ABSTRACT
In order to improve the accuracy for quantitating the bovine foamy virus(BFV)in vitro,we developed a baby hamster kidney cell(BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter(LTR,from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection(MOI)of BFV and the activated ratio of luciferase expression in BFVL. Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.
ABSTRACT
In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay. BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.
ABSTRACT
BACKGROUND: Granulocyte specific antibodies are associated with several clinical conditions including febrile transfusion reaction and transfusion-related acute lung injury as well as immune neutropenias. The identification of granulocyte specific antibodies is important for the diagnosis of these disorders. However, there have been rarely confirmed clinical reports in Korea since the testing techniques are complicated and difficult to maintain. In this study, development of in-house indicator cells and renewedly establishment of the mixed passive hemagglutination assay (MPHA) as a serologic test to detect and identify granulocyte specific antibodies were conducted. METHODS: The in-house indicator cells for MPHA were made by sensitizing human Rh(D) positive O RBCs with human IgG anti-Rh(D) (DiaMed AG, Switzerland) and then combining with AHG anti-IgG (Immucor Inc., USA). To determine the optimal conditions, various combinations of anti-Rh(D) IgG sensitization strengths of indicator cells, microwell coated antigens (intact granulocyte vs. extracted granulocyte) and reaction conditions were compared. RESULTS: The best test conditions for MPHA were as follows: optimal results were obtained with the anti-Rh(D) sensitization dilutions of 1/64-1/192 and the reaction condition of 4 hours incubation at room temperature in humid chamber. Extracted granulocytes coated at the plate showed better results than intact granulocytes. HLA antigens were completely removed from extracted granulocyte antigens after acidified chloroquine treatment. CONCLUSION: Granulocyte MPHA using in-house anti-Rh(D) sensitized indicator cells was developed for the first time in Korea. The newly established MPHA would be effectively used for the diagnosis and treatment of disorders associated with granulocyte specific antigen-antibody reactions in Korea.
Subject(s)
Humans , Acute Lung Injury , Antibodies , Antibodies, Anti-Idiotypic , Antigen-Antibody Reactions , Blood Group Incompatibility , Chloroquine , Granulocytes , Hemagglutination , HLA Antigens , Immunoglobulin G , Korea , Neutropenia , Serologic TestsABSTRACT
BACKGROUND: Platelet antibody test has been used in the diagnosis and management of immunological platelet disorders and platelet crossmatch. Mixed passive hemagglutination (MPHA) test is a cost-effective, reproducible, easy to perform and convenient method. Anti-IgG coated indicator red cells used for MPHA test have not been made in Korea and those cells have been exclusively donated by Dr. Shibata in Japan. This study was designed to produce domestic indicator cells and to determine its acceptability by comparing to the results obtained with Dr. Shibata's indicator cells. We produced homemade indicator cells by coating human RhD-positive O RBC with human IgG anti-D (check cell) and then coated with rabbit anti-human IgG. METHODS: Sixty three sera from healthy male donors, 58 sera tested positive in platelet suspension immunofluorescence test (PSIFT), and 61 sera tested negative in PSIFT were evaluated by MPHA employing both homemade and Dr. Shibata's indicator cells. RESULTS: The concordance rate between PSIFT and homemade MPHA was 74%. Test results of MPHA with homemade indicator cell showed excellent correlation with Shibata's indicator cell (P=0.002). All of 63 sera from healthy volunteer male blood donors without history of transfusion were tested negative with homemade MPHA method. Test results of MPHA employing homemade indicator cells showed excellent correlation with those of PSIFT (P=2.67x10-8). CONCLUSIONS: Homemade indicator cells we developed in this study were able to replace Dr. Shibata's indicator cells for the MPHA test.