CN107255727B - 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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- CN107255727B CN107255727B CN201710552588.5A CN201710552588A CN107255727B CN 107255727 B CN107255727 B CN 107255727B CN 201710552588 A CN201710552588 A CN 201710552588A CN 107255727 B CN107255727 B CN 107255727B
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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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Abstract
The invention discloses latexes than turbid freeze-dried reagent and preparation method thereof; method includes the following steps: latex particle and monoclonal antibody are coupled, protective agent is then added, to obtain latex antibody reagent; wherein, the protective agent is made of bovine serum albumin(BSA), vitamin C and glycine；The latex antibody reagent is subjected to cryogenic vacuum freeze-drying, to obtain dried frozen aquatic products, wherein cryogenic vacuum freeze-drying is realized by multigelation；And in atmosphere of inert gases, the dried frozen aquatic products are sealed, to obtain the latex than turbid freeze-dried reagent.For the latex prepared using this method than turbid freeze-dried reagent, stability is good；It is good to redissolve fast and homogeneity, without visible suspended matter；Performance is unaffected after redissolution, is swift in response with whole blood sample, can be reported in 1 minute as a result, and repeatability it is very good, as a result accurately and reliably.
Description
Technical field
The present invention relates to technical field of in vitro diagnostic reagents, and in particular to latex is than turbid freeze-dried reagent and preparation method thereof.
Background technique
External diagnosis reagent, which refers to, can be used alone or use 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 (object), the quality-control product (object) of vitro detection
Deng.The one kind of external diagnosis reagent as medical instrument is that reagent has with instrument and equipment class medical instrument maximum differential
The effect phase, i.e., declare condition of storage can stablize maintain reagent performance time limit.The validity period of reagent is mainly with reagent at grouping
It is related at, factors such as dosage form 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 that in 2~8 DEG C of low-temperature storages；Even if there is other individual non-latex immunoturbidimetry class examinations
Agent position dried frozen aquatic products, it is required that condition of storage be also substantially 2~8 DEG C or lower temperature.On the market there is not yet room temperature stores up
The latex deposited is sold than turbid reagent.
Thus, to can normal temperature storage latex still have than the research of turbid reagent it is to be strengthened.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention
It is to propose the latex for capableing of storage-stable at normal temperature than turbid reagent.
It should be noted that the present invention is following discovery based on inventor and work and completes:
On September 22nd, 2016, CFDA general bureau have issued the bulletin of " medical instrument cold chain (transport, storage) administration guide "
(2016 No. 154), to storage temperature require the medical device product within the scope of 2~8 DEG C and following temperature production,
Storage and the low-temperature protection measure of transportational process require to be standardized.The requirement itself is to the quality for reinforcing medical device product
There is very important meaning in terms of management and the effective reliability of promotion product safety, the risk of reduction patient, but undeniably must
The either cost of manufacturing enterprise or Sales Channel quotient will be promoted.Therefore, medical device product stability is promoted to make it
It can store and transport under normal temperature conditions, entreprise cost will can be greatly reduced and promote the effect of the links such as the manufacturing, transport
Rate.
The stability of reagent refers to that the performance of reagent when reagent reaches the validity period declared under condition of storage as required meets
The ability of pre-provisioning request, usual reagent during storage due to air contact or due to remaining air in reagent bottle,
Oxygen or carbon dioxide therein can occur chemical reaction with the ingredient in reagent or destroy the equilibrium condition of reagent solution,
Such as pH value, surface tension, chemical bond affinity, so that the performance of reagent be made to change, performance results are reagent test sample
This result shifts, and this destruction or influence are usually huge with storage temperature relationship, according to Arrhenius empirical equation,
Temperature is every to rise 10 DEG C, and reaction speed usually increases by 2~4 times, i.e. every 10 DEG C of the increase of reagent storage temperature, under the stability of reagent
It is down to original 1/2~1/4.
Thus, as one can imagine, the latex for capableing of storage-stable at normal temperature is obtained than turbid reagent, and difficulty is very big.And it sends out
Bright people 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 incorporated in latex antibody reagent carrying out cryogenic vacuum freeze-drying
The square technology of Shi Caiyong multigelation, and in atmosphere of inert gases carry out dried frozen aquatic products sealing, can prepare can reach it is pre-
The stabilizing effect of phase, can under room temperature storage-stable latex than turbid freeze-dried reagent.
Thus, in the first aspect of the present invention, method of the latex than turbid freeze-dried reagent is prepared the present invention provides a kind of.Root
According to the embodiment of the present invention, method includes the following steps: latex particle and monoclonal antibody are coupled, then addition is protected
Agent is protected, to obtain latex antibody reagent, wherein the protective agent is made of bovine serum albumin(BSA), vitamin C and glycine；
The latex antibody reagent is subjected to cryogenic vacuum freeze-drying, to obtain dried frozen aquatic products, wherein cryogenic vacuum freeze-drying is to pass through
What multigelation was realized；And in atmosphere of inert gases, the dried frozen aquatic products are sealed, to obtain the latex than turbid
Freeze-dried reagent.It is surprisingly found by the inventors that can effectively prepare latex than turbid freeze-dried reagent using this method.According to this hair
Bright embodiment, for the latex that the present invention prepares than turbid freeze-dried reagent, stability is good, at normal temperature can be storage-stable 12
Month or more in addition 14 months；It redissolves fastly, can be redissolved completely in 15 minutes and homogeneity is good, without visible suspended matter；Performance after redissolution
It is unaffected, be swift in response with whole blood sample, can be reported in 1 minute as a result, and repeatability it is very good, as a result accurately and reliably.
Wherein, it should be noted that when preparing latex antibody reagent, add by bovine serum albumin(BSA), vitamin C and sweet ammonia
The protective agent of acid composition, can make albumen and lipid in reagent be dispersed and be protected, and be unlikely to it in freeze-drying time variation
And conglomeration, and then subsequent obtained freeze-dried reagent can dissolve after adding water quickly when redissolving, that is, redissolve fastly, and reach homogeneity,
Without visible suspended matter.
According to an embodiment of the invention, the latex particle is provided in the form of latex solution, and the latex particle
Partial size is 200~300nm, and quality-volumetric concentration of latex particle is 0.1 ‰~1 ‰ in the latex solution.Latex as a result,
Particle and the coupling effect of monoclonal antibody are good, and the latex antibody reagent stability of acquisition is good.
According to an embodiment of the invention, further comprising that the latex particle is pre- advanced before carrying out the coupling
The step of row activation, and the activation is by the way that the latex solution and 1- (3- dimethylamino-propyl) -3- ethyl carbon two is sub-
Amide hydrochloride mixes in equal volume and is slowly stirred realization in 2 hours at room temperature.As a result, because of stable reagent, not easily to fall off,
Coupling effect is good, and the latex antibody reagent stability of acquisition is prominent.
Some specific examples according to the present invention, 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride
Quality-volumetric concentration of solution is 0.1 ‰~0.5 ‰.Latex particle activation effect is good as a result, subsequent and monoclonal antibody
Coupling effect is good, and the latex antibody reagent stability of acquisition is prominent.
According to an embodiment of the 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%.Good with the coupling effect of latex particle as a result, the latex antibody reagent stability of acquisition is prominent.
According to an embodiment of the invention, quality-volume of the bovine serum albumin(BSA) is dense in the latex antibody reagent
Degree is 1~5g/L, and the ascorbic quality-volumetric concentration is 1~10g/L, and quality-volumetric concentration of the glycine is
0.2-2g/L.Protective agent is good to the protecting effect of latex antibody reagent as a result, and obtained latex antibody reagent stability is prominent.
According to an embodiment of the invention, the multigelation be by not higher than 10Pa atmospheric pressure under, by the cream
Glue antibody reagent repeat freeze thawing treatment 3-5 times realization, the freeze thawing treatment be by the latex antibody reagent successively-
It is kept for 1 hour at 20 DEG C and is kept for 1 hour at 20 DEG C.
Wherein, latex is prepared in the prior art than turbid freeze-dried reagent, when latex antibody reagent is lyophilized, typically
Using disposable freeze-drying process, 2ml volume reagent at most can only effectively be lyophilized, be then not easy to be lyophilized more than 2ml, especially be lyophilized
Position among storehouse, liquid residue amount is more in the bottle of part, occasionally has liquid to overflow bottleneck；And extend freeze-drying time to 72 hours or more then
On the one hand increased costs be will lead to, on the other hand (freeze dryer continuous service 72 is small it will cause operational process of craft excessively complexity
When more than, risk is big, and special messenger is needed to keep an eye on).And method of the invention, using the technology of multigelation, by latex antibody reagent
Repeating freeze thawing treatment as described above, 3-5 times (freeze thawing treatment is successively to keep latex antibody reagent at -20 DEG C
1 hour and kept for 1 hour at 20 DEG C).Make as a result, 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 makes the built-in water effective frost of latex liquid with convenient
The distillation (i.e. moisture is easily discharged) of subsequent freeze-drying process, dried frozen aquatic products appearance uniform, without obvious bubble, moreover, even if loading amount is in 4ml
It still can be effectively lyophilized within 48 hours inside latex when above.Also, glue ratio is prepared using above-mentioned multigelation technology
When turbid freeze-dried reagent, the problems such as being not in albuminous degeneration, reagent conglomeration, latex surface molecule aquation damage layer, the cream 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
Can report as a result, and for diagnose when it is reproducible, as a result accurately and reliably.
According to an embodiment of the invention, further comprising: not high in atmospheric pressure before carrying out the cryogenic vacuum freeze-drying
Under conditions of 10Pa and temperature are -50 DEG C, the latex antibody reagent is subjected to pre-freeze processing no less than 6 hours.Freeze as a result,
Dry effect is good, and the latex of acquisition is more prominent than turbid freeze-dried reagent appearance, performance.
According to an embodiment of the invention, the atmosphere of inert gases is formed by the innocuous gas that density is greater than air.As a result,
It can effectively prevent influencing the entrance of the air of reagent stability when dried frozen aquatic products seal.It is more according to the present invention specifically to show
Example, the atmosphere of inert gases are formed by argon gas or carbon dioxide.The latex obtained as a result, is more prominent than turbid freeze-dried reagent stability
Out.
According to an embodiment of the invention, inert gas is accessed using freeze dryer air inlet, after obtaining dried frozen aquatic products to make
The inert gas forms atmosphere of inert gases full of freeze dryer.Thereby, it is possible to conveniently and effectively form inert gas atmosphere
It encloses.
In the second aspect of the present invention, the present invention provides a kind of latexes than turbid freeze-dried reagent.Implementation according to the present invention
Example, the latex are to prepare latex by mentioned-above and prepare than turbid freeze-dried reagent method than turbid freeze-dried reagent.According to
The embodiment of the present invention, latex of the invention is than turbid freeze-dried reagent appearance uniform, without obvious bubble；Stability is prominent, Neng Gouchang
Temperature storage, specifically, at normal temperature can be storage-stable 12 months or more or even 14 months；It redissolves fastly, it can be complete in 15 minutes
It redissolves and homogeneity is good, without visible suspended matter；Performance is good, and albuminous degeneration, reagent conglomeration, latex surface molecule hydrated sheath is not present
Phenomena such as destruction, performance is unaffected after redissolution, is swift in response with whole blood sample, can be reported in 1 minute as a result, and repeatability
It is very good, as a result accurately and reliably.
In addition, it should be noted that, 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 is carried out cryogenic vacuum freeze-drying, the dried frozen aquatic products appearance of acquisition is equal
It is even, without obvious bubble；
2) latex of the invention is more prominent 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 12 months or more under room temperature；
3) latex of the invention redissolves fast than turbid freeze-dried reagent, can redissolve completely in 15 minutes, and homogeneity is good, can not
See suspended matter；
4) latex of the invention is better than turbid freeze-dried reagent performance, and albuminous degeneration, reagent conglomeration, latex surface molecule is not present
Phenomena such as aquation damage layer, performance is unaffected after redissolution, is swift in response with whole blood sample, can be reported in 1 minute as a result, and
Repeatability is very good, as a result accurately and reliably.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 is to prepare flow diagram of the latex than the method for 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 reagent CRP1 of the invention that embodiment 1 is prepared；
Fig. 3 is the effect diagram after the CRP latex freeze-dried reagent CRP2 of the invention that embodiment 2 is prepared redissolves；
Fig. 4, which is the CRP latex freeze-dried reagent CRP2 of the invention that is prepared of embodiment 2, steps auspicious BC-5390's with compareing
Whole blood CRP measures the methodology comparison consistency detection result schematic diagram of whole blood sample.
Specific 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.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Embodiment 1
Referring to Fig.1, according to the present invention to prepare method of the latex than turbid freeze-dried reagent, latex CRP freeze-dried reagent is prepared, is had
Body is as follows:
1, latex reagent is prepared: being taken partial size 300nm, the latex solution 100ml that concentration is 0.1 ‰, is with 100ml concentration
0.5 ‰ 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) mixes, at room temperature gentle agitation 2 hours,
So that 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 for 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 make to be completely dissolved, and obtain CRP latex reagent.
2, profound hypothermia vacuum freeze-drying is carried out to latex reagent: CRP latex reagent is dispensed by every bottle of 2ml, is then put
Enter vacuum refrigeration freeze dryer (Jiangyin Science and Technology Ltd., model GD-2) starting vacuum pump, then setting freeze-drying program, makes true
Then reciprocal of duty cycle≤10Pa, then carries out multigelation, wherein multigelation is to repeat latex reagent at 6 hours of -50 DEG C of pre-freezes
Freeze thawing treatment 3 times realizations are carried out, which successively kept latex antibody reagent 1 hour at -20 DEG C and 20
Kept at DEG C 1 hour-, then complete remaining freeze-drying process, stop freeze dryer.
3, carry out inert gas shielding to dried frozen aquatic products: freeze dryer air inlet is connect 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 × 105Then Pa makes in reagent bottle pressure
Rubber plug sealing, obtains latex CRP freeze-dried reagent of the invention, is denoted as reagent C RP1.
Embodiment 2
Referring to Fig.1, according to the present invention to prepare method of the latex than turbid freeze-dried reagent, latex CRP freeze-dried reagent is prepared, is had
Body is as follows:
1, latex reagent is prepared: being taken partial size 200nm, the latex solution 100ml that concentration is 1 ‰, is with 500ml concentration
0.1 ‰ 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) mixes, at room temperature gentle agitation 2 hours,
So that latex particle is activated in advance；Then the CRP monoclonal antibody that 100ml concentration is 0.1g/L is added, is incubated at 37 DEG C
It educates 4 hours；BSA, 7.0gVc and 1.4g glycine of 3.5g is added, stirring makes to be completely dissolved, and obtains CRP latex reagent.
2, profound hypothermia vacuum freeze-drying is carried out to latex reagent: CRP latex reagent is dispensed by every bottle of 2ml, is then put
Enter vacuum refrigeration freeze dryer (Jiangyin Science and Technology Ltd., model GD-2) starting vacuum pump, then setting freeze-drying program, makes true
Then reciprocal of duty cycle≤10Pa, then carries out multigelation, wherein multigelation is to repeat latex reagent at 7 hours of -50 DEG C of pre-freezes
Freeze thawing treatment 5 times realizations are carried out, which successively kept latex antibody reagent 1 hour at -20 DEG C and 20
It is kept for 1 hour at DEG C, finally completes remaining freeze-drying process, stop freeze dryer.
3, carry out inert gas shielding to dried frozen aquatic products: freeze dryer air inlet is connect 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 × 105Then Pa makes in reagent bottle pressure
Rubber plug sealing, obtains latex CRP freeze-dried reagent of the invention, is denoted as reagent C RP2.
Embodiment 3
Referring to Fig.1, according to the present invention to prepare method of the latex than turbid freeze-dried reagent, latex CRP freeze-dried reagent is prepared, is had
Body is as follows:
1, latex reagent is prepared: being taken partial size 200nm, the latex solution 100ml that concentration is 0.5 ‰, is with 250ml concentration
0.3 ‰ 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) mixes, at room temperature gentle agitation 2 hours,
So that latex particle is activated in advance；Then the CRP monoclonal antibody that 20ml concentration is 0.1g/L is added, is incubated at 37 DEG C
4 hours；BSA, 3.0gVc and 0.5g glycine of 1.0g is added, stirring makes to be completely dissolved, and obtains CRP latex reagent.
2, profound hypothermia vacuum freeze-drying is carried out to latex reagent: CRP latex reagent is dispensed by every bottle of 2ml, is then put
Enter vacuum refrigeration freeze dryer (Jiangyin Science and Technology Ltd., model GD-2) starting vacuum pump, then setting freeze-drying program, makes true
Then reciprocal of duty cycle≤10Pa, then carries out multigelation, wherein multigelation is to repeat latex reagent at 6 hours of -50 DEG C of pre-freezes
Freeze thawing treatment 4 times realizations are carried out, which successively kept latex antibody reagent 1 hour at -20 DEG C and 20
It is kept for 1 hour at DEG C, finally completes remaining freeze-drying process, stop freeze dryer.
3, carry out inert gas shielding to vacuum freeze-drying product: freeze dryer air inlet is connect 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 × 105Then Pa makes reagent bottle
Top cement plug sealing is pressed, latex CRP freeze-dried reagent of the invention is obtained, is denoted as reagent C RP3.
Comparative example 1
Latex CRP freeze-dried reagent is prepared referring to the method for embodiment 3, difference is only that:
In step 3 without inert gas shielding, but directly pour into air, it may be assumed that freeze dryer air inlet directly with sky
Gas phase is logical, opens freeze dryer intake valve, so that air is gradually filled with freeze dryer to pressure and reach 1.1 × 105Then Pa makes reagent bottle
Top cement plug sealing is pressed, latex CRP freeze-dried reagent is obtained, is denoted as reagent C RP4.
The homogeneity detection of latex CRP reagent is lyophilized in embodiment 4
1,8 bottles of reagent C RP1 (being prepared by above-described embodiment 1) is randomly selected, the appearance after observing its freeze-drying, as a result
It was found that: reagent consolidation after freeze-drying and it is located at bottom of bottle, bubble-free generates.Its effect is shown in attached drawing 2.
2, it randomly selects 5 bottles of reagent C RP2 (being prepared by above-described embodiment 2), every bottle of addition 2.0ml purified water, 15 points
Interval is mildly mixed by inversion 5 times in clock, and set desktop, as a result observe: reagent is completely dissolved, and is visible by naked eyes suspended matter.It redissolves
Effect is shown in attached drawing 3 afterwards.
3,10 bottles of reagent C RP3 (being prepared by above-described embodiment 3) are extracted, with Shenzhen You Di Bioisystech Co., Ltd
(preparation method is see application No. is the realities of the Chinese patent application of CN201611218493.1 for the whole blood CRP hemolytic agent of production
Apply example 3) cooperation, respectively to the whole blood sample of two different levels (using stepping auspicious BC-5390 instrument and mating whole blood CRP reagent
Fresh whole blood sample after detection) each test is primary, as a result: the test result coefficient of variation of 10 bottles of reagents within 10%,
Concrete outcome is shown in Table 1.
Consistency result between the bottle of CRP latex reagent is lyophilized in table 1
| 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 |
| Mean value | 3.49 | 10.76 |
| CV | 4.04% | 3.65% |
The accuracy detection of latex CRP reagent is lyophilized in embodiment 5
The CRP latex freeze-dried reagent (reagent C RP1, CRP2, CRP3) of the invention that Example 1-3 is prepared, respectively
With Shenzhen You Di Bioisystech Co., Ltd production whole blood CRP hemolytic agent (preparation method see application No. is
Embodiment 3 in the Chinese patent application specification of CN201611218493.1) cooperation, with the whole blood CRP for stepping auspicious BC-5390
(compareing) compares, and tests from clinical fresh whole blood sample (in 2 hours), follow-on test 5 days, daily 8
A sample, totally 40 samples.As a result, it has been found that reagent C RP1, CRP2, CRP3 of the invention, to the testing results of 40 samples with
The consistency of control is very high.Wherein, by taking the testing result of CRP2 as an example, correlation Y=0.9778X-0.1356, R2=
0.998 (accuracy result is shown in Table 2, and correlation results are shown in Fig. 4).
The accuracy result of the freeze-drying CRP latex reagent of table 2
The stability of the freeze-drying latex CRP reagent of embodiment 6
CRP latex freeze-dried reagent --- 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 set room temperature storage respectively, then, the every two moon takes one bottle, with the excellent enlightening in Shenzhen
(preparation method is see application No. is CN201611218493.1's for the whole blood CRP hemolytic agent of Bioisystech Co., Ltd's production
The embodiment 3 of Chinese patent application) cooperation, Roche CRP quality-control product Precinorm U and the Precipath U of outsourcing is carried out
Test, and calculate 14th month testing result and the 0th month relative deviation.The results show that examination is lyophilized in CRP latex of the invention
Agent CRP1, CRP2, CRP3 result consistency after room temperature storage 14 months are still very high, and deviation is and right within 10%
The deviation of the CRP4 reagent of ratio just has exceeded 10% for 4th month room temperature storage, exceeds 40% or more within the 14th month.Tool
Volume data is shown in Table 3.
The stability result of the freeze-drying CRP latex reagent of table 3
It can be seen that the normal temperature storage stability of CRP latex freeze-dried reagent of the invention is prominent, and without inert gas
The normal temperature storage stability that protection will lead to freeze-dried reagent is remarkably decreased.
Disposably freeze-drying and multigelation effect compare embodiment 7
This test determines disposable freeze-drying and influence of the multigelation to the performance of latex freeze-dried reagent according to following operation:
1, the method referring to embodiment 2 prepares latex CRP freeze-dried reagent, and difference is only that: carrying out to latex reagent deep low
When warm vacuum freeze-drying, the technology of multigelation is not used, and uses disposable freeze-drying process: 1 hour of temperature is kept at -20 DEG C
(other lyophilisation conditions are constant).Freeze-dried reagent CRP5 is obtained as a result,.
2, the reagent C RP5 of above-mentioned preparation is taken, presses filling each 10 bottles of 2ml and 4ml respectively, and is divided into 2 groups: (10 bottles of group 1
2ml) and 2 (10 bottles of 4ml) are organized, amounts to 20 bottles.
3, the CRP latex reagent CRP2 prepared in Example 2 presses filling each 10 bottles of 2ml and 4ml respectively, and is divided into 2
Group: 4 (10 bottles of 4ml) of 3 (10 bottles of 2ml) of group and group amount to 20 bottles.
4, it weighs respectively to 4 groups of freeze-dried reagents, and calculates the homogeneity of weight after the freeze-drying of 4 group reagents, indicated with CV,
It the results are shown in Table 4.
Table 4 is disposably lyophilized and influence testing result of the multigelation to the performance of latex freeze-dried reagent
From the above results, using consistency and empty between bottle either after the freeze-drying of 2ml or 4ml loading amount after multigelation
Bottle is close；And disposable freeze-drying is used, differential nearly 10% between bottle when consistency is then higher by several times, especially 4ml loading amount between bottle, and
Weight shows and is not lyophilized seriously.Show the disposable freeze drying process used compared with the existing technology, the present invention uses multigelation
Method can effectively improve the performance and stability of latex freeze-dried reagent.
The influence of 8 pre-activate of embodiment and protective agent to the stability of latex freeze-dried reagent
This test compares according to following operation, determines pre-activate and protective agent to the shadow of the stability of latex freeze-dried reagent
It rings:
1, latex freeze-dried reagent is prepared without pre-activate: preparing latex CRP freeze-dried reagent, area referring to the method for embodiment 2
It is not only that: 500ml pH7.0 concentration being used to replace the 1- that 500ml concentration is 0.1 ‰ by the PBS buffer solution solution of 0.1mol/L
(3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC), namely latex particle is not activated in advance.As a result,
Obtain freeze-dried reagent CRP2-1.
2, it prepares the latex reagent of unprotect agent: preparing latex CRP freeze-dried reagent referring to the method for embodiment 2, difference is only
It is: does not add BSA, Vc and glycine.Freeze-dried reagent CRP2-2 is obtained as a result,.
3, reagent C RP2 (latex prepared by embodiment 2 is than turbid freeze-dried reagent), CRP2-1 and CRP2- are randomly selected
2 (being prepared by above-mentioned steps 1,2) are 5 bottles each, then set 37 DEG C of insulating boxs and carry out thermal stability failure test.
4, at interval of 1~2 day, CRP2, CRP2-1 and CRP2-2 freeze-dried reagent each one destroyed by thermal stability is taken out
Bottle carries out Detection of Stability: every bottle of addition 2.0ml purified water, and interval is mildly mixed by inversion 5 times in 15 minutes, is allowed to completely molten
Solution, the whole blood CRP hemolytic agent then produced respectively with Shenzhen You Di Bioisystech Co., Ltd cooperates, to two different levels-
20 DEG C of serum samples frozen are respectively tested once.Follow-on test 7 days, compare the test result that thermal stability destroys the 7th day reagent
And the deviation of test result, concrete outcome are shown in Table 5 before being lyophilized.
The influence testing result of 5 pre-activate of table 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 thermal stability,
Reagent test result error has good stability within 10%；Using the latex dried frozen aquatic products of non-EDC activation coupling method preparation
CRP2-1, thermal stability wretched insufficiency had dropped 30% or more in 7 days；The latex dried frozen aquatic products CRP2- of protective agent preparation is not added
The stability of 2 reagents is also decreased obviously, 37 DEG C processing 7 days in have dropped about 20%.Show compared with the existing technology, 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 specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable 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
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is defined by the claims and their equivalents.
Claims (9)
1. a kind of prepare method of the latex than turbid freeze-dried reagent, which comprises the following steps:
Latex particle and monoclonal antibody are coupled, protective agent is then added, to obtain latex antibody reagent, wherein
The protective agent is made of bovine serum albumin(BSA), vitamin C and glycine, in the latex antibody reagent, the cow's serum
Quality-volumetric concentration of albumin is 1~5g/L, and the ascorbic quality-volumetric concentration is 1~10g/L, the sweet ammonia
Quality-volumetric concentration of acid is 0.2-2g/L；
The latex antibody reagent is subjected to cryogenic vacuum freeze-drying, to obtain dried frozen aquatic products, wherein cryogenic vacuum freeze-drying is
It is realized by multigelation, wherein the multigelation is by the way that under the atmospheric pressure not higher than 10Pa, the latex is resisted
Body reagent repeats freeze thawing treatment 3-5 times realization, and the freeze thawing treatment is by the latex antibody reagent successively at -20 DEG C
It is lower to be kept for 1 hour and kept for 1 hour at 20 DEG C；And
In atmosphere of inert gases, the dried frozen aquatic products are sealed, to obtain the latex than turbid freeze-dried reagent,
Wherein, before carrying out the cryogenic vacuum freeze-drying, further comprise:
Under conditions of atmospheric pressure is not higher than 10Pa and temperature is -50 DEG C, the latex antibody reagent is subjected to pre-freeze processing not
Less than 6 hours.
2. the method according to claim 1, wherein the latex particle is provided in the form of latex solution, and
The partial size of the latex particle is 200~300nm, in the latex solution quality-volumetric concentration of latex particle be 0.1 ‰~
1‰。
3. according to the method described in claim 2, it is characterized in that, further comprising will be described before carrying out the coupling
The step of latex particle is activated in advance, and the activation is by by the latex solution and 1- (3- dimethylamino third
Base) -3- ethyl-carbodiimide hydrochloride solution mix in equal volume and be slowly stirred at room temperature 2 hours realize.
4. according to the method described in claim 3, it is characterized in that, the 1- (3- dimethylamino-propyl) -3- ethyl carbon two is sub-
Quality-volumetric concentration of amide hydrochloride is 0.1 ‰~0.5 ‰.
5. the method according to claim 1, wherein 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. the method according to claim 1, wherein the atmosphere of inert gases is greater than the harmless of air by density
Gas is formed.
7. according to the method described in claim 6, it is characterized in that, the atmosphere of inert gases is by argon gas or carbon dioxide shape
At.
8. the method according to claim 1, wherein being accessed after obtaining dried frozen aquatic products using freeze dryer air inlet
Inert gas, to make the inert gas form atmosphere of inert gases full of freeze dryer.
9. a kind of latex is prepared by the described in any item methods of claim 1-8.
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| 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 |
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| 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 |
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