit of detection of 0.1 ng/mL (1.56 pM) after concentration of the toxin from a 500 microL sample size. The variability of the assay was 10%. Finally, the method was applied successfully to milk and tap or bottled water samples.

Prion-like protein Doppel expression is not modified in scrapie-infected cells and in the brains of patients with Creutzfeldt-Jakob disease.

Doppel protein has been discovered in prnp knock-out mouse lines, with overproduction of this protein in the brain causing ataxia and neurodegeneration. We investigated whether Doppel expression (i) affected or was affected by the course of prion propagation in neuroblastoma cells, or (ii) modulated Creutzfeldt-Jakob disease pathogenesis. No change in Doppel production was detected in N2a cells, before or after infection. Transient murine Doppel gene expression had no effect on N2a viability or PrP(Sc) production. A sensitive immunometric assay revealed low levels of Doppel in human brain, reflecting weak transcription of the corresponding gene. No difference in brain Doppel levels was observed between Creutzfeldt-Jakob disease patients and controls, adding further evidence that Doppel is unlikely to be involved in prion disease pathogenesis.

Use of free radical chemistry in an immunometric assay for 17 beta-estradiol.

BACKGROUND: We wished to develop an enzyme immunometric assay for 17 beta-estradiol (E2) in human serum using solid-phase immobilized epitope immunoassay (SPIE-IA) technology and free radical chemistry. METHODS: We used an anti-estradiol monoclonal antibody as capture antibody and Fenton-like reagents to cross-link it to E2. The same antibody, labeled with acetylcholinesterase, was used for detection. Serum was diluted 10-fold before assay. RESULTS: After correction by the dilution factor, the detection limit was 5 ng/L for human serum and intra- and interassay CVs were <7% and 15%, respectively, at concentrations of 169-2845 ng/L. No cross-reactivity was seen with other natural steroids. In comparison with a competitive commercial RIA performed on 88 undiluted human sera, the slope (SD) of the regression line was 1.05 (+/- 0.02) and the intercept was 47 (+/-27) ng/L (S(y/x) = 186 ng/L) at concentrations of 20-5000 ng/L (r(2) = 0.97). CONCLUSIONS: The use of Fenton-like chemistry in SPIE-IA technology allows a sensitive measurement of E2 in human serum and could be a new approach for the development of sensitive immunoassays.

A solid-phase immobilized epitope immunoassay (SPIE-IA) permitting very sensitive and specific measurement of angiotensin II in plasma.

We have developed a new enzyme immunometric assay for angiotensin II (AII) based on SPIE-IA technology (solid-phase immobilized epitope-immunoassay). A monoclonal antibody with optimal properties (mAb3 131) was selected from a series of 19 anti-AII mAbs. The mAb had to be purified from ascitic fluid in a specific manner in order to remove endogenous AII from the antibody-binding sites. We established a sensitive (minimum detectable concentration 0.5 pg/ml) and precise (CV below 15% in the 2-100 pg/ml range) SPIE-IA. Using different AII-related peptides, we observed that this new assay has a specificity profile that compares favourably with the corresponding competitive immunoassay. We have used the assay to measure AII in 42 plasma samples, and demonstrated a good correlation with values obtained using a commercial radioimmunoassay. Assay specificity was supported by HPLC fractionation experiments, confirming the absence of interference induced by endogenous AII-related products.

A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application.

Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.

Two different approaches for developing immunometric assays of haptens.

To improve immunoassays of small haptens, we developed two different approaches for their measurement in a non-competitive format. We first devised two-site immunometric assays for small peptides (8-11 amino acids) by selecting two sets of antibodies specifically directed against C- and N-terminal moieties of the peptides. In each case, assay sensitivity improved substantially over that of the corresponding competitive assays. More interestingly, all of these new immunometric assays were much more specific than the competitive assays. In a second approach, we developed a new procedure, solid-phase-immobilized epitope immunoassay (SPIE-IA), in which a single monoclonal antibody uses the same epitope for capture and tracer binding and the hapten is covalently cross-linked to solid-phase proteins. To date, SPIE-IA have been successfully applied to the determination of haptens bearing primary amino groups, including substance P, thyroxine, leukotriene C4, endothelin, and angiotensin II. In each case, assay sensitivity was significantly improved.

Enzyme immunometric assay for leukotriene C4.

An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.

Subpicogram determination of oxytocin by an enzyme immunoassay using acetylcholinesterase as label.

The pure tetrameric form of Acetylcholinesterase (EC-3.1.1.7) from the electric eel electrophorus electricus has been covalently coupled to oxytocin. This conjugate has been used as tracer in a heterologous competitive immunoassay. Microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody were used to separate bound and free moieties of the tracer. Acetylcholinesterase activity bound to the solid phase was measured by a colorimetric assay. The minimum detectable concentration was 0.075 pg/well (ie 1.5 pg/ml) and precision was less than 8% at concentration above 0.15 pg/well. An extraction step improved sensitivity up to 10 times with good recoveries. To assess the validity of this assay, basal levels of oxytocin were measured during the oestrous cycle of a cow.

Influence of a solar eclipse on twilight.

The morning twilight of the presunrise sky was measured at the Hoher-List Observatory during the total eclipse of 22 July 1990. The location of observation was far away from the central eclipse zone. The luminance showed a deep minimum in twilight during the main phase of the solar eclipse compared with normal conditions. A first order scattering model explains the observations reasonably well and shows that the sky radiation during the first phase of twilight at a location far away from the central umbra depends primarily on the height profile of the air pressure between ~ 100 and 200 km.

Ulysses Radio Occultation Observations of the lo Plasma Torus During the Jupiter Encounter.

Radio signals from Ulysses were used to probe the lo plasma torus (IPT) shortly after the spacecraft's closest approach to Jupiter. The frequencies of the two downlinks at S-band (2.3 gigahertz) and X-band (8.4 gigahertz) were recorded, differenced, and integrated in order to derive the columnar electron density of the IPT. The measurements agree qualitatively with contemporary models of the IPT based on Voyager data, but significant differences are apparent as well. The overall level of the IPT electron density is approximately the same as the prediction, implying that the amount of gas (or plasma) injected from lo is similar to that observed during the Voyager era. On the other hand, the IPT seems to be less extended out of the centrifugal equator, implying a smaller plasma temperature than predicted.

PCM telemetry for physiological data.

PCM technology offers significant advantages in the application of telemetry to medical and physiological studies. The requirements for less complicated handling, standardized system layout, improvement of weight, size and power supply by commercial battery modules, as well as different wireless data links are met better by a PCM encoder which was specially developed for physiological applications. The advantages of PCM are illustrated in this paper by relating the experimental requirements to technical specifications for the elements of a telemetry link. A newly designed 8-channel PCM-telemetry system satisfying these requirements is described.