A modified SELEX approach to identify DNA aptamers with binding specificity to the major histocompatibility complex presenting ovalbumin model antigen.

Aptamers have sparked significant interest in cell recognition because of their superior binding specificity and biocompatibility. Cell recognition can be mediated by targeting the major histocompatibility complex (MHC) that presents short peptides derived from intracellular antigens. Although numerous antibodies have demonstrated a specific affinity for the peptide-MHC complex, the number of aptamers that exhibit comparable characteristics is limited. Aptamers are usually selected from large libraries via the Systemic Evolution of Ligands by Exponential Enrichment (SELEX), an iterative process of selection and PCR amplification to enrich a pool of aptamers with high affinity. However, the success rate of aptamer identification is low, possibly due to the presence of complementary sequences or sequences rich in guanine and cytosine that are less accessible for primers. Here, we modified SELEX by employing systemic consecutive selections with minimal PCR amplification. We also modified the analysis by selecting aptamers that were identified in multiple selection rounds rather than those that are highly enriched. Using this approach, we were able to identify two aptamers with binding specificity to cells expressing the ovalbumin alloantigen as a proof of concept. These two aptamers were also discovered among the top 150 abundant candidates, despite not being highly enriched, by performing conventional SELEX. Additionally, we found that highly enriched aptamers tend to contain fractions of the primer sequence and have minimal target affinity. Candidate aptamers are easily missed in the conventional SELEX process. Therefore, our modification for SELEX may facilitate the identification of aptamers for more application in diverse biomedical fields. Significance: we modify the conventional method to improve the efficiency in the identification of the aptamer, a single strand of nucleic acid with binding specificity to the target molecule, showing as a proof of concept that this approach is particularly useful to select aptamers that can selectively bind to cells presenting a particular peptide by the major histocompatibility complex (MHC) on the cell surface. Given that cancer cells may express mutant peptide-MHC complexes that are distinct from those expressed by normal cells, this study sheds light on the potential application of aptamers to cancer cell targeting.

Respiratory adenoviral infections in Taiwanese children: a hospital-based study.

BACKGROUND AND PURPOSE: Adenoviruses are a common etiology of respiratory tract infections in children, with several serotypes responsible for most epidemic respiratory infections. This study examined the molecular epidemiology and clinical features of pediatric adenoviral infections in a 1-year period. METHODS: From May 1999 to April 2000, virus specimens collected from children with respiratory tract infections were identified. The presence of adenovirus was confirmed by direct fluorescent staining, and viral types were determined by polymerase chain reaction sequencing. RESULTS: Adenoviruses were identified from 272 children (mean +/- standard deviation age, 48.3 +/- 30.5 months), 227 (83.5%) of whom were aged 6 years or younger. Inpatients were younger than outpatients (44.1 +/- 30.7 months vs 53.0 +/- 29.4 months; p = 0.006). The commonest serotype identified was serotype 3 (164 patients; 60.3%), 73.1% of which were identified between September 1999 and January 2000. Serotype 3 was more common in inpatients (p = 0.015), while serotypes 1, 2, 5, and 6 were more common in outpatients (p = 0.021). Patients with pneumonia were younger than those with other infections (31.8 +/- 20.2 months vs 50.3 +/- 31.0 months; p = 0.001). Most of the children (90.1%) had fever for a mean of 3.80 +/- 2.65 days before seeing a doctor. The clinical manifestations were similar regardless of the serotype. CONCLUSIONS: Adenovirus serotype 3 caused the most adenovirus infections in autumn and winter of 1999 to 2000. The children were mostly preschool age and required hospital admission.

Colonization of severe acute respiratory syndrome-associated coronavirus among health-care workers screened by nasopharyngeal swab.

STUDY OBJECTIVES: To report the efficacy and findings of a large-scale preventive screening program for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) using amplification of the virus from a nasopharyngeal swab (NPS) obtained from the health-care workers (HCWs). DESIGN: A prospective observational study. SETTING: A medical center in Taiwan. PARTICIPANTS: Two hundred thirty HCWs. INTERVENTION: NPS examination for the presence of SARS-CoV by two nested reverse transcription-polymerase chain reaction (RT-PCR) assays. MEASUREMENTS AND RESULTS: During the outbreak of severe acute respiratory syndrome (SARS), NPS polymerase chain reaction screening of HCWs for SARS-CoV was performed. SARS-CoV was examined by two nested RT-PCRs and a quantitative RT-PCR. Serum-specific antibodies were assessed by enzyme immunoassay and indirect immunofluorescence. We monitored 230 HCWs, including 217 first-line HCWs and 13 non-first-line HCWs. One hundred ninety first-line HCWs and 13 non-first-line HCWs had negative results in both nested RT-PCR assays. Two first-line HCWs who were positive on both nested RT-PCR assays had SARS. They had 16,900 +/- 7,920 copies (mean +/- SD) of RNA per milliliter in the NPS and had detectable anti-SARS antibodies. The remaining 25 first-line HCWs were negative for the first nested RT-PCR but positive for the second nested RT-PCR. Their corresponding titers were 338 +/- 227 copies of RNA per milliliter; antibodies developed in none of these 25 HCWs. The expression and function of angiotensin-converting enzyme-2 were not different among these HCWs. This study shows that colonization of SARS-CoV occurred in 25 of 217 well-protected first-line HCWs on a SARS-associated service, but they remained seronegative. CONCLUSION: With the second RT-PCR assay more sensitive than the first RT-PCR assay, we are able to show that approximately 11.5% of well-protected HCWs exposed to SARS patients or specimens may have colonization without seroconversion. Only those with significant clinical symptoms or disease would have active immunity. Thus, regular NPS screening for nested RT-PCR assays in conjunction with a daily recording of body temperature in all first-line HCWs may provide an effective way of early detection.

Capillary electrophoretic restriction fragment length polymorphism patterns for the Mycobacterial hsp65 gene.

PCR-restriction fragment length polymorphism (RFLP) analysis is a nonprobe method for the rapid identification of Mycobacterium species. We demonstrate the separation of DNA or restriction fragments digested from the mycobacterial gene encoding the 65-kDa heat shock protein (hsp65) by capillary electrophoresis (CE). By using a pair of unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, we investigated a total of 52 reference and clinical strains encompassing 12 Mycobacterium species. The electrophoretic separation of high-resolution CE required <20 min and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE, and the real sizes were deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. With the advantage of the complete RFLP pattern available from CE, it appears to be more convenient to use an electropherogram rather than performing the cumbersome slab gel electrophoresis plus diagnostic algorithm to identify Mycobacterium species. Beyond the agarose and polyacrylamide gel electrophoresis, high-resolution CE provides an alternative for rapid identification of Mycobacterium species that is feasible for automation and routine use without the need for costly probes.

Antibody detection of SARS-CoV spike and nucleocapsid protein.

Early detection and identification of SARS-CoV-infected patients and actions to prevent transmission are absolutely critical to prevent another SARS outbreak. Antibodies that specifically recognize the SARS-CoV spike and nucleocapsid proteins may provide a rapid screening method to allow accurate identification and isolation of patients with the virus early in their infection. For this reason, we raised peptide-induced polyclonal antibodies against SARS-CoV spike protein and polyclonal antibodies against SARS-CoV nucleocapsid protein using 6x His nucleocapsid recombinant protein. Western blot analysis and immunofluorescent staining showed that these antibodies specifically recognized SARS-CoV.