Direct Antigen Detection Research Articles

High Throughput Sequencing and Proteomics to Identify Immunogenic Proteins of a New Pathogen: The Dirty Genome Approach

BackgroundWith the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive.Methods/Principal FindingsWe thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA.Conclusions/SignificanceThis work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

Enhanced Viral Etiological Diagnosis of Respiratory System Infection Outbreaks by Use of a Multitarget Nucleic Acid Amplification Assay

A study was undertaken to assess the utility of the xTAG respiratory viral panel (RVP) for enhanced laboratory investigation of respiratory outbreaks. Specimens (n = 1,108) from 244 suspected respiratory virus outbreaks in 2006 and 2007 in Alberta, Canada, were included in the study. Testing by direct fluorescent antigen detection (DFA) and various in-house nucleic acid amplification tests (NATs) for common respiratory viruses provided an etiological diagnosis in 177 outbreaks (72.5%), with 524 samples testing positive (47.3%) for a respiratory virus. Two hundred samples from 51 unresolved outbreaks were further tested by RVP retrospectively. Fifty-eight samples from 30 unresolved outbreaks had a respiratory virus detected by RVP (47 picornavirus-positive, 9 coronavirus-positive, and 2 influenza virus A-positive samples). Overall, detection of a viral etiological agent was achieved in 90.8% of outbreaks using a combination of DFA, NATs, and RVP. Use of RVP enhances the laboratory investigation of respiratory virus outbreaks and facilitates appropriate patient and outbreak management.

Virologically Confirmed Population‐Based Burden of Hospitalization Caused by Influenza A and B among Children in Hong Kong

We sought to determine the virologically confirmed hospitalization rates associated with influenza virus infection among Hong Kong children. Patients <18 years of age who lived on Hong Kong Island (a separate island within Hong Kong) and were admitted to either of the only 2 public hospitals on the island for a febrile acute respiratory infection on 1 fixed day of the week in each hospital from October 2003 through September 2006 were prospectively recruited. These 2 hospitals together accounted for 72.5% of all general pediatric admissions in Hong Kong Island with a known population denominator. Nasopharyngeal aspirates were obtained from all recruited patients and were tested for influenza A and influenza B viruses by direct antigen detection and culture. All cases of influenza A during 2003-2004 were caused by H3N2 virus, whereas 85.7% of cases during 2004-2005 were due to H3N2 virus, and 93.5% during 2005-2006 were due to H1N1 virus. During 2004-2005, infants <1 year of age had the highest rate of hospitalization for influenza A (103.8 cases per 10,000 population), whereas children 1 year of age had the highest rate of hospitalization during the other 2 seasons (95.5 and 54.6 cases per 10,000 population during 2003-2004 and 2005-2006, respectively). A protection rate of 25%, presumably attributable to maternal antibodies, was seen in infants <1 year of age who were hospitalized during 2003-2004 with infection due to an H3N2 virus that had been in circulation. The hospitalization rates for influenza B were highest among children 2-4 years of age. This population-based study of hospitalizations due to virologically confirmed influenza demonstrated a very high burden of disease among young children in Hong Kong. The morbidity varied with virus type, subtype, and antigenic variants.

More and More Coronaviruses: Human Coronavirus HKU1

After human coronaviruses OC43, 229E and NL63, human coronavirus HKU1 (HCoV-HKU1) is the fourth human coronavirus discovered. HCoV-HKU1 is a group 2a coronavirus that is still not cultivable. The G + C contents of HCoV-HKU1 genomes are 32%, the lowest among all known coronaviruses with complete genome sequences available. Among all coronaviruses, HCoV-HKU1 shows the most extreme codon usage bias, attributed most importantly to severe cytosine deamination. All HCoV-HKU1 genomes contain unique tandem copies of a 30-base acidic tandem repeat of unknown function at the N-terminus of nsp3 inside the acidic domain upstream of papain-like protease 1. Three genotypes, A, B and C, of HCoV-HKU1 and homologous recombination among their genomes, are observed. The incidence of HCoV-HKU1 infections is the highest in winter. Similar to other human coronaviruses, HCoV-HKU1 infections have been reported globally, with a median (range) incidence of 0.9 (0 – 4.4) %. HCoV-HKU1 is associated with both upper and lower respiratory tract infections that are mostly self-limiting. The most common method for diagnosing HCoV-HKU1 infection is RT-PCR or real-time RT-PCR using RNA extracted from respiratory tract samples such as nasopharyngeal aspirates (NPA). Both the pol and nucleocapsid genes have been used as the targets for amplification. Monoclonal antibodies have been generated for direct antigen detection in NPA. For antibody detection, Escherichia coli BL21 and baculovirus-expressed recombinant nucleocapsid of HCoV-HKU1 have been used for IgG and IgM detection in sera of patients and normal individuals, using Western blot and enzyme-linked immunoassay.

Pneumocystis jiroveci Pneumonia (PCP) Diagnosis by Direct Fluorescent Antigen Detection

Pneumocystis jiroveci Pneumonia (PCP) Diagnosis by Direct Fluorescent Antigen Detection

Diagnóstico de las faringitis estreptocócicas

La faringoamigdalitis es una infección muy frecuente en nuestro medio. Su etiología es múltiple, especialmente viral. El diagnóstico etiológico únicamente tiene interés en las faringoamigadalitis causadas por Streptococcus pyogenes o estreptococo betahemolítico del grupo A, puesto que se deben tratar con antibióticos para evitar las complicaciones, especialmente la fiebre reumática.El diagnóstico se ha basado clásicamente en el aislamiento de la bacteria a partir del exudado faríngeo. No hay un método estandarizado para la realización de estos cultivos en cuanto al medio de aislamiento, la atmósfera de incubación o las pruebas mínimas de identificación que se deben realizar. Actualmente, en el mercado se dispone de múltiples reactivos que, por diferentes técnicas inmunológicas, permiten la detección del antígeno directamente en la muestra clínica. La mayoría de éstas presenta buenos valores de sensibilidad y de especificidad, aportando la ventaja de un resultado rápido, especialmente si se realiza en la misma consulta médica. Hay diferentes protocolos o estrategias para la atención de los pacientes con faringoamigadalitis. En cualquier caso, cada centro debe valorar el rendimiento de la técnica de detección de antígeno que se decida implementar, puesto que los resultados varían en función de las indicaciones y el cuidado en la obtención de la muestra.

Biología molecular en el diagnóstico de la infección respiratoria aguda de origen bacteriano

The bacteriological methods traditionally used in the diagnosis of acute respiratory infections (ARI) have limited sensitivity (culture, direct antigen detection, etc.) or require long periods to obtain results (appearance of antibodies). In the last few years, nucleic acid amplification techniques (NAAT) have been developed that allow pathogen-specific genetic targets to be detected in clinical samples. These techniques have been proven to be more sensitive than culture or direct detection and, unlike serological tests, are effective in the acute phase of the infection. However, NAAT also have certain limitations, such as the occasional presence of amplification inhibitors in clinical samples, the persistence of Mycoplasma pneumoniae or Chlamydophila pneumoniae in the mucosa of some individuals, and the lack of discrimination between pathogen infection and colonization in bacteria forming part of normal respiratory tract flora (Streptococcus pneumoniae). Recently developed real-time NAAT have raised expectations that some of these obstacles will be resolved, since these techniques allow bacterial load to be quantified. In the etiological diagnosis of ARI due to S. pneumoniae, the use of NAAT is still in an experimental phase. In M. pneumoniae and C. pneumoniae, combining NAAT with serological tests could potentially improve diagnosis. NAAT show good sensitivity and specificity in the detection of Legionella; however, the practical utility of these techniques should be weighed against that of antigenuria. NAAT provide advantages over other techniques in Bordetella pertussis. At present, these techniques are not useful in the diagnosis of Coxiella burnetii acute infections.

Direct and indirect immunolabelling of HeLa cells with quantum dots

With excellent optical properties, quantum dots (QDs) have been made as attractive molecular probes for labelling cells in biological research. In this study high-quality CdSe QDs prepared in a paraffin-oleic acid system were used as fluorescent labels in direct and indirect detection of carcinoembryonic antigen (CEA), a cancer marker expressed on the surface of HeLa cells. The primary antibody (Ab) (rabbit anti-CEA8) and secondary Ab (goat anti-rabbit IgG) were covalently linked to carboxyl-functioned CdSe QDs, and both the QDs-antibody and QDs-IgG probes were successfully used to label HeLa cells. The present study demonstrates the practicability of CdSe QDs as an attractive type of fluorescent labels for biological applications such as protein probes and cell imaging.

Use of Throat Swab or Saliva Specimens for Detection of Respiratory Viruses in Children

Background. Nasopharyngeal (NP) specimens are commonly used for the detection of respiratory viruses, but throat and saliva specimens are easier to obtain. The objective of this study was to compare the viral yield of direct fluorescent antigen detection of NP specimens and nucleic acid amplification tests (NAT) of direct fluorescent antigen-negative NP specimens with the viral yield of NAT of throat swab and saliva specimens. Methods. NP, throat swab, and saliva specimens were obtained from children and adolescents aged ⩽17 years. Direct fluorescent antigen testing of the NP specimen for respiratory syncytial virus, influenza A and B viruses, and parainfluenza virus was performed. If no virus was detected, NAT was performed for these 4 viruses, adenovirus, and human metapneumovirus. If a virus was detected by either method, NAT for the same virus was performed for the corresponding throat swab and saliva specimens. Results. A virus was detected in 105 of the 137 NP specimens. The same virus was detectable by NAT in 87 (83%) of 105 throat swab specimens and 77 (74%) of 104 saliva specimens. The likelihood of viral detection among throat swab and saliva swab specimens was higher when the NP specimen tested positive by direct fluorescent antigen testing, compared with NAT alone, and when the specimens were obtained within 3 days after symptom onset, compared with later in the illness. Conclusions. Throat swab and saliva specimens are inferior to NP specimens for the detection of respiratory viruses but might be acceptable for screening in a setting where it is impractical to obtain an NP specimen.

Virologie : l’apport de la biologie moléculaire dans le diagnostic microbiologique en pneumopédiatrie

Les outils traditionnels du diagnostic virologique comprennent la recherche antigénique directe par immunofluorescence (IF) ou test immunoenzymatique (EIA), et l’isolement en culture. Les techniques d’IF ou EIA ont une sensibilité en général légèrement inférieure à celle de la culture, mais elles permettent de détecter également une fraction d’échantillons faussement négatifs en culture. Les résultats de plusieurs équipes, utilisant des techniques de reverse transcription-polymerase chain reaction (RT-PCR), montrent que les méthodes moléculaires détectent plus d’échantillons positifs que les méthodes traditionnelles. Le travail est en cours pour élargir le nombre de virus identifiés par des RT-PCR multiplex et découvrir de nouveaux virus, tels le métapneumovirus humain, en cause dans les atteintes respiratoires de l’enfant. En conclusion, afin de concilier rapidité, contrôle des coûts et un fort taux de détection des virus respiratoires, l’utilisation combinée de l’IF et de la RT-PCR multiplex semble ce jour la meilleure façon de progresser dans le diagnostic des maladies respiratoires de l’enfant.

Culture-based methods for detection and identification of Streptococcus agalactiae in pregnant women—what are we missing?

We aimed to compare BD group B Streptococcus differential agar (GBSDA) with inoculation into LIM broth and subculture onto blood agar plates (LIM-BA) as a method for GBS screening in pregnant women. First, we compared the detection threshold and the ease of use of GBSDA with LIM-BA by inoculating known numbers of GBS mixed with approximately 10(5) cfu of other bacteria. Second, we tested the production of carotenoid pigment in 155 GBS blood culture isolates. Finally, we compared GBSDA, LIM broth with direct GBS antigen detection (LIM-AG) and LIM-BA as methods for GBS screening in pregnant women. GBS colonies were easily detected on GBSDA at a threshold of 20 cfu. In contrast, GBS was not detected in a mixed culture from LIM broth with initial inocula of up to 100 cfu. Orange pigment was produced in 146/155 GBS blood culture isolates. Pigment was not produced in eight non-hemolytic and two hemolytic strains. GBS was detected by GBSDA in 58 out of 297 parturient women (19.5%) but in only 50 (16.8%, P > 0.05) and 46 (15.4%, P = 0.079) women by LIM-BA and LIM-AG, respectively. GBSDA is a satisfactory medium for the rapid detection of most GBS isolates, but other methods may be needed if detection of all non-pigmented strains is required.

Rapid Detection of Oculopathogenic Adenovirus in Conjunctivitis

Ocular adenovirus (Ad) infections occur throughout the world in both sporadic and epidemic forms. Accurate laboratory diagnosis of Ad in conjunctival samples is always valuable. The present study was carried out to explore the presence of Ad as a causative agent in clinically suspected viral conjunctivitis and to compare the performance of conventional virus isolation on cell cultures, direct detection of Ad antigens in conjunctival cells by a direct fluorescence assay, Ad DNA detection by polymerase chain reaction (PCR), and specific IgM measurement by ELISA. Samples included scrapes from conjunctiva. Scrapes were subjected to study by direct immunofluorescence stain, culture on the Hep-2 cell line, and PCR for Ad detection. Blood samples were also taken and subjected to study for specific anti-Ad IgM determination. The culture for Ad was positive in 77.8%, direct antigen detection by fluorescent stain was positive in 72.2%, PCR was positive in 83.3%, and serology was positive in 88.9% of patients. Both determination of antibody IgM and PCR correctly identified a larger group of patients compared to cell culture. The most sensitive and specific method for diagnosis of Ad compared to culture was PCR (100%), followed by IgM detection (92.9%) then direct antigen detection by fluorescent stain (85.8%). From this study, we conclude that Ad is a common pathogen in sporadic cases of conjunctivitis. Screening of adenoviral conjunctivitis is possible by using specific IgM due to its high sensitivity. A confirmatory test can be done by PCR for diagnosis of Ad, as it is a rapid, specific, and accurate method.

Highly sensitive phage-based biosensor for the detection of β-galactosidase

Development of real-time sensor based on the target-specific probe that make possible sensitive, rapid and selective detection and monitoring of the particular antigen molecules could be of substantial importance to the many applications. Because of its high specificity to the target molecules, excellent temperature stability, and easy production, bacterial phage might serve as a powerful biorecognition probe in biosensor applications. Here, we report extremely sensitive and specific label-free direct detection of model antigen, β-galactosidase (β-gal), based on surface plasmon resonance (SPR) spectroscopy. The β-gal specific landscape phage 1G40 has been immobilized on the gold surface of SPR SPREETA™ sensor chip through physical adsorption [V. Nanduri, A.M. Samoylova, V.Petrenko, V. Vodyanoy and A.L.Simonian, Comparison of optical and acoustic wave phage biosensors, 206th Meeting of The Electrochemical Society, Honolulu, Hawaii, October 3–8, (2004)]. Another non-specific to the β-gal phage, a wild-type phage F8-5, was used in the reference channel. The concentration-dependent binding of β-gal in both channels were assessed by monitoring the sensor optical response as a function of time under different experimental conditions, and the concentration of β-gal was computed in differential mode. Concentrations of β-gal between 10 −12 M and 10 −7 M could be readily detected, with linear part of calibration curve between 10 −9 M and 10 −6 M. When β-gal was pre-incubated with different concentrations of free 1G40 phage prior to exposure to the biosensor, concentration-dependent inhibition was observed, indicating on biosensor high specificity toward β-gal. Apart from a flow through mode used to deliver the samples to the surface for the SPR sensor, batch mode sensing was also employed to study the binding of β-gal to immobilized phage on the SPR sensor surface. Experiments using a flow through mode provided more consistent results in the full dose range and showed higher sensitivity as opposed to the batch mode studies. The mean K d and binding valences for the flow through mode studies was 1.3 ± 0.001 nM and 1.5 ± 0.03, in comparison to 26 ± 0.003 nM and 2.4 ± 0.01 for the batch mode studies. The average thickness of phage 1G40 adlayer deposited through flow through and batch mode was 3 ± 0.002 and 0.66 ± 0.001 nm, respectively.

Direct electrical detection of antigen–antibody binding on diamond and silicon substrates using electrical impedance spectroscopy

The integration of biological molecules with semiconducting materials such as silicon and diamond has great potential for the development of new types of bioelectronic devices, such as biosensors and bioactuators. We have investigated the electrical properties of the antibody-antigen modified diamond and silicon surfaces using electrical impedance spectroscopy (EIS). Frequency dependent measurements at the open-circuit potential show: (a) significant changes in impedance at frequency >10(4) Hz when the surface immobilized IgG was exposed to anti-IgG, and (b) only little or no change when exposed to anti-IgM. Mott-Schottky measurements at high frequency (200 kHz) show that the impedance is dominated by the space charge layer of the semiconducting substrates. Silicon surfaces modified in a similar manner to the diamond surface are compared; n-type and p-type samples show complementary behavior, as expected for a field effect. We also show it is possible to directly observe antigen-antibody interaction at a fixed frequency in real time, and with no additional labeling.

Incremental detection of HIV infections by the HIV antigen/antibody combination assays: An Australian experience

Detection of acute cases of human immunodeficiency virus (HIV) infection by the direct detection of HIV antigen or HIV nucleic acid assays is well known with an estimated 5-9 days reduction in the pre-seroconversion 'window period' by the detection of HIV specific antibodies. The aim of this study was to observe the impact following routine introduction of a screening assay which simultaneously detects HIV (type 1 & 2) antigen and antibody on the yield of acute HIV infection in multiple sites servicing different patient populations with a varying range of risk factors associated with HIV acquisition. During the first year (2003-2004), a total of 27 cases of acute HIV-1 infection were identified by the HIV-1/2 Ab/Ag combo test which were confirmed to be detectable for HIV antigen only that may have gone undetected should an HIV-1/2 antibody only assay have been used. Specimens referred from higher HIV case load centers were more likely to have provided relevant clinical information consistent with acute retroviral syndrome and relevant history of risk however there were numerous cases where no clinical information was provided. This study shows that routine introduction of HIV-1/2 antigen/antibody screening assays increases the identification of acute cases of HIV infection in low prevalence setting and may represent an important tool for enhanced surveillance of incident HIV infection and opportunities for prevention.

Rapid Hybridization Probe Assay and PCR for Detection of Chlamydia trachomatis in Urinary Tract Infections: A Prospective Study

Chlamydia trachomatis is a widespread bacterium that causes trachoma and genital tract infections in humans. The fact that the growth of this pathogen does not normally occur outside living cells poses a challenge in its diagnosis. The present study aimed to compare the efficacies of different molecular and cultural methods in the detection of C. trachomatis in urine samples collected from patients with urinary tract infections. Examined detection methods involved the Gen-Probe C. trachomatis (GP-CT) assay, direct antigen detection by enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) method. The efficacies of these methods were compared to that of the cell culture technique depending on sensitivity, specificity, and accuracy. C. trachomatis was detected in 25 out of 50 (50%) of examined urine samples using the cell culture method. Compared with this standard technique, the GP-CT assay was the most sensitive procedure, being able to detect the pathogen in all positive samples, followed by PCR and ELISA, which showed 60% and 40% sensitivities, respectively. PCR and ELISA displayed the highest level of specificity (100%) compared to the cell culture method with the GP-CT assay showing 40% specificity. The rate of accuracy was comparable between the GP-CT, PCR, and ELISA methods ranging from 70-80% of the accuracy of the cell culture method. The above results suggest that C. trachomatis is a frequent pathogen associated with upper and lower urinary tract infections. Both the GP-CT assay and PCR method can be recommended as reliable detection methods for C. trachomatis, and the GP-CT can be used as a screening tool.

A nanotube array immunosensor for direct electrochemical detection of antigen–antibody binding

This paper describes the development of a label-free immunosensor based on carbon nanotube (CNT) array electrodes. Highly aligned multi-walled carbon nanotubes were grown on a Fe/Al 2O 3/SiO 2/Si substrate by chemical vapor deposition (CVD). The substrate was patterned with 100 μm square blocks and 100 μm spacing between blocks. Carbon nanotube towers up to 2 mm in height grew from the blocks, and the towers were easy to peel off the silicon substrate. The harvested towers were cast in epoxy and both ends were polished; one end for electrical connection, and the other end for use as an electrode. The nanotube electrode was then electrochemically activated to open the nanotube ends and to expose COOH groups on the surface. Anti-mouse IgG was then covalently immobilized on the nanotube array. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterise the binding of mouse IgG to its specific antibody already immobilized on the nanotube electrode surface. A non-linear calibration plot was constructed based on the change in the electron transfer resistance at the electrode surface as a function of mouse IgG concentrations. A detection limit of 200 ng/mL and a dynamic range up to a 100 μg/mL range were obtained. Overall, it was found that the nanotube array immunosensor was easy to fabricate and has good sensitivity.

Diagnostic Procedures in Tularaemia with Special Focus on Molecular and Immunological Techniques

Tularaemia is a severe bacterial zoonosis caused by the highly infectious agent Francisella tularensis. It is endemic in countries of the northern hemisphere ranging from North America to Europe, Asia and Japan. Very recently, Francisella-like strains causing disease in humans were described from tropical northern Australia. In the last decade, efforts have been made to develop sensitive and specific immunological and molecular techniques for the laboratory diagnosis of tularaemia and also for the definite identification of members of the species F. tularensis and its four subspecies. Screening for the keyword 'Francisella' a Medline search over the last decade was performed and articles describing diagnostic methods for tularaemia and its causative agent were selected. Besides classical microbiological techniques (cultivation, biochemical profiling, susceptibility testing) several new immunological and molecular approaches to identify F. tularensis have been introduced employing highly specific antibodies and various polymerase chain reaction (PCR)-based methods. Whereas direct antigen detection by enzyme-linked immunosorbent assay (ELISA) or immunofluorescence might allow early presumptive diagnosis of tularaemia, these methods--like all PCR techniques--still await further evaluation. Therefore, diagnosis of tularaemia still relies mainly on the demonstration of specific antibodies in the host. ELISA and immunoblot methods started to replace the standard tube or micro-agglutination assays. However, the diagnostic value of antibody detection in the very early clinical phase of tularaemia is limited. Francisella tularensis is regarded as a 'highest priority' biological agent (category 'A' according to the CDC, Atlanta, GA, USA), thus rapid and reliable diagnosis of tularaemia is required not only for a timely onset of therapy, the handling of outbreak investigations but also for the surveillance of endemic foci. Only very recently, evaluated test kits for serological diagnosis of human tularaemia became available, while the introduction of standardized molecular techniques for detection and typing is still missing.

Discordant detection of Chlamydia pneumoniae in patients with carotid artery disease using polymerase chain reaction, immunofluorescence microscopy and serological methods

To determine the presence of Chlamydia pneumoniae in carotid plaque and peripheral blood mononuclear cells (PBMCs) using a combination of serology, direct antigen detection by immunofluorescence (IF) microscopy and polymerase chain reaction (PCR), and to compare the results obtained from each assay. A total of 54 atherosclerotic carotid plaques were tested for the presence of Chlamydia by PCR and IF methods. Of these 54 patients with carotid artery disease (CAD), 43 were also tested for the presence of C. pneumoniae DNA in PBMCs and for Chlamydia antibodies using two methods, the Medac Chlamydien rELISA and Focus Chlamydia microimmunofluorescence (MIF) methods. Eighteen of the 54 (33%) carotid specimens were positive for the presence of C. pneumoniae DNA, whereas only two of 43 (5%) patients had C. pneumoniae DNA present within their PBMC fraction. Chlamydial antibodies were detected by MIF and/or rELISA in 56% (24/43) of the patients tested. None of the 43 patients was C. pneumoniae positive in all of the test specimens (plaque, PBMCs and serum). Chlamydia pneumoniae is commonly found in Australian patients with CAD. Serology and PCR-based detection of C. pneumoniae in PBMCs and plaque give highly discordant results.

A sensitive adenovirus immunoassay as a model for using nanoparticle label technology in virus diagnostics

Currently, PCR-based hybridization assays are widely applied in adenovirus diagnostics. However, the technology requires tedious sample preparation, and the amplification phase is susceptible to various contaminants leading to inconvenient and time-consuming assay procedure. Methods relying on viral antigen detection, e.g. immunofluorometric assays (IFMAs) and enzyme immunoassays (EIAs), are less complicated to carry out, but they provide limited sensitivity. Our aim was to develop a simple and sensitive adenovirus assay based on direct antigen detection via sandwich-forming immunoreaction. The assay employed highly fluorescent europium(III)-chelate-doped nanoparticle labels and selection of high affinity monoclonal antibodies (anti-hexon) coated on label particles and microtitration wells. The extremely high specific activity of the nanoparticle labels enabled the detection limit over 5000 virus particles per millilitre with purified virus particles. The sensitivity was improved by three orders of magnitude (800-fold) compared to concurrent time-resolved IFMA. Furthermore, the nanoparticle assay showed reasonably low coefficients of variation (4.0-20%) and excellent linearity of more than four orders of magnitude (from below 10(5) to 10(9) virus particles per millilitre). Analyzed nasopharyngeal patient specimens revealed a minor disturbance of matrix components, which could be avoided by dilution. The average signal difference between negative and positive samples was nearly four orders of magnitude. The developed assay was sensitive and more convenient approach to adenovirus screening compared to available assays. In addition, the study demonstrates the potential of nanoparticles in sensitive screening of viral analytes.