CN105628941A - Method for preparing IgM serological test quality control material - Google Patents
Method for preparing IgM serological test quality control material Download PDFInfo
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- CN105628941A CN105628941A CN201510971625.7A CN201510971625A CN105628941A CN 105628941 A CN105628941 A CN 105628941A CN 201510971625 A CN201510971625 A CN 201510971625A CN 105628941 A CN105628941 A CN 105628941A
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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
The invention discloses a method for preparing an IgM serological test quality control material, and relates to a coupling method that polysaccharide is used as a carrier and sodium periodate-sodium borohydride is reacted. As the polysaccharide with a large molecular weight is used as the carrier, hydroxyl in polysaccharide is oxidized into aldehyde groups by using a sodium periodate method, and activated aldehyde groups on one polysaccharide molecule are far greater than two activated aldehyde groups on a glutaraldehyde molecule, the condition that two types of impurities are generated in glutaraldehyde two-step method marking is avoided, and the use activity of a final product can be improved. A coupled product prepared by using the method disclosed by the invention can be applied to quality control in IgM serological tests, and the effect of the coupled product is prior to that of a coupled product obtained through glutaraldehyde two-step method marking.
Description
Technical field
The present invention relates to immune labeled analysis field, the preparation method being specifically related to a kind of IgM Serologic detection Quality Control thing.
Background technology
The positive needed during infectious disease detection and negative quality-control product are for detecting the process control of test, it is ensured that the correctness of final result output and accuracy. Negative quality-control product, generally by the serum for normal person, does not comprise any purpose antibody that need to detect. Positive quality control product usually is diluted being prepared from by the negative quality-control product of the serum containing the purpose antibody that need to detect in a large number obtained from the patient or blood plasma.
It is known that IgM antibody can produce when infectious disease antigen invades the first time immunne response produced after body. IgM antibody only can exist in one shorter time period of immunne response early stage, generally can at several thoughtful some months. The immunne response of IgG antibody produces and duration all in the relatively long time, can be generally several years.
IgM antibody is that primary immune response produces, and therefore, its persistent period is very of short duration, but the detection of IgM antibody is but very important, can detect that when patient's early infection. But, short owing to producing the IgM persistent period after patient infection, it is difficult to the IgM antibody positive blood obtaining abundance from individuality for the infectious disease IgM Quality Control detected. It is thus desirable to the specific IgG antibodies of use animal is as the positive quality control (less understanding, seemingly can delete) in detection herein, described in WO89/10980. The method describing the operation preparation IgM antibody adopting immune animal in WO89/10980, wherein needs to prepare corresponding IgM antibody up to the time of 15 days, and to need to feed corresponding animal body, required time and cost be all extremely huge.
What this area was common measures IgM antibody in serum such as prize law, it is possible to IgM and animal derived specific antibody carry out the positive reference as Quality Control of prepared product that coupling obtains. Owing to this prepared product needs the coupling of animal derived special IgG and the non-specific IgM in people source, the classical way of method many employings albumen coupling of current coupling and glutaraldehyde coupling, the method adopts a kind of conventional homotype bi-functional cross-linking agent glutaraldehyde to carry out coupling, its two aldehyde radicals can form Schiff key (-N=C-) with the amino of albumen, they is coupled together with five carbon bridges. But owing to it is non-specific two-way cross-linking agent, therefore there is the ratio between corsslinking molecular not strict, size also differs, the shortcoming that specificity Conjugate ratio is low. When adopting glutaraldehyde method to carry out animal derived special IgG and the non-specific IgM coupling in people source, except generating the animal derived non-specific IgM product in special IgG people source, also inevitably generate animal derived special IgG animal derived for special IgG and people source non-specific IgM people source two kinds of impurity product of non-specific IgM, and be difficult to separate purification, have a strong impact on activity during final utilization.
Summary of the invention
Coupled product can be made to produce impurity the defect of activity when affecting the final utilization of coupled product further for the coupling method adopted in prior art. The present invention provides a kind of new coupling technology, new coupling technology provided by the invention to can be used for animal derived special IgG and the coupling of the non-specific IgM in people source.
When this coupling technology can overcome glutaraldehyde coupling, the ratio between corsslinking molecular is not strict, size also differs, the shortcoming that specificity Conjugate ratio is low, when making animal derived special IgG and people source non-specific IgM coupling, product is all almost the animal derived special non-specific IgM in IgG people source, and seldom there are special IgG animal derived for animal derived special IgG and people source non-specific IgM people source two kinds of impurity of non-specific IgM, thus improve the activity of end product. Adopting the coupled product that the method for the invention prepares to may be used for the SD quality control of IgM, its effect is better than the coupled product that glutaraldehyde method labelling obtains.
The present invention adopts the following technical scheme that
The invention discloses a kind of coupled complex, this coupled complex includes coupling carrier and at least two antibody being coupled, and described coupling carrier has more than 2 with described antibody coupling conjugation sites.
In a detailed description of the invention, described antibody includes first antibody and second antibody, and described first antibody is animal derived special IgG, and the non-specific IgM in described second antibody behaviour source, described coupling carrier is polysaccharide.
The preparation method that the invention discloses a kind of IgM Serologic detection Quality Control thing, it is characterised in that adopt polysaccharide as carrier, animal derived special IgG and the non-specific IgM in people source is carried out coupling.
The preparation method that the invention discloses a kind of IgM Serologic detection Quality Control thing, it is characterised in that after the hydroxyl oxygen of polysaccharide is melted into aldehyde radical by described method, carries out coupling by the polysaccharide after animal derived special IgG and the non-specific IgM in people source and oxidation.
The preparation method of IgM Serologic detection Quality Control thing disclosed by the invention adopts a kind of polysaccharide as crosslinking carrier, and by adopting sodium metaperiodate-sodium borohydride method to cross-link, described method comprises the steps:
(1) polysaccharide and sodium metaperiodate (NaIO are weighed4), dissolve standby with carbonate buffer solution respectively;
(2) it is placed on agitator by polysaccharide solution in 28 DEG C at the uniform velocity to stir, then NaIO4 solution is slowly added dropwise in polysaccharide solution, lucifuge stirring reaction 60min;
(3) by ethylene glycol dilute with water 10 times, step (2) well-oxygenated polysaccharide is taken out from refrigerator, add the ethylene glycol diluted, room temperature lucifuge reaction 30min;
(4) polysaccharide solution that aspiration step (3) has activated joins in the bag filter equipped with animal derived special IgG and the non-specific IgM in people source, mix homogeneously, it is lucifuge dialysis in 0.05M carbonate buffer solution in pH9.6 concentration, change a buffer after 2 hours, react 15 17 hours;
(5) by conjugate sucking-off to clean glass container, with backward container adds sodium borohydride (NaHB4), mix homogeneously, 28 DEG C of lucifuges are reacted 2 hours;
(6), after the reactant concentrating and precipitating that step (5) is obtained, redissolve and namely prepare coupled product.
Wherein, the method for reactant concentrating and precipitating can be adopted ammonium sulfate precipitation.
Wherein, the step of described ammonium sulfate precipitation is as follows: reactant step (5) obtained is placed on agitator in 28 DEG C and at the uniform velocity stirs, it is slowly added dropwise the saturated ammonium sulfate of equal-volume pH7.4, after mix homogeneously, lucifuge, standing 60min, load centrifuge tube by the reactant mix homogeneously after precipitation, 4 DEG C of centrifugal 10min of 12000rpm/min, abandon supernatant, stay precipitation.
Preferably, polysaccharide in the step (1) of the inventive method is glucosan, and mean molecule quantity is from 1000 to 2800000 all can use, and best molecular weight ranges is 40000 to 1000000, after dissolving, concentration range can be 5-50mg/ml, and optium concentration is 20mg/ml.
Preferably, in the method for the invention step (1), after the dissolving of NaIO4 used, concentration range can be 5-50mg/ml, and optium concentration is 25mg/ml.
Preferably, in the method for the invention step (4), the molar ratio of zoogenous special IgG and the non-specific IgM in people source can from 1:10 to 10:1, and optimum molar ratio is 1:1.
Preferably, in the method for the invention step (5), the ultimate density scope of NaHB4 can be 0.0001 to 0.01mg/ml, and optium concentration is 0.025mg/ml.
It addition, the present invention specifically provides a kind of method utilizing glucosan, as carrier, little for animal derived monoclonal antibody mouse-anti HEV monoclonal antibody S-13 and nonspecific people IgM are carried out coupling.
It addition, adopt the coupled product that method of the present invention prepares to have higher activity compared with the conjugate that conventional glutaraldehyde method prepares, therefore, the coupled product that the method for the invention prepares is adopted to fall within the summary of the invention of the present invention.
The present invention passes through the polysaccharide adopting macromolecule as carrier, the hydroxyl in polysaccharide is aoxidized with sodium periodate method, it is made to be oxidized to aldehyde radical, one molecular weight be 40000 to 1000000 dextran molecule can form the aldehyde radical of 245-6125 activation in theory, activation aldehyde radical on one molecule is significantly larger than 2 active aldehyde radicals on glutaraldehyde molecules, after being added into zoogenous special IgG and the non-specific IgM in people source, the poly skeleton that hardly possible formation is all cross-linked by specific IgG and the poly carbon skeleton all cross-linked by the non-specific IgM in people source, therefore two kinds of impurity during similar glutaraldehyde two-step method labelling are avoided the occurrence of, thus improving the use activity of end product.
Detailed description of the invention
Technical solution of the present invention can adopt following operating procedure to be carried out.
(1) appropriate glucosan and appropriate sodium metaperiodate (NaIO are first weighed4), dissolve polysaccharide with the carbonate buffer solution (CB) of the pH=9.6 of 0.05M and NaIO4 is standby, wherein glucosan mean molecule quantity is from 1000 to 2800000 all can use, best molecular weight ranges is 40000 to 1000000, after dissolving, concentration range can be 5-50mg/ml, optium concentration be 20mg/ml, NaIO4 dissolving after concentration range can be 5-50mg/ml, optium concentration is 25mg/ml;
(2) it is placed on polysaccharide solution on agitator (2 8 DEG C) at the uniform velocity to stir, then the NaIO4 solution prepared drop by drop is slowly added in polysaccharide solution with one milliliter of sample loading gun, lucifuge stirring reaction 60min;
(3) shift to an earlier date 5min by standby for ethylene glycol water for injection dilution 10 times, then well-oxygenated polysaccharide is taken out from refrigerator, and add the ethylene glycol diluted, room temperature (16 25 DEG C) lucifuge reaction 30min;
(4) draw the polysaccharide solution activated in right amount and join in the bag filter equipped with animal derived special IgG and the non-specific IgM in people source, mix homogeneously, put into lucifuge dialysis in the 0.05M carbonate buffer solution of pH9.62L, a buffer is changed after 2 hours, lucifuge dialysed overnight, reacting 15 17 hours, wherein the molar ratio of zoogenous special IgG and the non-specific IgM in people source can from 1:10 to 10:1, and optimum molar ratio is 1:1;
(5) in conjugate sucking-off to clean glass container, being added thereto to the sodium borohydride (NaHB4) that appropriate concentration is subsequently, 28 DEG C of lucifuges of mix homogeneously are reacted 2 hours; Wherein the ultimate density scope of NaHB4 can be 0.0001 to 0.01mg/ml, and optium concentration is 0.025mg/ml.
(6) it is placed on conjugate on agitator (2 8 DEG C) at the uniform velocity to stir, is drop by drop slowly added to the saturated ammonium sulfate of equal-volume pH7.4 simultaneously with one milliliter of sample loading gun, after mix homogeneously, lucifuge, standing 60min;
(7) the label mix homogeneously after precipitation is loaded centrifuge tube, centrifuge is arranged to the centrifugal 10min of 4 DEG C of 12000rpm/min, abandon supernatant, stay precipitation;
(8) conjugate of precipitation 20%NBS (new-born calf serum) is redissolved to 1mg/ml.
Below in conjunction with specific embodiment, the present invention is described in detail. Following example will assist in those skilled in the art and are further appreciated by the present invention, but do not limit in any form. It should be pointed out that, to those of ordinary skill in the art, under the premise without departing from the design of the present invention, it is also possible to some deformation and improvement. These broadly fall into protection scope of the present invention.
Carry out the coupling of hepatitis E IgM below by this method and conventional glutaraldehyde method simultaneously, and the product of coupling is carried out activity identification. Specific animal sources antibody is little mouse-anti HEV monoclonal antibody S-13, purchasing from Abcam company of Britain, article No.: ab19053, nonspecific people IgM purchases from Millipore Corp., two kinds of antibody purity are all higher than 90%, and all the other all reagent are all purchased from sigma company.
Embodiment 1
The inventive method technique is as follows:
(1) first weighing glucosan that appropriate mean molecule quantity is 750000 and appropriate NaIO4, dissolve polysaccharide with the CB of the pH=9.6 of 0.05M and NaIO4 is standby, concentration is 20mg/ml and 25mg/ml respectively;
(2) it is placed on polysaccharide solution on agitator (2 8 DEG C) at the uniform velocity to stir, then one milliliter of sample loading gun of molten for the NaIO4 prepared use is drop by drop slowly added in polysaccharide solution, lucifuge stirring reaction 60min;
(3) shift to an earlier date 5min by standby for ethylene glycol water for injection dilution 10 times, then well-oxygenated glucosan is taken out from refrigerator, and add the ethylene glycol diluted, room temperature (16 25 DEG C) lucifuge reaction 30min;
(4) drawing the polysaccharide solution activated in right amount is 5:1 by joining the mass ratio of two kinds of antibody in the bag filter equipped with little mouse-anti HEV monoclonal antibody S-13 and people IgM, mix homogeneously, put in the 0.05M carbonic acid buffer of pH9.62L) lucifuge dialysis, a buffer is changed after 2 hours, lucifuge dialysed overnight, reacts 15 17 hours;
(5) in conjugate sucking-off to clean glass container, being added thereto to the NaHB4 that appropriate concentration is subsequently, 28 DEG C of lucifuges of mix homogeneously are reacted 2 hours;
(6) it is placed on conjugate on agitator (2 8 DEG C) at the uniform velocity to stir, is drop by drop slowly added to the saturated ammonium sulfate of equal-volume pH7.4 simultaneously with one milliliter of sample loading gun, after mix homogeneously, lucifuge, standing 60min;
(7) the label mix homogeneously after precipitation is loaded centrifuge tube, centrifuge is arranged to the centrifugal 10min of 4 DEG C of 12000rpm/min, abandon supernatant, stay precipitation. The conjugate of precipitation is redissolved to 1mg/ml by the 8th step with 20%NBS.
Conventional glutaraldehyde coupling method is as follows:
(1) little mouse-anti HEV monoclonal antibody S-13 and people IgM is handled well in cleaned bag filter by respective quality 5:1 loading, and whether procuratorial work rubber band ties up, determine and after tying up, be placed in the 0.1M phosphate buffer of pH6.8,2L 4 DEG C of dialysed overnight, period, within every 2 hours, change a buffer;
(2) the little mouse-anti HEV monoclonal antibody S-13 and people IgM after dialysis is taken out in the centrifuge tube putting into dimension from bag filter;
(3) by the little mouse-anti HEV monoclonal antibody S-13 after dialysis and 1% glutaraldehyde adding respective volume in people's IgM mixed liquor, (such as, the antibody of labelled protein content 1mg need to add 1% glutaraldehyde of 5ul) places 37 DEG C, rotating speed 200rpm3h on shaking table after being sufficiently mixed;
(4) by the solution after coupling adds respective volume water for injection prepare 2M glycine solution close, make glycine concentration be finally reached 0.1M, room temperature place 2h, shake gently per half an hour even once;
(5) the mixed solution loading after closing is handled well in cleaned bag filter, and whether procuratorial work rubber band ties up, determine and after tying up, be placed in the 0.01MTris-hcl buffer of pH8.0,2L 4 DEG C of dialysed overnight, period, within every 2 hours, change a buffer, get final product dialysed overnight after changing 2 times, redissolve to 1mg/ml with 20%NBS after taking out sulfur ammonium precipitation next day.
Two kinds of method conjugate 20%NBS are diluted, dilution ratio is 1:100,1:500,1:1000,1:5000 and 1:10000, being operated the prepared product after detection dilution according to the HEV-IgM reagent description of ten thousand safe biological Pharmaceutical limited companies, concrete data are in Table 1:
The Performance comparision of 12 kinds of coupling protocols of table
Table 1 can be seen that the product using the glucosan coupling of the present invention to obtain can test positive A value be still 0.156 after 1:10000 dilutes on HEV-IgM test kit, and adopting the product of traditional glutaraldehyde coupling only can detect when 1:500, two kinds of different coupling method activity differ nearly 20 times.
Embodiment 2
The glucosan of different molecular weight in marking process of the present invention is studied, and all the other techniques are consistent with above-described embodiment 1.
The different mean molecule quantity polysaccharide coupling ratio of table 2 is relatively
Can be seen that from table 2 data, mean molecule quantity is used equally to crosslinking carrier at the glucosan of 1000-2800000 scope, and glucosan mean molecule quantity is when 700000, and activity is the highest, and mean molecule quantity is excessive or the too small activity that all can affect final conjugate.
Embodiment 3
Glucosan in marking process of the present invention being added concentration study, all the other techniques are consistent with above-described embodiment 1.
The Performance comparision of the Different adding amount of table 3 poly carbon skeleton
Can be seen that from table 3 data, between glucosan interpolation concentration is from 5mg/ml to 50mg/ml, activity presents a process fallen after rising, and when concentration is 20mg/ml, activity is maximum, finally determines that concentration is 20mg/ml.
Embodiment 4
The medium and small mouse-anti HEV monoclonal antibody S-13 of marking process of the present invention is studied with people's IgM mass ratio, and all the other techniques are consistent with the above.
The Performance comparision of the little mouse-anti HEV monoclonal antibody S-13 of table 4 and people's IgM different quality ratio
Be can be seen that by data above, little mouse-anti HEV monoclonal antibody S-13 and the people's IgM mass ratio activity when 5:1 is better, in view of the mass difference of monoclonal antibody and IgM about 5 times, therefore the mol ratio of little mouse-anti HEV monoclonal antibody S-13 and people IgM is approximately 1:1 at this moment.
Above specific embodiments of the invention are described. It is to be appreciated that the present invention is limited to above-mentioned particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, and this has no effect on the flesh and blood of the present invention.
Claims (10)
1. a coupled complex, this coupled complex includes coupling carrier and at least two antibody with coupling carrier coupling, and described coupling carrier has more than 2 with described antibody coupling conjugation sites.
2. coupled complex according to claim 1, it is characterised in that described antibody includes first antibody and second antibody, described first antibody is animal derived special IgG, and the non-specific IgM in described second antibody behaviour source, described coupling carrier is polysaccharide.
3. an IgM Serologic detection Quality Control thing, it is characterised in that include the coupled complex described in claim 2.
4. the preparation method of an IgM Serologic detection Quality Control thing, it is characterised in that adopt polysaccharide as carrier, animal derived special IgG and the non-specific IgM in people source is carried out coupling.
5. method according to claim 4, it is characterised in that after the hydroxyl oxygen of polysaccharide is melted into aldehyde radical by described method, carries out coupling by the polysaccharide after animal derived special IgG and the non-specific IgM in people source and oxidation.
6. preparation method as claimed in claim 5, it is characterised in that described method comprises the steps:
(1) weighing polysaccharide and sodium metaperiodate, dissolve standby with carbonate buffer solution respectively, the concentration of the polysaccharide solution wherein prepared is 5-50mg/ml, and the concentration of sodium periodate solution is 5-50mg/ml;
(2) it is placed on agitator by polysaccharide solution in 28 DEG C at the uniform velocity to stir, then sodium periodate solution is slowly added dropwise in polysaccharide solution, lucifuge stirring reaction;
(3) by ethylene glycol dilute with water 10 times, being taken out from refrigerator by step (2) well-oxygenated polysaccharide, and add the ethylene glycol diluted, room temperature lucifuge is reacted;
(4) polysaccharide solution that aspiration step (3) has activated, join in the bag filter equipped with animal derived special IgG and the non-specific IgM in people source, mix homogeneously, lucifuge dialysis in carbonate buffer solution, a buffer, hemodialysis reaction 15 17 hours is changed after 2 hours;
(5) in conjugate sucking-off to clean glass container, sodium borohydride, mix homogeneously, 28 DEG C of lucifuge reactions will be added in container;
(6), after step (5) being obtained reactant concentrating and precipitating, redissolve.
7. the preparation method as described in claim 5-6 any one claim, it is characterised in that wherein said polysaccharide is glucosan, the mean molecule quantity of described glucosan is 1000 to 2800000.
8. preparation method as claimed in claim 6, it is characterized in that, the lucifuge stirring reaction time that wherein step (2) is addressed is 60min, and the lucifuge response time that step (3) is addressed is 30min, and the lucifuge response time that step (5) is addressed is 2 hours.
9. preparation method as claimed in claim 6, it is characterised in that the concentrating and precipitating wherein addressed in step (6) is ammonium sulfate precipitation.
10. a coupled product, its preparation method described in claim 4-9 any one claim prepares.
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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 |
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