ABSTRACT
A double-antigen sandwich ELISA was developed for the detection of antibodies to influenza A viruses. A recombinant nucleoprotein (rNP) of influenza A virus was used as a capture antigen and an HRP-conjugate for detecting the antibodies. A total of 125 serum samples from birds of different species including chickens, geese, open-billed storks, Khaki Campbell ducks, lesser whistling ducks, and pigeons with known antibodies were tested by ELISA. The sensitivity and the specificity of ELISA were found to be 98% and 97.3%, respectively. The assay was able to detect the presence of influenza A antibodies as early as the fourth day post-inoculation in ducks infected experimentally with influenza A (H5N1) virus. Excellent agreement (97.6%) was obtained between this sandwich ELISA and the hemagglutination inhibition (HI) tests (kappa=0.95). The double-antigen sandwich ELISA correlated well with a commercial avian influenza (AI) multispecies ELISA and was slightly more sensitive than the AI multispecies ELISA. These findings indicate that the double-antigen sandwich ELISA based on rNP may offer an effective screening method for serodiagnosis of influenza A virus. The double-antigen sandwich ELISA also enables the detection of antibodies to influenza A viruses in different species without the need for species-specific secondary antibodies.
Subject(s)
Antibodies, Viral/blood , Antigens, Viral , Bird Diseases/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Influenza A virus/immunology , Influenza in Birds/diagnosis , Poultry Diseases/diagnosis , RNA-Binding Proteins , Viral Core Proteins , Animals , Antigens, Viral/genetics , Birds , Hemagglutination Inhibition Tests , Nucleocapsid Proteins , Poultry , RNA-Binding Proteins/genetics , Recombinant Proteins/genetics , Sensitivity and Specificity , Statistics as Topic , Viral Core Proteins/geneticsABSTRACT
Umbilical cord blood (UCB) is being increasingly used as an alternative source of hematopoietic stem cells for allogeneic bone marrow transplantation. UCB transplantation has been successfully used to treat a variety of genetic, hematological, and oncological disorders in children and adults. The objectives of this study was to establish a closed-system technique for UCB collection and buffy coat separation by Optipress I device. Thirty-four UCB were collected by triple-bag system from pregnant mothers whose fetuses were not affected by thalassemic diseases after prenatal diagnosis. The mean volumn of UCB collection were 120 +/- 5 ml (range 65-180 ml). Total WBC, CD34+ cells, the progenitor cell erythroid burst-forming unit (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) in the UCB units were (9.36 +/- 0.84) x 10(8), (3.61 +/- 0.52) x 10(6), (9.12 +/- 1.60) x 10(5), and (5.32 +/- 1.23) x 10(5), respectively. Good correlation between the nucleated cell and net cord blood volume could be demonstrated (p < 0.0001). The correlation between CD34+ cells and the following parameters: nucleated cell, BFU-E or CFU-GM were also demonstrated (p = 0.001, 0.0105 or 0.0001, respectively). Buffy coat was subsequently separated from 18 UCB units by Optipress I device. 70 +/- 3 ml of buffy coat were collected and cryoprocessing was done by automatic controlled-rate freezer. Good recovery of total WBC, CD34+ cells, progenitor cells BFU-E and CFU-GM after buffy coat separation were observed 89 per cent, 95 per cent, 109 per cent, and 102 per cent respectively. There was no aerobic bacterial or fungal contamination in the separated blood products. By using this technique, the UCB units were easily collected, rapidly separated within one hour, and high recovery of the hematopoietic progenitor cells could be obtained.
