One of the key benefits in using chickens for immunization is the high yield of antibodies obtainable. It is known that egg production decreases over time, while animal maintenance costs remain stable. It would, however, be desirable to keep hens as long as possible to obtain maximal amounts of antibodies. To identify a suitable length of time that animals can be kept and to optimize the cost:yield ratio, we monitored the number of eggs laid, the total amount of chicken IgY, and the specific antibody titer from individually prepared eggs over a 2-yr period. The plant toxin ricin and the Clostridium botulinum neurotoxins type A and B were used to immunize 4 chickens. The number of eggs laid in 2 yr was approximately 600 per hen (about 80% of the maximum egg number), yielding about 20 to 40 g of total IgY per hen. A stable antibody titer of 1:100,000 to 1:1,000,000, as measured by ELISA, was obtained following up to 11 injections of 10 to 20 microg of immobilized native toxin. Laying capacities were found to decrease, on average, from 7 eggs/wk at the point of first immunization to 2 eggs/wk after more than 2 yr. In parallel, the yield of total and specific IgY increased over time, so that the antibody recovery remained high, even after prolonged immunization times. Using purified IgY preparations, classical immunological assays such as ELISA and Western blotting were performed. Furthermore, the IgY showed neutralizing capacity when used to block the functional activity of the toxins both in vitro and in vivo. Analysis of the total IgY content over time demonstrated a complex biological oscillation (and the antigen-specific titer), with a shorter time period of around 7 d (circaseptan rhythm). In summary, we successfully immunized chickens with ricin and botulinum neurotoxins and monitored laying capacity, IgY concentration, and specific antibody titer over an extended period of 2 yr.
Available rabbit anti-NANOS2 antibodies failed to recognize the NANOS2 protein of goat origin though they showed better reactivity towards the same protein of human and mice origin. A specific antibody against goat NANOS2 is necessary to explore the biological functions of dairy goat NANOS2 protein. Recombinant NANOS2 protein (dairy goat origin) was expressed in E. coli (BL21). Anti-NANOS2 immunoglobulin Y (IgY; in chicken) was developed and evaluated. NANOS2 protein sequences between mice and dairy goat were compared using bioinformatics tools. The specific IgY titer attained the peak (≥ 1 : 128 000) after the 3 rd booster injection, then it gradually decreased to 1 : 16 000 and remained stable. The specificity and sensitivity of anti-NANOS2 IgY was superior to rabbit anti-NANOS2 IgG. Though, the functional domain 63-116 or 62-115 of NANOS2 protein is highly conserved with 100% identity in dairy goat and mice, the considerable difference in sequence at region 18-61 was identified from the predicted epitopes. According to our survey, this is the first report on preparation of chicken IgY against NANOS2 protein and indicating the prerequisite of species specific NANOS2 antibodies due to epitopes difference in functionally conserved NANOS2 protein. Anti-NANOS2 IgY could be applied as a novel research tool in developmental biology.
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