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Immune affinity precipitation method for purification of antibodies

A precipitation method and immunophilic technology, applied in the research field of immunochemistry, can solve the problems of expensive agarose gel, highly toxic, and high purchase cost, and achieve the effect of being suitable for mass production, easy to operate, and low in preparation cost

Active Publication Date: 2013-02-27
BEIJING NORTH INST OF BIOLOGICAL TECH
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Problems solved by technology

[0003] Disadvantages of immunoaffinity chromatography for antibody purification: ① When preparing immunosorbent, the coupling agent cyanogen bromide is used, which is highly toxic, and special protection is required during operation; ② Activated agarose gel The glue is relatively expensive, and the purchase cost is high during production and preparation

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  • Immune affinity precipitation method for purification of antibodies
  • Immune affinity precipitation method for purification of antibodies
  • Immune affinity precipitation method for purification of antibodies

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example

[0015] Example: Purification of rabbit IgG antibodies by immunoaffinity precipitation.

[0016] ①Purification of rabbit IgG (at this time, rabbit IgG is regarded as an antigen, and the corresponding antibody is: antibody of rabbit IgG): take 10ml rabbit serum, use ammonium sulfate precipitation method for rough extraction, and buffer with 0.01MPB (pH7.4) Liquid dialysis, 4500ml / time, change the dialysate three times altogether, obtain the rabbit IgG13.3ml that concentration is 13.54mg / ml.

[0017] ②The connection between the solid-phase carrier and rabbit IgG: Take 1 g of the solid-phase carrier and centrifuge (3500r / min) with 0.01MPB (pH7.4) buffer to wash 3 times for 10 minutes. 4) buffer solution, the volume of the precipitate after centrifugation of the solid phase carrier is about 19ml. Resuspend the solid-phase carrier with 19ml of 0.01MPB (pH7.4) buffer, take 100μl of the solid-phase carrier suspension that has not been linked to rabbit IgG, and then add 10ml of the ra...

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Abstract

The invention discloses an immune affinity precipitation method for purification of antibodies. The immune affinity precipitation method comprises that copolymer solid-phase microballoons containing acrylamide and acrylic acid as monomers are used as solid-phase carriers; antigens are connected to the solid-phase carriers by a coupling agent of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) so that an immunosorbent is formed; an antibody stock solution needing to be purified is mixed with the immunosorbent and the mixture undergoes a full reaction to produce an antibody-immunosorbent conjugate; the antibody-immunosorbent conjugate is subjected to centrifugal washing so that other unbound mixtures are removed and a pure antibody-immunosorbent conjugate is obtained; a system pH value is reduced so that antibodies are dissociated in the solution; the solution with the dissociated antibodies is subjected to centrifugal separation; and a supernatant is collected and then is dialyzed so that the purified antibodies are obtained. The immune affinity precipitation method has simple processes, is suitable for volume production and realizes preparation of antibodies of which purity reaches to an immune affinity chromatography level.

Description

technical field [0001] The invention is a new method for purifying antibodies in immunochemistry, and mainly relates to the fields of immunochemistry research and medical and health production. Background technique [0002] In many immunochemical research and medical and health production processes, purified antibodies of immunoaffinity chromatography are often required. In order to obtain antibodies with good affinity, strong specificity and high purity, the commonly used purification method is immunoaffinity chromatography. The process of immunoaffinity chromatography is: under normal circumstances, the antigen is first connected to the solid phase carrier - agarose gel (Sepharose 4B or 6B) as an immunosorbent, and then put the immunosorbent into the chromatography column, wash off the unlinked antigen and linker with eluent, then load the antibody mixture to be purified, when the mixture When the antibody is close to the antigen connected to the immunosorbent, the antig...

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Application Information

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IPC IPC(8): C07K16/00C07K1/32
Inventor 贺佑丰袁鹏飞吕东川谷泽亮张庚宽王丁泉
Owner BEIJING NORTH INST OF BIOLOGICAL TECH
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