A diagnostic biochip for the comprehensive analysis of MLL translocations in acute leukemia.

Reciprocal rearrangements of the MLL gene are among the most common chromosomal abnormalities in both Acute Lymphoblastic and Myeloid Leukemia. The MLL gene, located on the 11q23 chromosomal band, is involved in more than 40 recurrent translocations. In the present study, we describe the development and validation of a biochip-based assay designed to provide a comprehensive molecular analysis of MLL rearrangements when used in a standard clinical pathology laboratory. A retrospective blind study was run with cell lines (n=5), and MLL positive and negative patient samples (n=31), to evaluate assay performance. The limits of detection determined on cell line data were 10(-1), and the precision studies yielded 100% repeatability and 98% reproducibility. The study shows that the device can detect frequent (AF4, AF6, AF10, ELL or ENL) as well as rare partner genes (AF17, MSF). The identified fusion transcripts can then be used as molecular phenotypic markers of disease for the precise evaluation of minimal residual disease by RQ-PCR. This biochip-based molecular diagnostic tool allows, in a single experiment, rapid and accurate identification of MLL gene rearrangements among 32 different fusion gene (FG) partners, precise breakpoint positioning and comprehensive screening of all currently characterized MLL FGs.

Flow cytometric microsphere-based immunoassay: analysis of secreted cytokines in whole-blood samples from asthmatics.

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-gamma (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-gamma than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.

Assessment of the Th1/Th2 paradigm in whole blood in atopy and asthma. Increased IFN-gamma-producing CD8(+) T cells in asthma.

Atopy is characterized by an immune system that is biased to T helper cell, type 2 (Th2) activation. This condition predisposes to asthma, a disease in which a Th2 activation was found in blood and lungs. However, most blood studies have considered purified cells, which might give an incomplete view of immune reactions. In this study, we assessed in whole blood cultures the Th1/Th2 paradigm in atopy and asthma. Sixty-nine subjects (31 atopic asthmatics, six nonatopic asthmatics, 13 atopic nonasthmatics, and 19 control subjects) were included in this study. Interleukin-4 (IL-4), interferon gamma (IFN-gamma), and IL-12 were assayed in stimulated whole blood culture supernatants by using a flow cytometer microsphere-based assay. Intracellular IL-4 and IFN-gamma were detected in T cells and CD8(+) T cells by flow cytometry. Atopy was characterized by a higher production of IL-4, which was correlated to total IgE levels, and by an impairment of the T-cell capacity to produce IFN-gamma. This impairment was correlated to the number of positive skin tests. In asthma, the overproduction of IL-4 was still found if atopy was present. Unexpectedly, an overproduction of IFN-gamma was found, which was related to an increased capacity of CD8(+) T cells to produce IFN-gamma. The number of IFN-gamma-producing CD8(+) T cells was related to asthma severity, to bronchial hyperresponsiveness, and to blood eosinophilia. In addition, this number was correlated to IL-12 production. These results show that in addition to the well-known Th2 inflammation in asthma, there are IFN-gamma-producing CD8(+) T cells in the blood, possibly controlled by IL-12.

Simultaneous detection of multiplex-amplified human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA using a flow cytometer microsphere-based hybridization assay.

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from the gag region of the HIV-1 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from the 5' noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.

Relationships between natural T cells, atopy, IgE levels, and IL-4 production.

BACKGROUND: Th2 cells govern allergic disorders. Mechanisms leading to the Th2 commitment are dominated by the requirement of IL-4. A potential source of this triggering IL-4 could be the CD4 + subset of a small population of T cells, natural T (NT) cells. Indeed, this subset is involved in IgE responses in mice and produces promptly high amounts of IL-4 in both mice and man. METHODS: NT cells were identified in peripheral blood by flow cytometry with antibodies against Valpha24 and Vbeta11, recognizing the T-cell receptor specific for NT cells. Simultaneous staining with anti-CD3, anti-CD4, or anti-CD8 antibodies was performed. The frequency of NT cells in man was studied according to the presence of atopy defined by the positivity of skin tests, according to total IgE levels in serum, and according to IL-4 concentration of whole-blood culture supernatants determined by a flow cytometer microsphere-based assay. RESULTS: Seventy subjects were included, of whom 30 were atopic. The number of CD4+ NT cells was higher in atopics than in nonatopics (P=0.009). This number was correlated to the total IgE levels (r = 0.34, P = 0.03). In addition, the number of CD4 + NT cells, but also of CD8 + NT cells, was correlated to the levels of IL-4 (r=0.71, P=0.01, and r=0.6, P=0.03, respectively). CONCLUSIONS: These results show that the number of NT cells, particularly the CD4+ subset, is related to atopy, IL-4 production, and IgE levels. Therefore, this population of T cells is likely to play a role in the Th2 commitment initiating atopic diseases.

Antigenic mapping of human thyroglobulin--topographic relationship between antigenic regions and functional domains.

We characterized 26 mAb to human thyroglobulin to obtain a topographic map of the thyroglobulin antigenic surface. Among these mAb, three bind thyroglobulin peptides that are located in the primary sequence of thyroglobulin at either the N terminus or in the middle part of the molecule, three bind thyroglobulin via epitopes comprising the thyroid-hormone moiety, and three bind thyroglobulin through epitopes involved in the recognition of the molecule by its receptor. The 18 remaining mAb bind thyroglobulin through undetermined epitopes; most of these epitopes are resistant to trypsinization. We used two methods to map the antigenic regions of thyroglobulin: all 26 mAb were grouped, by means of cross-inhibition experiments, in 11 clusters corresponding to 11 antigenic regions of the thyroglobulin surface; by means of thyroglobulin peptides of decreasing size, obtained by time-controlled tryptic digestion, we analyzed the relative distance between pairs of epitopes in sandwich immunoassays. By combining these two methods, we organized most of the 11 antigenic regions on a topographic representation of the thyroglobulin surface. This new topographic map of thyroglobulin led us to some unexpected features of the thyroglobulin structure. First, antigenic region 8 located far from the N-terminal region is in close contact with two remote N-terminal antigenic regions (1 and 4), both involved in hormone formation. This antigenic region is likely to play a role in the correct positioning of hormonogenic tyrosines so as to optimize iodination-coupling reactions. Secondly, the domain involved in the binding of thyroglobulin to its receptor, probed by three mAb, is shared by two distinct mid-molecule antigenic regions, one being the main autoantigenic region of thyroglobulin.

Autoantibodies to thyroid hormones: the role of thyroglobulin.

Autoantibodies against thyroid hormones (THAA) are frequently detected in the sera of patients with thyroid disorders together with autoantibodies against thyroglobulin (TGAA). THAA are considered to be a subset of TGAA, but alternative possibilities have not been excluded. We hypothesize that if THAA arise through an immune response to iodothyronines carried by circulating thyroglobulin (hTg), THAA should be found together with autoantibodies against the peptide backbone of hTg (TPAA) close to the hormone-forming sites. We measured TPAA in 178 serum samples, obtained from healthy subjects and patients with thyroid disorders, using two hormone-forming peptides isolated from hTg. The occurrence of TPAA was much lower than that of TGAA. Autoantibodies to the hormone-rich peptide, P3, were significantly more common than autoantibodies to the hormone-poor peptide, P1 (111/178 = 62.3% for TGAA versus 21/178 = 11.8% for anti-P3 TPAA and 7/178 = 3.9% for anti-P1 TPAA). The presence of autoantibodies to thyroid hormones was investigated in 25 TPAA+ and 26 TPAA- sera. THAA were found more frequently in TPAA+ sera (10/25 = 40% for TPAA+ and 4/26 = 15.3% for TPAA-). Correlation analysis shows that the anti-P3, but not the anti-P1 binding activity, correlates positively with the THAA-binding activity (P < 0.001 for anti-T4 THAA; P < 0.01 for anti-T3 THAA). Specificity of anti-P3 TPAA indicates that a subset of the anti-P3 antibodies is directed against the thyroid hormone moiety and another subset is directed against the peptide backbone near the hormone-forming peptide, according to our hypothesis. These results indicate that the THAA response is an anti-hTg response directed, in a significant number of cases, against the hormone-forming site included in the P3 peptide. This response seems to be elicited by either native hormone-rich hTg or by hTg fragments.

Carbohydrate and protein determinants are involved in thyroglobulin recognition by FRTL 5 cells.

To avoid premature lysosomal degradation, thyrocytes have a system able to recycle internalized immature thyroglobulin molecules (Tg) to the follicular lumen via the Golgi apparatus. It has been shown that this quality control system depends on recognition of exposed N-acetylglucosamine (GlcNAc) determinants (Miquelis et al., J Cell Biol, 1993, 123, 1695) present on immature Tg (Bastiani et al., 1995, Endocrinology, 1995, 136, 4204). However, the same in vitro kinetics studies also showed that GlcNAc residues alone induce only weak recycling. The latter finding led us to investigate the possibility that protein determinants might also be involved in binding. For this purpose, we studied binding of Tg to FRTL 5 cells, a widely available TSH-dependent cell line and found that binding to plasma membranes occurred at acidic pH in the presence of calcium, i.e. under conditions previously reported for binding of GlcNAc-BSA to porcine thyroid cell membranes. As expected, binding was GlcNAc- and oligosaccharide-dependent because Bandeiraea Simplificiola II affinity column analysis indicated that GlcNAc-bearing Tg were preferentially bound and N-glycanase treatment of Tg inhibited interaction. Ovomucoid, GlcNAc-BSA, and porcine Tg oligosaccharides did not inhibit binding, indicating that carbohydrates were not the sole determinants for binding. The fact that pronase digestion of Tg totally abolished binding implied that peptide determinants were involved in the interaction. This involvement is supported by the observation that porcine, rat, bovine, and human Tg bound FRTL 5 cell membranes and that monoclonal antibodies raised against human Tg interfered with the binding of both human and porcine Tg. Based on these findings we conclude that, besides the involvement of GlcNAc-bearing oligosaccharides, Tg receptors form a stable bond with peptide determinants.

Rapid and specific enzyme immunoassay of serotonin.

A new, highly sensitive enzyme immunoassay (EIA) of serotonin (5HT) is described. The assay is based on the competition between N-succinyl-glycinamide-serotonin (N-SGA-5HT, obtained by acylation of the 5HT in the sample to be assayed) and an enzymic tracer, N-succinyl-5HT-acetylcholinesterase, for binding to rabbit polyclonal antibody coated onto the wells of microtiter plates. The antibody is directed against an immunogen obtained by coupling N-succinyl-5HT to glycyl-bovine serum albumin. The EIA permits the accurate measurement of as little serotonin as 0.5 nmol/L (1.8 pg per well) in blood, plasma, serum, cerebrospinal fluid, urine, platelets, and other tissues, with no significant cross-reactivity with other compounds. The results obtained correlate well with those obtained by HPLC after extraction. The assay has the advantage of permitting the measurement of 5HT in up to 500 samples in as little as 3 h.

Preparation and characterization of xanthine oxidase-antibody and -hapten conjugates for use in sensitive chemiluminescent immunoassays.

Methods have been developed to label haptens or antibodies with xanthine oxidase for use in chemiluminescent enzyme immunoassays. We have optimised coupling reactions involving the use of heterobifunctional cross-linkers, the introduction of sulfhydryl groups and the utilization of accessible cysteine residues on the native enzyme. The versatility of xanthine oxidase as a label in immunoanalysis was studied in five assay systems, including both competition procedures (TT4 and direct serum estradiol assays) and immunometric assays (TSH, IgE, hCG). In all of the assay systems, the performance of the conjugates was excellent, demonstrating that the chelate enhanced luminometric detection of xanthine oxidase should have a wide potential in many immunoassays.

Application of a long-term enhanced xanthine oxidase-induced luminescence in solid-phase immunoassays.

The use of xanthine oxidase (XO) as a label in immunoanalysis has not been previously reported. This can be explained by the difficulties encountered in XO assays (poor sensitivity and versatility) and the competitive inhibition of the enzyme by allopurinol, a widely used hypouricemic agent. We demonstrate here that both difficulties can be circumvented. (i) The XO-dependent luminescent signal related to the oxidation of luminol is dramatically enhanced in the presence of iron-EDTA complex and sodium perborate in alkaline buffer. The mechanism of this enhancement is consistent with an O2-driven Fenton reaction, leading to the production of highly reactive OH radical. (ii) Residual inhibition of solid-phase bound XO by serum allopurinol and its metabolites is spontaneously reversible and can be prevented by the presence of folic acid or azahypoxanthine in the incubation buffer. With these two problems solved, XO can be classified as a choice label in immunoanalysis with the following properties: (i) high detection sensitivity (3 amol label), (ii) long-term luminescent signal (several days), (iii) versatile preparation and stability of conjugates, and (iv) long-term stability of the luminescence reagent. As an example of application, some data concerning total IgE and direct 17 beta-estradiol assays are described. Several other luminescent immunoassays of large and small molecules have been developed using XO conjugates as tracer (free and total T4, ultrasensitive thyroid stimulating hormone, CA 19.9, prolactin, hCG, specific IgE, anti-toxoplasma, and anti-chlamydia IgG), thus proving that XO can be classified as a universal label.

T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay.

The use of xanthine oxidase in immunoanalysis has never been reported. We describe here a procedure in which the xanthine oxidase dependent luminescence of luminol is enhanced in the presence of Fe-EDTA complex, providing an highly sensitive assay (3 amol of enzyme) and a long-term signal. This specific amplification has been applied to T4 and ultrasensitive TSH solid phase immunoassays, with T4-XO and anti-TSH monoclonal antibody-XO conjugates as tracers. The performances of these assays are at least equivalent to those obtained with iodinated tracers, using the same solid phases and the same calibrators. The major advantages of these immunoassays are: (1) the long-term signal which can be repeatedly recorded over several days, (2) the high detection sensitivity, (3) the long-term stability of the luminescence reagent and (4) the stability of the conjugates.

Immunopurification of human factor VIII/vWF complex from plasma.

In this study we describe a process for immunopurification of FVIII/vWF complex directly from plasma. A mAb against vWF has been selected that is able to bind, under physiologic conditions, the FVIII/vWF complex and to release it in slightly alkaline conditions while preserving its activity. After investigating the influence of solid supports and of coupling methods on the recovery of active FVIII we produced an immunoadsorbent by immobilisation of the selected mAb onto a Sephacryl S-1000 support using a benzoquinone coupling method. With this immunoadsorbent we developed a purification process directly from plasma with an excellent recovery (50%) of both FVIII and vWF activities. The product obtained is very enriched (the FVIII:C specific activity is 20 IU/mg of protein) and is stable after lyophilization.