Oral yolk-specific antibodies have been successfully used to treat bacterial and viral infections in animals. It is a convenient and effective method to control E. coli in early weaned piglets by adding an anti-E. coli yolk antibody to the diet. Yokoyama et al (1992) confirmed that oral IgY (anti-pathogenic Escherichia coli K88, K99, 987P) can prevent E. coli disease in early weaned piglets, reduce diarrhea rate and mortality of piglets, and effectively prevent bacteria and viruses. Digestive and non-digestive diseases, and increase the daily weight gain of early weaned piglets. A large amount of data (Yokoyama et al., 1992; 1993; 1997; 1998; Imberechts et al., 1997; Jin et al., 1998; Marquardt et al., 1999;) showed that IgY can increase feed intake in early weaned piglets, promote piglet growth, and improve weaned piglets. Feed conversion efficiency, reduce diarrhea and mortality, reduce the incidence of suckling weaning syndrome, and improve economic efficiency. It can be seen from the above that IgY research and development may replace antibiotics, hormones and chemical synthetic drugs, and is a safe and reliable green feed additive.
1 Formation of egg yolk antibody
The humoral immune system of poultry is controlled by the supra-cavity capsule. When the body is stimulated by external specific antigens (especially inactivated antigens), it induces a series of immune response, which stimulates B cells to differentiate into plasma cells that secrete specific antibodies, and secrete a large amount of specific antibodies into the blood. In laying poultry, specific antibodies in the blood gradually migrate into the ovaries and fallopian tubes and accumulate in the yolk. Rose et al. (1974) found that IgY in egg yolk was significantly higher than that in serum because of the cumulative effect of egg yolk.
Williams et al. (1962) found that immunization of laying hens with certain antigens produced corresponding antibodies in chicken serum, while laying hens effectively transferred serum immunoglobulins to the yolk by laying eggs. Immunoglobulins transferred from chicken blood to egg yolk are only immunoglobulin IgG, and immunoglobulins IgA and IgM have not been found in egg yolk (Hu Qinglin et al., 1997). Locken (1983) demonstrated that the process by which IgG is transferred to ovarian follicles is determined by ovarian IgG receptors, which only allow selective transfer of all IgG subpopulations present in maternal blood to ovarian follicles. IgA and IgM enter the chicken egg along with the secreted ovalbumin in the fallopian tubes, so IgA and IgM are only present in the protein. The IgG in the yolk is called yolk antibody or yolk immunoglobulin, referred to as IgY (Leslie and Clem, 1969).
2 characteristics of egg yolk antibody
Chicken IgY is functionally equivalent to mammalian IgG, but differs from mammalian IgG in many functions. Chicken IgY consists of two light chains and two heavy chains with a molecular weight of approximately 180 kDa, a heavy chain molecular weight of 66 kDa and a light chain of 22 kDa. Studies on the physical and chemical properties of IgY confirmed that the heavy chain of IgY entering the yolk contains 4 constant regions (3 mammalian IgGs), but no hinge region (Fc), which makes it resistant to gastric acid in young animals. The ability of the barrier to bind to rheumatoid factors in complement and serum. Egg yolk antibody (IgY) has good stability and is good in heat resistance, acid resistance and alkali resistance.
Shimizu et al. (1988, 1992, 1993) studied the thermal stability of IgY and mammalian IgG and found that IgY has approximately the same thermal stability as mammalian IgG. When hot (>75 ° C) or acid (pH < 3.0), the antibody activity of IgY will be lowered. At 65 ° C, the activity can be maintained for more than 24 h, but the activity is significantly reduced after 90 min treatment at 70 ° C.
Larsson et al (1993) stored the IgY preparation at 4 ° C for 5 years or at room temperature for 6 months, and stored at 37 ° C for 1 month. The IgY activity was not significantly decreased by immunodiffusion assay.
Yokoyama et al. (1993) reported that IgY is resistant to the gastric acid barrier of young animals and resists digestion of trypsin and chymotrypsin in the gut. Hatta et al. (1993) examined the effects of three digestive enzymes against HRV (rotavirus) IgY. After incubating pepsin and IgY for 1 h at pH 2.0, almost all IgY was lost, while incubation at pH 4.0 for 1 h. After maintaining 91% activity, there was still 63% activity after 10 hours of incubation. IgY was incubated with trypsin and chymotrypsin for 8 h, respectively, and the activity was maintained at 39% and 41%, respectively.
IgY is relatively stable at pH 4.0 to 11.0; it is rapidly inactivated at a pH of 3.0 to 3.5; when the pH is 12.0, its activity is also lowered.
Studies by Schmidt et al. (1989) and Wiedemann et al. (1990) have shown that IgY is well resistant to the digestive process, especially in the form of whole eggs.
3 Preparation of antibodies
3.1 Selection of
The chickens used to prepare IgY should meet the following basic conditions: 1 There should be no vertical infectious diseases such as chicken white diarrhea, chronic respiratory disease, leukemia, infectious anemia, egg drop syndrome, etc.; 2 farms should be free from strong infections. The medical history of the disease; 3 the flocks have good egg production performance. Generally speaking, the chickens used for the production of egg yolk antibodies are preferably inbred chickens. In addition, according to the use of the produced egg yolk antibody, the requirements for chicken varieties are also different. For example, SPF chicken should be used for the production of egg yolk antibody for therapeutic purposes, and commercial egg laying can be used for the production of egg yolk antibody for prevention purposes. .
3.2 Immunization Program
The general immunization procedure for preparing IgY is to use attenuated vaccine for 2 to 3 basic immunizations during the breeding of female birds to induce an immune response, and inactivate the antigen by intramuscular or subcutaneous injection of an oil emulsion containing a high concentration of antigen one month before the start of production. 0.5 ~ 1.0ml / feather, once every 10 ~ 15d boosted immunization, the dose is gradually increased by 1 to 2 times, a total of 2 to 3 times of enhanced immunity. 15 days after the last immunization, the egg-like titer was sampled and collected as a high-free egg. After 4°C storage, IgY was prepared within 7 days. The antibody level was periodically detected and boosted once every 1 to 2 months. , you can use high-level eggs continuously.
3.3 Preparation of high-IgY-free
The extraction and purification methods used should also be different for different application purposes. According to the application purpose of IgY, it can be generally divided into oral, injection, immunological detection and the like. If taken orally, the immune egg yolk can be directly made into egg yolk powder. If injection is needed, it is necessary to refine IgY to effectively remove lipids and avoid vertical contamination of pathogenic microorganisms. If the lipids in the yolk cannot be effectively removed, the lipids will play a sustained release effect, which may induce the body to produce an immune response to IgY, and Lipids are prone to spoilage and are not conducive to preservation. If IgY is applied to immunological detection, it requires high purity, strong reactivity, and higher requirements for separation and extraction.
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