| Coli phage f2 is an RNA virus and its character is very similar with enterovirus (EV), which exist in intestinal tract of people and animal just like fecal coliform. The survival time of coli phage f2 in water and the resistance to chlorine sterilization are nearly as the same as that of poliovirus (PV). Since the difficulty of direct detection and high incidence of EV which exist in water every year, we select the coli phage f2 as one of the detection indicator for water virus. Previous studies have shown that the research for coliphage f2 immunoassay has been undertaken less and it is necessary to do some work for it. The objective of our study is to prepare polyclonal anti-coliphage f2 antibody, which is the basic biological material for various application, especially in environmental monitoring.In the present study, high purity of coli phage f2 was made by using ultra centrifugation. Then, we immuned rabbits with the phage f2 and got the polyclonal antibody and relatively purified IgG antibody. With high titer and high concentration, the prepared polyclonal antibodies provided the necessary material for further study. Finally, we constructed the immunomagnetic beads (IMB) to adsorb and separate coliphage f2 in water for establishing a fast and economic method for viral determination. Partâ… Preparation and purification of specific polyclonal antibody against coliphage f2Objective: To prepare rabbit anti-coli phage f2 polyclonal antibody, separate and purify IgG.Methods: After multiplicating coli phage f2, we separated and purify phage f2 by using ultracentrifugation; then we selected three healthy Japanese rabbit, injected purified phage f2 subcutaneously at different points and repeated the injections every two weeks for 4 times. After that, the titre of antiserum was tested by using serum neutralization test (SN test) and indirect-ELISA method. When titre exceeded l:3200, the blood was collected from aorta. The IgG was separated and purified with saturated ammonium sulfate and DEAE-cellulose and the purified IgG concentration was scanned and determined by ultraviolet spectrophotometer.Results: Antiserum was obtained, the titer of which exceeded 1:16000 by using serum neutralization test and the titer for indirect-ELISA was above 1:5000. Furthermore, rabbit globulin prepared as an ammonium sulfate precipitate of whole serum and rabbit IgG with DEAE-cellulose showed the same bingding ability to phage f2 as that of antiserum, which were determined by SN test and indirect-ELISA. The IgG concentration was 23.11mg/ml.Conclusion: The antiserum abtained in the study showed good specificity and sensitivity for phage f2 antigen and the polyclonal antibody could be used in the further research and application related to environmental coli phage f2 detection.Partâ…¡Adsorption and separation of coliphage f2 in water by immunomagnetic beadsObjective: Construction of the immunomagnetic beads (IMB) to adsorb and separate coliphage f2 in water for establishing a fast and economic method for viral determination.Methods: Immunomagnetic beads were made through coating the antibody to magnetic beads. Bacteriophage f2 in water samples was adsorbed by IMB. Then, eluting the f2 phages from immunomagnetic beads for Plaque assay and observing the elution effects at different pH after being absorbed.Results: Good effect of coating the antibody to magnetic beads had been achieved by using chemical method, so had the adsorption of f2 phages by IMB. But the elution efficiency with which less than 30% bacteriophage f2 was eluted was not desirable; Solutions with various pH had different elution effect and the maximum elution values could be obtained at about pH 2.7.Conclusion: In this experiment, construction of IMB by means of chemical coating had a good effect. However, choosing of the eluent played a key role in improving elution efficiency of virus from IMB.
|
PDF Full Text Request