Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells

The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.     

No association between VNTR polymorphisms of dopamine transporter gene and attention deficit hyperactivity disorder in Chinese children.

Dopamine transporter (DAT) gene is implicated in the pathogenesis of attention deficit hyperactivity disorder (ADHD). Previously a meta-analysis concluded no association between the variable-number-of-tandem-repeats (VNTR) polymorphisms of the DAT gene and ADHD. However, significant heterogeneity was present among studies and no conclusion can be drawn about the association in any single ethnicity given the small number of studies. There were also conflicting results in Chinese populations. We therefore perform the present study to investigate the association in Chinese children in Hong Kong. In this prospective family-based and case-control study during January to June 2004, we recruited consecutive Chinese children diagnosed with ADHD by DSM-IV criteria, their family members, and sex-matched controls admitted for acute upper respiratory infection, excluding those with perinatal brain insults, mental retardation, or neurological deficits. VNTR polymorphisms of the DAT gene were determined by standard PCR followed by agarose gel electrophoresis. Sixty-four ADHD cases (52 boys, 12 girls), their family members and 64 normal controls were recruited. The 10-repeat allele (92.6%) and the 10/10 repeat genotype (85.2%) were the most prevalent. Both family-based and case-control analyses showed no association between the DAT gene polymorphisms and ADHD (transmission dysequilibrium test: P = 0.99; odds ratio of 10-repeat allele = 0.89 (95%CI 0.35-2.28), P = 0.81; odds ratio of 10/10 repeat genotype = 0.69 (95%CI 0.26-1.84), P = 0.46). We concluded that VNTR polymorphism of the DAT gene is not associated with ADHD in Chinese children, and further studies are needed to clarify the polygenic and environmental influences for pathogenesis of ADHD.     

Iduronate-2-sulfatase gene mutations in 16 patients with mucopolysaccharidosis type II (Hunter syndrome)

Mutations of the iduronate-2-sulfatase gene were identifiedin 16 patients with mucopolysaccharidosis type II (Hunter syndrome).Together with another 10 cases reported by us earlier it emergesthat about 20% of the patients have deletions of the whole geneor other major structural alterations. One, two or three basepair deletions are found in about 23% of the cases while theremaining about 57% carry point mutations predicting amlno acidreplacement, premature termination of translation, or aberrantsplicing. Molecular analysis of mRNA in splice site mutantsshowed that these latter defects frequently resulted in useof cryptic splice sites in exons or introns. 62% of the smalldeletions and point mutations have occurred in 3 of the 9 iduronate-2-sulfatasegene exons. Knowledge of the primary genetic defect allows fastand reliable carrier detection and prenatal diagnosis as wellas insight into the relationship between genotype and phenotype.     

Longitudinal Profile of Immunoglobulin G (IgG), IgM, and IgA Antibodies against the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in Patients with Pneumonia Due to the SARS Coronavirus

By using a recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein-based enzyme-linked immunosorbent assay (ELISA) and serum specimens serially collected (from day 0 to day 240 after symptom onset) from patients with pneumonia due to SARS-CoV, we analyzed the longitudinal profiles of immunoglobulin G (IgG), IgM, and IgA antibodies against the SARS-CoV nucleocapsid protein in patients with pneumonia due to SARS-CoV. For IgG, the median optical density at 450 nm (OD₄₅₀) turned positive at day 17 and a biphasic response was observed. At day 240, all patients were still positive for anti-nucleocapsid protein IgG antibody. For IgM, the median OD₄₅₀ turned positive at day 20.5, peaked at about day 80, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgM antibody. For IgA, the median OD₄₅₀ turned positive at day 17, peaked at about day 50, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgA antibody. The time of seroconversion detected by the recombinant SARS-CoV nucleocapsid protein-based ELISA and that detected by indirect immunofluorescence assay were similar. The median times of seroconversion for IgG, IgM, and IgA detected by the indirect immunofluorescence assay were 17 days (17 days by ELISA), 16.5 days (20.5 days by ELISA), and 17.5 days (17 days by ELISA), respectively, after disease onset. One, four, and one of the six patients who died did not produce any IgG, IgM, and IgA antibodies against the nucleocapsid protein of SARS-CoV, respectively, although these antibodies were detected in all six patients by the indirect immunofluorescence assay. Further studies should be performed to see whether SARS-CoV nucleocapsid protein antibody positivity has any prognostic significance.     

Typhoid fever associated with acute appendicitis caused by an H1-j strain of Salmonella enterica serotype Typhi

While most strains of Salmonella enterica serotype Typhi, the etiologic agent of typhoid fever, have only a phase 1 flagellar antigen, H1-d, variations of the flagellar antigen have been observed. Although H1-j strains (one of the flagellar antigen variants) account for 10 to 50% of S. enterica serotype Typhi strains found in Indonesia, there have been no published data to suggest its existence in other parts of the world. We describe a case of typhoid fever associated with acute appendicitis caused by an S. enterica serotype Typhi H1-j strain in a Chinese woman in Hong Kong. A gram-negative, motile rod was recovered from her blood and stool cultures. Conventional biochemical tests and the Vitek system (GNI+) showed that the bacterium was S. enterica serotype Typhi. The isolate agglutinated with poly(O), 9O, Vi and H1-j Salmonella antisera but not with poly(H) antisera. The patient developed antibodies against only S. enterica serotype Typhi O antigens but not against H1-d antigen by the Widal test. Flagellin C gene (fliC) sequencing showed a 261-bp deletion in the fliC gene of the isolate, confirming that the isolate possessed the H1-j antigen. The patient had no past history of travel to Indonesia or personal contact with any Indonesian. She recovered with appendectomy and antibiotic treatment. Further studies should be performed to determine the prevalence of this unusual S. enterica serotype Typhi strain in our locality.     

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.