Background: Recombinant adeno-associated virus vectors based on serotype 2 (AAV-2) have become leading vehicles for gene therapy. Most humans in the general population have anti-AAV-2 antibodies as a result of naturally acquired infections. Pre-existing immunity to AAV-2 might affect the functional and safety consequences of AAV-2 vector-mediated gene transfer in clinical applications. Methods: An enzyme-linked immunosorbent assay (ELISA) method was developed using microwell plates coated with intact particles of recombinant AAV-2 vectors, and horseradish peroxidase-conjugated anti-human immunoglobulin G (HRP-IgG). Neutralizing antibody titres were analysed by assessing the ability of serum antibody to inhibit transduction into HEK293 cells of AAV vectors that express b-galactosidase. Results: Anti-AAV-2 antibodies were detected by ELISA in two of 20 healthy subjects. The positivity criterion (optical density .0.5) in ELISA corresponded to the cut-off value (320-fold dilution of serum) in the AAV-2 neutralization assay. Influences of interfering substances were not observed. Conclusion: This ELISA method may be useful for rapid screening of anti-AAV-2 neutralizing antibodies in candidates for gene therapy.

A switch of viral hemagglutinin receptor binding specificity from bird-type α2,3- to human-type α2,6-linked sialic acid is necessary for an avian influenza virus to become a pandemic virus. In this study, an easy-to-use strip test to detect receptor binding specificity of influenza virus was developed. A biotinylated anti-hemagglutinin antibody that bound a broad range of group 1 influenza A viruses and latex-conjugated α2,3 (blue) and α2,6 (red) sialylglycopolymers were used in an immunochromatographic strip test, with avidin and lectin immobilized on a nitrocellulose membrane at test and control lines, respectively. Accumulation of a sialylglycopolymer-virus-antibody complex at the test line was visualized by eye. The strip test could be completed in 30min and did not require special equipment or skills, thereby avoiding some disadvantages of current methods for analyzing receptor binding specificity of influenza virus. The strip test could detect the receptor binding specificity of a wide range of influenza viruses, as well as small increases in the binding affinity of variant H5N1 viruses to α2,6 sialylglycans at viral titers >128 hemagglutination units. The strip test results were in agreement with those of ELISA virus binding assays, with correlations >0.95. In conclusion, the immunochromatographic strip test developed in this study should be useful for monitoring potential changes in the receptor binding specificity of group 1 influenza A viruses in the field.

The SSX family proteins have been considered new members of the cancer/testis antigens because of the restricted expression in testis among normal tissues and the activation in a wide range of cancers. Thus, they would be potential molecular targets for immunotherapeutic strategies. We have developed a competitive nucleic acid sequence-based amplification (NASBA) assay to analyze SSX mRNA expression in 211 bone and soft tissue tumors. The copy numbers of SSX mRNA per mug of total RNA in tumor tissues were widely distributed, ranging logarithmically from 0.6 to 6.6. We found that malignant tumors showed significantly higher expression of SSX mRNA than benign tumors (P < 0.0001). Further, SSX mRNA expression in stage III tumors was significantly higher than that in stage I or II tumors (P < 0.005). This NASBA assay was also more sensitive compared to immunohistochemistry using newly affinity-purified polyclonal antibody against SSX. Collectively, these results suggest that the SSX quantitative NASBA assay could provide useful information to select eligible patients for SSX-specific cancer vaccines.

No abstract

Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4pol from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.