CN107255727A - Latex is than turbid freeze-dried reagent and preparation method thereof - Google Patents
Latex is than turbid freeze-dried reagent and preparation method thereof Download PDFInfo
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- CN107255727A CN107255727A CN201710552588.5A CN201710552588A CN107255727A CN 107255727 A CN107255727 A CN 107255727A CN 201710552588 A CN201710552588 A CN 201710552588A CN 107255727 A CN107255727 A CN 107255727A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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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
The invention discloses latex than turbid freeze-dried reagent and preparation method thereof, this method comprises the following steps：Latex particle is coupled with monoclonal antibody, protective agent is then added, to obtain latex antibody reagent, wherein, the protective agent is made up of bovine serum albumin(BSA), vitamin C and glycine；Latex antibody reagent progress cryogenic vacuum is freezed, to obtain dried frozen aquatic products, realized wherein the cryogenic vacuum is lyophilized by multigelation；And in atmosphere of inert gases, the dried frozen aquatic products is sealed, to obtain the latex than turbid freeze-dried reagent.The latex prepared using this method is than turbid freeze-dried reagent, and stability is good；Redissolve soon and homogeneity is good, without visible suspension；Performance is unaffected after redissolution, is swift in response with whole blood sample, and result can be reported in 1 minute, and repeatability is very good, as a result accurately and reliably.
Description
Technical field
The present invention relates to external diagnosis reagent technical field, and in particular to latex is than turbid freeze-dried reagent and preparation method thereof.
Background technology
External diagnosis reagent refers to can be used alone or used with instrument, utensil, equipment or system in combination, in the pre- of disease
During anti-, diagnosis, Treatment monitoring, Observation On The Prognosis, health status evaluation and the prediction of genetic disease, for human body sample
This (various body fluid, cell, tissue samples etc.) carry out reagent, kit, calibration object (thing), the quality-control product (thing) of vitro detection
Deng.External diagnosis reagent is that reagent has as one kind of medicine equipment with instrument and equipment class medicine equipment maximum differential
The effect phase, i.e., declare condition of storage can stablize maintain reagent performance time limit.The term of validity of reagent is mainly with reagent into packet
It is related into, the factor such as formulation state, condition of storage.The immune ratio of the external diagnosis reagent sold on Vehicles Collected from Market, especially latex
Turbid reagent, is substantially liquid condition, it is desirable in 2~8 DEG C of low-temperature storages；Even if there is other indivedual non-latex immunoturbidimetry class examinations
Agent position dried frozen aquatic products, its desired condition of storage is also substantially 2~8 DEG C or lower temperature.On the market there is not yet normal temperature is stored up
The latex deposited is sold than turbid reagent.
Thus, pair can research of the latex than turbid reagent of normal temperature storage still have to be strengthened.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.Therefore, one object of the present invention
It is to propose the latex for being capable of storage-stable at normal temperatures than turbid reagent.
It should be noted that the present invention is following discovery and work based on inventor and completed：
On September 22nd, 2016, CFDA general bureaus issue《Medicine equipment cold chain (transport, storage) administration guide》Bulletin
(2016 No. 154), its to medical device product of the storage temperature requirement in the range of 2~8 DEG C and temperature below production,
Storage and the low-temperature protection measure of transportation requirement have carried out specification.The quality of the requirement to reinforcement medical device product in itself
There is very important meaning in terms of management and the effective reliability of lifting product safety, the risk of reduction patient, but undeniably must
The cost of either manufacturing enterprise or Sales Channel business will be lifted.Therefore, medical device product stability is lifted to make it
It can store and transport under normal temperature condition, by the effect of the link such as great reduction entreprise cost and the lifting manufacturing, transport
Rate.
The performance that the stability of reagent refers to reagent when reagent reaches the term of validity declared under condition of storage on request is met
The ability of pre-provisioning request, usual reagent during storage due to air contact or due to remaining air in reagent bottle,
The equilibrium condition of chemical reaction or destruction reagent solution can occur with the composition in reagent for oxygen or carbon dioxide therein,
Such as pH value, surface tension, chemical bond affinity, so that the performance of reagent changes, performance results are reagent test sample
This result shifts, and this destruction or influence are generally huge with storage temperature relation, according to Arrhenius empirical equation,
Temperature often rises 10 DEG C, and reaction speed generally increases by 2~4 times, i.e. reagent storage temperature and often increased under 10 DEG C, the stability of reagent
It is down to original 1/2~1/4.
Thus, well imagine, obtain the latex for being capable of storage-stable at normal temperatures than turbid reagent, difficulty is very big.And send out
A person of good sense passes through a series of theoretical research and experimental exploring, surprisingly finds：When preparing latex antibody reagent, addition is by ox blood
The specific protective agent of pure albumen, vitamin C and glycine composition, is freezed with reference to by latex antibody reagent progress cryogenic vacuum
The square technology of Shi Caiyong multigelations, and in atmosphere of inert gases carry out dried frozen aquatic products sealing, can prepare can reach it is pre-
The latex of storage-stable is than turbid freeze-dried reagent under the stabilizing effect of phase, energy normal temperature.
Thus, in the first aspect of the present invention, method of the latex than turbid freeze-dried reagent is prepared the invention provides a kind of.Root
According to embodiments of the invention, this method comprises the following steps：Latex particle is coupled with monoclonal antibody, then addition is protected
Agent is protected, to obtain latex antibody reagent, wherein, the protective agent is made up of bovine serum albumin(BSA), vitamin C and glycine；
Latex antibody reagent progress cryogenic vacuum is freezed, to obtain dried frozen aquatic products, wherein it is to pass through that the cryogenic vacuum is lyophilized
What multigelation was realized；And in atmosphere of inert gases, the dried frozen aquatic products is sealed, to obtain the latex than turbid
Freeze-dried reagent.It is surprisingly found by the inventors that, latex can be effectively prepared than turbid freeze-dried reagent using this method.According to this hair
Bright embodiment, the latex that the present invention is prepared is than turbid freeze-dried reagent, and stability is good, at normal temperatures being capable of storage-stable 12
More than month, or even 14 months；Redissolve soon, can completely be redissolved in 15 minutes and homogeneity is good, without visible suspension；Performance after redissolution
It is unaffected, it is swift in response with whole blood sample, result can be reported in 1 minute, and repeatability is very good, as a result accurately and reliably.
, wherein it is desired to which explanation, when preparing latex antibody reagent, is added by bovine serum albumin(BSA), vitamin C and sweet ammonia
The protective agent of acid composition, can make albumen and lipid in reagent be disperseed and protected, it is unlikely in lyophilized time variation
And conglomeration, and then the freeze-dried reagent subsequently obtained can dissolve quickly when redissolving after adding water, that is, redissolve it is fast, and up to homogeneity,
Without visible suspension.
Embodiments in accordance with the present invention, the latex particle is provided in the form of latex solution, and the latex particle
Particle diameter is that quality-volumetric concentration of latex particle in 200~300nm, the latex solution is 0.1 ‰~1 ‰.Thus, latex
Particle and the coupling effect of monoclonal antibody are good, and the latex antibody reagent stability of acquisition is good.
Embodiments in accordance with the present invention, before the coupling is carried out, further comprise the latex particle is pre- advanced
The step of row activation, and the activation is by the way that the latex solution and 1- (3- dimethylamino-propyls) -3- ethyls carbon two is sub-
Amide hydrochloride mixes and is slowly stirred what is realized within 2 hours at room temperature in equal volume.Thus, because of stable reagent, difficult for drop-off,
Coupling effect is good, and the latex antibody reagent stability of acquisition is protruded.
According to some specific examples of the present invention, 1- (3- the dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
Quality-volumetric concentration of solution is 0.1 ‰~0.5 ‰.Thus, latex particle activation effect is good, subsequently with monoclonal antibody
Coupling effect is good, and the latex antibody reagent stability of acquisition is protruded.
Embodiments in accordance with the present invention, the monoclonal antibody is provided in the form of antibody-solutions, and the antibody-solutions
Quality-volumetric concentration of middle monoclonal antibody be the latex solution in latex particle quality-volumetric concentration 5%~
10%.Thus, the coupling effect with latex particle is good, and the latex antibody reagent stability of acquisition is protruded.
Embodiments in accordance with the present invention, in the latex antibody reagent, quality-volume of the bovine serum albumin(BSA) is dense
Spend for 1~5g/L, the ascorbic quality-volumetric concentration is 1~10g/L, quality-volumetric concentration of the glycine is
0.2-2g/L.Thus, protective agent is good to the protecting effect of latex antibody reagent, and obtained latex antibody reagent stability is protruded.
Embodiments in accordance with the present invention, the multigelation is by under not higher than 10Pa atmospheric pressure, by the breast
Glue antibody reagent repeats the realization of freeze thawing treatment 3-5 times, the freeze thawing treatment be by the latex antibody reagent successively-
Kept for 1 hour and kept for 1 hour at 20 DEG C at 20 DEG C.
Wherein, latex is prepared in the prior art than turbid freeze-dried reagent, when carrying out lyophilized to latex antibody reagent, is typically all
Using disposable freeze-drying process, it at most can only effectively freeze 2ml volume reagents, then be difficult to freeze more than 2ml, especially freeze
Liquid residue amount is more in storehouse middle part, part bottle, occasionally has liquid to overflow bottleneck；And extend freeze-drying time to more than 72 hours then
On the one hand cost can be caused to increase, on the other hand then causing operational process of craft excessively complexity, (freeze dryer continuous service 72 is small
When more than, risk is big, and needs special messenger to keep an eye on).And the method for the present invention, using the technology of multigelation, by latex antibody reagent
Repeating freeze thawing treatment as described above 3-5 times, (freeze thawing treatment is to keep latex antibody reagent at -20 DEG C successively
1 hour and holding 1 hour at 20 DEG C).Thus, make latex antibody reagent between -20 DEG C and 20 DEG C back and forth freeze thawing several times, energy
Enough make latex antibody reagent molecular structure arrangement more consolidation regular, and make the built-in water effective frost of latex liquid with convenient
The distillation (i.e. moisture is easily discharged) of follow-up freeze-drying process, dried frozen aquatic products appearance uniform, without obvious bubble, even if moreover, loading amount is in 4ml
Still can effectively it be freezed in 48 hours inside latex during the above.Also, glue ratio is prepared using above-mentioned multigelation technology
During turbid freeze-dried reagent, the problems such as be not in albuminous degeneration, reagent conglomeration, latex surface molecule aquation damage layer, the breast of acquisition
Glue is than turbid freeze-dried reagent, and relative to not freeze-dried liquid reagent, performance indifference is swift in response with whole blood sample, in 1 minute
Result can be reported, and it is reproducible when being used to diagnose, as a result accurately and reliably.
Embodiments in accordance with the present invention, before the progress cryogenic vacuum is lyophilized, further comprise：It is not high in atmospheric pressure
Under conditions of 10Pa and temperature is -50 DEG C, the latex antibody reagent is subjected to pre-freeze processing and is no less than 6 hours.Thus, freeze
Dry effect is good, and the latex of acquisition is protruded than turbid freeze-dried reagent outward appearance, performance.
Embodiments in accordance with the present invention, the innocuous gas that the atmosphere of inert gases is more than air by density is formed.Thus,
It can effectively prevent from influenceing the entrance of the air of reagent stability when dried frozen aquatic products is sealed.Specifically shown according to some of the invention
Example, the atmosphere of inert gases is formed by argon gas or carbon dioxide.Thus, the latex of acquisition is more prominent than turbid freeze-dried reagent stability
Go out.
Embodiments in accordance with the present invention, after dried frozen aquatic products is obtained, access inert gas, to make using freeze dryer air inlet
The inert gas forms atmosphere of inert gases full of freeze dryer.Thereby, it is possible to conveniently and effectively form inert gas atmosphere
Enclose.
In the second aspect of the present invention, the invention provides a kind of latex than turbid freeze-dried reagent.According to the implementation of the present invention
Example, the latex is prepared than turbid freeze-dried reagent by the foregoing latex for preparing than turbid freeze-dried reagent method.According to
Embodiments of the invention, latex of the invention is than turbid freeze-dried reagent appearance uniform, without obvious bubble；Stability is protruded, Neng Gouchang
Temperature storage, specifically, at normal temperatures being capable of storage-stable more than 12 months, or even 14 months；Redissolve soon, can be complete in 15 minutes
Redissolve and homogeneity is good, without visible suspension；Performance is good, in the absence of albuminous degeneration, reagent conglomeration, latex surface molecule hydrated sheath
The phenomenons such as destruction, performance is unaffected after redissolution, is swift in response with whole blood sample, and result, and repeatability can be reported in 1 minute
It is very good, as a result accurately and reliably.
Furthermore, it is necessary to explanation, according to some embodiments of the present invention, latex of the invention than turbid freeze-dried reagent and its
Preparation method has at least one of following advantages：
1) method of the invention, after latex antibody reagent progress cryogenic vacuum is lyophilized, the dried frozen aquatic products outward appearance of acquisition is equal
It is even, without obvious bubble；
2) latex of the invention is protruded than turbid freeze-dried reagent stability, storage-stable at least 30 days under the conditions of 37 DEG C,
And (0~35 DEG C) storage-stable more than 12 months under normal temperature；
3) latex of the invention redissolves soon than turbid freeze-dried reagent, can be redissolved completely in 15 minutes, and homogeneity is good, can not
See suspension；
4) latex of the invention is better than turbid freeze-dried reagent performance, in the absence of albuminous degeneration, reagent conglomeration, latex surface molecule
Performance is unaffected after the phenomenons such as aquation damage layer, redissolution, is swift in response with whole blood sample, and result can be reported in 1 minute, and
Repeatability is very good, as a result accurately and reliably.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become from description of the accompanying drawings below to embodiment is combined
Substantially and be readily appreciated that, wherein：
Fig. 1 is the schematic flow sheet for preparation method of the latex than turbid freeze-dried reagent according to the embodiment of the present invention；
Fig. 2 is the effect schematic diagram for the CRP latex freeze-dried reagents CRP1 of the invention that embodiment 1 is prepared；
Fig. 3 is the effect diagram after the CRP latex freeze-dried reagents CRP2 of the invention that embodiment 2 is prepared redissolves；
Fig. 4 is that the CRP latex freeze-dried reagent CRP2 of the invention that embodiment 2 is prepared step auspicious BC-5390's with compareing
Whole blood CRP, determines the methodology contrast consistency detection result schematic diagram of whole blood sample.
Embodiment
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carried out according to the technology or condition described by document in the art or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market.
Embodiment 1
Reference picture 1, prepares method of the latex than turbid freeze-dried reagent according to the present invention, prepares latex CRP freeze-dried reagents, has
Body is as follows：
1st, latex reagent is prepared：The latex solution 100ml for taking particle diameter 300nm, concentration to be 0.1 ‰, be with 100ml concentration
0.5 ‰ 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) are mixed, at room temperature gentle agitation 2 hours,
So as to which latex particle is activated in advance；Then CRP (enhancing C reactive protein) monoclonal that 5ml concentration is 0.1g/L is added
Antibody, is incubated 4 hours at 37 DEG C；The bovine serum albumin(BSA) (BSA), 0.21 vitamin C (Vc) and 0.042g for adding 0.21g are sweet
Propylhomoserin, stirring makes to be completely dissolved, and obtains CRP latex reagents.
2nd, profound hypothermia vacuum freeze-drying is carried out to latex reagent：CRP latex reagents are dispensed for every bottle by 2ml, Ran Houfang
Enter vacuum refrigeration freeze dryer (Jiangyin Science and Technology Ltd., model GD-2) and start vavuum pump, then set lyophilized program, make true
Reciprocal of duty cycle≤10Pa, then 6 hours of -50 DEG C of pre-freezes, then carries out multigelation, wherein multigelation is to repeat latex reagent
3 realizations of freeze thawing treatment are carried out, the freeze thawing treatment is to keep latex antibody reagent 1 hour and 20 at -20 DEG C successively
Kept at DEG C 1 hour-, then complete remaining freeze-drying process, stop freeze dryer.
3rd, inert gas shielding is carried out to dried frozen aquatic products：Freeze dryer air inlet is connected with argon tanks gas outlet, opens argon tanks
Valve and freeze dryer intake valve, make argon gas be gradually filled with freeze dryer to pressure and reach 1.1 × 105Pa, then makes in reagent bottle pressure
Plug is sealed, and is obtained the latex CRP freeze-dried reagents of the present invention, is designated as reagent C RP1.
Embodiment 2
Reference picture 1, prepares method of the latex than turbid freeze-dried reagent according to the present invention, prepares latex CRP freeze-dried reagents, has
Body is as follows：
1st, latex reagent is prepared：The latex solution 100ml for taking particle diameter 200nm, concentration to be 1 ‰, be with 500ml concentration
0.1 ‰ 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) are mixed, at room temperature gentle agitation 2 hours,
So as to which latex particle is activated in advance；Then add and incubated at the CRP monoclonal antibody that 100ml concentration is 0.1g/L, 37 DEG C
Educate 4 hours；3.5g BSA, 7.0gVc and 1.4g glycine is added, stirring makes to be completely dissolved, and obtains CRP latex reagents.
2nd, profound hypothermia vacuum freeze-drying is carried out to latex reagent：CRP latex reagents are dispensed for every bottle by 2ml, Ran Houfang
Enter vacuum refrigeration freeze dryer (Jiangyin Science and Technology Ltd., model GD-2) and start vavuum pump, then set lyophilized program, make true
Reciprocal of duty cycle≤10Pa, then 7 hours of -50 DEG C of pre-freezes, then carries out multigelation, wherein multigelation is to repeat latex reagent
5 realizations of freeze thawing treatment are carried out, the freeze thawing treatment is to keep latex antibody reagent 1 hour and 20 at -20 DEG C successively
Kept for 1 hour at DEG C, finally complete remaining freeze-drying process, stop freeze dryer.
3rd, inert gas shielding is carried out to dried frozen aquatic products：Freeze dryer air inlet is connected with argon tanks gas outlet, opens argon tanks
Valve and freeze dryer intake valve, make argon gas be gradually filled with freeze dryer to pressure and reach 1.1 × 105Pa, then makes in reagent bottle pressure
Plug is sealed, and is obtained the latex CRP freeze-dried reagents of the present invention, is designated as reagent C RP2.
Embodiment 3
Reference picture 1, prepares method of the latex than turbid freeze-dried reagent according to the present invention, prepares latex CRP freeze-dried reagents, has
Body is as follows：
1st, latex reagent is prepared：The latex solution 100ml for taking particle diameter 200nm, concentration to be 0.5 ‰, be with 250ml concentration
0.3 ‰ 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) are mixed, at room temperature gentle agitation 2 hours,
So as to which latex particle is activated in advance；Then add and be incubated at the CRP monoclonal antibody that 20ml concentration is 0.1g/L, 37 DEG C
4 hours；1.0g BSA, 3.0gVc and 0.5g glycine is added, stirring makes to be completely dissolved, and obtains CRP latex reagents.
2nd, profound hypothermia vacuum freeze-drying is carried out to latex reagent：CRP latex reagents are dispensed for every bottle by 2ml, Ran Houfang
Enter vacuum refrigeration freeze dryer (Jiangyin Science and Technology Ltd., model GD-2) and start vavuum pump, then set lyophilized program, make true
Reciprocal of duty cycle≤10Pa, then 6 hours of -50 DEG C of pre-freezes, then carries out multigelation, wherein multigelation is to repeat latex reagent
4 realizations of freeze thawing treatment are carried out, the freeze thawing treatment is to keep latex antibody reagent 1 hour and 20 at -20 DEG C successively
Kept for 1 hour at DEG C, finally complete remaining freeze-drying process, stop freeze dryer.
3rd, inert gas shielding is carried out to vacuum freeze-drying product：Freeze dryer air inlet is connected with argon tanks gas outlet, opens argon
Gas tank valve and freeze dryer intake valve, make argon gas be gradually filled with freeze dryer to pressure and reach 1.1 × 105Pa, then makes reagent bottle
Top cement plug sealing is pressed, the latex CRP freeze-dried reagents of the present invention is obtained, is designated as reagent C RP3.
Comparative example 1
Latex CRP freeze-dried reagents are prepared with reference to the method for embodiment 3, are differed only in：
In step 3 without inert gas shielding, but air is directly poured into, i.e.,：Freeze dryer air inlet directly with sky
Gas phase is led to, and is opened freeze dryer intake valve, air is gradually filled with freeze dryer to pressure and reach 1.1 × 105Pa, then makes reagent bottle
Top cement plug sealing is pressed, latex CRP freeze-dried reagents is obtained, is designated as reagent C RP4.
Embodiment 4 freezes the homogeneity detection of latex CRP reagents
1st, 8 bottles of reagent C RP1 (being prepared by above-described embodiment 1) are randomly selected, its outward appearance after freezing are observed, as a result
It was found that：Reagent consolidation after lyophilized and positioned at bottom of bottle, bubble-free is produced.Its effect is shown in accompanying drawing 2.
2nd, 5 bottles of reagent C RP2 (being prepared by above-described embodiment 2), every bottle of addition 2.0ml purified water, 15 points are randomly selected
Gentle overturn in interval mixes 5 times in clock, puts desktop, as a result observes：Reagent is completely dissolved, and is visible by naked eyes suspension.Redissolve
Effect is shown in accompanying drawing 3 afterwards.
3rd, 10 bottles of reagent C RP3 (being prepared by above-described embodiment 3) are extracted, with Shenzhen You Di Bioisystech Co., Ltd
Whole blood CRP hemolytic agents (reality of its preparation method see Application No. CN201611218493.1 Chinese patent application of production
Apply example 3) coordinate, (auspicious BC-5390 instruments advanced in years and supporting whole blood CRP reagents are utilized to the whole blood sample of two varying levels respectively
Fresh whole blood sample after detection) respectively test once, as a result：The test result coefficient of variation of 10 bottles of reagents within 10%,
Concrete outcome is shown in Table 1.
Table 1 freezes uniformity result between the bottles of CRP latex reagents
| Reagent C RP3 | Whole blood sample 1 | Whole blood sample 2 |
| 1st bottle | 3.28 | 11.15 |
| 2nd bottle | 3.33 | 11.08 |
| 3rd bottle | 3.68 | 10.08 |
| 4th bottle | 3.49 | 10.44 |
| 5th bottle | 3.52 | 10.35 |
| 6th bottle | 3.53 | 10.86 |
| 7th bottle | 3.31 | 11.28 |
| 8th bottle | 3.54 | 10.89 |
| 9th bottle | 3.66 | 10.51 |
| 10th bottle | 3.55 | 10.95 |
| Average | 3.49 | 10.76 |
| CV | 4.04% | 3.65% |
Embodiment 5 freezes the accuracy detection of latex CRP reagents
The CRP latexes freeze-dried reagent (reagent C RP1, CRP2, CRP3) of the invention that Example 1-3 is prepared, respectively
(its preparation method is see Application No. for the whole blood CRP hemolytic agents produced with Shenzhen You Di Bioisystech Co., Ltd
Embodiment 3 in CN201611218493.1 Chinese patent application specification) coordinate, the whole blood CRP with stepping auspicious BC-5390
(compareing) is contrasted, to being tested from clinical fresh whole blood sample (in 2 hours), follow-on test 5 days, daily 8
Individual sample, totally 40 samples.As a result find, reagent C RP1, CRP2, CRP3 of the invention, to the testing results of 40 samples with
The uniformity of control is very high.Wherein, by taking CRP2 testing result as an example, correlation is Y=0.9778X-0.1356, R2=
0.998 (accuracy result is shown in Table 2, and correlation results are shown in Fig. 4).
Table 2 freezes the accuracy result of CRP latex reagents
Embodiment 6 freezes the stability of latex CRP reagents
CRP latex freeze-dried reagents --- reagent C RP1, CRP2, CRP3 of the invention that embodiment 1-3 is prepared, with
And the CRP4 that comparative example 1 is prepared, four kinds of reagents put room temperature storage respectively, then, and each two month takes one bottle, with the excellent enlightening in Shenzhen
(its preparation method is see Application No. CN201611218493.1's for the whole blood CRP hemolytic agents of Bioisystech Co., Ltd's production
The embodiment 3 of Chinese patent application) coordinate, Roche CRP quality-control product Precinorm U and the Precipath U of outsourcing are carried out
Test, and calculate the testing result of 14th month and the relative deviation of the 0th month.As a result show, the lyophilized examination of CRP latexes of the invention
Agent CRP1, CRP2, CRP3 result uniformity after room temperature storage 14 months are still very high, and deviation is and right within 10%
The deviation of the CRP4 reagents of ratio just exceeded more than 40% in 4th month room temperature storage beyond 10%, the 14th month.Tool
Volume data is shown in Table 3.
Table 3 freezes the stability result of CRP latex reagents
As can be seen here, the normal temperature storage stability of CRP latex freeze-dried reagents of the invention is protruded, and without inert gas
Protection can cause the normal temperature storage stability of freeze-dried reagent to be remarkably decreased.
Disposably lyophilized and multigelation effect compares embodiment 7
This experiment determines disposable lyophilized and influence of the multigelation to the performance of latex freeze-dried reagent according to following operation：
1st, the method with reference to embodiment 2 prepares latex CRP freeze-dried reagents, differs only in：Latex reagent is carried out deep low
During warm vacuum freeze-drying, the technology of multigelation is not used, and uses disposable freeze-drying process：1 hour of keeping temperature at -20 DEG C
(other lyophilisation conditions are constant).Thus, freeze-dried reagent CRP5 is obtained.
2nd, the reagent C RP5 of above-mentioned preparation is taken, respectively filling each 10 bottles by 2ml and 4ml, and is divided into 2 groups：(10 bottles of group 1
2ml) with 2 (10 bottles of 4ml) of group, altogether 20 bottles.
3rd, the CRP latex reagent CRP2 prepared in Example 2, respectively filling each 10 bottles by 2ml and 4ml, and are divided into 2
Group：Organize 3 (10 bottles of 2ml) and organize 4 (10 bottles of 4ml), altogether 20 bottles.
4th, 4 groups of freeze-dried reagents are weighed respectively, and calculate the homogeneity of the lyophilized rear weight of 4 group reagents, represented with CV,
It the results are shown in Table 4.
Table 4 is disposably freezed and influence testing result of the multigelation to the performance of latex freeze-dried reagent
From the above results, using uniformity between bottle after either 2ml or 4ml loading amounts are lyophilized after multigelation with it is empty
Bottle is close；And use disposable lyophilized, differential nearly 10% between bottle when uniformity is then higher by several times, especially 4ml loading amounts between bottle, and
Weight shows and not freezed seriously.Show, the disposable freeze drying process used relative to prior art, the present invention uses multigelation
Method can effectively improve the performance and stability of latex freeze-dried reagent.
The influence of the pre-activate of embodiment 8 and protective agent to the stability of latex freeze-dried reagent
This experiment compares according to following operation, determines the shadow of pre-activate and protective agent to the stability of latex freeze-dried reagent
Ring：
1st, latex freeze-dried reagent is prepared without pre-activate：Latex CRP freeze-dried reagents, area are prepared with reference to the method for embodiment 2
It is not only that：500ml pH7.0 concentration is used to replace the 1- that 500ml concentration is 0.1 ‰ for 0.1mol/L PBS solution
(3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), namely latex particle do not activated in advance.Thus,
Obtain freeze-dried reagent CRP2-1.
2nd, the latex reagent of unprotect agent is prepared：Latex CRP freeze-dried reagents are prepared with reference to the method for embodiment 2, difference is only
It is：Without BSA, Vc and glycine.Thus, freeze-dried reagent CRP2-2 is obtained.
3rd, reagent C RP2 (latex prepared by embodiment 2 is than turbid freeze-dried reagent), CRP2-1 and CRP2- are randomly selected
2 each 5 bottles of (being prepared by above-mentioned steps 1,2), then puts 37 DEG C of insulating boxs and carries out heat endurance failure test.
4th, at interval of 1~2 day, CRP2, CRP2-1 and CRP2-2 freeze-dried reagent each one destroyed by heat endurance is taken out
Bottle, carries out Detection of Stability：Every bottle of addition 2.0ml purified water, is spaced gentle overturn and mixes 5 times, be allowed to completely molten in 15 minutes
Solution, then respectively with Shenzhen You Di Bioisystech Co., Ltd produce whole blood CRP hemolytic agents coordinate, to two varying levels-
20 DEG C of serum samples frozen are respectively tested once.Follow-on test 7 days, compares the test result that heat endurance destroys the 7th day reagent
And the deviation of test result before lyophilized, concrete outcome is shown in Table 5.
The influence testing result of the pre-activate of table 5 and protective agent to the stability of latex freeze-dried reagent
From the above results, latex freeze-dried reagent CRP2 of the invention is destroyed 7 days in 37 DEG C of conditions by heat endurance,
Reagent test result error has good stability within 10%；Latex dried frozen aquatic products prepared by coupling method is activated using non-EDC
CRP2-1, heat endurance wretched insufficiency have dropped more than 30% in 7 days；The latex dried frozen aquatic products CRP2- prepared without protective agent
The stability of 2 reagents is also decreased obviously, 37 DEG C processing 7 days in have dropped about 20%.Show, relative to prior art, the present invention
Method using EDC activation coupling, addition protective agent, the stability of latex freeze-dried reagent can be effectively improved.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or the spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
Claims (12)
1. a kind of prepare method of the latex than turbid freeze-dried reagent, it is characterised in that comprises the following steps：
Latex particle is coupled with monoclonal antibody, protective agent is then added, to obtain latex antibody reagent, wherein,
The protective agent is made up of bovine serum albumin(BSA), vitamin C and glycine；
Latex antibody reagent progress cryogenic vacuum is freezed, to obtain dried frozen aquatic products, wherein the cryogenic vacuum is lyophilized is
Realized by multigelation；And
In atmosphere of inert gases, the dried frozen aquatic products is sealed, to obtain the latex than turbid freeze-dried reagent.
2. according to the method described in claim 1, it is characterised in that the latex particle is provided in the form of latex solution, and
The particle diameter of the latex particle is 200~300nm, in the latex solution quality-volumetric concentration of latex particle for 0.1 ‰~
1‰。
3. method according to claim 2, it is characterised in that before the coupling is carried out, further comprising will be described
The step of latex particle is activated in advance, and the activation is by by the latex solution and 1- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochlorides solution in equal volume mix and be slowly stirred at room temperature 2 hours realization.
4. method according to claim 3, it is characterised in that 1- (3- the dimethylamino-propyls) -3- ethyls carbon two is sub-
Quality-volumetric concentration of amide hydrochloride is 0.1 ‰~0.5 ‰.
5. according to the method described in claim 1, it is characterised in that the monoclonal antibody is provided in the form of antibody-solutions,
And quality-volumetric concentration of monoclonal antibody is quality-volume of latex particle in the latex solution in the antibody-solutions
The 5%~10% of concentration.
6. according to the method described in claim 1, it is characterised in that in the latex antibody reagent, the bovine serum albumin
White quality-volumetric concentration is 1~5g/L, and the ascorbic quality-volumetric concentration is 1~10g/L, the glycine
Quality-volumetric concentration is 0.2-2g/L.
7. according to the method described in claim 1, it is characterised in that the multigelation is by not higher than 10Pa air
Pressure, repeats 3-5 realization of freeze thawing treatment, the freeze thawing treatment is to resist the latex by the latex antibody reagent
Body reagent is kept for 1 hour and kept for 1 hour at 20 DEG C at -20 DEG C successively.
8. according to the method described in claim 1, it is characterised in that before the progress cryogenic vacuum is lyophilized, further wrap
Include：
Under conditions of atmospheric pressure is not higher than 10Pa and temperature for -50 DEG C, the latex antibody reagent is subjected to pre-freeze processing not
Less than 6 hours.
9. according to the method described in claim 1, it is characterised in that the atmosphere of inert gases is more than the harmless of air by density
Gas is formed.
10. method according to claim 9, it is characterised in that the atmosphere of inert gases is by argon gas or carbon dioxide shape
Into.
11. according to the method described in claim 1, it is characterised in that after dried frozen aquatic products is obtained, accessed using freeze dryer air inlet
Inert gas, to make the inert gas form atmosphere of inert gases full of freeze dryer.
12. a kind of latex is than turbid freeze-dried reagent, it is prepared by the method described in claim any one of 1-11.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110716048A (en) * | 2019-10-30 | 2020-01-21 | 烟台芥子生物技术有限公司 | Dry immunoturbidimetric reagent and preparation method and application thereof |
| CN111505317A (en) * | 2020-04-21 | 2020-08-07 | 江西乐成生物医疗有限公司 | Adiponectin determination reagent quality control product and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104502600A (en) * | 2014-11-10 | 2015-04-08 | 江苏宏泰格尔生物医学工程有限公司 | Pepsinogen I detection method and kit thereof |
| CN104949980A (en) * | 2015-07-01 | 2015-09-30 | 南京普朗医疗设备有限公司 | C reaction protein immune turbidimetry kit and preparation method thereof |
| CN105362958A (en) * | 2015-12-17 | 2016-03-02 | 别会荣 | Traditional Chinese medicine composition for preventing ABO hemolysis of fetuses and newborns |
| CN105709235A (en) * | 2016-03-31 | 2016-06-29 | 深圳市国赛生物技术有限公司 | Cryoprotectant and lyophilization method for latex coupled antibody |
-
2017
- 2017-07-07 CN CN201710552588.5A patent/CN107255727B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104502600A (en) * | 2014-11-10 | 2015-04-08 | 江苏宏泰格尔生物医学工程有限公司 | Pepsinogen I detection method and kit thereof |
| CN104949980A (en) * | 2015-07-01 | 2015-09-30 | 南京普朗医疗设备有限公司 | C reaction protein immune turbidimetry kit and preparation method thereof |
| CN105362958A (en) * | 2015-12-17 | 2016-03-02 | 别会荣 | Traditional Chinese medicine composition for preventing ABO hemolysis of fetuses and newborns |
| CN105709235A (en) * | 2016-03-31 | 2016-06-29 | 深圳市国赛生物技术有限公司 | Cryoprotectant and lyophilization method for latex coupled antibody |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110716048A (en) * | 2019-10-30 | 2020-01-21 | 烟台芥子生物技术有限公司 | Dry immunoturbidimetric reagent and preparation method and application thereof |
| CN110716048B (en) * | 2019-10-30 | 2021-08-13 | 烟台芥子生物技术有限公司 | Dry immunoturbidimetric reagent and preparation method and application thereof |
| CN111505317A (en) * | 2020-04-21 | 2020-08-07 | 江西乐成生物医疗有限公司 | Adiponectin determination reagent quality control product and preparation method thereof |
| CN111505317B (en) * | 2020-04-21 | 2023-09-29 | 江西乐成生物医疗有限公司 | Quality control product of adiponectin determination reagent and preparation method thereof |
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