Preparation method of apolipoprotein b antiserum

A technology for apolipoprotein and antiserum, which is applied in the field of preparation of apolipoprotein B antiserum, can solve the problems of low success rate, high requirements, and difficulty in protein reconstitution, and achieve the improvement of potency and success rate, frequency and time The effect of saving and improving biosecurity

Active Publication Date: 2020-02-28
玉环市南方试剂有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The traditional method of preparing apolipoprotein B is directly centrifuged from human peripheral blood, which requires many times and takes a long time, and the protein is precipitated with half-saturated ammonium sulfate, which makes it difficult to redissolve the protein and requires high requirements. low price, low success rate

Method used

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preparation example Construction

[0031] The preparation steps of the present invention can be specifically divided into: 1) serum pretreatment; 2) extraction of low-density lipoprotein containing apolipoprotein B; 3) preparation of apolipoprotein B antiserum.

[0032] The specific implementation is as follows:

Embodiment 1

[0033] Embodiment one: 1, the extraction of LDL crude product: collect mixed human serum 5L, [after detecting hepatitis B virus surface antigen (HBsAg), hepatitis C virus surface antibody (HCV antibody) and human immunodeficiency virus type Ⅰ and type Ⅱ Antibodies (HIV antibodies) are all negative], add 25ml of 10% polyanions and 250ml of 2M calcium chloride, mix well, place for 30 minutes, 4000 rpm, centrifuge for 20 minutes, the precipitate is LDL and VLDL (the crude product of HDL can be used) Precipitate in the first step of extraction), collect the precipitate and add 200ml of 0.5M potassium oxalate to precipitate polyanions and separate LDL+VLDL. After centrifugation, the supernatant is dialyzed with normal saline and set aside.

[0034] 2. Further purification of LDL components: adjust the dialyzed LDL+VLDL solution with KBr to make the density reach 1.030, use BECKMAN-COULTER L-90K preparative ultracentrifuge, special centrifuge tube (30ml / tube), 50000 RPM, 8°C, centri...

Embodiment 2

[0036] Embodiment two: 1, the extraction of LDL crude product: collect mixed human serum 5L, [after detecting hepatitis B virus surface antigen (HBsAg), hepatitis C virus surface antibody (HCV antibody) and human immunodeficiency virus type Ⅰ and type Ⅱ Antibodies (HIV antibodies) are all negative], add 8% polyanion 50ml and 2M calcium chloride 500ml, mix well, place for 30 minutes, 4000 rpm, centrifuge for 20 minutes, the precipitate is LDL and VLDL (the crude product of HDL can be used) Precipitate in the first step of extraction), collect the precipitate and add 400ml of 0.5M potassium oxalate to precipitate polyanions and separate LDL+VLDL. After centrifugation, the supernatant is dialyzed with normal saline and set aside.

[0037] 2. Further purification of LDL components: Use KBr to adjust the dialyzed LDL+VLDL solution to make the density reach 1.030, use BECKMAN-COULTER L-90K preparative ultracentrifuge, special centrifuge tube (30ml / tube), 45000 RPM, 8°C, centrifuge f...

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Abstract

The invention discloses a method for preparing apolipoprotein B antiserum. According to the technical scheme, the method comprises the following steps: (1) treating serum by using a polyanion compound and divalent metal ions to obtain low density lipoprotein; (2) removing the foreign protein in the low density lipoprotein to leave apolipoprotein B only; and (3) immunizing immune animals by using the apolipoprotein B as an antigen. The method has the advantages that firstly, as a concentrated LDL (Low Density Lipoprotein) rough product is prepared by using the polyanion and the divalent metal ions, the times and the time for later ultracentrifugation are greatly reduced (reduced by 20 times), and a condition is provided for on-scale ApoB extraction; secondly, the dosage of the antigen for the immune animals is reasonably improved, the preparation time of the antiserum is shortened, namely, only a half time of a conventional method is taken, and moreover the immune titer and the success rate are improved when being compared with that of the conventional method.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing an apolipoprotein B antiserum. Background technique [0002] Apolipoprotein B (ApoB) is the main structural protein of low-density lipoprotein (LDL) in human serum, accounting for about 97% of the protein content of LDL. Its content can reflect the level of LDL. The increase of ApoB in serum is the risk of cardiovascular and cerebrovascular diseases factor. Clinically, ApoB determination is often used as an estimation of the risk of cardiovascular and cerebrovascular diseases. All ApoB immunoassay techniques require the use of ApoB antiserum. At present, the international and domestic ApoB assay kits all use the immunoturbidimetric assay (ITA), because it is fast, accurate, precise and suitable for various types of assays. Automatic biochemical analyzer, especially suitable for the determination of large batches of samples. In order to meet the needs of clinic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K16/06
Inventor 杨昌国
Owner 玉环市南方试剂有限公司
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