CN1680446A

CN1680446A - Extraction of immunoglobulin

Info

Publication number: CN1680446A
Application number: CN 200510011244
Authority: CN
Inventors: 宋怀燕
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-01-24
Filing date: 2005-01-24
Publication date: 2005-10-12

Abstract

The invention opened a method of exacting IgG. The method is to put the serum into the ABS then to adjust the pH by the NaOH, next to add the ethyl and PBS, last to add the (NH4)2SO4. The purity of the product is enhanced from 65% to 97%. The method can be used for the serum of rabbit, horse, sheep, chicken and mouse.

Description

A kind of extracting method of immunoglobulin (Ig)

Technical field

The invention discloses the extracting method of a kind of immunoglobulin (Ig) (IgG), specifically is raw material extracts the higher degree immunoglobulin (Ig) under simple condition method with the animal serum.

Background technology

(immunoglobulim Ig) is meant to have antibody activity or the chemical structure sphaeroprotein similar to antibody to immunoglobulin (Ig).Because there is the human electrophoresis to prove that antibody activity is in the gamma Globulin part, so once antibody was referred to as two kinds of (γ) sphaeroprotein in the serum at first.Prove that afterwards antibody is also not all in the γ district; And be positioned at the sphaeroprotein in γ district, also differing to establish a capital has antibody activity.1964, the World Health Organization held ad hoc meeting, will have antibody activity and the sphaeroprotein relevant with antibody and be referred to as immunoglobulin (Ig) (Ig).As myeloma protein, the abnormal immunoglobulin that exists among the patients serums such as macroglobulinemia, cryoglobulinemia and " normal people " naturally occurring immunoglobulin (Ig) subunit etc.Thereby immunoglobulin (Ig) is the notion of structure and chemistry, and antibody is the notion of biology and function.We can say that all antibody all are immunoglobulin (Ig)s, but be not all immunoglobulin (Ig)s all be antibody.

Immunoglobulin (Ig) is prevalent in the Mammals and the mankind's blood, tissue juice and the external secretion, and is widely used in biomedical each field.But in the prior art, what routine was used is ammonium sulfate precipitation method, this method production cost height, production cycle is long, production efficiency is low, and purity difference is in a large amount of leaching process, this method descends difficult simultaneously IgG other foreign protein in addition of removing because of serum haemolysis too much causes the bad quality product that causes of product appearance.In addition, number be 200410029860.4 as Chinese patent application, denomination of invention is: " high-activity immune globulin extracting method in the blood ", and open day is on January 12nd, 2005, publication number CN1563091A, and dna purity has only 65%.

Summary of the invention

Purpose of the present invention is exactly the problem that exists at above-mentioned prior art, and a kind of method of extracting high immunoglobulinlg that provides.

Serum is added acetate buffer in 1: 3 ratio, and mixing stirs, and transfers PH to 4-4.5 with NaOH, press 25-40ul/ml add sad, dilute serum, it is sad to drip with suction pipe, the tinfoil sealed vessel is used in stirring simultaneously.Balance in centrifugal bottle, 4000rpm-6000rpm is centrifugal, elimination buoyant fat in filter, it is standby that precipitation is kept at refrigerator.The weighing liquor capacity is put into container, press the 10%-12% volume ratio and adds PBS by rising, and transfers PH to 7.2-7.6 with NaOH, surveys the amount of NaOH, the calculating overall solution volume: liquor capacity+add NaOH volume=cumulative volume of PBS volume+accent PH.Add dry powder ammonium sulfate, cumulative volume: ml * 0.227g/ml=g was left standstill 8-24 hour by adding volume of ammonium sulfate.Solution to going into centrifuge tube, balance, 4000rpm-6000rpm is centrifugal, outwells supernatant solution, collecting precipitation, dialysis.Solution precipitation adds PBS in the 10%-15% ratio, adds the damping fluid dilution in the 75%-85% ratio, and 8000rpm-6000rpm is centrifugal in the dialysis back, measures protein content behind the membrane filtration.

Use this immunoglobulin (Ig) that extracts of improving one's methods after measured and bring up to 97% from 65%, can be widely used in the immunoglobulin (Ig) that extracts in rabbit anteserum, horse serum, sheep blood serum, chicken serum and the mouse serum than the purity of using the resulting immunoglobulin (Ig) of conventional extracting method.

Advantage of the present invention and positively effect are:

1, present method is simple and easy to do, especially in a large amount of leaching process when using conventional ammonium sulfate precipitation method can't remove effectively that the product appearance that too much causes owing to serum haemolysis is bad to cause quality product decline, use this extracting method can effectively remove the protoheme that exists owing to haemolysis in the serum, significantly improve product appearance.

2, present method can effectively be removed IgG other foreign protein in addition, significantly improves the dna purity of immunoglobulin (Ig).

3, use this extracting method effectively to improve the quality of products, reduce production costs, shorten the production cycle, enhance productivity, help exploiting market rapidly.

4, the immunoglobulin (Ig) primary products of present method extraction are purified according to the needs of different tests research in the biomedical research field again, extensively are used as detection reagent.

5, the immune globulin products that extracts of present method, is convenient to store and carry the purity height.

Embodiment:

The present invention will further specify by embodiment:

Embodiment one

1, serum volume V---1.0L;

2,1 volume serum adds 3 volume acetate buffers;

3, transfer PH to 4 with NaOH;

4, add sad 25ul/ml, dilute serum;

Cumulative volume V---ml * 25ul/ml=---ul

5, sad with the suction pipe dropping, stir the tinfoil sealed vessel simultaneously;

6, with liquid to going in the centrifugal bottle balance, centrifugal 4000rpm, 30 minutes;

7, with supernatant liquor to advancing filter, elimination buoyant fat, precipitation is taken out from bottle and is kept at refrigerator;

8, the weighing liquor capacity is put into appropriate containers, rises (V)---

9, by volume 10% add PBS;

Volume V---x 0.10=---volume V is by being added the PBS amount;

10, transfer PH to 7.2 with NaOH, survey the amount of NaOH---

11, calculate overall solution volume:

Liquor capacity+add NaOH volume=cumulative volume of PBS volume+accent PH;

12, add dry powder ammonium sulfate 0.225g/ml cumulative volume: ml * 0.227g/ml=g is by being added volume of ammonium sulfate;

13, leave standstill 8 hours;

14, solution to going into centrifuge tube, balance, centrifugal 4000rpm, 30 minutes;

15, to falling supernatant solution, collect all precipitations, dialysis;

16, solution precipitation PBS;

10% of about original serum volume

Such as: 1L * 0.10-0.15=100-150ml

1.2L×0.10-0.15＝120-180ml

With the dilution of 75% damping fluid, remainingly be used for washing centrifuge tube;

17, dialysis back 8000rpm is centrifugal;

18, use membrane filtration;

19, measure protein content.

Embodiment two;

1, serum volume V---1.0L;

2,1 volume serum adds 3 volume acetate buffers;

3, transfer PH to 5 with NaOH;

4, add sad 40ul/ml, dilute serum;

Cumulative volume V---ml * 40ul/ml=---ul

5, sad with the suction pipe dropping, stir simultaneously, use the tinfoil sealed vessel;

6, with liquid to going in the centrifugal bottle balance, centrifugal 6000rpm, 30 minutes;

7, with supernatant liquor to advancing filter, elimination buoyant fat, precipitation is kept at refrigerator;

9, add PBS, by volume 12% adds;

Volume V---* 0.10=---volume V is by being added the PBS amount;

10, transfer PH to 7.6 with NaOH, survey the amount of NaOH---

11, calculate overall solution volume:

Liquor capacity+add NaOH volume=cumulative volume of PBS volume+accent PH;

12, add dry powder ammonium sulfate 0.229g/ml cumulative volume: ml * 0.227g/ml=g is by being added volume of ammonium sulfate;

13, leave standstill 24 hours;

14, solution to going into centrifuge tube, balance, centrifugal 6000rpm, 30 minutes;

15,, collect all precipitation dialysis to falling supernatant solution;

16, solution precipitation PBS

15% of about original serum volume

Such as: 1L * 0.10-0.15=100-150ml

2.2L×0.10-0.15＝120-180ml

With the dilution of 85% damping fluid, remainingly be used for washing centrifuge tube;

17, dialysis back 6000rpm/ branch is centrifugal;

18, use membrane filtration;

24, measure protein content.

Claims

1. the extracting method of an immunoglobulin (Ig) is characterized in that: serum is added acetate buffer in proportion, and mixing stirs, and adjusts pH value with NaOH.

2. the extracting method of a kind of immunoglobulin (Ig) according to claim 1, it is characterized in that: serum and acetate salt buffer liquid proportional are 1: 3.

3. the extracting method of a kind of immunoglobulin (Ig) according to claim 1 is characterized in that: adjust pH value 4-4.5 with NaOH.

4. the extracting method of an immunoglobulin (Ig), it is characterized in that: it is sad to add, and balance, centrifugal in centrifugal bottle by rising volume ratio adding PBS, is adjusted pH value with NaOH.

5. the extracting method of a kind of immunoglobulin (Ig) according to claim 4 is characterized in that: press 25-40ul/ml add sad, centrifugal 4000rpm-6000rpm.

6. the extracting method of a kind of immunoglobulin (Ig) according to claim 4 is characterized in that: press the 10%-12% volume ratio and add PBS.

7. the extracting method of a kind of immunoglobulin (Ig) according to claim 4 is characterized in that adjusting pH value 7.2-7.6 with NaOH.

8. the extracting method of an immunoglobulin (Ig) is characterized in that: add dry powder ammonium sulfate by cumulative volume ml * 0.227g/ml=g by being added volume of ammonium sulfate, left standstill 8-24 hour

9. the extracting method of an immunoglobulin (Ig), it is characterized in that: solution is to going into centrifuge tube, and balance, centrifugal 4000rpm-6000rpm outwell supernatant solution, collecting precipitation, dialysis.

10. the extracting method of an immunoglobulin (Ig), it is characterized in that: solution precipitation adds PBS in proportion, adds the damping fluid dilution in proportion, measures protein content behind the centrifugal membrane filtration in dialysis back.

11. the extracting method of a kind of immunoglobulin (Ig) according to claim 10 is characterized in that: solution precipitation is pressed 10%-15% and is added PBS.

12. pressing 75%-85%, the extracting method of a kind of immunoglobulin (Ig) according to claim 10, solution precipitation add damping fluid.