CN105628914A - Diluent enabling stability for acridinium ester antigen-antibody conjugate and preparation method of diluent - Google Patents
Diluent enabling stability for acridinium ester antigen-antibody conjugate and preparation method of diluent Download PDFInfo
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- CN105628914A CN105628914A CN201610081312.9A CN201610081312A CN105628914A CN 105628914 A CN105628914 A CN 105628914A CN 201610081312 A CN201610081312 A CN 201610081312A CN 105628914 A CN105628914 A CN 105628914A
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- diluent
- acridinium ester
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
Abstract
The invention discloses a diluent enabling stability for acridinium ester antigen-antibody conjugate. The diluent is prepared by adding a carbohydrate, polyalcohol, amino acids, a protein stabilizer, a chelating agent, a surfactant, a preservative and an additive into a buffer solution; the diluent prepared by the invention overcomes the defect that acridinium ester is unstable and easily hydrolyzes in the buffer solution, can protect the acridinium ester antigen-antibody conjugate to improve its stability and prolong its service life, and also can be fully used in in-vivo diagnostic kits.
Description
Technical field
The invention belongs to chemical analysis technology field, be specifically related to a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable and preparation method thereof.
Background technology
Chemiluminescence immunoassay is the immunoassay with the direct labelled antigen of chemiluminescence agent or antibody, acridinium ester is ideal luminous substrate, in the basic conditions, acridinium ester molecule is subject to hydrogen peroxide attack can generate dichloroethane, and dichloroethane is unstable and is decomposed into CO2With the N-methylacridine ketone of excited electronic state, send the light that wavelength is 430nm when it returns to ground state.
Acridinium ester label has the specific groups producing luminescence in chemical constitution, directly participates in luminescence-producing reaction in luminescence immunoassay process. Usual this kind of material does not have background Iuminescence, can be used for detecting the sample of low concentration or micro-concentrations in the reaction, is the significantly high luminous agent of a class luminous efficiency. Acridinium ester I, II molecule and acridinium carboxamide III all can combine with antibody (or antigen), generate and have the traget antibody (or antigen) that chemiluminescence activity is strong, immunoreation specificity is high.
From current technology level, acridinium ester is unstable, facile hydrolysis in general buffer, when conjugate is diluted to working concentration, owing to conjugate stability reducing caused reagent of concentration in diluent declines, thus causing that the storage life of reagent shortens. From external diagnosis reagent industry market, the stability quality of acridinium ester antigen-antibody conjugate and use effect phase length, the performance indications that test kit is overall also can be directly affected.
Summary of the invention
For the deficiencies in the prior art; it is an object of the invention to provide a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable; this diluent overcomes the acridinium ester shortcoming in buffer instability, facile hydrolysis; acridinium ester antigen-antibody conjugate is played a protective role; improve the stability of acridinium ester antigen-antibody conjugate; extend the effect phase of use, moreover it is possible to make full use of in external diagnosis reagent case simultaneously.
Another object of the present invention is to the preparation method that a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable is provided.
Realize the purpose of the present invention can pass through to adopt the following technical scheme that and reach:
A kind of diluent that acridinium ester antigen-antibody conjugate can be made stable, its component being prepared by mass percentage is prepared from:
Preferably, described carbohydrate is made up of sucrose and trehalose, and the weight ratio of described sucrose and trehalose is (1-3): 1.
Preferably, described polyhydric alcohol is sorbitol, inositol, isopropanol or glycerol one or more mixture therein; Described aminoacid is serine, glycine, alanine or lysine one or more mixture therein.
Preferably, described protein stabiliser is bovine serum albumin (BSA), defatted milk powder, casein, calf serum, cattle IgG or Mus IgG one or more mixture therein.
Preferably, the described protein stabiliser one or more mixture in the casein of BSA, 0.1-1.5% that mass percent is 0.1-2.5% or the cattle IgG of 0.01-0.1%.
Preferably, described chelating agen is EDTA or DTPA one or both mixture therein; Described surfactant is Tween-20, Tween-40, Nonidet P40 (NP-40), TritonX-100 or sodium lauryl sulphate one or more mixture therein; Described preservative is thimerosal, Proclin300, sodium azide or hibitane one or more mixture therein.
Preferably, described additive selected from mass percent be the polyethylene glycol 6000 (PEG6000) of 0.1-1%, the gelatin of 0.05-0.5%, 0.9% sodium chloride one or more mixture therein.
Preferably, described buffer is the citrate buffer of 0.01-0.2mol/L, 2-(N-morpholine) ethanesulfonic acid buffer (MES buffer), Tris-HCl buffer or phosphate buffer one therein selected from concentration.
Preferably, described buffer is 0.05-0.2mol/L citrate buffer or 0.01-0.1mol/LMES buffer.
A kind of preparation method that can make the stable diluent of acridinium ester antigen-antibody conjugate, it specifically comprises the following steps that
1) adding in buffer by each thing of carbon aquation of formula ratio, polyhydric alcohol, aminoacid, protein stabiliser, chelating agen, surfactant, preservative and additive, stirring, to being completely dissolved, obtains mixed liquor;
2) by step 1) the mixed liquor stirring and evenly mixing that obtains, adding HCl or NaOH, to regulate pH value be 6-7, namely obtains diluent.
The beneficial effects of the present invention is:
1, the diluent of the present invention acridinium ester antigen-antibody conjugate can be made to remain stable for, not facile hydrolysis, extend the storage life of reagent;
2, the diluent of the present invention can keep the stability of acridinium ester antigen-antibody conjugate and use the effect phase, solves the problem that test kit uses the effect phase short, it is ensured that the performance indications that test kit is overall.
Detailed description of the invention
A kind of diluent that acridinium ester antigen-antibody conjugate can be made stable, its component being prepared by mass percentage is prepared from:
Wherein, carbohydrate: be made up of sucrose and trehalose, the weight ratio of sucrose and trehalose is (1-3): 1, it is preferred to 2:1;
Polyhydric alcohol: select sorbitol, inositol, isopropanol or glycerol one or more mixture therein, it is preferred to glycerol;
Aminoacid: select serine, glycine, alanine or lysine one or more mixture therein, it is preferred to glycine;
Protein stabiliser: select BSA, defatted milk powder, casein, calf serum, cattle IgG or Mus IgG one or more mixture therein, it is preferred to weight/mass percentage composition is one or more the mixture in 0.1-2.5%BSA, 0.1-1.5% casein or 0.01-0.1% cattle IgG;
Chelating agen: select EDTA or DTPA one or both mixture therein, it is preferred to DTPA;
Surfactant: select Tween-20, Tween-40, NP-40, TritonX-100 or sodium lauryl sulphate one or more mixture therein, it is preferred to weight/mass percentage composition is one or both the mixture in Tween-20 or TritonX-100 of 0.01��0.5%;
Preservative: select thimerosal, Proclin300, sodium azide or hibitane one or more mixture therein;
Additive: sodium chloride selected from the gelatin of PEG6000,0.05-0.5% that mass percent is 0.1-1%, 0.9% one or more mixture therein, it is preferred to weight/mass percentage composition is 0.05-0.5%PEG6000 and 0.9% sodium chloride;
Buffer: be the citrate buffer of 0.01-0.2mol/L, MES buffer, Tris-HCl buffer or phosphate buffer one therein selected from concentration, it is preferred to the citrate buffer of 0.05-0.2mol/L or the MES buffer of 0.01-0.1mol/L.
The compound method of diluent is as follows:
1) being mixed by above-mentioned raw materials, stirring, to being completely dissolved, obtains mixed liquor;
2) by step 1) the mixed liquor stirring and evenly mixing that obtains, adding HCl or NaOH, to regulate pH value be 6.4 �� 0.1, namely obtains diluent.
Embodiment 1:
A kind of diluent that acridinium ester antigen-antibody conjugate can be made stable, its component being prepared by mass percentage is prepared from:
The compound method of diluent is as follows:
1) adding in 0.05mol/L citrate buffer by the sucrose of formula ratio, trehalose, glycerol, glycine, BSA, DTPA, Tween-20, sodium azide, PEG6000 and sodium chloride, stirring, to being completely dissolved, obtains mixed liquor;
2) by step 1) the mixed liquor stirring and evenly mixing that obtains, adding NaOH, to regulate pH value be 6.4 �� 0.1, namely obtains diluent.
It is diluted below in conjunction with thing with the diluent of embodiment 1 preparation, conjugate is acridinium ester-FSH antibody and acridinium ester-E2 antigen respectively, it is diluted to working concentration, obtains acridinium ester-FSH antibody diluent (1) and acridinium ester-E2 antigenic dilution (1). Conjugate after two kinds of dilutions respectively separates 13 bottles, every bottle of general 1ml. Wherein 37 DEG C of baking ovens of 3 bottles of placements are accelerated experiment, place 2-8 DEG C of Refrigerator store for other 5 bottles, also have 5 bottles to place room temperature preservation.
Embodiment 2:
A kind of diluent that acridinium ester antigen-antibody conjugate can be made stable, its component being prepared by mass percentage is prepared from:
The compound method of diluent is as follows:
1) adding in 0.2mol/L citrate buffer by the sucrose of formula ratio, trehalose, glycerol, glycine, BSA, DTPA, Tween-20, sodium azide, PEG6000 and sodium chloride, stirring, to being completely dissolved, obtains mixed liquor;
2) by step 1) the mixed liquor stirring and evenly mixing that obtains, adding NaOH, to regulate pH value be 6.4 �� 0.1, namely obtains diluent.
It is diluted below in conjunction with thing with the diluent of embodiment 2 preparation, conjugate is acridinium ester-FSH antibody and acridinium ester-E2 antigen respectively, it is diluted to working concentration, obtains acridinium ester-FSH antibody diluent (2) and acridinium ester-E2 antigenic dilution (2). Conjugate after two kinds of dilutions respectively separates 13 bottles, every bottle of general 1ml. Wherein 37 DEG C of baking ovens of 3 bottles of placements are accelerated experiment, place 2-8 DEG C of Refrigerator store for other 5 bottles, also have 5 bottles to place room temperature preservation.
Embodiment 3:
A kind of diluent that acridinium ester antigen-antibody conjugate can be made stable, its component being prepared by mass percentage is prepared from:
The compound method of diluent is as follows:
1) adding in 0.1mol/L citrate buffer by the sucrose of formula ratio, trehalose, glycerol, glycine, BSA, DTPA, Tween-20, sodium azide, PEG6000 and sodium chloride, stirring, to being completely dissolved, obtains mixed liquor;
2) by step 1) the mixed liquor stirring and evenly mixing that obtains, adding NaOH, to regulate pH value be 6.4 �� 0.1, namely obtains diluent.
It is diluted below in conjunction with thing with the diluent of embodiment 3 preparation, conjugate is acridinium ester-FSH antibody and acridinium ester-E2 antigen respectively, it is diluted to working concentration, obtains acridinium ester-FSH antibody diluent (3) and acridinium ester-E2 antigenic dilution (3). Conjugate after two kinds of dilutions respectively separates 13 bottles, every bottle of general 1ml. Wherein 37 DEG C of baking ovens of 3 bottles of placements are accelerated experiment, place 2-8 DEG C of Refrigerator store for other 5 bottles, also have 5 bottles to place room temperature preservation.
Embodiment 4:
A kind of diluent that acridinium ester antigen-antibody conjugate can be made stable, its component being prepared by mass percentage is prepared from:
The compound method of diluent is as follows:
1) add in 0.1mol/LMES buffer by the sucrose of formula ratio, trehalose, glycerol, glycine, BSA, cattle IgG, DTPA, Tween-20, TritonX-100, sodium azide, Proclin300, PEG6000 and sodium chloride, stirring, to being completely dissolved, obtains mixed liquor;
2) by step 1) the mixed liquor stirring and evenly mixing that obtains, adding NaOH, to regulate pH value be 6.4 �� 0.1, namely obtains diluent.
It is diluted below in conjunction with thing with the diluent of embodiment 4 preparation, conjugate is acridinium ester-FSH antibody and acridinium ester-E2 antigen respectively, it is diluted to working concentration, obtains acridinium ester-FSH antibody diluent (4) and acridinium ester-E2 antigenic dilution (4). Conjugate after diluting two kinds respectively separates 13 bottles, every bottle of general 1ml. Wherein 37 DEG C of baking ovens of 3 bottles of placements are accelerated experiment, place 2-8 DEG C of Refrigerator store for other 5 bottles, also have 5 bottles to place room temperature preservation.
The stability test of the conjugate after dilution:
According to the Check-Out Time formulated, respectively the acridinium ester antigen-antibody conjugate after above-mentioned dilution is detected, conjugate after 100 �� l dilutions is added in reaction cup, light-emitting appearance is automatically added to the 100 �� l preexciting liquid (acid solutions containing hydrogen peroxide, pH<2.0) and the 100 �� l exciting liquid (alkaline solutions containing sodium hydroxide, pH>13.0), conjugate is luminous at once after adding exciting liquid, and light-emitting appearance shows the relative light unit (RLUs) measured. The following is the testing result that the specific embodiment of the invention obtains.
The conjugate after table 1 dilution Detection of Stability result under 37 DEG C of conditions
The conjugate after table 2 dilution Detection of Stability result under 2-8 DEG C of condition
Conjugate after table 3 dilution Detection of Stability result at ambient temperature
Being analyzed drawing to the assay of above three forms, acridinium ester antigen-antibody conjugate is all had protective effect by four kinds of diluents that specific embodiment is prepared out so that the luminous efficiency under different conditions of storage of the conjugate reagent after dilution does not decline. This is due to along with the rising of temperature or reduction; the key meeting that antigen or antibody and acridinium ester connect is unstable and ruptures; and the material such as sucrose, BSA such as the saccharide added in diluent, albumen can keep stablizing of key; make the key that antigen or antibody and acridinium ester connect will not rupture because of the change of temperature; thus protect acridinium ester antigen-antibody conjugate, and maintain the luminous efficiency under different conditions of storage of the conjugate reagent after dilution. After wherein placing 7 days under 37 DEG C of conditions, 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 2-8 DEG C of condition, four kinds of acridinium ester-FSH antibody reagents and four kinds of acridinium ester-E2 antigenic agents luminous efficiencies all only have dropped 3.27-6.49%; And after placing 5 weeks at ambient temperature, four kinds of acridinium ester-FSH antibody reagents and four kinds of acridinium ester-E2 antigenic agents luminous efficiencies only have dropped 1.75-5.89%.
Therefore the diluent that acridinium ester antigen-antibody conjugate can be made stable provided by the invention, acridinium ester conjugate is diluted, the reagent preservation effect phase after dilution is, can preserve more than 12 months under 2-8 DEG C of condition, can preserve under 37 DEG C of conditions more than 7 days, at least can place 4 weeks under room temperature condition.
For a person skilled in the art, can technical scheme as described above and design, make other various corresponding changes and deformation, and all these change and deformation all should belong within the protection domain of the claims in the present invention.
Claims (10)
1. one kind can make the diluent that acridinium ester antigen-antibody conjugate is stable, it is characterised in that its component being prepared by mass percentage is prepared from:
2. a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable according to claim 1, it is characterised in that described carbohydrate is made up of sucrose and trehalose, and the weight ratio of described sucrose and trehalose is (1-3): 1.
3. a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable according to claim 1, it is characterised in that described polyhydric alcohol is sorbitol, inositol, isopropanol or glycerol one or more mixture therein; Described aminoacid is serine, glycine, alanine or lysine one or more mixture therein.
4. a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable according to claim 1, it is characterized in that, described protein stabiliser is bovine serum albumin, defatted milk powder, casein, calf serum, cattle IgG or Mus IgG one or more mixture therein.
5. a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable according to claim 4, it is characterized in that, described protein stabiliser is one or more the mixture in the bovine serum albumin of 0.1-2.5%, the casein of 0.1-1.5% or the cattle IgG of 0.01-0.1% selected from mass percent.
6. a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable according to claim 1, it is characterised in that described chelating agen is EDTA or DTPA one or both mixture therein; Described surfactant is Tween-20, Tween-40, Nonidet P40, TritonX-100 or sodium lauryl sulphate one or more mixture therein; Described preservative is thimerosal, Proclin300, sodium azide or hibitane one or more mixture therein.
7. a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable according to claim 1, it is characterized in that, described additive selected from mass percent be the polyethylene glycol 6000 of 0.1-1%, the gelatin of 0.05-0.5%, 0.9% sodium chloride one or more mixture therein.
8. a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable according to claim 1, it is characterized in that, described buffer is the citrate buffer of 0.01-0.2mol/L, 2-(N-morpholine) ethanesulfonic acid buffer, Tris-HCl buffer or phosphate buffer one therein selected from concentration.
9. a kind of diluent that acridinium ester antigen-antibody conjugate can be made stable according to claim 8, it is characterized in that, described buffer is 0.05-0.2mol/L citrate buffer or 0.01-0.1mol/L2-(N-morpholine) ethanesulfonic acid buffer.
10. a kind of preparation method that can make the stable diluent of acridinium ester antigen-antibody conjugate according to claim 1, it is characterised in that it specifically comprises the following steps that
1) adding in buffer by each thing of carbon aquation of formula ratio, polyhydric alcohol, aminoacid, protein stabiliser, chelating agen, surfactant, preservative and additive, stirring, to being completely dissolved, obtains mixed liquor;
2) by step 1) the mixed liquor stirring and evenly mixing that obtains, adding HCl or NaOH, to regulate pH value be 6-7, namely obtains diluent.
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