CN114200125B - Protein-free alkaline phosphatase-antibody conjugate diluent and preparation method thereof - Google Patents

Protein-free alkaline phosphatase-antibody conjugate diluent and preparation method thereof Download PDF

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CN114200125B
CN114200125B CN202111481036.2A CN202111481036A CN114200125B CN 114200125 B CN114200125 B CN 114200125B CN 202111481036 A CN202111481036 A CN 202111481036A CN 114200125 B CN114200125 B CN 114200125B
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alkaline phosphatase
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antibody conjugate
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周旭淼
华权高
甘华铭
来祥兵
舒芹
张韦
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Wuhan Life Origin Biotech Joint Stock Co ltd
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    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

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Abstract

The invention relates to a protein-free alkaline phosphatase-antibody conjugate diluent and a preparation method thereof, wherein the diluent comprises, by mass, 0.1-1% of a buffer system, 2-10% of a water-soluble salt, 0.01-1% of allantoin, 0.1-2.1% of amino acid, 1-5% of a saccharide compound, 0-5% of an alcohol compound, 0.01-0.2% of a preservative, 0.01-0.5% of a surfactant and 0.001-0.01% of a reducing agent, and does not contain protein components. The diluent does not contain protein components, and can keep the stability of the alkaline phosphatase-antibody conjugate through the coordination of the components, and can keep the activity of nearly 100 percent under the conditions of shaking at 42 ℃ for 3 days and standing at 37 ℃ for 7 days to be at least 83 percent; the activity can be maintained at more than 95% and at least 72% at the highest after standing for one year under the environment of 4 ℃.

Description

Protein-free alkaline phosphatase-antibody conjugate diluent and preparation method thereof
Technical Field
The invention relates to the field of chemical reagents, in particular to a protein-free alkaline phosphatase-antibody conjugate diluent and a preparation method thereof.
Background
Alkaline phosphatase (ALP, EC 3.1.3.1) is a plasma membrane-bound glycoprotein and is widely distributed in nature, including prokaryotes and higher eukaryotes. The active site of the enzyme comprises two zinc ions and one magnesium ion, and the metal ions of the three sites are critical to the activity of the enzyme. Alkaline phosphatase can hydrolyze various monophosphates and release inorganic phosphate. The 1, 2-dioxetane compound AMPPD is used as an ultrasensitive alkaline phosphatase substrate, and phosphate groups are hydrolyzed under the catalysis of alkaline phosphatase to form an unstable intermediate, and a quaternary ring is broken to release a large amount of energy after self-decomposition, so that chemiluminescent reaction is excited. Therefore, the magnetic bead-antibody conjugate and alkaline phosphatase-antibody conjugate can be used as a solid-phase capture reagent and an enzyme-labeled reagent in a chemiluminescent kit for detecting trace antigens in serum.
Alkaline phosphatase-antibody conjugates are used as a conjugated protein, and their inactivation in the dilution is mainly divided into physical degradation and chemical degradation. Protein substances such as Bovine Serum Albumin (BSA) are usually added to the dilution solution in order to prevent aggregation or adsorption of the conjugate and physical degradation of the solid phase surface, and chemical degradation such as deamidation and disulfide bond cleavage.
For alkaline phosphatase alone, the method for stabilizing alkaline phosphate, which was filed earlier than 18/8 of 2002, was used in patent publication No. CN1522298A, in which sugar and albumin were mixed and then stored by freeze-drying. Regarding alkaline phosphatase markers, patent "reagent and method for stabilizing alkaline phosphatase or its markers" filed by Shenzhen Maire biomedical electronics Co., ltd.12.31 earlier in 2009 (CN 102115737A), wherein mainly contains protein, magnesium ion, calcium ion activator, zinc ion, sodium ion, saccharide additive and alcohol additive. On this basis, patents CN102628863a and CN109212180a, which are the dilutions of alkaline phosphatase antigen antibodies and the preservation solutions for small molecule antigen and alkaline phosphatase labeled conjugates, and their preparation methods, amplify the range of additives in the dilutions, including metal ions, carbohydrates, alcohols, polymers, surfactants, amino acids, and proteins. The patent of dilution liquid special for alkaline phosphatase antigen antibody (CN 108663511B) adds self-made macromolecular hydrophilic compound and modified amino acid into the dilution liquid. Patent (dilution of alkaline phosphatase marker, preparation method and application) (CN 110540978A) adds amphiphilic polymer with unknown structure into the dilution. The above patents all show that the addition of proteinaceous substances to the diluent is required to prevent aggregation and adsorption of alkaline phosphatase conjugates.
Alkaline phosphatase-antibody conjugates must be soluble in aqueous solution as a reagent component and be stable in aqueous solution under a variety of possible limiting conditions. At the same time, the solution should not contain other protein components which may affect immune response, such as bovine serum albumin, casein and gelatin. Because the added protein may aggregate itself or with alkaline phosphatase-antibody conjugates after long term or extreme environmental storage, a non-specific reaction occurs during the assay, primarily reflected in a blank value-rising or abnormal clinical test result.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a protein-free alkaline phosphatase-antibody conjugate diluent and a preparation method thereof, wherein the diluent does not contain protein components, and the stability of the alkaline phosphatase-antibody conjugate can be maintained through the cooperation of components.
In order to achieve the above purpose, the technical scheme of the diluent of the invention is as follows:
comprises 0.1-1% of buffer system, 2-10% of water soluble salt, 0.01-1% of allantoin, 0.1-2.1% of amino acid, 1-5% of sugar compound, 0-5% of alcohol compound, 0.01-0.2% of preservative, 0.01-0.5% of surfactant and 0.001-0.01% of reducing agent by mass percent, and does not contain protein component.
Further, the buffer system comprises MES buffer solution, bis-tris buffer solution, ADA buffer solution, ACES buffer solution, MOPSO buffer solution, MOPS buffer solution, phosphate buffer solution or acetic acid buffer solution, and the pH value is 5-7.
Further, the water-soluble salt includes (NH) 4 ) 2 SO 4 、K 2 SO 4 、Na 2 SO 4 、MgSO 4 、NaCl、KCl、CaCl 2 、MgCl 2 、KNO 3 、NaNO 3 、ZnCl 2 、Mg(NO 3 ) 2 And Ca (NO) 3 ) One or two or more of them are mixed in an arbitrary ratio.
Further, the amino acid includes one or two or more of histidine, glycine, serine, tryptophan, alanine, glutamic acid, arginine, cysteine, methionine and proline in an arbitrary ratio.
Further, the alcohol compound includes one or two or more of ethylene glycol, glycerol, mannitol, sorbitol and 2-amino-2-methyl-1, 3-propanediol, which are mixed in any ratio.
Further, the saccharide compound includes one or two or more of trehalose, sucrose, dextran, fructose, glucose, pullulan and (2-hydroxypropyl) -beta-cyclodextrin, which are mixed in an arbitrary ratio.
Further, the preservative comprises one or more of sodium azide, proclin300 and BND-10 mixed in any ratio.
Further, the surfactant comprises one or more of Tween 20, tween 80, tritonx-100, poloxamer 188, glycine betaine, 3-sulfopropyl hexadecyl dimethyl betaine, tetronic-1307, NP-40 and CHAPS, which are mixed in any ratio.
Further, the reducing agent comprises one or more than two of sodium sulfite, vitamin C, DTT, sodium thiosulfate and beta-mercaptoethanol which are mixed in any proportion.
The preparation method of the invention has the technical scheme that: uniformly mixing a buffer system, soluble salt, allantoin, amino acid, saccharide compounds, a preservative, a surfactant and a reducing agent, and regulating the pH value to 5-7 to obtain the protein-free alkaline phosphatase-antibody conjugate diluent.
Compared with the prior art, the invention has the following beneficial technical effects:
the present invention includes water soluble salts to increase the concentration of ions in the solution to enhance the solubility of the conjugate and screens appropriate ion pairs to increase the stability of the conjugate according to the Hofmeister effect. The invention uses the surfactant to adsorb on the surface of protein and the surface of solid phase, fully disperses the conjugate, prevents the conjugate from aggregating, and prevents the conjugate from adsorbing on the solid phase. The reducing substances are included in the present invention to ensure that the conjugate is in a reducing environment, thereby reducing potential peptide chain oxidation and disulfide bond cleavage. In the invention, allantoin and amino acid are added, and can be cooperatively adsorbed on the protein surface, so that the aggregation of the conjugate is prevented through electrostatic action and steric hindrance, the thermal stability of the conjugate is improved, and the deamidation reaction is reduced. The saccharide compound is added to the invention for protein excipient, and the preservative prevents bacterial pollution so as to increase the long-term stability of the conjugate. The diluent does not contain protein components, and can keep the stability of the alkaline phosphatase-antibody conjugate through the coordination of the components, and the activity of the alkaline phosphatase-antibody conjugate can be kept to be 100% at most under the conditions of 42-degree vibration for 3 days and 37-degree standing for 7 days, and is at least 83%; the activity can be maintained at more than 95% and at least 72% at the highest after standing for one year under the environment of 4 ℃.
Detailed Description
The invention is further described in connection with the following detailed description.
The bottleneck of alkaline phosphatase-antibody conjugates is their stability, and in order to maintain the stability of alkaline phosphatase-antibody conjugates and prevent their aggregation or degradation, the additives of conjugate dilutions are screened and optimized according to the invention, which mainly comprise:
buffer system, soluble salt, amino acid, allantoin, alcohol compound, saccharide compound, antiseptic, surfactant and reducing agent, but does not contain protein component-containing substances such as bovine serum albumin, casein, gelatin or other various animal serum.
In the diluent, the concentration of a buffer system is 0.1-1%, the mass fraction of soluble salt is 2-10%, the mass concentration of amino acid is 0.1-2.1%, the mass concentration of allantoin is 0.01-1%, the mass concentration of alcohol compound is 0-5%, the mass concentration of saccharide compound is 1-5%, the mass concentration of preservative is 0.01-0.2%, and the mass concentration of surfactant is 0.01-0.5%; 0.001 to 0.01 percent of reducing agent;
the buffer system is one of MES, bis-tris, ADA, ACES, MOPSO, MOPS, phosphate and acetic acid, and the pH is 5-7;
the water-soluble salt includes (NH) 4 ) 2 SO 4 、K 2 SO 4 、Na 2 SO 4 、MgSO 4 、NaCl、KCl、CaCl 2 、MgCl 2 、KNO 3 、NaNO 3 、Mg(NO 3 ) 2 、Ca(NO 3 ) One or more than two of them are mixed in any proportion;
the amino acid comprises one or more of histidine, glycine, serine, tryptophan, alanine, glutamic acid, arginine, cysteine, methionine and proline, which are mixed in any proportion;
the alcohol compound comprises one or more than two of ethylene glycol, glycerol, mannitol, sorbitol and 2-amino-2-methyl-1, 3-propanediol which are mixed in any proportion;
the saccharide compound comprises one or more than two of trehalose, sucrose, dextran, fructose, glucose, pullulan and (2-hydroxypropyl) -beta-cyclodextrin;
the preservative comprises one or more than two of sodium azide, proclin300 and BND-10 which are mixed in any proportion;
the surfactant comprises one or more of Tween 20, tween 80, tritonx-100, poloxamer 188, glycine betaine, 3-sulfopropyl hexadecyl dimethyl betaine, tetronic-1307, NP-40, and CHAPS;
the reducing agent comprises one or more than two of sodium sulfite, vitamin C, DTT, sodium thiosulfate and beta-mercaptoethanol which are mixed in any proportion;
the preparation method of a preferred scheme comprises the following steps:
preparing 0.2-1% acetic acid or MOPSO or MES buffer solution, adding 1-5% NH 4 SO 4 、1～5％KNO 3 、0.01～1％MgCl 2 、0.0001～0.01％ZnCl 2 0.01 to 0.1 percent of Tween-80, 0.01 to 0.1 percent of poloxamer 188, 0.01 to 0.1 percent of CHAPS, 0.001 to 0.01 percent of vitamin C, 0.001 to 0.01 percent of cysteine, 0.1 to 1 percent of allantoin, 0.5 to 2 percent of arginine, 0.01 to 0.2 percent of Proclin300 and 1 to 5 percent of trehalose, and adjusting the pH value of the buffer solution to 5 to 7 by hydrochloric acid or sodium hydroxide and then fixing the volume.
The present invention uses nonionic surfactants and amphoteric surfactants to prevent adsorption of the conjugate to a solid phase surface, including but not limited to Tween-80, poloxamer 188, CHAPS. In order to enhance the solubility of the conjugate, the invention comprises ammonium sulfate or potassium nitrate to increase the concentration of solution ions, so that the surfactant is better adsorbed on the surface of the protein, and the conjugate is fully dispersed to prevent the conjugate from aggregating or adsorbing on a solid phase. Since the antibody and alkaline phosphatase contain-S-S-bonds, and both are coupled by-S-S-, the present invention contains a reducing substance such as cysteine or vitamin C to ensure that the conjugate is in a reducing environment, thereby reducing the potential for oxidation of peptide chains or cleavage of-S-S-bonds. In the invention, allantoin and arginine are added, and can be synergistically adsorbed on negative charge groups on the surface of protein, so that aggregation of the conjugate is prevented through electrostatic action and steric hindrance, and the thermal stability of the conjugate is improved. In addition, trehalose is added for protein excipients and Proclin300 is added for preservatives in the present invention.
The invention is further illustrated by the following specific examples.
Example 1
The buffer system, soluble salt, amino acid, allantoin, saccharide compound, preservative and surfactant are mixed uniformly, and the pH of the buffer solution is regulated to 6 by sodium hydroxide and then the volume is fixed.
The buffer system adopts acetic acid buffer solution; the concentration was 0.2%.
The water soluble salt comprises NH with a final concentration of 2.5% 4 SO 4 KNO of 2.5% 3 0.1% MgCl 2 ZnCl 0.001% 2 。
Amino acids include cysteine at a final concentration of 0.005% and 1% arginine.
The final allantoin concentration was 0.5%.
The saccharide compound included trehalose at a final concentration of 2.5%.
The preservative comprises Proclin300 at a final concentration of 0.1%.
The surfactant included tween 80 at a final concentration of 0.05%, poloxamer 188 at 0.05% and CHAPS at 0.05%.
The reducing agent comprises vitamin C at a final concentration of 0.005%.
The final concentrations herein refer to the concentration of each substance in the alkaline phosphatase-antibody conjugate buffer.
Application example 1
The protein-free diluent for storing the alkaline phosphatase-antibody conjugate is optimized, so that the alkaline phosphatase-antibody conjugate in CA125 and G17 projects can be diluted in the diluent to keep good stability under certain environmental pressure.
1 Experimental materials
1.1 preparation of antibody coated magnetic beads
1.1.1 preparation of antibody coated magnetic particle buffer
After preparing a phosphate buffer of 0.5%, adding 0.9% NaCl, 0.05% EDTA, 0.05% Proclin300 and 5% trehalose, the pH of the phosphate buffer was adjusted to 7.4 with hydrochloric acid, and 0.1% BSA was added to fix the volume.
1.1.2 magnetic beads coated CA125 (gastrin 17) antibody
An appropriate amount of the magnetic beads were washed 3 times with 0.1mol/L MES, and the magnetic beads were resuspended to a concentration of 2mg/ml with 0.1mol/L MES. And taking the EDC solution which is prepared at present, adding the EDC solution into the resuspended magnetic particles according to the mass ratio of EDC to the magnetic particles of 1:100, uniformly mixing by vortex, and uniformly mixing at room temperature level for 20 minutes, wherein the obtained solution is the activated magnetic particle solution. Adding the CA125 (gastrin 17) antibody to be coated into the activated magnetic particle solution according to the coating proportion of the CA125 (gastrin 17) antibody to the magnetic particle mass ratio of 1:500, mixing uniformly by vortex, mixing uniformly at room temperature level for 12 hours, continuously adding 5% BSA solution, mixing uniformly at room temperature level for 2 hours, removing the supernatant by magnetic separation, adding the magnetic particle buffer solution, mixing uniformly and preserving.
1.2 preparation of alkaline phosphatase-CA 125 (gastrin 17) antibody conjugates
10mg of alkaline phosphatase was dissolved in 1ml of PBS (0.01 mol/L pH 7.0) containing 1mg of the anti-gastrin 17 antibody, after complete dissolution, 4ml of a 1% glutaraldehyde solution was added to give a final glutaraldehyde concentration of 0.2%, and after removing excess glutaraldehyde by dialysis, 1ml of the alkaline phosphatase-antibody conjugate buffer prepared in example one was added and stored at 4 ℃.
1.3 preparation of calibration Material
1.3.1 preparation of Gastrin 17 and CA125 calibrator buffers
Phosphate buffer of 0.5% was prepared, added with 0.9% NaCl, 0.1% Tween-20, 0.05% Proclin300, pH was adjusted to 7.4, and then added with 0.1% BSA to fix the volume.
1.3.2 calibration Material
The calibrator was prepared by diluting commercial glycoprotein CA125 and gastrin 17 at known concentrations with calibrator buffer, wherein CA125 concentrations were 0, 20, 100, 500U/mL and gastrin 17 concentrations were 0, 5, 15, 50pmol/L.
1.4 Mixed clinical samples
Different serum samples collected in hospitals are mixed, sub-packaged and frozen in a refrigerator at the temperature of minus 80 ℃ for later use.
2 experimental methods or results
2.1.1 transport stability test
The alkaline phosphatase-antibody conjugate obtained in step 1.2 was diluted 1000-fold with the alkaline phosphatase-antibody conjugate buffer prepared in example one, and the alkaline phosphatase-CA 125 (gastrin 17) antibody conjugate was packaged in 2 bottles, one bottle was stored at 4℃and the other bottle was placed in a shaker at 42℃to accelerate shaking. And 3 days later, taking out the magnetic beads simultaneously, and testing a plurality of calibrators and mixed clinical samples simultaneously with the antibody coated magnetic beads newly prepared in the step 1.1, wherein the test results are shown in the following tables 1 and 2.
TABLE 1 transport stability of alkaline phosphatase-CA 125 antibody conjugates after dilution in the buffer of the invention
TABLE 2 transport stability of alkaline phosphatase-gastrin 17 antibody conjugates after dilution in the buffer of the invention
2.1.2 thermal stability test
The buffer solution of the invention is diluted 1000 times with alkaline phosphatase-CA 125 (gastrin 17) antibody conjugate, and is split into 2 bottles, one bottle is stored at 4 ℃, and the other bottle is stored in an incubator at 37 ℃. After 7 days, the sample was removed simultaneously, and a plurality of calibrator and samples were tested simultaneously with the freshly prepared antibody-coated magnetic beads, and the results were as shown in tables 3 and 4 below.
TABLE 3 thermal stability of alkaline phosphatase-CA 125 antibody conjugates after dilution in the buffer of the invention
TABLE 4 thermal stability of alkaline phosphatase-gastrin 17 antibody conjugates after dilution in buffer according to the invention
2.1.3 Long term stability
The buffer solution of the invention is diluted 1000 times to alkaline phosphatase-CA 125 (gastrin 17) antibody conjugate, and the conjugate is split into 5 bottles, and one bottle is placed at 4 ℃ for preservation. And taking out at the 2 nd day, 3 rd month, 6 th month and 12 th month respectively, and testing a plurality of calibrator and samples simultaneously with the newly prepared antibody-coated magnetic beads, and performing testing and analysis respectively, with the results shown in tables 5 to 8 below.
TABLE 5 results of long-term stability of alkaline phosphatase-CA 125 antibody conjugates after dilution in buffer according to the invention
TABLE 6 Long-term stability analysis of alkaline phosphatase-CA 125 antibody conjugates after dilution in the buffer of the invention
TABLE 7 results of long-term stability of alkaline phosphatase-gastrin 17 antibody conjugates after dilution in buffer according to the invention
TABLE 8 Long term stability analysis of alkaline phosphatase-gastrin 17 antibody conjugates after dilution in buffer according to the invention
As is clear from the test results shown in tables 1 to 8, the alkaline phosphatase-CA 125 antibody (gastrin 17) conjugate is stabilized without adding a protective protein such as BSA, and the activity is maintained at approximately 100% in a 3-day 42-degree shaking and 7-day 37-degree standing environment, and at least 95% in a 4-degree standing environment for one year.
Example two
The buffer system, soluble salt, amino acid, allantoin, saccharide compound, preservative and surfactant are mixed uniformly, and the pH of the buffer solution is regulated to 6 by sodium hydroxide and hydrochloric acid and then the volume is fixed.
The buffer system adopts MOPSO buffer solution; the concentration was 0.4%.
The water-soluble salt comprises NH with a final concentration of 1% 4 SO 4 1% KNO 3 0.1% MgCl 2 ZnCl 0.001% 2 。
Amino acids include cysteine at a final concentration of 0.001% and arginine at 0.5%.
The final allantoin concentration was 0.1%.
The saccharide compound includes trehalose at a final concentration of 1%.
The preservative included Proclin300 at a final concentration of 0.01%.
The surfactant included tween 80 at a final concentration of 0.01%, poloxamer 188 at 0.01% and CHAPS at 0.01%.
The reducing agent comprises vitamin C at a final concentration of 0.001%.
The final concentrations herein refer to the mass concentration of each substance in the alkaline phosphatase-antibody conjugate buffer.
Through testing, the test method and conditions are the same as those of application example I, the diluent obtained in the embodiment keeps the activity of approximately 83% in the environment of shaking at 42 ℃ for 3 days and standing at 37 ℃ for 7 days, and the activity can keep more than 72% in the environment of standing for one year at 4 ℃.
Example III
The buffer system, soluble salt, amino acid, allantoin, saccharide compound, preservative and surfactant are mixed uniformly, and the pH of the buffer solution is regulated to 6.5 by sodium hydroxide and hydrochloric acid and then the volume is fixed.
The buffer system adopts MES buffer solution; the final concentration was 0.5%.
The water-soluble salt comprises NH with a final concentration of 2% 4 SO 4 KNO of 2% 3 0.5% MgCl 2 ZnCl 0.001% 2 。
Amino acids include cysteine at a final concentration of 0.01% and 2% arginine.
The final allantoin concentration was 1%.
The saccharide compound includes trehalose at a final concentration of 5%.
The preservative included Proclin300 at a final concentration of 0.2%.
The surfactant included tween 80 at a final concentration of 0.1%, poloxamer 188 at 0.1% and CHAPS at 0.1%.
The reducing agent comprises vitamin C at a final concentration of 0.01%.
The final concentrations herein refer to the mass concentration of each substance in the alkaline phosphatase-antibody conjugate buffer.
Through testing, the test method and conditions are the same as those of application example I, the diluent obtained in the embodiment keeps the activity of approximately 87% in the environment of shaking at 42 ℃ for 3 days and standing at 37 ℃ for 7 days, and the activity can keep more than 75% in the environment of standing for one year at 4 ℃.
Example IV
The buffer system, the soluble salt, the amino acid, the allantoin, the alcohol compound, the saccharide compound, the preservative and the surfactant are uniformly mixed, and the pH value of the buffer solution is adjusted to 5 and then the volume is fixed.
The buffer system adopts phosphate buffer solution; the final concentration was 0.1%.
The water-soluble salt comprises NH with a final concentration of 2% 4 SO 4 。
The amino acids included histidine at a final concentration of 1.5%.
The final allantoin concentration was 0.05%.
The alcohol compound included ethylene glycol at a final concentration of 3%.
The saccharide compound comprises dextran with a final concentration of 1%, fructose with a final concentration of 1% and glucose with a final concentration of 1%.
The preservative comprises BND-10 at a final concentration of 0.15%.
The surfactant included tween 20 at a final concentration of 0.5%.
The reducing agent included sodium sulfite at a final concentration of 0.003% and sodium thiosulfate at a final concentration of 0.003%.
Through testing, the test method and conditions are the same as those of application example I, the diluent obtained in the embodiment keeps the activity of approximately 84% in the environment of shaking at 42 ℃ for 3 days and standing at 37 ℃ for 7 days, and the activity can keep more than 72% in the environment of standing for one year at 4 ℃.
Example five
The buffer system, the soluble salt, the amino acid, the allantoin, the alcohol compound, the saccharide compound, the preservative and the surfactant are uniformly mixed, and the pH of the buffer solution is adjusted to 7 and then the volume is fixed.
The buffer system adopts Bis-tris buffer solution; the final concentration was 1%.
The water soluble salt comprises K with a final concentration of 5% 2 SO4, 4% NaNO 3 0.5% of Mg (NO 3 ) 2 0.1% Ca (NO 3 )。
Amino acids include tryptophan at a final concentration of 0.1% and alanine at a final concentration of 0.1%.
The final allantoin concentration was 0.01%.
The alcohol compound includes glycerol and mannitol with a final concentration of 3% and 2%.
The saccharide compound comprises pullulan with a final concentration of 1.5% and (2-hydroxypropyl) -beta-cyclodextrin with a final concentration of 1.5%.
The preservative comprises BND-10 at a final concentration of 0.1% and sodium azide at a final concentration of 0.05%.
The surfactant included 3-sulfopropyl hexadecyl dimethyl betaine at a final concentration of 0.01%.
The reducing agent comprises beta-mercaptoethanol at a final concentration of 0.003%.
Through testing, the test method and conditions are the same as those of application example I, the diluent obtained in the embodiment keeps the activity of approximately 83% in the environment of shaking at 42 ℃ for 3 days and standing at 37 ℃ for 7 days, and the activity can keep more than 73% in the environment of standing for one year at 4 ℃.
Comparative example one
Adding common diluent such as 0.5% phosphate buffer solution, 0.9% NaCl, 0.1% MgCl 2 、0.001％ZnCl 2 0.05% proclin300, 5% trehalose, pH adjusted to 6.5.
Through testing, the testing method and the conditions are the same as the application example I; the dilution obtained in this example has an activity of less than 50% in a 3 day 42 degree shaking and 7 day 37 degree standing environment.
Comparative example two
Bovine serum albumin was added at 0.1% and the other conditions were the same as in comparative example one.
Through testing, as shown in the following table 9, the stability of the common diluent added with bovine serum albumin is improved to more than 70% after the oscillation for 42 degrees for three days, but the abnormal rise of zero point occurs, and the coefficient of variation of the same sample or calibrator is repeatedly measured to be high, which indicates that the uniformity of the diluent is reduced.
TABLE 9 transport stability of alkaline phosphatase-CA 125 antibody conjugates after dilution with common dilutions of bovine serum albumin
As can be seen from the above examples and comparative examples, the components and amounts of the present invention are compatible with each other, and the alkaline phosphatase-antibody conjugate is sensitive to the pH value of the diluent, so that the present invention can achieve the effect of ensuring the stability of the diluent without adding any protein substances and without causing zero point abnormality and uniformity decrease due to the addition of bovine serum albumin by using specific components, amounts and specific pH values. In addition, since polymers such as polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, and the like are generally negative effects in the present invention, no polymer is added to the components of the present invention.

Claims (3)

1. A protein-free alkaline phosphatase-antibody conjugate diluent, characterized in that: comprises 0.1 to 1 percent of buffer system, 2 to 10 percent of water soluble salt, 0.01 to 1 percent of allantoin, 0.1 to 2.1 percent of amino acid, 1 to 5 percent of sugar compound, 0 to 5 percent of alcohol compound, 0.01 to 0.2 percent of preservative, 0.01 to 0.5 percent of surfactant and 0.001 to 0.01 percent of reducing agent according to mass percent, and does not contain protein components;
the buffer system comprises MES buffer solution, bis-tris buffer solution, ADA buffer solution, ACES buffer solution, MOPSO buffer solution, MOPS buffer solution, phosphate buffer solution or acetic acid buffer solution, and the pH value is 5-7;
the water-soluble salt includes (NH) 4 ) 2 SO 4 、K 2 SO 4 、Na 2 SO 4 、MgSO 4 、NaCl、KCl、CaCl 2 、MgCl 2 、KNO 3 、NaNO 3 、ZnCl 2 、Mg(NO 3 ) 2 And Ca (NO) 3 ) One or more than two of them are mixed in any proportion;
the amino acid comprises one or more than two of histidine, glycine, serine, tryptophan, alanine, glutamic acid, arginine, cysteine, methionine and proline which are mixed in any proportion;
the alcohol compound comprises one or more than two of ethylene glycol, glycerol, mannitol, sorbitol and 2-amino-2-methyl-1, 3-propanediol which are mixed in any proportion;
the saccharide compound comprises one or more than two of trehalose, sucrose, dextran, fructose, glucose, pullulan and (2-hydroxypropyl) -beta-cyclodextrin which are mixed in any proportion;
the surfactant comprises one or more of Tween 20, tween 80, tritonx-100, poloxamer 188, glycine betaine, 3-sulfopropyl hexadecyl dimethyl betaine, tetronic-1307, NP-40 and CHAPS;
the reducing agent comprises one or more than two of sodium sulfite, vitamin C, DTT, sodium thiosulfate and beta-mercaptoethanol which are mixed in any proportion;
the alkaline phosphatase-antibody conjugate is alkaline phosphatase-gastrin 17 antibody conjugate or alkaline phosphatase-CA 125 antibody conjugate.
2. A protein-free alkaline phosphatase-antibody conjugate diluent according to claim 1, wherein: the preservative comprises one or more of sodium azide, proclin300 and BND-10 mixed in any proportion.
3. A method of preparing a protein-free alkaline phosphatase-antibody conjugate diluent according to claim 1, wherein: uniformly mixing a buffer system, soluble salt, allantoin, amino acid, saccharide compounds, a preservative, a surfactant and a reducing agent, and regulating the pH value to 5-7 to obtain the protein-free alkaline phosphatase-antibody conjugate diluent.
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