Development of indirect enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection and quantification of antibodies against avian influenza virus.

Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. In the present study, we developed an indirect enzyme-linked immunosorbent assay (ELISA) employing E. coli-expressed full-length nucleoprotein (NP) of H9N2 avian influenza virus for the detection and quantification of antibodies against AIV nucleoprotein. The NP-ELISA was compared with the AI agar gel propagation (AGP) test, haemagglutination inhibition (HI) test, and IDEXX-FlockChek ELISA using 263 sera. The NP-ELISA was significantly more sensitive than the AGP and HI tests, and showed 96.2% agreement ratio with IDEXX-FlockChek ELISA. With results obtained using the NP-ELISA, an ELISA titre (ET) prediction equation, with which the ELISA titres of a flock or individual chickens can be determined, was derived from a positive/negative (P/N) ratio standard curve. The NP-ELISA enables an alternative rapid serological diagnosis and is suitable for influenza A antibody screening, especially in species that harbour several influenza subtypes.

Microfluorometric assessment of the DNA-DNP complex in human spermatozoa.

During spermiogenesis, the DNA-nucleoprotein complex undergoes alterations that are reflected in a decreasing capacity for binding DNA-specific dyes, such as ethidium bromide (EB). Human spermatozoa with a low or high capacity for EB binding were depleted of RNA and most nuclear proteins by exposure to RNAse, EDTA and trypsin, with and without additional high salt buffer (HSB) treatment. When treated with RNAse, EDTA and trypsin only, the haploid DNA fluorescence value (calculated from the diploid value of the standard cell population) was found at EB concentrations of 6.5 to 12.5 micrograms/mL. At these EB concentrations, a significantly lower fluorescence was found in the material also treated with HSB, probably reflecting an unwinding of the highly negatively supercoiled DNA loops that are induced by HSB treatment. Maximal fluorescence was not found until a concentration of 50 micrograms EB/mL. This may be due to an overwinding of the DNA by the positive supercoiling caused by EB. The significant difference in EB uptake initially found between the two groups whose spermatozoa showed low and high capacities for EB binding disappeared after removal of the nucleoproteins, suggesting that this compartment of the nucleoprotein-DNA complex is responsible for the different uptakes of EB.

Histochemical assessment of nuclear protein content and enzyme activity in confluent nonarrested and low-serum arrested WI-38 fibroblasts at mid and late population doubling levels.

Cultures of WI-38 fibroblasts maintained in low-serum medium have little mitotic activity. Such arrested cultures can serve as a useful adjunct to the normal-serum proliferating fibroblast model system of senescence since the reduced cycling activity more accurately reflects in vivo conditions. Normal-serum plateau phase cultures and 17-day low-serum arrested plateau phase cultures were examined at both mid and late population doubling levels (PDL). Acidic, basic and total nuclear protein contents were determined by staining with toluidine blue O, alkaline fast green and acid fast green, respectively. Succinate dehydrogenase and glucose-6-phosphate dehydrogenase were localized with the nitroblue tetrazolium staining reaction. Quantification of individual cell staining was done on a Vickers M85 microdensitometer. Low-serum arrested cells at both mid and late PDL showed a significant increase in acidic protein staining and a decrease in basic protein staining when compared with normal-serum nonarrested cells. Enzyme activities in arrested cells were lower than nonarrested cells at mid PDL but equivalent to nonarrested cells at late PDL. This study demonstrates that the use of selected quantitative histochemical staining in combination with cytophotometry can be employed to differentiate arrested from nonarrested fibroblast populations as well as mid PDL from late PDL cultures.