CN105628941B - A kind of preparation method of IgM Serologic detections Quality Control thing - Google Patents
A kind of preparation method of IgM Serologic detections Quality Control thing Download PDFInfo
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
It is a kind of by the use of polysaccharide as carrier, the coupling method reacted by using sodium metaperiodate sodium borohydride the invention discloses a kind of preparation method of IgM Serologic detections Quality Control thing.Carrier is used as by using the polysaccharide of macromolecule, the hydroxyl in polysaccharide is aoxidized with sodium periodate method, it is set to be oxidized to aldehyde radical, activation aldehyde radical on one poly glycan molecule is significantly larger than 2 active aldehyde radicals on glutaraldehyde molecules, therefore two kinds of impurity during glutaraldehyde two-step method mark are avoided the occurrence of, so that it is active to improve using for final product.The coupled product prepared using the method for the invention can be used for the quality control of IgM Serologic detection, and its effect marks obtained coupled product better than glutaraldehyde method.
Description
Technical field
The present invention relates to immune labeled analysis field, and in particular to a kind of preparation method of IgM Serologic detections Quality Control thing.
Background technology
The positive and negative quality-control product that infectious disease needs when detecting are used for the process control for detecting experiment, it is ensured that most terminate
The correctness and accuracy of fruit output.Negative quality-control product is generally by the serum for normal person, not comprising any purpose that need to be detected
Antibody.Positive quality control product is typically to be used by the serum containing the purpose antibody that need to largely detect or blood plasma obtained from the patient
Negative quality-control product, which is diluted, to be prepared from.
It is well known that IgM antibody can be produced when communicable disease antigen invades the first time immune response produced after body
It is raw.IgM antibody can only exist in the period one shorter immune response early stage, typically can be in several thoughtful some months.IgG antibody
Immune response produce and duration all can be in relatively long time, generally several years.
IgM antibody is that primary immune response is produced, therefore, and its duration is very of short duration, but the inspection of IgM antibody
Survey is very important, be can detect that in patient's early infection.However, when continuing due to producing IgM after patient's infection
Between it is short, it is difficult to obtained from individual abundance IgM antibody positive blood be used for infectious disease IgM detect Quality Control.Therefore need to use
The specific IgG antibodies of animal as the positive quality control (less understanding, can delete seemingly) in detection herein, just as
Described in WO89/10980.The method of the operation preparation IgM antibody using immune animal is recorded in WO89/10980, wherein needing
Can prepare corresponding IgM antibody up to the times of 15 days, and need to feed corresponding animal body, required time and
Cost is all extremely huge.
IgM antibody in the common measure serum such as prize law in this area, also can be by IgM and animal derived specific antibody
Obtained prepared product be coupled as the positive reference of Quality Control.Needed animal derived special IgG due to the prepared product and
The non-specific IgM in people source coupling, is coupled more than the method being coupled at present, this method using the classical way i.e. glutaraldehyde of albumen coupling
It is coupled using a kind of conventional homotype bi-functional cross-linking agent-glutaraldehyde, its two aldehyde radicals can be formed with the amino of albumen
Schiff keys (- N=C-), they are connected with five carbon bridges.But because it is non-specific two-way crosslinking agent, therefore exist
Ratio between corsslinking molecular is not strict, and size also differs, the low shortcoming of specific Conjugate ratio.Animal sources are carried out using glutaraldehyde method
Property special IgG and people source non-specific IgM couplings when, except generating the animal derived non-specific IgM products in special IgG-people source
Outside, animal derived special IgG-animal derived special IgG and the non-specific IgM in people source-people source is also inevitably generated
Non-specific two kinds of impurity products of IgM, and be difficult to isolate and purify, have a strong impact on activity during final use.
The content of the invention
For the coupling method used in the prior art coupled product can be made to produce impurity, and further influence coupled product
Final active defect when using.The present invention provides a kind of new coupling technology, the new coupling technology that the present invention is provided
Coupling available for animal derived special IgG and the non-specific IgM in people source.
Ratio when this coupling technology can overcome the glutaraldehyde to be coupled between corsslinking molecular is not strict, and size also differs, specifically
Property the low shortcoming of Conjugate ratio, product almost all is animal sources when making animal derived special IgG and the non-specific IgM couplings in people source
Special IgG-non-specific IgM in people source of property, and seldom there is animal derived special IgG-animal derived special IgG and people
The non-specific IgM in source-people source two kinds of impurity of non-specific IgM, so as to improve the activity of final product.Using the method for the invention
The coupled product prepared can be used for the quality control of IgM Serologic detection, and its effect is marked better than glutaraldehyde method
The coupled product arrived.
The present invention is adopted the following technical scheme that：
The invention discloses a kind of coupled complex, the coupled complex includes coupling carrier and be coupled at least two
Antibody is planted, the coupling carrier has the conjugation sites with the antibody coupling of more than 2.
In a detailed embodiment, the antibody includes first antibody and secondary antibody, and the first antibody is
The special IgG of material resource, the non-specific IgM in secondary antibody behaviour source, the coupling carrier are polysaccharide.
The invention discloses a kind of preparation method of IgM Serologic detections Quality Control thing, it is characterised in that is made using polysaccharide
For carrier, animal derived special IgG and the non-specific IgM in people source are coupled.
The invention discloses a kind of preparation method of IgM Serologic detections Quality Control thing, it is characterised in that methods described will be many
The hydroxyl of glycan is oxidized to after aldehyde radical, and the animal derived special IgG and non-specific IgM in people source is carried out with the polysaccharide after oxidation
Coupling.
The preparation method of IgM Serologic detections Quality Control thing disclosed by the invention uses a kind of polysaccharide as crosslinking carrier,
It is crosslinked by using sodium metaperiodate-sodium borohydride method, methods described comprises the following steps：
(1) polysaccharide and sodium metaperiodate (NaIO are weighed4), dissolve standby with carbonate buffer solution respectively；
(2) polysaccharide solution is placed in 2-8 DEG C on agitator and at the uniform velocity stirred, then NaIO4 solution is slowly dropped to many
In glycan solution, lucifuge stirring reaction 60min；
(3) ethylene glycol is diluted with water 10 times, step (2) well-oxygenated polysaccharide is taken out from refrigerator, addition dilutes
Ethylene glycol, room temperature lucifuge reaction 30min；
(4) polysaccharide solution that aspiration step (3) has been activated is added to equipped with animal derived special IgG and the non-spy in people source
In different IgM bag filter, it is well mixed, lucifuge is dialysed in pH9.6 concentration is 0.05M carbonate buffer solutions, is changed after 2 hours
Buffer solution, reacts 15-17 hours；
(5) conjugate is suctioned out into clean glass container, then to sodium borohydride (NaHB4) is added in container, mixed
Close uniform, 2-8 DEG C of lucifuges are reacted 2 hours；
(6) after the reactant concentrating and precipitating for obtaining step (5), redissolve and prepare coupled product.
Wherein, the method for reactant concentrating and precipitating can be used into ammonium sulfate precipitation.
Wherein, the step of described ammonium sulfate precipitation is as follows：The reactant that step (5) is obtained is placed on stirring in 2-8 DEG C
At the uniform velocity stirred on device, isometric pH 7.4 saturated ammonium sulfate is slowly added dropwise, after being well mixed, lucifuge, standing 60min will be heavy
The well mixed loading centrifuge tube of reactant behind shallow lake, 4 DEG C of 12000rpm/min centrifuge 10min, abandon supernatant, stay precipitation.
It is preferred that, polysaccharide the step of the inventive method in (1) is glucan, mean molecule quantity from 1000 to
2800000 can be used, and optimal molecular weight ranges are 40000 to 1000000, concentration range can be 5- after dissolving
50mg/ml, optium concentration is 20mg/ml.
It is preferred that, concentration range can be 5-50mg/ after NaIO4 used dissolving in the method for the invention step (1)
Ml, optium concentration is 25mg/ml.
It is preferred that, the special IgG of animal sources and the non-specific IgM in people source mol ratio in the method for the invention step (4)
Example scope can be from 1:10 to 10:1, optimum molar ratio is 1:1.
It is preferred that, in the method for the invention step (5) NaHB4 ultimate density scope can be 0.0001 to
0.01mg/ml, optium concentration is 0.025mg/ml.
Animal derived monoclonal antibody mouse is resisted by the use of glucan as carrier in addition, the present invention specifically provides one kind
The method that HEV monoclonal antibodies S-13 and nonspecific people IgM is coupled.
In addition, what the coupled product prepared using method of the present invention was prepared with conventional glutaraldehyde method
Conjugate, which is compared, has higher activity, and therefore, the coupled product prepared using the method for the invention falls within this hair
The bright content of the invention.
The present invention, as carrier, the hydroxyl in polysaccharide is aoxidized with sodium periodate method by using the polysaccharide of macromolecule
Base, makes it be oxidized to aldehyde radical, and a molecular weight can form 245-6125 in theory for 40000 to 1000000 dextran molecule
Activation aldehyde radical on the aldehyde radical of individual activation, a molecule is significantly larger than 2 active aldehyde radicals on glutaraldehyde molecules, is added into animal
After the special IgG and the non-specific IgM in people source in source, as a consequence it is hardly possible to form the poly skeleton that is all crosslinked by specific IgG and entirely
The poly carbon skeleton that portion is crosslinked by the non-specific IgM in people source, therefore two kinds avoided the occurrence of when similar glutaraldehyde two-step method is marked are miscellaneous
Matter, so that it is active to improve using for final product.
Embodiment
Technical solution of the present invention can be implemented using following operating procedure.
(1) appropriate glucan and appropriate sodium metaperiodate (NaIO are weighed first4), with 0.05M pH=9.6 carbonate
Buffer solution (CB) dissolves polysaccharide and NaIO4 is standby, and wherein glucan mean molecule quantity can make from 1000 to 2800000
With optimal molecular weight ranges are 40000 to 1000000, and concentration range can be 5-50mg/ml after dissolving, and optium concentration is
Concentration range can be 5-50mg/ml after 20mg/ml, NaIO4 dissolving, and optium concentration is 25mg/ml；
(2) polysaccharide solution is placed on agitator (2-8 DEG C) and at the uniform velocity stirred, then by the NaIO4 solution prepared with one milli
Rise sample loading gun to be drop by drop slowly added into polysaccharide solution, lucifuge stirring reaction 60min；
(3) shift to an earlier date 5min by ethylene glycol water for injection dilute 10 times it is standby, then by well-oxygenated polysaccharide from refrigerator
Take out, and add the ethylene glycol diluted, room temperature (16-25 DEG C) lucifuge reaction 30min；
(4) the appropriate polysaccharide solution activated of absorption is added to non-specific equipped with animal derived special IgG and people source
In IgM bag filter, it is well mixed, is put into lucifuge in pH9.62L 0.05M carbonate buffer solutions and dialyses, one is changed after 2 hours
Secondary buffer solution, lucifuge dialysed overnight reacts 15-17 hours, the wherein special IgG of animal sources and the non-specific IgM in people source mole
Proportion can be from 1:10 to 10:1, optimum molar ratio is 1:1；
(5) conjugate is suctioned out into clean glass container, the sodium borohydride that appropriate concentration is then is added thereto
(NaHB4), 2-8 DEG C of lucifuges are well mixed to react 2 hours；Wherein NaHB4 ultimate density scope can be 0.0001 to
0.01mg/ml, optium concentration is 0.025mg/ml.
(6) conjugate is placed on agitator (2-8 DEG C) and at the uniform velocity stirred, while drop by drop being delayed with one milliliter of sample loading gun
Slowly isometric pH 7.4 saturated ammonium sulfate is added, after being well mixed, lucifuge, standing 60min；
(7) load centrifuge tube by the label after precipitation is well mixed, by centrifuge set to 4 DEG C of 12000rpm/min from
Heart 10min, abandons supernatant, stays precipitation；
(8) conjugate of precipitation 20%NBS (NBCS) is redissolved to 1mg/ml.
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but do not limit in any form.It should be pointed out that coming to one of ordinary skill in the art
Say, can be with several modifications and improvements on the premise of the design of the present invention is not departed from.These belong to the protection model of the present invention
Enclose.
Carry out viral hepatitis type E IgM coupling simultaneously below by this method and conventional glutaraldehyde method, and the product of coupling is entered
Row activity identification.Specific animal sources antibody is the anti-HEV monoclonal antibodies S-13 of mouse, is purchased from Abcam companies of Britain, goods
Number：Ab19053, nonspecific people IgM buyings are from Millipore Corp., and two kinds of antibody purities are all higher than 90%, remaining all examination
Agent is purchased from sigma companies.
Embodiment 1
The inventive method technique is as follows：
(1) glucan and appropriate NaIO4 that appropriate mean molecule quantity is 750000 are weighed first, with 0.05M pH=
9.6 CB dissolving polysaccharides and NaIO4 is standby, and concentration is respectively 20mg/ml and 25mg/ml；
(2) polysaccharide solution is placed on agitator (2-8 DEG C) and at the uniform velocity stirred, then the NaIO4 prepared is molten with one milliliter
Sample loading gun is drop by drop slowly added into polysaccharide solution, lucifuge stirring reaction 60min；
(3) shift to an earlier date 5min by ethylene glycol water for injection dilute 10 times it is standby, then by well-oxygenated glucan from refrigerator
Take out, and add the ethylene glycol diluted, room temperature (16-25 DEG C) lucifuge reaction 30min；
(4) draw appropriate polysaccharide solution activate and the anti-HEV monoclonal antibodies S-13 of mouse and people are housed by being added to
The mass ratio of two kinds of antibody is 5 in IgM bag filter:1, be well mixed, be put into pH9.62L 0.05M carbonic acid buffers) keep away
Light is dialysed, and a buffer solution is changed after 2 hours, and lucifuge dialysed overnight is reacted 15-17 hours；
(5) conjugate is suctioned out into clean glass container, the NaHB4 that appropriate concentration is then is added thereto, mixed
Uniform 2-8 DEG C of lucifuges are closed to react 2 hours；
(6) conjugate is placed on agitator (2-8 DEG C) and at the uniform velocity stirred, while drop by drop being delayed with one milliliter of sample loading gun
Slowly isometric pH 7.4 saturated ammonium sulfate is added, after being well mixed, lucifuge, standing 60min；
(7) load centrifuge tube by the label after precipitation is well mixed, by centrifuge set to 4 DEG C of 12000rpm/min from
Heart 10min, abandons supernatant, stays precipitation.8th step redissolves the conjugate of precipitation 20%NBS to 1mg/ml.
Conventional glutaraldehyde coupling method is as follows：
(1) mouse anti-HEV monoclonal antibodies S-13 and people IgM is pressed into respective quality 5:1 loads and handles cleaned saturating well
Analyse in bag, and whether procuratorial work rubber band tie up, dialysed it is determined that being placed in pH6.8,2L 0.1M phosphate buffers 4 DEG C after tying up
At night, during which, change a buffer solution within every 2 hours；
(2) the mouse anti-HEV monoclonal antibodies S-13 and people IgM after dialysis is taken out from bag filter and is put into dimension
Centrifuge tube in；
(3) the 1% of respective volume will be added in the anti-HEV monoclonal antibodies S-13 of mouse and people's IgM mixed liquors after dialysis
Glutaraldehyde, (for example, labelled protein content 1mg antibody need to add 5ul 1% glutaraldehyde) is placed 37 on shaking table after being sufficiently mixed
DEG C, rotating speed 200rpm 3h；
(4) the 2M glycine solutions for preparing the water for injection that respective volume is added in the solution after coupling are closed,
Glycine concentration is finally reached 0.1M, room temperature places 2h, gently shaken per half an hour it is even once；
(5) mixed solution after closing is fitted into the bag filter handled well and cleaned, and whether procuratorial work rubber band ties up,
It is determined that being placed on 4 DEG C of dialysed overnights in pH8.0,2L 0.01M Tris-hcl buffer solutions after tying up, during which, one is changed within every 2 hours
Secondary buffer solution, change 2 times after can dialysed overnight, next day take out sulphur ammonium precipitation after is redissolved with 20%NBS to 1mg/ml.
Two methods conjugate is diluted with 20%NBS, dilution ratio is 1:100、1:500、1:1000、1:5000
With 1:10000, carried out according to the HEV-IgM reagents specification of ten thousand safe biological medicine company limited companies after operation detection dilution
Prepared product, specific data are shown in Table 1：
The performance comparision of 12 kinds of coupling protocols of table
Table 1 is it can be seen that the product obtained using the glucan coupling of the present invention is 1:In HEV-IgM examinations after 10000 dilutions
Still can be using test positive A values as 0.156 on agent box, and the product that traditional glutaraldehyde is coupled is used only 1:When 500
It can detect, two kinds of different nearly 20 times of coupling method activity differences.
Embodiment 2
The glucan of different molecular weight in marking process of the present invention is studied, in remaining technique and above-described embodiment 1
Unanimously.
The different mean molecule quantity polysaccharide coupling ratios of table 2 compared with
It can be seen that from the data of table 2, mean molecule quantity is used equally for being crosslinked carrier in the glucan of 1000-2800000 scopes,
Glucan mean molecule quantity is at 700000, active highest, and mean molecule quantity is excessive or too small can influence final conjugate
Activity.
Embodiment 3
Glucan addition concentration in marking process of the present invention is studied, it is consistent in remaining technique and above-described embodiment 1.
The performance comparision of the Different adding amount of the poly carbon skeleton of table 3
It can be seen that from the data of table 3, between glucan adds concentration from 5mg/ml to 50mg/ml, an elder generation is presented in activity
The process dropped after rising, when concentration is 20mg/ml, activity is maximum, final to determine that concentration is 20mg/ml.
Embodiment 4
Both HEV monoclonal antibodies S-13 anti-to mouse in marking process of the present invention and people IgM mass ratioes are studied, its
Remaining technique is consistent with the above.
The anti-HEV monoclonal antibodies S-13 of the mouse of table 4 and people IgM different qualities than performance comparision
The anti-HEV monoclonal antibodies S-13 of mouse and people's IgM mass ratioes are 5 it can be seen from data above:When 1 activity compared with
It is good, in view of about 5 times of monoclonal antibody and IgM mass difference, therefore the anti-HEV monoclonal antibodies S-13 of mouse and people IgM mole
Than being about 1 at this moment:1.
The specific embodiment of the present invention is described above.It is to be appreciated that the present invention is limited to above-mentioned specific
Embodiment, those skilled in the art can make various deformations or amendments within the scope of the claims, and this has no effect on this
The substantive content of invention.
Claims (8)
1. a kind of coupled complex, the coupled complex includes coupling carrier and at least two antibody being coupled with coupling carrier,
The coupling carrier has the conjugation sites with the antibody coupling of more than 2,
The antibody includes first antibody and secondary antibody, and the first antibody is animal derived special IgG, and described second resists
The non-specific IgM in body behaviour source, the coupling carrier is polysaccharide.
2. a kind of IgM Serologic detections Quality Control thing, it is characterised in that including the coupled complex described in claim 1.
3. a kind of preparation method of IgM Serologic detections Quality Control thing as claimed in claim 2, it is characterised in that use poly
Animal derived special IgG and the non-specific IgM in people source are coupled by sugar as carrier；
The hydroxyl of polysaccharide is oxidized to after aldehyde radical by methods described, by animal derived special IgG and the non-specific IgM in people source and oxygen
Polysaccharide after change is coupled.
4. preparation method as claimed in claim 3, it is characterised in that methods described comprises the following steps：
(1) polysaccharide and sodium metaperiodate are weighed, dissolves standby with carbonate buffer solution respectively, wherein the polysaccharide solution prepared
Concentration is 5-50mg/ml, and the concentration of sodium periodate solution is 5-50mg/ml；
(2) polysaccharide solution is placed in 2-8 DEG C on agitator and at the uniform velocity stirred, then sodium periodate solution is slowly dropped to many
In glycan solution, lucifuge stirring reaction；
(3) ethylene glycol is diluted with water 10 times, step (2) well-oxygenated polysaccharide taken out from refrigerator, and add what is diluted
Ethylene glycol, the reaction of room temperature lucifuge；
(4) polysaccharide solution that aspiration step (3) has been activated, is added to non-specific equipped with animal derived special IgG and people source
In IgM bag filter, it is well mixed, lucifuge is dialysed in carbonate buffer solution, a buffer solution is changed after 2 hours, dialysis is anti-
Answer 15-17 hours；
(5) conjugate is suctioned out into clean glass container, sodium borohydride is added into container, be well mixed, 2-8 DEG C are kept away
Light reaction；
(6) step (5) is obtained after reactant concentrating and precipitating, redissolution.
5. preparation method as claimed in claim 4, it is characterised in that wherein described polysaccharide is glucan, the Portugal gathers
The mean molecule quantity of sugar is 1000 to 2800000.
6. preparation method as claimed in claim 4, it is characterised in that the lucifuge stirring reaction time that wherein step (2) is addressed
For 60min, the lucifuge reaction time that step (3) is addressed is 30min, and the lucifuge reaction time that step (5) is addressed is 2 hours.
7. preparation method as claimed in claim 4, it is characterised in that the concentrating and precipitating wherein addressed in step (6) is sulfuric acid
Ammonium is precipitated.
8. a kind of coupled product, it is prepared as the preparation method described in any one of claim 3-6 claim.
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|---|---|---|---|---|
| EP0429218A1 (en) * | 1989-11-17 | 1991-05-29 | Baxter Diagnostics Inc. | Nonhuman primate antibodies for use as immunoglobulin assay positive control |
| EP0636886A2 (en) * | 1993-06-29 | 1995-02-01 | Behring Diagnostics Inc. | A calibrator or control composition for an IgM serology assay |
| US5846738A (en) * | 1993-10-20 | 1998-12-08 | Boehringer Mannheim Gmbh | Synthetic standard for immunoassays |
| US6015662A (en) * | 1996-01-23 | 2000-01-18 | Abbott Laboratories | Reagents for use as calibrators and controls |
| US6114180A (en) * | 1995-07-06 | 2000-09-05 | Bayer Aktiengesellschaft | Synthetic calibrators for use in immunoassays, comprising the analytes or partial sequences thereof which are conjugated to inert carrier molecules |
| CN102206280A (en) * | 2011-04-25 | 2011-10-05 | 江苏昶迅生物科技有限公司 | IgG-IgM polymer and polymerization method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050048468A1 (en) * | 2003-08-27 | 2005-03-03 | Bio-Rad Laboratories, Inc. | Multiconstituent liquid lgG and IgM calibrators |
-
2015
- 2015-12-22 CN CN201510971625.7A patent/CN105628941B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0429218A1 (en) * | 1989-11-17 | 1991-05-29 | Baxter Diagnostics Inc. | Nonhuman primate antibodies for use as immunoglobulin assay positive control |
| EP0636886A2 (en) * | 1993-06-29 | 1995-02-01 | Behring Diagnostics Inc. | A calibrator or control composition for an IgM serology assay |
| US5846738A (en) * | 1993-10-20 | 1998-12-08 | Boehringer Mannheim Gmbh | Synthetic standard for immunoassays |
| US6114180A (en) * | 1995-07-06 | 2000-09-05 | Bayer Aktiengesellschaft | Synthetic calibrators for use in immunoassays, comprising the analytes or partial sequences thereof which are conjugated to inert carrier molecules |
| US6015662A (en) * | 1996-01-23 | 2000-01-18 | Abbott Laboratories | Reagents for use as calibrators and controls |
| CN102206280A (en) * | 2011-04-25 | 2011-10-05 | 江苏昶迅生物科技有限公司 | IgG-IgM polymer and polymerization method thereof |
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