Preparation And Characterization Of IgY Against Human Glioma

Objective: glioma is one of the most common malignant neoplasms difficult to cure in central nervous system. Traditional surgical operation, radiotherapy and chemotherapy are not evry effective . The dynamics research of glioma express that because the tumor present gradually growth, a equal part of tumorses to be placed in quiet appearance temporarily , even cut off 99% tumor tissue,it can propagate to the original size after eight weeks. Along with the technical development of cell biology, molecular biology and the biology engineerings, it brings new turning point for the treatment of glioma. The immunological treatment led by antibody has become one of the hotspots of tumour therapy.The antibody of IgY is a kind of immunoglobulin which is Purified from egg yolk,it has special biological function. Because hen belongs to the non- mammalian, the relative relation is far from human, so it has strong immunological response to human pathogeny. The anti-glioma IgY can identify the brain glioma tissue especially and selectly,so it can be a kind of small scale and efficient guide carrier ,which can transport the toxin to the tumor cell teleologily, when the carrier identify the tumor cell,it can combine with the tumor cell immediately and then release the toxin, in order to kill the tumor cell but don't affect other normal cell around the glioma tissue. In addition the preparation of IgY is low cost and high output, as well as no toxin side effect, so it will become a kind of valid means of large-scale antibody's preparation. This research's aim is separating and purifying the anti-glioma IgY, as well as analyseing and authenticateing it's basic characteristic, so it can give a new idea to diagnoseing and treatingbrain tumor.Methods: 1. The human glioma cell line U251 which was presented by Neuromedical institute of the fourth military medical university were cultured and passaged with routine methods in 37℃,5%CO2's condition.2. The hens we used were named JingBai hen which were provided by YiKang biology products factory in LiaoYang city, after a week's normal feeding, the hens were ready for immunizing.3.Collected the cell to prepare the suspension containing with the NS, according to 109 cells a hen immunized the JingBai hens. noticing the first immunity needed to add the same quantity of Freund complete adjuvant, then injected the hens at different spots,such as both wing skin and both back skin.Gathered its eggs after a week everyday, then mark them, keep them in 4℃ready for using.4. This experiment used the eggs before immunized and the 15th,20th,25th,30th,35th,40th,45th days', wiped off the egg white, collected the yolk liquid , then through distilled water extracted , 50% saturation ammonium sulfate salt out,dialysis, PEG concentrate and other steps to get to turn the IgY antibody purely,We named the antibody as AG-IgY.Then protein ration,Finally, the obtained protein antibodys were identified through immunohistochemistry, immunity cell chemistry,SDS-polyacrylamide gel electrophoresis (PAGE),Western blot and enzyme- linked immunosorbent assay (ELISA).Results: In about 10 days a special IgY antibody could be detected from the hen's yolk, the quantity of antibody attains a high peak on about 35 days, each egg could harvest 100-200 mg antibody. It had strong heat-proof and acid-proof, moreover its effect could last 2-6 months.the results of immunohistochemistry staining demonstrated that IgY antibody and glioma cell U251 presented a strong positive reaction, and the positive position located at the cellular membranes and cytoplasm mainly. The purified IgY were shown two straps with SDS-PAGE, and the molecular weight (MW) of them were estimated to be approximately 70kD and 20kD respectively. shows that no cross reaction was observed between IgY and other antigenses, such as liver cancer, lung cancer, mammary glands cancer, the glioma cell line U87 and so on. Similar results as ELISA were seen in the Western blot.Conclusion: 1.In this study, the against human glioma IgY were isolated and purified successfully.2.Through researching on it's physics and chemistry property, SDS- PAGE, Western blot and ELISA, the result indicated that IgY is a kind of new antibody which had the character of strong acid-proof and heat-proof,low cost, high output and easy to make.It had better particularity, had no cross reaction with other irrelevant antigen.3.This research would develop a new method for the prevention and diagnoses of human brain glioma.