Process for purifying human retinol binding protein and preparation process of polyclonal antibody thereof

A polyclonal antibody and protein-binding technology, applied in the field of medical immunity, can solve the problems of complex treatment of prealbumin antibody, difficult industrialization, and unsatisfactory extraction effect.

Active Publication Date: 2014-12-10
桂林英美特生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing purification techniques for human RBP mainly include: use ammonium sulfate salting out, and then separate and purify human plasma RBP through DEAE-Sephadex A25 column, SephadexG200, Ultrogel ACA44 column and Sephadex G100 column chromatography, or separate and purify human plasma RBP The complex of RBP and prealbumin was purified by Sepharose 4B gel column containing prealbumin antibody. The yield of antigen obtained by this method is high, but the treatment of prealbumin antibody is complicated and requires a lot of experiments. Finding the best eluent components is not easy for industrialization
The other is to subject the RBP protein liquid derived from urine to acetyl-dextran gel column chromatography and DEAE-dextran gel column chromatography to obtain human RBP antigen, but the extraction effect is not ideal

Method used

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  • Process for purifying human retinol binding protein and preparation process of polyclonal antibody thereof
  • Process for purifying human retinol binding protein and preparation process of polyclonal antibody thereof
  • Process for purifying human retinol binding protein and preparation process of polyclonal antibody thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Purification of Immunogen (RBP Antigen)

[0045] 1. Instruments and columns used:

[0046] 1.1 Instruments: simple protein purification system (including constant flow pump, UV detector, recorder) (Amersham Pharmacia Biotech Inc), GradiFrac TM Programming (Amersham Pharmacia Biotech Inc), Waters600 high-phase liquid chromatography (Waters, USA), etc. .

[0047] 1.2 Column: ion exchange column DEAE-Sepharose Fast Flow (DEAE-S), molecular sieve (Superdex 75, Sephacryls-200), hydrophobic column Phenyl Sepharose TM High Performance (PSHP) and CNBr-activated Sepharose 4B are produced by GE Healthcare; Protein Pak Glass 300SW is produced by waters.

[0048] 2. Pretreatment of urine in patients with renal impairment

[0049] 2.1 Centrifuge the patient's urine for 30 minutes (4000r / min), and take the supernatant;

[0050] 2.2 Add solid SAS (ammonium sulfate) to the supernatant to make the saturation reach 60%, and let stand overnight;

[0051] 2.3 Centrifuge the...

Embodiment 2

[0083] Example 2 Purification of Immunogen (RBP Antigen)

[0084] 1. The pretreatment of urine from patients with renal impairment is the same as in Example 1

[0085] 2. Separation and purification:

[0086] 2.1 Preparation of mobile phase:

[0087] Buffer A: 25mmol / L Tris pH=7.5+0.01%NaN 3 +1mM EDTA;

[0088] Measure 312.5mL 200mmol / L Tris pH=7.5, 1.25mL 20%aN 3 , 5mL 500mM EDTA mixed, add ultrapure water to 2.5L, mix well, that is.

[0089] Buffer B1: 20mmol / L Tris pH=8.0+50mmol / L NaCl+0.03%NaN 3 +1mM EDTA;

[0090] Measure 250mL 200mmol / L Tris pH=8.0, 31.25mL 4mol / L NaCl, 3.75mL 20%NaN 3 , 5mL 500mM EDTA mixed, add ultrapure water to 2.5L, mix well, that is.

[0091] Buffer B2: 15mmol / L Tris pH=8.0+250mmol / L NaCl+0.01%NaN 3 +1mM EDTA;

[0092] Measure 250mL 187.5mmol / L Tris pH=8.0, 156.25mL 4mol / L NaCl, 1.25mL 20%NaN 3 , 5mL 500mM EDTA mixed, add ultrapure water to 2.5L, mix well, that is.

[0093] Buffer C: 20mmol / L Tris pH=8.0+120mmol / L NaCl+0.03%NaN 3 +1mM ...

Embodiment 3

[0114] Example 3 Preparation of goat anti-human RBP polyclonal antibody

[0115] 1. Immunization of goats: Boer goats were selected for subcutaneous immunization (implementation of RBP antigen prepared in 1+Freund's adjuvant) injection.

[0116]2. Collect goat antiserum: collect goat antiserum and perform immunoelectrophoresis on the antiserum. If there are bands of immunoglobulins and albumin, remove the collected antiserum by CNBr-activated Sepharose 4B column coupled with normal human serum. Impurity bands of immunoglobulins and albumin are obtained to obtain goat antiserum after removal of impurities.

[0117] 3. Purification of the goat anti-human RBP polyclonal antibody: Precipitate the goat anti-human ascites globulin in the goat antiserum after the impurity removal with ammonium sulfate solution with a saturation of 50%, centrifuge at 8000r / min for 30min, take the precipitate and dialyze it for desalination. Purified by DEAE-Sepharose Fast Flow (DEAE-S) ion exchange c...

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Abstract

The invention discloses a process for purifying a human retinol binding protein and a preparation process of a polyclonal antibody thereof. According to the invention, by using a separation technology by multiple chromatographic columns such as a DEAE-Sepharose Fast Flow (DEAE-S) ion exchange column, a molecular sieve (Superdex 75, Sephacryls-200) and a hydrophobic column Phenyl SepharoseTM High Performance (PHSP), the human retinol binding protein is successfully purified from urine of a patient suffering from renal injury, and the immunogenicity of the RBP protein is remained to the greatest degree. The obtained human RBP antigen is used for immunizing animals, thus obtaining a polyclonal antibody of the human retinol binding protein. The obtained polyclonal antibody of the human retinol binding protein can be applied to an immunonephelometry kit.

Description

technical field [0001] The invention relates to the technical field of medical immunity, in particular to the purification process of human retinol binding protein and the preparation process of polyclonal antibody. Background technique [0002] Retinol Binding Protcin (RBP) is a transporter of retinol (vitamin A) in blood. In 1961, Berggard discovered in immunoelectrophoresis a protein that can form a long precipitation line in the α2-globulin region, which was once called long α2-globulin. In the following decades, people conducted a comprehensive study on the molecular structure and biological characteristics of this protein, and found that this protein is widely distributed in normal human body fluids, belongs to α1-globulin, and has the ability to transport from liver cells. Function of retinol to surrounding tissues. At present, it is believed that RBP in the blood mainly exists in the form of complexes bound to retinol and prealbumin. When retinol in the complex bin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/36C07K1/30C07K1/16C07K16/18
CPCC07K14/4702C07K16/06C07K16/18Y02P20/54
Inventor 蔡豪斌李珏燕粟晓玲
Owner 桂林英美特生物技术有限公司
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