CN105628914B - A kind of dilution and preparation method thereof that acridinium ester antigen-antibody conjugate can be made stable - Google Patents
A kind of dilution and preparation method thereof that acridinium ester antigen-antibody conjugate can be made stable Download PDFInfo
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Abstract
The invention discloses a kind of dilutions that acridinium ester antigen-antibody conjugate can be made stable, are added in buffer by carbohydrate, polyalcohol, amino acid, protein stabiliser, chelating agent, surfactant, preservative and additive and are prepared;Dilution produced by the present invention overcomes acridinium ester the shortcomings that buffer is unstable, facile hydrolysis; it plays a protective role to acridinium ester antigen-antibody conjugate; improve the stability of acridinium ester antigen-antibody conjugate; the effect phase used is extended simultaneously, moreover it is possible to be made full use of in external diagnosis reagent case.
Description
Technical field
The invention belongs to chemical analysis technology fields, and in particular to a kind of that acridinium ester antigen-antibody conjugate can be made stable
Dilution and preparation method thereof.
Background technique
Chemiluminescence immunoassay is the immunoassay with the direct labelled antigen of chemiluminescent agent or antibody, acridinium ester
It is ideal luminous substrate, under alkaline condition, acridine ester molecule is attacked by hydrogen oxide produces dichloroethane, and two
Oxidative ethane is unstable and is decomposed into CO2With the N- methylacridine ketone of excited electronic state, issuing wavelength when it returns to ground state is
The light of 430nm.
Acridinium ester label has in chemical structure generates luminous specific groups, during luminescence immunoassay directly
Participate in luminescence-producing reaction.This usual substance does not have background Iuminescence, can be used to detect the sample of low concentration or micro-concentrations in the reaction
Product are the very high luminous agents of a kind of luminous efficiency.Acridinium ester I, II molecule and acridinium carboxamide III can be tied with antibody (or antigen)
It closes, generates the labelled antibody (or antigen) for having that chemiluminescence activity is strong, immune response specificity is high.
From the point of view of current technology level, acridinium ester unstable, facile hydrolysis, conjugate in general buffer are diluted to
When working concentration, the stability of the reagent due to caused by conjugate reduction of concentration in dilution declines, so as to cause examination
The storage life of agent shortens.From the point of view of external diagnosis reagent industry market, the stability quality of acridinium ester antigen-antibody conjugate and
Using effect phase length, the performance indicator of kit entirety also will have a direct impact on.
Summary of the invention
In view of the deficiencies of the prior art, acridinium ester antigen-antibody conjugate can be made steady the purpose of the present invention is to provide a kind of
Fixed dilution, which overcomes acridinium ester the shortcomings that buffer is unstable, facile hydrolysis, to acridinium ester antigen-antibody knot
It closes object to play a protective role, improves the stability of acridinium ester antigen-antibody conjugate, while extending the effect phase used, moreover it is possible to
It makes full use of in external diagnosis reagent case.
Another object of the present invention is to provide a kind of systems of dilution that acridinium ester antigen-antibody conjugate can be made stable
Preparation Method.
Achieving the object of the present invention can be reached by adopting the following technical scheme that:
A kind of dilution that acridinium ester antigen-antibody conjugate can be made stable, is by the component of following mass percentage
It is prepared:
Preferably, the carbohydrate is made of sucrose and trehalose, and the weight ratio of the sucrose and trehalose is (1-
3):1。
Preferably, the polyalcohol is sorbierite, inositol, isopropanol or the one such or two or more mixing of glycerol
Object;The amino acid is serine, glycine, alanine or the one such or two or more mixture of lysine.
Preferably, the protein stabiliser is bovine serum albumin(BSA) (BSA), skimmed milk power, casein, calf serum, ox
IgG or mouse IgG one such or two or more mixture.
Preferably, the protein stabiliser is selected from the casein of BSA, 0.1-1.5% that mass percent is 0.1-2.5%
Or the mixture of one or more of ox IgG of 0.01-0.1%.
Preferably, the chelating agent is that EDTA or DTPA is one such or two kinds of mixture;The surfactant
Wherein for Tween-20, Tween-40, Nonidet P40 (NP-40), Triton X-100 or lauryl sodium sulfate
One or more kinds of mixtures;The preservative is that thimerosal, Proclin 300, Sodium azide or chlorohexidene are therein
One or more kinds of mixtures.
Preferably, the additive is selected from Macrogol 6000 (PEG6000), the 0.05- that mass percent is 0.1-1%
0.5% gelatin, 0.9% the one such or two or more mixture of sodium chloride.
Preferably, the buffer is selected from citrate buffer, the 2- (N- morpholine) that concentration is 0.01-0.2mol/L
Ethanesulfonic acid buffer (MES buffer), Tris-HCl buffer or phosphate buffer are one such.
Preferably, the buffer is that 0.05-0.2mol/L citrate buffer or 0.01-0.1mol/L MES are buffered
Liquid.
A kind of preparation method for the dilution that acridinium ester antigen-antibody conjugate can be made stable, the specific steps of which are as follows:
1) it presses each object of carbon aquation of formula ratio, polyalcohol, amino acid, protein stabiliser, chelating agent, surfactant, prevent
Rotten agent and additive are added in buffer, stir to being completely dissolved, obtain mixed liquor;
2) mixed liquor for obtaining step 1) stirs and evenly mixs, and it is 6-7 to get to dilution that HCl or NaOH, which is added, and adjusts pH value
Liquid.
The beneficial effects of the present invention are:
1, dilution of the invention can make acridinium ester antigen-antibody conjugate keep stabilization, not facile hydrolysis, extend examination
The storage life of agent;
2, dilution of the invention is able to maintain the stability of acridinium ester antigen-antibody conjugate and uses the effect phase, solves
The kit problem short using the effect phase, ensure that the performance indicator of kit entirety.
Specific embodiment
A kind of dilution that acridinium ester antigen-antibody conjugate can be made stable, is by the component of following mass percentage
It is prepared:
Wherein, carbohydrate: being made of sucrose and trehalose, and the weight ratio of sucrose and trehalose is (1-3): 1, preferably
For 2:1;
Polyalcohol: sorbierite, inositol, isopropanol or the one such or two or more mixture of glycerol are selected, preferably
For glycerol;
Amino acid: selecting serine, glycine, alanine or the one such or two or more mixture of lysine,
Preferably glycine;
Protein stabiliser: select BSA, skimmed milk power, casein, calf serum, ox IgG or mouse IgG it is one such or
Two or more mixtures, preferably mass percentage are 0.1-2.5%BSA, 0.1-1.5% casein or 0.01-0.1%
The mixture of one or more of ox IgG;
Chelating agent: select EDTA or DTPA one such or two kinds of mixture, preferably DTPA;
Surfactant: select Tween-20, Tween-40, NP-40, Triton X-100 or lauryl sodium sulfate its
One or more of mixture, preferably mass percentage be 0.01~0.5% Tween-20 or Triton
The mixture of one or both of X-100;
Preservative: select thimerosal, Proclin 300, Sodium azide or chlorohexidene one such or two or more mixed
Close object;
Additive: selected from mass percent be 0.1-1% PEG6000,0.05-0.5% gelatin, 0.9% chlorination
The one such or two or more mixture of sodium, preferably mass percentage are 0.05-0.5%PEG6000 and 0.9% chlorine
Change sodium;
Buffer: citrate buffer, MES buffer, the Tris-HCl buffering for being 0.01-0.2mol/L selected from concentration
Liquid or phosphate buffer are one such, preferably the citrate buffer of 0.05-0.2mol/L or 0.01-0.1mol/L
MES buffer.
The preparation method of dilution is as follows:
1) above-mentioned raw materials are mixed, stirs to being completely dissolved, obtains mixed liquor;
2) mixed liquor for obtaining step 1) stirs and evenly mixs, and it is 6.4 ± 0.1 to get arriving that HCl or NaOH, which is added, and adjusts pH value
Dilution.
Embodiment 1:
A kind of dilution that acridinium ester antigen-antibody conjugate can be made stable, is by the component of following mass percentage
It is prepared:
The preparation method of dilution is as follows:
1) sucrose, trehalose, glycerol, glycine, BSA, DTPA, Tween-20, Sodium azide, the PEG6000 of formula ratio are pressed
It is added in 0.05mol/L citrate buffer with sodium chloride, stirs to being completely dissolved, obtain mixed liquor;
2) mixed liquor for obtaining step 1) stirs and evenly mixs, be added NaOH adjust pH value be 6.4 ± 0.1 to get to dilution
Liquid.
It is diluted with the dilution that embodiment 1 is prepared to below with reference to object, conjugate is acridinium ester-FSH antibody respectively
With acridinium ester-E2 antigen, it is diluted to working concentration, obtains acridinium ester-FSH antibody diluent (1) and acridinium ester-E2 antigen diluent
Liquid (1).Conjugate after two kinds of dilutions respectively separates 13 bottles, every bottle of general 1ml.Wherein 3 bottles of placements, 37 DEG C of baking ovens accelerate real
It tests, in addition places 2-8 DEG C of refrigerator preservation for 5 bottles, there are also 5 bottles of placement room temperature preservations.
Embodiment 2:
A kind of dilution that acridinium ester antigen-antibody conjugate can be made stable, is by the component of following mass percentage
It is prepared:
The preparation method of dilution is as follows:
1) sucrose, trehalose, glycerol, glycine, BSA, DTPA, Tween-20, Sodium azide, the PEG6000 of formula ratio are pressed
It is added in 0.2mol/L citrate buffer with sodium chloride, stirs to being completely dissolved, obtain mixed liquor;
2) mixed liquor for obtaining step 1) stirs and evenly mixs, be added NaOH adjust pH value be 6.4 ± 0.1 to get to dilution
Liquid.
It is diluted with the dilution that embodiment 2 is prepared to below with reference to object, conjugate is acridinium ester-FSH antibody respectively
With acridinium ester-E2 antigen, it is diluted to working concentration, obtains acridinium ester-FSH antibody diluent (2) and acridinium ester-E2 antigen diluent
Liquid (2).Conjugate after two kinds of dilutions respectively separates 13 bottles, every bottle of general 1ml.Wherein 3 bottles of placements, 37 DEG C of baking ovens accelerate real
It tests, in addition places 2-8 DEG C of refrigerator preservation for 5 bottles, there are also 5 bottles of placement room temperature preservations.
Embodiment 3:
A kind of dilution that acridinium ester antigen-antibody conjugate can be made stable, is by the component of following mass percentage
It is prepared:
The preparation method of dilution is as follows:
1) sucrose, trehalose, glycerol, glycine, BSA, DTPA, Tween-20, Sodium azide, the PEG6000 of formula ratio are pressed
It is added in 0.1mol/L citrate buffer with sodium chloride, stirs to being completely dissolved, obtain mixed liquor;
2) mixed liquor for obtaining step 1) stirs and evenly mixs, be added NaOH adjust pH value be 6.4 ± 0.1 to get to dilution
Liquid.
It is diluted with the dilution that embodiment 3 is prepared to below with reference to object, conjugate is acridinium ester-FSH antibody respectively
With acridinium ester-E2 antigen, it is diluted to working concentration, obtains acridinium ester-FSH antibody diluent (3) and acridinium ester-E2 antigen diluent
Liquid (3).Conjugate after two kinds of dilutions respectively separates 13 bottles, every bottle of general 1ml.Wherein 3 bottles of placements, 37 DEG C of baking ovens accelerate real
It tests, in addition places 2-8 DEG C of refrigerator preservation for 5 bottles, there are also 5 bottles of placement room temperature preservations.
Embodiment 4:
A kind of dilution that acridinium ester antigen-antibody conjugate can be made stable, is by the component of following mass percentage
It is prepared:
The preparation method of dilution is as follows:
1) sucrose, trehalose, glycerol, glycine, BSA, the ox IgG, DTPA, Tween-20, Triton X- of formula ratio are pressed
100, Sodium azide, Proclin 300, PEG6000 and sodium chloride are added in 0.1mol/LMES buffer, stirring to being completely dissolved,
Obtain mixed liquor;
2) mixed liquor for obtaining step 1) stirs and evenly mixs, be added NaOH adjust pH value be 6.4 ± 0.1 to get to dilution
Liquid.
It is diluted with the dilution that embodiment 4 is prepared to below with reference to object, conjugate is acridinium ester-FSH antibody respectively
With acridinium ester-E2 antigen, it is diluted to working concentration, obtains acridinium ester-FSH antibody diluent (4) and acridinium ester-E2 antigen diluent
Liquid (4).Conjugate after two kinds of dilutions is respectively separated 13 bottles, every bottle of general 1ml.Wherein 3 bottles of placements, 37 DEG C of baking ovens are accelerated
In addition experiment places 2-8 DEG C of refrigerator preservation for 5 bottles, there are also 5 bottles of placement room temperature preservations.
The stability test of conjugate after dilution:
According to the Check-Out Time of formulation, the acridinium ester antigen-antibody conjugate after above-mentioned dilution is detected respectively, it will
Conjugate after 100 μ l dilution is added in reaction cup, and light-emitting appearance is automatically added to the 100 μ l preexciting liquid (acid containing hydrogen peroxide
Property solution, pH<2.0) and 100 μ l exciting liquids (alkaline solution containing sodium hydroxide, pH>13.0), conjugate excites being added
It shines at once after liquid, light-emitting appearance shows the relative light unit (RLUs) measured.It is that the specific embodiment of the invention obtains below
Testing result.
Detection of Stability result of conjugate under the conditions of 37 DEG C after the dilution of table 1
Detection of Stability result of conjugate under the conditions of 2-8 DEG C after the dilution of table 2
The Detection of Stability result of conjugate at room temperature after the dilution of table 3
It carries out analysis to the inspection result of three tables above to obtain, it is equal that specific embodiment makes the four kinds of dilutions come
There is protective effect to acridinium ester antigen-antibody conjugate, so that the shining under different conditions of storage of the conjugate reagent after dilution
Efficiency does not decline.This is because as the temperature rises or reduce, the key that antigen or antibody are connected with acridinium ester can it is unstable and
The substances such as carbohydrate, the albumen for being broken, and adding in dilution such as sucrose, BSA are able to maintain the stabilization of key, make antigen or
The key that antibody is connected with acridinium ester will not because of temperature variation and be broken, thus protect acridinium ester antigen-antibody conjugate,
And maintain luminous efficiency of the conjugate reagent under different conditions of storage after dilution.Wherein placed 7 days under the conditions of 37 DEG C
Afterwards, four kinds of acridinium ester-FSH antibody reagents and four kinds of acridinium ester-E2 antigenic agents luminous efficiencies are reduced only by 6.34-7.62%;
After placing 14 months under the conditions of 2-8 DEG C, four kinds of acridinium ester-FSH antibody reagents and four kinds of acridinium ester-E2 antigenic agents shine and imitate
Rate only has dropped 3.27-6.49%;And after placing 5 weeks at room temperature, four kinds of acridinium ester-FSH antibody reagents and four kinds
Acridinium ester-E2 antigenic agents luminous efficiency only has dropped 1.75-5.89%.
Therefore the dilution provided by the invention that acridinium ester antigen-antibody conjugate can be made stable, to acridinium ester conjugate into
Row dilution, the reagent preservation effect phase after dilution is that can save 12 months or more under the conditions of 2-8 DEG C, can be protected under the conditions of 37 DEG C
It deposits 7 days or more, can at least place 4 weeks under room temperature.
For those skilled in the art, it can make other each according to the above description of the technical scheme and ideas
Kind is corresponding to be changed and deforms, and all these change and deform the protection model that all should belong to the claims in the present invention
Within enclosing.
Claims (3)
1. a kind of stable purposes of dilution for acridinium ester antigen conjugates or acridinium ester antibody conjugates, feature exist
In the dilution is prepared by the component of following mass percentage:
Carbohydrate 0.1-2%
Polyalcohol 1-10%
Amino acid 0.1-1%
Protein stabiliser 0.01-2.5%
Chelating agent 0.001-0.1%
Surfactant 0.01-0.5%
Preservative 0.01-0.1%
Additive 0.1-1%
Buffer surplus;
The carbohydrate is made of sucrose and trehalose, and the weight ratio of the sucrose and trehalose is (1-3): 1;
In ox IgG of the protein stabiliser selected from bovine serum albumin(BSA), 0.01-0.1% that mass percent is 0.1-2.5%
One or two kinds of mixtures;
It is one or both of the Macrogol 6000 of 0.1-1%, 0.9% sodium chloride that the additive, which is selected from mass percent,
Mixture;
The polyalcohol is glycerol;The amino acid is glycine;The chelating agent is DTPA;The surfactant is
The mixture of one or both of Tween-20, Triton X-100;The preservative is Proclin 300, in Sodium azide
One or two kinds of mixtures;The buffer is selected from the citrate buffer or 0.01- that concentration is 0.01-0.2mol/L
2- (N- morpholine) ethanesulfonic acid buffer of 0.2mol/L.
2. dilution according to claim 1 is stable for acridinium ester antigen conjugates or acridinium ester antibody conjugates
Purposes, which is characterized in that the buffer is 0.05-0.2mol/L citrate buffer or 0.01-0.1mol/L2- (N-
Coffee quinoline) ethanesulfonic acid buffer.
3. the stable dilution according to claim 1 for being used for acridinium ester antigen conjugates or acridinium ester antibody conjugates
Preparation method, which is characterized in that the specific steps of which are as follows:
1) each object of carbon aquation, polyalcohol, amino acid, protein stabiliser, chelating agent, the surfactant, preservative of formula ratio are pressed
It is added in buffer with additive, stirs to being completely dissolved, obtain mixed liquor;
2) mixed liquor for obtaining step 1) stirs and evenly mixs, and it is 6-7 to get dilution is arrived that HCl or NaOH, which is added, and adjusts pH value.
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