Microplate Hybridization Assay Research Articles

A Rapid and Accurate Detection Approach for Multidrug-Resistant Tuberculosis Based on PCR-ELISA Microplate Hybridization Assay.

Rapid and accurate diagnosis of multidrug-resistant tuberculosis (MDR-TB) is important for timely and appropriate therapy. In this study, a rapid and easy-to-perform molecular test that integrated polymerase chain reaction (PCR) amplification and a specific 96-well microplate hybridization assay, called PCR-ELISA (enzyme-linked immunosorbent assay), were developed for detection of mutations in rpoB, katG, and inhA genes responsible for rifampin (RIF) and isoniazid (INH) resistance and prediction of drug susceptibility in Mycobacterium tuberculosis clinical isolates. We evaluated the utility of this method by using 32 multidrug-resistent (MDR) isolates and 22 susceptible isolates; subsequently, we compared the results with data obtained by conventional drug susceptibility testing and DNA sequencing. The sensitivity and specificity of the PCR-ELISA test were 93.7% and 100% for detecting RIF resistance, and 87.5% and 100% for detecting INH resistance, respectively. These results were comparable to those yielded by commercially available molecular tests such as the GenoType MTBDRplus assay. Based on the aforementioned results, we conclude that the PCR-ELISA microplate hybridization assay is a rapid, inexpensive, convenient, and reliable test that will be useful for rapid diagnosis of MDR-TB, for improved clinical care.

Implication of antigen-driven mechanisms in the pathogenesis of interstitial pneumonia associated with polymyositis

Event Abstract Back to Event Implication of antigen-driven mechanisms in the pathogenesis of interstitial pneumonia associated with polymyositis Akira Takeda1, 2*, Yasutsugu Fukushima3, Takaji Matsutani4, Yoichiro Haji2, Ryo Rokutanda2, Yasuhiro Suyama2, Mitsumasa Kishimoto2, Kenichi Yamaguchi2 and Masato Okada2 1 International University of Health and Welfare Hospital, Division of Clinical Immunology & Rheumatology, Japan 2 St. Luke's International Hospital, Division of Allergy & Rheumatology, Japan 3 Dokkyo University School of Medicine, Department of Pulmonary Medicine and Clinical Immunology, Japan 4 Wakayama Medical University, Laboratory of Immune Regulation, Japan Objective: Connective tissue diseases (CTDs) frequently involve the lung. Polymyositis (PM) is a major CTD characterized by chronic inflammatory lesions of muscle and other organs, including critical pulmonary involvement. Interstitial lung diseases, mainly interstitial pneumonia (IP), have been recognized in 30% - 70% of PM patients and have a poor prognosis. While the presence of myositis-specific autoantibodies suggests an autoimmune etiology of PM, the pathogenesis of PM-associated IP remains unclear. The aim of this study was to elucidate the role of T cells in this pulmonary complication. Methods: We took advantage of a rare opportunity to carefully study the cases of earliest-stage IP associated with PM by utilizing lung biopsy tissue. We characterized the phenotype of lung-infiltrating lymphocytes from lung biopsy specimens obatained by video-assisted thoracoscopy, and analyzed T-cell receptor a-chain (TCR Va) and TCR b-chain (TCR Vb) variable region repertoires of T-cells infiltrating the lung tissues using a validated adaptor ligation polymerase chain reaction (PCR)-based microplate hybridization assay, comparing these to peripheral blood lymphocytes (PBL). As expected, no TCR signals were detected from non-IP lung tissue controls. Results: The cases demonstrated substantial CD3+ T cell lung infiltrates. The usage of repertoires of TCR Va/Vb in the lung differed from those in PBL, with certain TCR V gene families detected more frequently in lung tissue, suggesting a pivotal role for T cells in the pathogenesis of IP associated with PM. This is the first robust demonstration of selective TCR repertoire usage and its differential expression in lung tissue versus PBL. Conclusion: These findings strongly suggest a pathogenic contribution of organ-specific oligoclonal T cell accumulation through antigen-driven immune mechanisms, implying potential development of immunospecific treatments such as molecular-targeted therapies. Acknowledgements The authors thank G. Deshpande, MD, for help with editing. This study was supported by the Ministry of Education, Science and Technology in Japan. Keywords: Interstitial Lung Disease, Interstitial pneumonia, Polymyositis, Dermatomyositis, Lung biopsy, Cell infiltrates, phenotypes, T cell, T cell receptor (TCR), T cell receptor repertoir, PCR, hybridization assay, Humans Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Takeda A, Fukushima Y, Matsutani T, Haji Y, Rokutanda R, Suyama Y, Kishimoto M, Yamaguchi K and Okada M (2013). Implication of antigen-driven mechanisms in the pathogenesis of interstitial pneumonia associated with polymyositis. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00414 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Akira Takeda, International University of Health and Welfare Hospital, Division of Clinical Immunology & Rheumatology, Nasushiobara, Tochigi, 329-2763, Japan, atakeda123@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Akira Takeda Yasutsugu Fukushima Takaji Matsutani Yoichiro Haji Ryo Rokutanda Yasuhiro Suyama Mitsumasa Kishimoto Kenichi Yamaguchi Masato Okada Google Akira Takeda Yasutsugu Fukushima Takaji Matsutani Yoichiro Haji Ryo Rokutanda Yasuhiro Suyama Mitsumasa Kishimoto Kenichi Yamaguchi Masato Okada Google Scholar Akira Takeda Yasutsugu Fukushima Takaji Matsutani Yoichiro Haji Ryo Rokutanda Yasuhiro Suyama Mitsumasa Kishimoto Kenichi Yamaguchi Masato Okada PubMed Akira Takeda Yasutsugu Fukushima Takaji Matsutani Yoichiro Haji Ryo Rokutanda Yasuhiro Suyama Mitsumasa Kishimoto Kenichi Yamaguchi Masato Okada Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A new method for quantitative analysis of the T cell receptor V region repertoires in healthy common marmosets by microplate hybridization assay

The common marmoset, Callithrix jacchus, is one of the smallest primates and is increasingly used for an experimental nonhuman primate model in many research fields. Analysis of T cell receptor (TCR) repertoires is a powerful tool to investigate T cell immunity in terms of antigen specificity and variability of TCR expression. However, monoclonal antibodies specific for many TCR Vα or Vβ chains have not been created. We have recently identified a large number of TCRα chain variable (TRAV) and TCRβ chain variable (TRBV) sequences from a cDNA library of common marmosets. The purpose of this study is to develop a new method for analysis of TCR repertoires in the common marmoset using this sequence information. This method is based on a microplate hybridization technique using 32 TRAV-specific and 32 TRBV-specific oligoprobes following an adaptor-ligation PCR. This enables the easy quantitation of the respective TRAV and TRBV expression levels. No cross-hybridization among specific-oligoprobes and very low variances in repeated measures of the same samples was found, demonstrating high specificity and reproducibility. Furthermore, this method was validated by an antihuman Vβ23 antibody which specifically bound to marmoset Vβ23. Using this method, we analyzed TCR repertoires from various tissue samples (PBMCs, spleen, lymph node and thymus) and isolated T cell subpopulations (CD4+CD8+, CD4+CD8− and CD4−CD8+) from the thymus of 10 common marmosets. Neither tissue-specific nor T cell subpopulation-specific differences was found in TRAV and TRBV repertoires. These results suggest that, unlike mice, TCR repertoires in the common marmoset are not affected by endogenous superantigens and are conserved among individuals, among tissues, and among T cell subpopulations. Thus, TCR repertoire analysis with high specificity and reproducibility is a very useful technique, with the potential to replace flow cytometric analysis using a panel of TRV-specific antibodies, many of which remain unavailable.

Microplate electrochemical DNA detection for phosphinothricin acetyltransferase gene sequence with cadmium sulfide nanoparticles

An electrochemical DNA detection method for the phosphinothricin acetyltransferase (PAT) gene sequence from the transgenetic plants was established by using a microplate hybridization assay with cadmium sulfide (CdS) nanoparticles as oligonucleotides label. The experiment included the following procedures. Firstly target PAT ssDNA sequences were immobilized on the polystyrene microplate by physical adsorption. Then CdS nanoparticle labeled oligonucleotide probes were added into the microplate and the hybridization reaction with target ssDNA sequences took place in the microplate. After washing the microplate for three times, certain amounts of HNO 3 were added into the microplate to dissolve the CdS nanoparticles anchored on the hybrids and a solution containing Cd 2+ ion was obtained. At last differential pulse anodic stripping voltammetry (DPASV) was used for the sensitive detection of released Cd 2+ ion. Based on this principle a sensitive electrochemical method for the PAT gene sequences detection was established. The voltammetric currents of Cd 2+ were in linear range with the target ssDNA concentration from 5.0 × 10 − 13 to 1.0 × 10 − 10 mol/L and the detection limit was estimated to be 8.9 × 10 − 14 mol/L (3 σ). The proposed method showed a good promise for the sensitive detection of specific gene sequences with good selectivity for the discrimination of the mismatched sequences.

Microplate hybridization assay for detection of isoniazid resistance in Mycobacterium tuberculosis

Early and accurate detection of drug resistant Mycobacterium tuberculosis can improve both the treatment outcome and public health control of tuberculosis. A number of molecular-based techniques have been developed including ones using probe molecules that target drug resistance-related mutations. Although these techniques are highly specific and sensitive, mixed signals can be obtained when the drug resistant isolates are mixed with drug susceptible isolates. In order to overcome this problem, we developed a new drug susceptibility test (DST) for one of the most effective anti-tuberculosis drug, isoniazid. This technique employed a microplate hybridization assay that quantified signals from each probe molecule, and was evaluated using clinical isolates. The evaluation analysis clearly showed that the microplate hybridization assay was an accurate and rapid method that overcame the limitations of DST based on conventional molecular techniques.

Electrochemical DNA hybridization assay for the FMV 35S gene sequence using PbS nanoparticles as a label

An electrochemical DNA detection method was developed for the 35S promoter from Figwort mosaic virus (FMV 35S) gene sequence by using a microplate hybridization assay with PbS nanoparticles as labels. There are four main steps in the procedure including target FMV 35S ssDNA immobilization on the microplate, the hybridization of ssDNA with a PbS NPs-labeled oligonucleotide probe, the dissolution of the PbS anchored on the hybrids by the addition of HNO3 and the detection of released Pb (II) by differential pulse anodic stripping voltammetry. Based on this principle, the voltammetric response of Pb (II) was linearly related to the logarithm of the target FMV 35S concentration in the range from 8.0 × 10−13 to 1.6 × 10−10 mol L−1. The detection limit is 2.5 × 10−13 mol L−1 (3σ).

Prevalence of herpes B virus genome in the trigeminal ganglia of seropositive cynomolgus macaques

Herpes B virus infection is almost asymptomatic in macaques (Macaca spp.), which are the natural hosts of this pathogen, but is the cause of high mortality in humans. Reactivation of the latent virus in the trigeminal ganglia (TG) results in the shedding of infectious particles into the oral mucosal membrane. Saliva contaminated with the reactivated virus from the ganglia of the natural host is considered to be important for viral transmission to humans and other monkeys. In the present study, we investigated the prevalence of the herpes B virus genome in the left and right TG of seropositive asymptomatic cynomolgus macaques. The latent virus genome was detected using a polymerase chain reaction and microplate hybridization assay. We found that the virus DNA was present in one or both TG of 12 of the 30 macaques (40%) tested, with the virus being detected from both TG in five of the 12 macaques and from a single TG in the remaining seven.

New Commercially Available PCR and Microplate Hybridization Assay for Detection and Differentiation of Human Polyomaviruses JC and BK in Cerebrospinal Fluid, Serum, and Urine Samples

JC and BK human polyomaviruses (family Polyomaviridae) may cause severe neurological or urinary tract pathologies in immunocompromised hosts. In the present study, we evaluated a new commercially available PCR and microplate colorimetric hybridization assay for the standardized differential detection of JC virus (JCV) and BK virus (BKV) genomes in clinical samples. This JC/BK Consensus test was first evaluated by testing serial dilutions of JCV or BKV plasmid DNA standards and was then compared with an in-house reference PCR assay for the detection of JC and BK virus genomes in 70 cerebrospinal fluid (CSF) samples of patients with neurological disorders and in 75 serum or plasma samples and 125 urine samples of renal graft recipients. This new test allowed a limit of detection of 10 copies and 1 copy of JC and BK virus genomes, respectively, and was able to differentiate various levels of JCV, BKV, and mixed JCV and BKV DNA genomes in a single reaction tube. Our results showed 100% specificity and sensitivity for the JC/BK Consensus test with CSF samples. With serum or plasma samples, this test had a sensitivity and a specificity of 100% for both JCV and mixed JCV and BKV DNA detection and a sensitivity and a specificity of 100 and 97.8% for BKV DNA detection, respectively. With urine samples, the sensitivity and specificity were 100 and 96.6%, respectively, for JCV DNA detection; 100 and 89.4%, respectively, for BKV DNA detection; and 44.4 and 100%, respectively, for mixed JCV and BKV DNA detection. In conclusion, our data indicate that this new test, the JC/BK Consensus test, is valuable for the sensitive and specific differential detection of single JCV and BKV infections in CSF, serum or plasma, and urine samples. The use of this reliable PCR assay would improve the routine virological diagnosis as well as the clinical care of immunocompromised patients with polyomavirus-related pathologies.

Detection of foot and mouth disease virus by RT-PCR and microplate hydridization assay using inactivated viral antigens.

A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.

Novel assay of competitively differentiated polymerase chain reaction for screening point mutation of hepatitis B virus.

Point mutation, one of the commonest gene mutations, is the most important molecular pathogenesis of cancer and chronic infection. The commonest methods for detection of point mutation are based on polymerase chain reaction (PCR). These techniques, however, cannot be used in large scale screening since they are neither accurate nor simple. For this reason, this study established a novel method of competitively differentiated PCR (CD-PCR) for screening point mutation in clinical practice. Two competitively differentiated primers for mutant-type and wild-type templates respectively with an identically complemented region in 3' end except for last 2 base pairs and a different non-complemented region in 5' end were designed. Thus, competitive amplification might be carried out at a lower annealing temperature at first, and then differentiated amplification at a higher annealing temperature when primers could not combine with initial templates. The amplification was performed in one-tube. The products of CD-PCR were detected using microplate hybridization assay. CD-PCR was evaluated by detecting G1896A variant of hepatitis B virus (HBV) in form of recombinant plasmids and in sera from patients with hepatitis B, and compared with allele-specific PCR (AS-PCR) and competitive AS-PCR. CD-PCR was successfully established. It could clearly distinguish wild-type and mutant-type plasmid DNA of G1896A variant when the amount of plasmid DNA was between 10(2)-10(8)copies/reaction, while for AS-PCR and competitive AS-PCR, the DNA amount was between 10(2)-10(4)copies/reaction. CD-PCR could detect one copy of G1896A variant among 10-100 copies of wild-type plasmid DNA. The specificity of CD-PCR was higher than those of AS-PCR and competitive AS-PCR in the detection of HBV G1896A variant in sera from patients with hepatitis B. CD-PCR was independent of the amount of HBV DNA in serum. HBV G1896A variant was more often found in HBeAg (-) patients with a lower level of detectable viremia than that with a higher level of detectable viremia (P=0.0192). CD-PCR is more specific since it is less influenced by the amount of initial templates and the cross amplification between mutant- and wild-type amplified products. It is also simple and time-saving. Thus, CD-PCR might be useful in routine gene typing and point mutation screening. HBV G1896A or other more important mutations have to be routinely detected in patients with a detectable level of viremia after HBeAg/antibody conversion in clinical practice.

Does Streptococcal Pyrogenic Exotoxin-C Trigger the Onset of Kawasaki Disease? -Association between Superantigens and Autoantibody against Vascular Smooth Muscle Cells-

I) T cell receptor (TCR) usage We investigated TCR usage and antibody responses to streptococcal pyrogenic exotoxin-C (SPE-C) in order to determine whether an association exists between SPE-C and the onset of Kawasaki disease (KD). Patients and Method Fifty-four patients with KD were studied. Analysis of TCR usage was performed by adaptor-ligation PCR and microplate hybridization assay. Serum levels of antibody against SPE-C were assessed by ELISA using purified recombinant protein. Results Forty (74.1%) of the patients with KD showed significant polyclonal activation of Vβ2- and/or Vβ-6.5-bearing T cells. In 22 of 35 paired patients with KD, serum levels of anti-SPE-C antibodies (IgM) in the convalescent phase were lower than those in the acute phase. II) Detection of autoantibodies against vascular smooth muscle cells To investigate the pathogenesis of vasculitis in KD, we tested for the presence of autoantibodies against vascular smooth muscle cells in the patients. Patients and Methods Sera from 48 patients with KD were examined for reactivity with both coronary arterial wall tissues and cultured smooth muscle cells (CSMC) derived from human coronary artery, using immunofluorescence and Western immunoblotting. Results Sera from 16 of 48 patients with KD gave positive immunoreactions with the smooth muscle cells of coronary artery with anti-human IgA antibodies. Western immunoblotting revealed positive reactions of sera from 15 of 34 and 10 of 31 patients with KD against a 70-kDa protein from CSMC with IgA and IgM antibodies, respectively. Positive immunoreactivity of sera from KD patients was detected more frequently in patients with coronary arterial lesions (CAL) than in those without CAL (p <0.05). Conclusions These results suggest that an association may exist between SPE-C and the onset of KD. In addition, autoantibody against vascular smooth muscle cells may cause systemic vasculitis in KD.

The detection of bovine leukemia virus proviral DNA by PCR–ELISA

A sensitive non-radioactive microplate hybridization assay for the detection of proviral DNA of bovine leukemia virus (BLV)-specific polymerase chain reaction (PCR) product is described. The PCR products are labeled by adding digoxigenin-dUTP to the nested PCR reaction and are captured by a microtitre plate coated with oligonucleotide probe, which is complementary to the inner region of the amplification product. Captured products are reacted with an anti-DIG Fab fragment conjugated to peroxidase, and detected using a colorimetric reaction. The PCR-enzyme linked immunosorbent assay (ELISA), detecting as low as 10 −4 ng of proviral DNA in a background of 1 μg of BLV-negative DNA, was up to 100-fold more sensitive than ethidium bromide staining, and showed equal sensitivity to Southern blot hybridization. Using this method it was possible to monitor the presence of proviral DNA in four sheep infected experimentally with BLV, over a 10 months postinfection period, as well as in 29 cattle infected naturally. The test is rapid and highly sensitive and is a useful additional tool for the detection of BLV-infected animals.

Restricted usage of T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation.

We analysed T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo-BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6-7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1-4 (26%, 11 of 41 recipients), VA3-1 (32%) and VB24-1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity-determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0.05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T-cell regeneration via a thymic-dependent pathway in children.

Detection of Legionella pneumophila DNA in urine and serum samples from patients with pneumonia

albicans strains adhere better to epithelial cells of the oral cavity or the esophagus, but it is accepted that esophageal candidiasis represents a form of invasive candidiasis which requires higher dosages of antifungal drugs compared to OPC [11]. In this patient, replacement of a fluconazole-resistant C. albicans strain by a genetically different fluconazole-sensitive strain was observed; this finally caused further recurrences of OPC, which responded well to intermittent therapy with itraconazole solution. However, it is unclear whether the disappearance of the resistant genotype is due to the temporary increase of the CD4 cell count or because of therapy with itraconazole despite highlevel resistance to azoles in vitro. It may be concluded that serial DNA typing of C. albicans strains, together with antifungal susceptibility testing, may help us to monitor the susceptibility of clinical isolates to azole therapy.

Respiratory coronavirus infections in children younger than two years of age.

Respiratory coronavirus infections in children younger than two years of age.

Detection of beer spoilage bacteria Megasphaera and Pectinatus by polymerase chain reaction and colorimetric microplate hybridization

Anaerobic bacteria of the genera Megasphaera and Pectinatus cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. A polymerase chain reaction (PCR) method and a colorimetric microplate hybridization assay were developed for the rapid and specific detection of Megasphaera cerevisiae and Pectinatus spp. A biotinylated primer pair was designed for the amplification of a 403 base pair (bp) fragment of the M. cerevisiae 16S rRNA gene and a primer pair from literature was used for the amplification of an 816 bp fragment of Pectinatus 16S rRNA gene. Amplified PCR products were analyzed by the colorimetric microplate hybridization method in which a biotinylated PCR product was captured by streptavidin and hybridized with a digoxigenin-labelled oligonucleotide probe. In the final step an enzyme-linked antibody and a colorimetric reaction were utilized. A simple and rapid sample treatment was set up for the PCR detection of contaminants in beer. Detection of M. cerevisiae (≥5·10 3 colony forming units [cfu]/100 ml) and Pectinatus frisingensis (≥5·10 5 cfu/100 ml) in beer was successful, but the sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

Detection and identification of human papilloma viral DNA, types 16, 18, and 33, by a combination of polymerase chain reaction and a colorimetric solid phase capture hybridisation assay

A colorimetric microplate hybridization assay was developed previously to simplify detection procedures of DNA fragments resulting from polymerase chain reactions (PCR). This format has now been adapted for the simultaneous detection and identification of three human papillomavirus (HPV), types 16, 18 and 33, associated frequently with cervical cancer. This post-PCR detection system uses three type-specific capture oligonucleotides linked covalently to a single microplate well and three type-specific multibiotinylated oligonucleotidic probes for detection. It therefore offers a double specificity; the first is conferred by pairs of primers, specific of each type of virus tested, and the second, by the sets of capture and detection probes which are complementary to internal regions of the amplified DNA fragments. The detection format outperformed agarose gel electrophoresis of amplified DNA products in sensitivity and specificity. The rapidity and simplicity of this hybridisation system would justify its use in routine diagnostic examination of cervical specimens (smears and biopsies).

PCR ELISA for the quantitative detection of Epstein–Barr virus genome

A highly sensitive and nonradioactive microplate hybridization assay for the detection of Epstein–Barr virus (EBV)-specific polymerase chain reaction (PCR) product was developed. The PCR product is labelled by adding digoxigenin-dUTP directly to the reaction mixture and, after denaturation, is captured by a microtitre plate coated with an extravidin-linked biotinylated probe. Captured products are reacted with a peroxidase-conjugated anti-digoxigenin antibody and detected using tetramethylbenzidine. The assay detected less than ten EBV genomes in a background EBV-negative DNA of 0.75 μg and, when tested on clinical samples, it was able to define the viral load in throat washings of patients with acute infectious mononucleosis, immunosuppressed patients with HIV infection, and rare normal individuals who shed the virus in the oropharynx.

Analysis of TCRAV and TCRBV Repertoires in Healthy Individuals by Microplate Hybridization Assay

ABSTRACT: We have developed an adaptor ligation PCR-based microplate hybridization assay (MHA) for analysis of T cell receptor α chain variable region (TCRAV) and T cell receptor β chain variable region (TCRBV) repertoires. Forty three TCRAV and thirty eight TCRBV-specific probes were immobilized onto microplate wells in water-soluble carbodiimide. After hybridization of 5′-biotinylated PCR products, quantitative ELISA was carried out and followed by automated colorimetric reading. The conditions for immobilization and hybridization were optimized using representative TCRBV-specific probes. The sensitivity of MHA allows us to detect as low as 40 pg of biotinylated PCR products. The frequencies of individual V segments obtained by MHA were consistent with those obtained by FACS analysis and reverse dot blot assays. Analysis of the entire TCRAV and TCRBV repertoires could be done using a single 96-well plate, and completed in less than 6 h. Simplicity and reproducibility of this method make it suitable for routine laboratory use. The expression of TCRAV and TCRBV segments was next studied in peripheral blood mononuclear cells (PBMC) of 14 healthy donors using the newly developed MHA method. TCRAV8S1, TCRAV23S1, TCRBV2S1, TCRBV3S1, TCRBV4S1, and TCRBV6S5 were highly expressed in PBMC. Further, the TCRAV repertoires among individuals were less variable compared to the TCRBV repertoires. Interestingly, considerable variations in the expression levels of BV3S1, BV4S1, and BV17S1 were observed among individuals. One polymorphic site was found at the coding region of BV4S1, and there were two alleles. These results suggest that variable expression among individuals may be associated with unknown allelic polymorphism in coding and/or regulatory regions of these TCRBV segments, or with disparity in HLA genes.

Antibody-enhanced microplate hybridization assays.

A method for signal enhancement in a colorimetric, nonradioactive and quantitative microplate hybridization assay is described. The signal enhancement provides for the direct detection of viral DNA in serum samples without the use of DNA amplification.