Preparation Of Egg Yolk Antibodies Against Avian Influenza Virus (H5N1) And Preliminary Study Of Their Biological Propertie

The Avian Influenza (AI) is an acute and high contact infectious disease caused by the A-type influenza virus. In Italy, it erupted for the first time in 1878. In the 20th century, the epidemic of the highly pathogenic Avian Influenza (HPAI) had been reported nearly 20 times in the world. In the initial period of the 21st century, the Avian Influenza seems to erupt .Since 2004, the epidemic of the Avian Influenza of more than 50 countries and the areas have successively been reported in the world. Until March 29 2007, it had been reported that 285 cases of people had infected 170 cases had died in the world. The mortality rate of AI was higher than the Non-typical pneumonia by far, which caused the shock and attention of the entire world. In china, since the first human-AIV case on November 12, 2005 in Xiangtan County in the Hunan Province, to March 29 2007, 24 cases had been diagnosed and determined, 15cases had died. The host boundary of the species has been broken through by H5N1 AIV virus for several times, which causes the highest rate of human infection and the casualty of the entire AIV virus. If the variation of the H5N1 occurs, the infection of person-to-person will start and the prevalence is coming in the world. The AIV virus originates from the fowls and birds. It is important to prevent, monitor and treat to fowl in epidemic situation. As a method of passive immune, antibody therapy has already been an important method of clinical. The researching to develop effective antibody-medicine could reduce the death of infected cases, the control of the spread of epidemic, which could be positive. the Immunoglobulin of yolk (IgY) come from hens lead by the acceptor, which is cheap, high-effective and stable, the distance of biological phylogenic between the human, mammal and birds is far, which is the important function and be highly valued. Based on the investigations before, the protection effect of IgY as a passive immune method was confirmed, this paper try out a new method of purification, the key is preparation, purifying and study the biological properties of the IgY against AIV-H5N1 preliminarily.This paper researches mainly: first, the investigation of the method of immune, withdraw, purification and the preparation of IgY against AIV-H5N1, which is suitable for preparation of massive IgY, as a purification method which is easy, cheap high-effective; second, study the biological properties of the purified IgY against AIV-H5N1 preliminarily and have a research for the interdiction to H1N1 flu virus FM1 strain proliferating on MDCK cells.First , the laying hens were immunized with the AIV(H5N1, RE-1) oil inactivated vaccine to confirm the dose and the immune scheme and determinate the suitable immune program; while the dose was 1.5ml,the average titer of special antibodies of HI reach to 11.73, along with the enhance of dose, the increasing of the titer is not prominent, although the influence to the hens is increasing. According to the principle to protecting the animal and utilizing the vaccine efficiently, the dose was definited. Comparisons of the different diluent , depuration methods and the depuration effect, it was indicated that the best immune program to the foundation immunization is 1.5ml, the enhance immunizations were taken (1.5ml)every 10 days later, for 3 times. The average efficiency of HI reaches to 11.58. The result showed the titer of special antibody is higher than 9log2, in the extent of protected, the titer of special antibody is stable for a long period. The preparation and purification of IgY : diluted by acetic acid sodium acetate buffer (0.1mol/l pH5.0), isolated by caprylate acid (3.5μl/ml),two-step salt precipitation with solid ammonium sulfate and filtered by Sephadex G-150.The protein value is 13.5%,the average titer (log2) of HI is 5.33. IgY was examined by SDS-PAGE, the purity is very high. Moreover Caprylic Acid could deactivate the virus. This test is establishing the method of IgY depuration which is economical, simple and effective. It will lay an experiment basis for the preparation of massive IgY and the industrial preparation of immunoglobulin products.The stability of IgY against AIV-H5N1 is good at pH 5.0-11.0; IgY against AIV-H5N1 is awfully sensitive to the temperature and still stable at 65℃, heated for 30min, the rate of denature was 38.2%; But at 80℃for 2min, IgY was denatured completely; at 75℃or pH2.0, the protectants of the sugar compound is directly correlated with the proportion of their quality. It was concluded that the order of protection effects is Sucrose, Fructose, Glucose,Maltose,Lactose. It may be the experiment basis for the preservation of IgY against AIV-H5N1. The result showed the TD0 value of IgY to MDCK cell is 0.2205mg/ml, the biological security to the MDCK cell of the IgY against AIV-H5N1 is very good. The properties of security and affectivity have been applied in examination method, treatment and clinical diagnosis and so on. When 4×TCID50 FM1 strain of H1N1 virus attack the MDCK cell, the IC50 value of IgY is 0.0828mg/ml. It showed that the biological properties were stable and the inhibitory to FM1 strain can be examined. Although the pathogenesis mechanism of AIV is not clear completely, the way to inducing programmed cell death (PCD) is multi-means, but it may be concluded that since there is the similarity between H5N1 and H1N1 Flu virus and it showed cross-reaction inducing PCD of MDCK cells. The mechanism is required for further investigated clearly.