The Preparation Of Chicken Immunoglobulin Y Antibodies And Study Its Uses For Depleting Partial Plasma High Abundant Protein

The high abundance proteins of human plasma has strong cover function, which will have effect on the detection of some low abundance proteins (ng/mL or pg/mL). However, these low abundance proteins maybe functions as key proteins for signal conduction and control and some serious diseases's key indication molecules. It place an important part in the early diagnosis and curative effect examination. Therefore, established a method of depleting the plasma high abundance protein and enhancing the detection sensitivity of plasma low abundance protein is very important.AIM to prepare specific egg yolk immunoglobulin (IgY) against partial high abundance protein of human plasma, using immune affinity principle, to research human plasma high abundance protein depletion. METHODS we use pre-laying Roman hens(100 d) and laying Roman hens(150 d) which all have been immunized with different antigens, notably HSA, IgG, mixtures of these proteins and human plasma. And study the best condition of isolating the IgY antibody . The antibody titre-developments are examined using indirect ELISA and Agar gel double immunodiffusion. Purify IgY using the water-diluted method, the saturated ammonium sulfate gradient precipitation and the anionic exchange column separation. Subsequently, the specific IgY is bound to the surface of GoldMag for high abundance protein depletion research. RESU1TS The pre-laying Roman hens can produce perfect antibody-titre. Sixty to hundred and fifty days after immunization, the antibody titer in the plasma for the different antigens can reach to the level of: 1:10 000～1: 25 000; Obtained the IgY separation purification technical route: The yolk fluid and the deionized water mixed, stirred (1V: 9V) , centrifugated, filtrated, added the saturated ammonium sulfate to the final concentration 30% and 19% respectively. After the steps of centrifugation and dialysis, take use of the method of DEAE-fast-flow anionic exchange column to do the purification. The purified IgY titre can reach to 1:10 000～1:25 000, the purity can reach up to above 98% . Make use of the golden magnetic particle carrier bounded with IgY, can deplete the plasma HSA and IgG. specially. One the other hand, anti-human plasma IgY can deplete macromolecular weight protein(>83K) in plasma. Immuno electrophoresis analysis, there are at least four kinds of specific anti-human plasma protein in the hens' yolk. CONCLUSION The usage of HSA ,IgG and human plasma as antigens to immunize laying hens can produce high quantities of pure IgY. The GoldMag carrier bound with IgY can be used to deplete the relevant HSA and IgG from human plasma effectively. As a new method of proteome of plasma study, it has great application prospect.