CN102206280A - IgG-IgM polymer and polymerization method thereof - Google Patents
IgG-IgM polymer and polymerization method thereof Download PDFInfo
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Abstract
The invention discloses an IgG-IgM polymer and a polymerization method thereof. The IgG-IgM polymer comprises immunoglobulins of IgG and IgM. Schiff alkali chemical bonds are formed through combinations of aldehyde groups produced by oxidation of hydroxyl groups in polysaccharide groups of one of IgG and IgM, and amino groups of the other one of IgG and IgM. The IgG-IgM polymer is prepared through processes of dialysis, oxidation, terminate oxidation, antibody cross-linking, reduction, purification, etc., and can inhibit effectively non-specific reactions thereby improving an anti-interference capability of a reaction system.
Description
Technical field
The present invention relates to a kind of IgG-IgM polymkeric substance and polymerization process thereof, particularly a kind of IgG-IgM polymkeric substance and polymerization process thereof that strengthens the immunity from interference of external immune response system belongs to biology field.
Background technology
Antigen (antigen) is meant the material that can produce specific immune response in body, after it enters body, can stimulate body to produce antibody (antibody), activated cell immunity.Antigenic molecular weight is generally greater than 5000, and micromolecular haptens (hapten) must with can cause that just body produces specific antibody after macro-molecular protein combines.Antigenic reactivity depends on antigenic determinant (antigenic determinant), or epi-position (epitope).According to antigen molecular size and its protein structure, an antigen molecule can have different determinants.
Antibody be meant can with antigen-specific bonded immunoglobulin (Ig) (immunoglobulin Ig), is divided into five classes, i.e. IgG, IgM, IgA, IgD and IgE, that relevant with immunoassay is IgG and IgM.The IgG monomer of each Y type shape has two antigen-binding sites or valency, and molecular weight is 150000.And the pentamer that IgM is made up of five monomers has 10 antigenic valences, and because the influence of locus only shows as five antigenic valences, the molecular weight of IgM is 900000.
The serial antibody that produces at the same body of the different determinants of same antigen is called polyclonal antibody.And be called monoclonal antibody at the antibody that the same body of same antigen, same determinant produces.The animal derived animal species that is meant that antibody produces of antibody.
Thereby the principle of utilizing the antibody antigen association reaction detects the content of determinand and has been widely used in the vitro diagnostic techniques many decades.And color-developing compounds adds the Enzyme-multiplied immune technique that produces, and makes sensitivity obtain large increase.The adding of isotope tracer and the radioimmunology that produces more steps a stage sensitivity, the processing of consequent radwaste and radioactive rays have hindered further developing of this technology to may injuring that human body causes.The chemiluminescence immunoassay method that the eighties of last century the nineties grows up, the chain type chemical reaction that utilizes enzyme or other markers to cause at the antibody antigen association reaction is measured the photon that discharges in the chemical energy switching process, thereby obtains the content of determinand.Because this chain type chemical reaction can amplify photon signal in how much order of magnitude ground, make that measuring sensitivity has obtained very big raising, thereby enlarged the accuracy that linearity range has been improved high low side simultaneously, this has established the theoretical basis of chemoluminescence in immunodetection.But because each detects the diversity of sample, each is different for its inclusion, and many small molecules or resemblance can disturb normal antibody antigen specific reaction, thereby cause the non-specific binding reaction of antibody.Because chemiluminescent hypersensitivity, if can not effectively suppress this non-specific responding, so inevitably can produce false sun, false yin constipation fruit, particularly in the false positive inaccuracy of linear low side.
Summary of the invention
The invention provides a kind of IgG-IgM polymkeric substance and polymerization process thereof, the interference of small molecules or resemblance when purpose is the reduction immunoassay.
For achieving the above object, first kind of technical scheme that the present invention adopts is: a kind of IgG-IgM polymkeric substance, and described polymkeric substance is made up of IgG and two kinds of immunoglobulin (Ig)s of IgM; The aldehyde radical that generates behind the hydroxyl oxidize in the polysaccharide group of one in two kinds of immunoglobulin (Ig)s of described IgG and IgM combines with another person's amino and generates Schiff alkalization bonding.
For achieving the above object, second kind of technical scheme that the present invention adopts is: a kind of polymerization process of IgG-IgM polymkeric substance, mainly form by the following step:
The first step, dialysis
It is to obtain the IgM immunoglobulin (Ig) damping fluid that concentration is 2mg/ml in 7.2 the phosphate buffered saline buffer that the IgM immunoglobulin (Ig) is dissolved in the pH value, and in the dialysis tubing of packing into then, under 2～8 ℃ of conditions, the pH value is to dialyse 8～15 hours in 7.2 the phosphate buffered saline buffer;
Second step, oxidation
Shift out the IgM immunoglobulin (Ig) damping fluid after the dialysis in the first step, adding the concentration that equates with its volume in the IgM immunoglobulin (Ig) damping fluid after described dialysis then is the sodium periodate aqueous solution of 43mg/ml, make the hydroxyl oxidize reaction back generation aldehyde radical in the polysaccharide group in the IgM immunoglobulin (Ig) obtain the oxidation IgM immunoglobulin (Ig) aqueous solution, the time of described oxidizing reaction is 25～35 minutes, and the temperature condition of described oxidizing reaction is 2～8 ℃;
The 3rd step, termination oxidation
In the second oxidation IgM immunoglobulin (Ig) aqueous solution that obtains of step, add the mass concentration that equates with described sodium periodate aqueous solution volume and be 3% aqueous glycol solution, under 2～8 ℃ of conditions, keep then obtaining stopping the oxidation IgM immunoglobulin (Ig) aqueous solution to stop described oxidizing reaction in 25～35 minutes;
The 4th step, antibody linked
Go on foot adding IgG immunoglobulin (Ig) in the termination oxidation IgM immunoglobulin (Ig) aqueous solution that obtains to the 3rd, pack into then in the dialysis tubing, placing concentration is that 0.05mol/l, pH value are that 9.6 carbonate buffer solution is dialysed and obtained IgG-IgM immunoglobulin (Ig) polymer dope in 8～15 hours, wherein, described IgM immunoglobulin (Ig) and IgG immunoglobulin (Ig) mol ratio between the two are 1:1;
The 5th step, reduction
Take out the IgG-IgM immunoglobulin (Ig) polymer dope that obtains in the 4th step, adding concentration to it is the sodium borohydride aqueous solution of 10mg/ml, carry out reduction reaction and obtained IgG-IgM polymer reduction liquid in 1.5～2.5 hours under 2～8 ℃ of conditions, described IgG-IgM polymer dope and described sodium borohydride aqueous solution volume ratio between the two are 12.5:1;
The 6th step: purify
In the 5th IgG-IgM polymer reduction liquid that obtains of step, add and equate with its volume, and the pH value is 25 ℃ of ammonium sulfate saturated aqueous solutions of 7.6, place to react under 2～8 ℃ of conditions to produce precipitation in 1.5～2.5 hours, adopt the mode of centrifugation to take out this precipitation then;
The 7th step, the 6th precipitation that obtain of step is dissolved in the Tris-HCl damping fluid that the pH value is 7.4 0.05mol/l, be diluted to concentration and be and pack into behind the 2mg/ml in the dialysis tubing, then under 2～8 ℃ of conditions, placing the pH value is that the Tris-HCl damping fluid of 7.4 0.05mol/l is dialysed and promptly got the IgG-IgM polymkeric substance in 8～15 hours.
Related content in the technique scheme is explained as follows:
In the such scheme, the IgG-IgM polymkeric substance that obtains in described the 7th step is mixed with the glycerine that its volume equates, preserve then with under subzero 20 ℃ of conditions.
Principle of work of the present invention is:
Because the technique scheme utilization, the present invention compared with prior art has following advantage and effect:
What prior art generally adopted is to add in reaction system and the common immunoglobulin (Ig) that detects antibody same animals source property, principle by the non-material-specific of competitive adsorption, at utmost suppress the generation of non-specific responding, owing to spatial configuration of molecules, its effect is unsatisfactory in the time of many.The IgM-IgG polymerization physical efficiency that the present invention obtains suppresses non-specific responding effectively, thereby improves the immunity from interference of reactive system.
Description of drawings
Accompanying drawing 1 is the influence synoptic diagram of IgG monomer to typical curve for the IgG-IgM polymkeric substance.
Embodiment
Below in conjunction with drawings and Examples the present invention is further described:
Embodiment: a kind of IgG-IgM polymkeric substance and polymerization process thereof
A kind of polymerization process of IgG-IgM polymkeric substance, mainly form by the following step:
The first step, dialysis
It is to obtain the IgM immunoglobulin (Ig) damping fluid that concentration is 2mg/ml in 7.2 the phosphate buffered saline buffer that the IgM immunoglobulin (Ig) is dissolved in the pH value, in the dialysis tubing of packing into then, and under 2～8 ℃ of conditions, dialysed overnight in the phosphate buffered saline buffer of pH value 7.2 (12 hours);
Second step, oxidation
Shift out the IgM immunoglobulin (Ig) aqueous solution after the dialysis in the first step, adding the concentration that equates with its volume in the IgM immunoglobulin (Ig) aqueous solution after described dialysis then is the sodium periodate aqueous solution of 43mg/ml, make the hydroxyl oxidize reaction back generation aldehyde radical in the polysaccharide group in the IgM immunoglobulin (Ig) obtain the oxidation IgM immunoglobulin (Ig) aqueous solution, the time of described oxidizing reaction is 30 minutes, and the temperature condition of described oxidizing reaction is 4 ℃;
The 3rd step, termination oxidation
Add the mass concentration that equates with described sodium periodate aqueous solution volume and be 3% aqueous glycol solution in the second oxidation IgM immunoglobulin (Ig) aqueous solution that obtains of step, maintenance 30 minutes obtains stopping the oxidation IgM immunoglobulin (Ig) aqueous solution to stop described oxidizing reaction under 4 ℃ of conditions then;
The 4th step, antibody linked
Go on foot adding mouse IgG immunoglobulin (Ig) in the termination oxidation IgM immunoglobulin (Ig) aqueous solution that obtains to the 3rd, pack into then in the dialysis tubing, placing concentration is that 0.05mol/l, pH value are that 9.6 carbonate buffer solution dialysed overnight (12 hours) obtains IgG-IgM immunoglobulin (Ig) polymer dope, wherein, described IgM immunoglobulin (Ig) and IgG immunoglobulin (Ig) mol ratio between the two are 1:1;
The 5th step, reduction
Take out the IgG-IgM immunoglobulin (Ig) polymer dope that obtains in the 4th step, adding concentration to it is the sodium borohydride aqueous solution of 10mg/ml, carry out reduction reaction and obtained IgG-IgM polymer reduction liquid in 2 hours under 4 ℃ of conditions, described IgG-IgM polymer dope and described sodium borohydride aqueous solution volume ratio between the two are 12.5:1;
The 6th step: purify
In the 5th IgG-IgM polymer reduction liquid that obtains of step, adds and equate, and the pH value is 7.6 ammonium sulfate saturated aqueous solution, place and react generation in 2 hours under 4 ℃ of conditions and precipitate, adopt the mode of centrifugation to take out this precipitation then with its volume;
The 7th step, the 6th precipitation that obtain of step is dissolved in the Tris-HCl damping fluid that the pH value is 7.4 0.05mol/l, be diluted to concentration and be and pack into behind the 2mg/ml in the dialysis tubing, then under 4 ℃ of conditions, placing the pH value is that the Tris-HCl damping fluid dialysed overnight (12 hours) of 7.4 0.05mol/l promptly gets the IgG-IgM polymkeric substance.
The 8th goes on foot, the IgG-IgM polymkeric substance that obtains in described the 7th step is mixed with the glycerine that its volume equates, preserves then with under subzero 20 ℃ of conditions.
The structure of described IgG-IgM polymkeric substance is: described polymkeric substance is made up of IgG and two kinds of immunoglobulin (Ig)s of IgM; The aldehyde radical that generates behind the hydroxyl oxidize in the polysaccharide group of one in two kinds of immunoglobulin (Ig)s of described IgG and IgM combines with another person's amino and generates Schiff alkalization bonding.
The pH value is the tris-HCI buffer compound method of 7.4 0.05mol/l: behind 50ml 0.1mol/L Tutofusin tris (Tris) solution and the 42.0 ml 0.1mol/L hydrochloric acid mixings, thin up is to 100ml.Saturated (NH4)
2SO
4Be preformulation, saturated (NH4)
2SO
4Need to transfer PH to 7.6 with ammoniacal liquor.
Precaution:
When one, taking by weighing Powdered pharmaceutical chemicals, it is accurate to guarantee weighing to notice that gesture is slowly closed exhaust system as far as possible in case of necessity.
Two, check that the required pharmaceutical chemicals that takes by weighing is whether all in time limit of service.
Three, check whether some special pharmaceutical chemicals makes moist or lump.
Four, treat that all pharmaceutical chemicals weighings finish after, the arrangement weighing room to keep clean.
Five, NaIO
4, ethylene glycol, NaBH
4All need fresh preparation.
Six, drip NaIO
4The time, NaIO
4Need to drip uniformly while vibrating.
Seven, pre-operational check:
Whether electronic weighing scale surface pallet is clean.
Whether weighing scoop is clean.
Whether required reagent is all in time limit of service.
Verification method:
A) absorbance method: cross-linking products, IgG-IgM polymkeric substance, monomer I gG and IgM have different absorption peaks.Can carry out the crosslinked result of preliminary identification thus.
B) purifying: gel column and affinity column method are carried out purifying, are verified the purity of possibility polymkeric substance again by electrophoretic technique.
C) functional experiment: polymkeric substance is added antibody antigen reaction system (chemoluminescence method) to verify its immunity from interference in system.Adding common homoimmune sphaeroprotein, and do not add any immunoglobulin (Ig) and be contrast.
The result:
This reaction system adopts double antibody sandwich method as detection means.Two antibody wherein are respectively mouse and cavy source property.
Double antibody sandwich method: the mark of the immobilization of antigen or antibody and antigen or antibody.The antigen or the antibody that are combined in surface of solid phase carriers still keep its immunologic competence, and the antigen of mark or antibody had both kept its immunologic competence, again the catalytic activity of retention marker thing.Antigen or the antibody of being examined sample and surface of solid phase carriers react.With the method for washing the immune complex that forms on the solid phase carrier and other materials in the liquid are separated.The antigen or the antibody that add mark also are combined on the solid phase carrier by reaction.After adding the substrate of marker, substrate is produced chain reaction by catalysis and launches photon, and the amount that photon produces is directly related with the amount of examined object matter in the sample, carries out qualitative or quantitative analysis according to measuring relative photon unit.The catalytic efficiency of marker is very high, has greatly amplified immunoreactive result, makes measuring method reach very high susceptibility.
Competition law: principle is that antigen and the competition of a certain amount of enzyme-labelled antigen in the sample combines with insolubilized antibody.In the sample antigen amount content the more, the enzyme-labelled antigen that is combined on the solid phase is fewer, last colour developing is also more shallow, or the photon that produces in chemiluminescence reaction is fewer.
The result is shown in following five forms and accompanying drawing 1:
Table 1: standard control (no immunoglobulin (Ig)) (RLU*)
| Standard antigen mg/ml | ? | ? | ? | ? | On | % | |
| 0 | 36909 | 19336 | 30714 | 28936 | 28974 | 4.16% | |
| 0.1 | 33483 | 38495 | 29549 | 39573 | 35275 | 5.06% | |
| 1 | 107736 | 86689 | 95708 | 114214 | 101087 | 14.50% | |
| 10 | 737074 | 698688 | 664681 | 688792 | 697309 | 100.00% |
Table 2:IgG monomer (0.1mg/ml) (RLU*)
| Standard antigen mg/ml | ? | ? | On | % | |
| 0 | 23365 | 41860 | 32613 | 5.80% | |
| 0.1 | 43272 | 56195 | 49734 | 8.84% | |
| ?1 | 97131 | 92634 | 94883 | 16.86% | |
| 10 | 582185 | 543300 | 562743 | 100.00% |
Table 3:IgG-IgM (not purifying) is (RLU*) (0.1mg/ml)
| Standard antigen mg/ml | ? | ? | On | % | |
| 0 | 24390 | 56151 | 40271 | 7.03% | |
| 0.1 | 39547 | 45945 | 42746 | 7.46% | |
| 1 | 96332 | 68353 | 82343 | 14.37% | |
| 10 | 569838 | 575837 | 572838 | 100.00% |
Table 4:IgG-IgM (purifying) is (RLU*) (0.1mg/ml)
| Standard antigen mg/ml | 0.1mg/ml | 0.1mg/ml | On | % | |
| 0 | 20755 | 20300 | 20528 | 2.70% | |
| 0.1 | 31240 | 28217 | 29729 | 3.92% | |
| 1 | 84722 | 77532 | 81127 | 10.68% | |
| 10 | 843986 | 674684 | 759335 | 100.00% |
| Difficult sample | Standard control (RLU*) | Standard control (RLU*) | Standard control (RLU*) | The IgG monomer | IgG-IgM | IgG-IgM |
| ? | ? | ? | On average | Purifying not | Purifying | |
| 31# | 64905 | 110619 | 87762 | 20667 | 15457 | 12096 |
| 116# | 27350 | 39257 | 33304 | 13023 | 11176 | 7649 |
| 118# | 19520 | 16631 | 18076 | ------- | ------- | 6817 |
| 312# | 25003 | 18058 | 21531 | 21105 | 14608 | 9627 |
| 828# | 32125 | 76014 | 54070 | 16709 | 14993 | 13426 |
Annotate: what RLU represented is relative photon unit.
Can see from the result of top five forms and accompanying drawing 1:
Have three oblique lines in the accompanying drawing 1 from top to bottom, going up one most is IgG-IgM polymkeric substance (purifying), the centre be standard control (no immunoglobulin (Ig)) next root be the coincidence line of two lines of IgG monomer and IgG-IgM polymkeric substance (purifying).
For the typical curve that is come by standard antigen: the reactive system that adds IgG purification-IgM polymkeric substance has the curve of maximum slope.The sensitiveest at low concentration region, and linearity range is the widest.
All difficult samples show false positive or approximate false positive results in the standard reaction system that does not have immunoglobulin (Ig) to add.But add in the reaction system of different immunoglobulin (Ig)s at all, these samples all demonstrate negative essence.And it is best in this effect of inhibition nonspecific immunity reaction that wherein adds IgG purification-IgM.Generally, the immunity from interference of each material: IgG-IgM (purifying)〉IgG-IgM (not purifying)〉the IgG monomer.
Conclusion: the IgG-IgM polymkeric substance can strengthen the immunity from interference of external immune response system effectively.
The foregoing description only is explanation technical conceive of the present invention and characteristics, and its purpose is to allow the personage who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalences that spirit is done according to the present invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (3)
1. IgG-IgM polymkeric substance, it is characterized in that: described IgG-IgM polymkeric substance is made up of IgG and two kinds of immunoglobulin (Ig)s of IgM; The aldehyde radical that generates behind the hydroxyl oxidize in the polysaccharide group of one in two kinds of immunoglobulin (Ig)s of described IgG and IgM combines with another person's amino and generates Schiff alkalization bonding.
2. the polymerization process of an IgG-IgM polymkeric substance is characterized in that: mainly be made up of the following step:
The first step, dialysis
It is to obtain the IgM immunoglobulin (Ig) damping fluid that concentration is 2mg/ml in 7.2 the phosphate buffered saline buffer that the IgM immunoglobulin (Ig) is dissolved in the pH value, and in the dialysis tubing of packing into then, under 2～8 ℃ of conditions, the pH value is to dialyse 8～15 hours in 7.2 the phosphate buffered saline buffer;
Second step, oxidation
Shift out the IgM immunoglobulin (Ig) damping fluid after the dialysis in the first step, adding the concentration that equates with its volume in the IgM immunoglobulin (Ig) damping fluid after described dialysis then is the sodium periodate aqueous solution of 43mg/ml, make the hydroxyl oxidize reaction back generation aldehyde radical in the polysaccharide group in the IgM immunoglobulin (Ig) obtain the oxidation IgM immunoglobulin (Ig) aqueous solution, the time of described oxidizing reaction is 25～35 minutes, and the temperature condition of described oxidizing reaction is 2～8 ℃;
The 3rd step, termination oxidation
In the second oxidation IgM immunoglobulin (Ig) aqueous solution that obtains of step, add the mass concentration that equates with described sodium periodate aqueous solution volume and be 3% aqueous glycol solution, under 2～8 ℃ of conditions, keep then obtaining stopping the oxidation IgM immunoglobulin (Ig) aqueous solution to stop described oxidizing reaction in 25～35 minutes;
The 4th step, antibody linked
Go on foot adding IgG immunoglobulin (Ig) in the termination oxidation IgM immunoglobulin (Ig) aqueous solution that obtains to the 3rd, pack into then in the dialysis tubing, placing concentration is that 0.05mol/l, pH value are that 9.6 carbonate buffer solution is dialysed and obtained IgG-IgM immunoglobulin (Ig) polymer dope in 8～15 hours, wherein, described IgM immunoglobulin (Ig) and IgG immunoglobulin (Ig) mol ratio between the two are 1:1;
The 5th step, reduction
Take out the IgG-IgM immunoglobulin (Ig) polymer dope that obtains in the 4th step, adding concentration to it is the sodium borohydride aqueous solution of 10mg/ml, carry out reduction reaction and obtained IgG-IgM polymer reduction liquid in 1.5～2.5 hours under 2～8 ℃ of conditions, described IgG-IgM polymer dope and described sodium borohydride aqueous solution volume ratio between the two are 12.5:1;
The 6th step: purify
In the 5th IgG-IgM polymer reduction liquid that obtains of step, add and equate with its volume, and the pH value is 25 ℃ of ammonium sulfate saturated aqueous solutions of 7.6, place to react under 2～8 ℃ of conditions to produce precipitation in 1.5～2.5 hours, adopt the mode of centrifugation to take out this precipitation then;
The 7th step, the 6th precipitation that obtain of step is dissolved in the Tris-HCl damping fluid that the pH value is 7.4 0.05mol/l, be diluted to concentration and be and pack into behind the 2mg/ml in the dialysis tubing, then under 2～8 ℃ of conditions, placing the pH value is that the Tris-HCl damping fluid of 7.4 0.05mol/l is dialysed and promptly got the IgG-IgM polymkeric substance in 8～15 hours.
3. the polymerization process of IgG-IgM polymkeric substance according to claim 2 is characterized in that: the IgG-IgM polymkeric substance that obtains in described the 7th step is mixed with the glycerine that its volume equates, preserve then with under subzero 20 ℃ of conditions.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104193825A (en) * | 2014-08-28 | 2014-12-10 | 上海昶达生物科技有限公司 | Novel IgM-IgG polymer and polymerization method thereof |
| CN105628941A (en) * | 2015-12-22 | 2016-06-01 | 北京康彻思坦生物技术有限公司 | Method for preparing IgM serological test quality control material |
-
2011
- 2011-04-25 CN CN201110103790.2A patent/CN102206280B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 邓光辉 等: "毛细管电泳免疫分析法研究鼠IgM", 《理化检验-化学分册》 * |
| 郑南才 等: "双特异性嵌合抗体的制备及斑点红细胞免疫技术的建立", 《安徽医科大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104193825A (en) * | 2014-08-28 | 2014-12-10 | 上海昶达生物科技有限公司 | Novel IgM-IgG polymer and polymerization method thereof |
| CN105628941A (en) * | 2015-12-22 | 2016-06-01 | 北京康彻思坦生物技术有限公司 | Method for preparing IgM serological test quality control material |
| CN105628941B (en) * | 2015-12-22 | 2017-09-22 | 北京康彻思坦生物技术有限公司 | A kind of preparation method of IgM Serologic detections Quality Control thing |
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Effective date of registration: 20170720 Address after: 315699 Zhejiang city of Ningbo province Ninghai County Yuelong streets Xinghai Road No. 439 Patentee after: Ningbo reach Biological Technology Co., Ltd. Address before: 200082 room 56, building 2103, No. 608, Pingliang Road, Shanghai, Yangpu District Patentee before: SHANGHAI CHANGDA BIOLOGICAL TECHNOLOGY CO., LTD. |