CN115436621A - Diluent of acridinium ester antibody conjugate - Google Patents
Diluent of acridinium ester antibody conjugate Download PDFInfo
- Publication number
- CN115436621A CN115436621A CN202211201418.XA CN202211201418A CN115436621A CN 115436621 A CN115436621 A CN 115436621A CN 202211201418 A CN202211201418 A CN 202211201418A CN 115436621 A CN115436621 A CN 115436621A
- Authority
- CN
- China
- Prior art keywords
- acridinium ester
- diluent
- antibody conjugate
- content
- buffer solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229940127121 immunoconjugate Drugs 0.000 title claims abstract description 47
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 239000003085 diluting agent Substances 0.000 title claims abstract description 31
- 239000012895 dilution Substances 0.000 claims abstract description 26
- 238000010790 dilution Methods 0.000 claims abstract description 26
- 239000002518 antifoaming agent Substances 0.000 claims abstract description 13
- 239000007853 buffer solution Substances 0.000 claims abstract description 12
- 239000003755 preservative agent Substances 0.000 claims abstract description 12
- 230000002335 preservative effect Effects 0.000 claims abstract description 12
- 239000000654 additive Substances 0.000 claims abstract description 11
- 230000000996 additive effect Effects 0.000 claims abstract description 11
- 239000004094 surface-active agent Substances 0.000 claims abstract description 11
- 229940124272 protein stabilizer Drugs 0.000 claims abstract description 10
- 239000002738 chelating agent Substances 0.000 claims abstract description 9
- DZBUGLKDJFMEHC-UHFFFAOYSA-O acridine;hydron Chemical compound C1=CC=CC2=CC3=CC=CC=C3[NH+]=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-O 0.000 claims description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- -1 casein sodium salt Chemical class 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical group CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 3
- 239000005018 casein Substances 0.000 claims description 3
- 235000021240 caseins Nutrition 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical group OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 2
- RWZXIUKKMTXBQY-UHFFFAOYSA-M sodium dodecanoylazanide Chemical compound C(CCCCCCCCCCC)(=O)[NH-].[Na+] RWZXIUKKMTXBQY-UHFFFAOYSA-M 0.000 claims description 2
- 239000003550 marker Substances 0.000 abstract description 10
- 239000007788 liquid Substances 0.000 abstract description 8
- 238000000605 extraction Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 22
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 20
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 230000003247 decreasing effect Effects 0.000 description 9
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000005284 excitation Effects 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000009472 formulation Methods 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000006260 foam Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000003929 acidic solution Substances 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 239000013530 defoamer Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920000259 polyoxyethylene lauryl ether Polymers 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000010907 mechanical stirring Methods 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000012430 stability testing Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
Classifications
-
- 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
- G01N33/531—Production of immunochemical test materials
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a diluent of an acridinium ester antibody conjugate, which consists of a specific protein stabilizer, a chelating agent, a surfactant, a preservative, an additive, a buffer solution and a defoaming agent, wherein the pH value is 7.2 +/-0.2. The dilution liquid provided by the invention can ensure the stability and the reaction performance of the acridinium ester marker, ensure the accuracy of sample extraction, and effectively reduce the light-emitting value jump. In addition, the dilution prepared by the invention does not influence the luminous efficiency of the acridinium ester.
Description
Technical Field
The application relates to the technical field of immunodetection analysis, in particular to a diluent of an acridinium ester antibody conjugate.
Background
The acridinium ester luminescent marker is likely to be partially decomposed under the condition of illumination, so that the luminescent effect and the stability of a chemiluminescent reagent are influenced, and the experimental operation and storage of the acridinium ester and the marker thereof are recommended to be carried out under the condition of keeping out of the sun, so that a reagent bottle for subpackaging the acridinium ester marking working solution in the chemiluminescent kit adopts a brown bottle, the bottle mouth is designed to be small in consideration of the volatilization of the solution, and the condition of the reagent in the bottle cannot be seen clearly after the acridinium ester marking working solution is subpackaged.
In order to enhance the stability and the reaction performance of the acridinium ester marker, a protein protective agent, a surfactant and other reagents which are easy to generate bubbles are added into a diluent of the acridinium ester marker, and a diluent formula which can stabilize the acridinium ester antigen-antibody conjugate disclosed in the patent application CN201610081312.9 also comprises a carbohydrate, an amino acid and other reagents, so that the process difficulty and the controllability of raw material sources are increased, and the diluent of the acridinium ester marker is easy to grow bacteria, thereby influencing the stability of the acridinium ester marker.
When the acridinium ester marking working solution is used, bubbles floating on the liquid surface can interfere the liquid level detection accuracy of the sample adding needle, so that the liquid sample adding amount is inaccurate, the dissolving uniformity of the acridinium ester marker can be influenced by the bubbles in the liquid, the inaccuracy (CV) of the kit in sample detection is increased, and the performance of the kit is influenced.
The physical methods for eliminating the foam mainly include placing a baffle or a screen, mechanical stirring, static electricity, freezing, heating, steam, radiation irradiation, high-speed centrifugation, pressurization and depressurization, high-frequency vibration, instantaneous discharge, and ultrasonic waves (acoustic liquid control), and these methods make the stability factor of the foam smaller than the attenuation factor to various degrees, so that the amount of the foam gradually decreases. However, these methods have a common disadvantage that the use is restricted by environmental factors, the defoaming rate is not high, and the time efficiency and the operability of the use of the reagent are affected.
Disclosure of Invention
In order to solve the problem of inaccurate sample addition caused by adding an easily-foaming reagent into an acridinium ester marker working solution, the invention provides a diluent of an acridinium ester antibody conjugate, wherein the diluent consists of a protein stabilizer, a chelating agent, a surfactant, a preservative, an additive, a buffer solution and an antifoaming agent;
wherein the protein stabilizer is selected from one or more of bovine IgG, bovine serum albumin, casein sodium salt and mouse IgG, and the content is 5-30g/L;
the chelating agent is EDTA with the content of 10mg/L;
the surfactant is selected from brij35 (polyoxyethylene lauryl ether) and/or sodium lauroyl amide, and the content is 0.2-2g/L;
the preservative is selected from one or a mixture of two of Proclin 300 and sodium azide, and the content of the preservative is 0.05-0.5 percent by mass;
the additive is sodium chloride, and the content of the additive is 9g/L;
the buffer solution is Tris-HCl buffer solution, and the final concentration of the buffer solution in the diluent solution is 10-100mM;
the defoaming agent is selected from DMF (N, N-dimethylformamide), absolute ethyl alcohol or a mixture of DMF and absolute ethyl alcohol, and the mass percentage content of the defoaming agent is 0.1-0.3%;
the pH value of the diluent is 7.2 +/-0.2.
Further, the pH value of the diluted solution is adjusted to 7.2 +/-0.2 by adopting HCl or NaOH.
And adding the protein stabilizer, the chelating agent, the surfactant, the preservative, the additive and the defoaming agent into the buffer solution, stirring until the protein stabilizer, the chelating agent, the surfactant, the preservative, the additive and the defoaming agent are completely dissolved, and adjusting the pH value to obtain the diluent.
The acridinium ester antibody conjugate is an acridinium ester-S100 antibody conjugate.
The beneficial effects of the invention include:
the dilution solution of the acridinium ester antibody conjugate provided by the invention can ensure the stability and the reaction performance of the acridinium ester marker, can ensure the accuracy of the sample extraction amount, and can effectively reduce the light-emitting value jump value. In addition, the diluent prepared by the invention does not influence the luminous efficiency of the acridinium ester.
Under different temperature conditions, the bond between the antibody protein and the acridine ester can be unstable and broken along with the lapse of storage time, the structure of the acridine ester is changed, and further the photon yield is influenced, although protein protection components such as saccharides, amino acids and the like are not added in the diluent of the acridine ester antibody conjugate provided by the invention, under the condition that the pH value is ensured to be in the range of 7.2 +/-0.2 by adding an antifoaming agent DMF, absolute ethyl alcohol or a mixture of the two, the synergistic effect among the components can keep the stability of the bond of the acridine ester antibody and the structure of the acridine ester, the diluted reagent of the acridine ester antibody conjugate provided by the invention is adopted to dilute the acridine ester antibody conjugate, and the storage period of the diluted reagent is as follows: can be stored at 2-8 deg.C for more than 18 months, at 37 deg.C for more than 10 days, and at room temperature for at least 6 weeks. Meanwhile, the formula of the dilution liquid of the acridine ester antibody conjugate provided by the invention is relatively simple, and the realizability and controllability of the production process are improved.
Detailed Description
The present invention will be further illustrated and described with reference to the following examples, but the examples described are only a part of the examples of the present invention, and not all of the examples. All other inventions and embodiments based on the present invention and obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 dilution of an acridinium ester antibody conjugate
The formulation of dilutions of acridinium ester antibody conjugates is shown in table 1 below.
TABLE 1 content of Components in dilutions of acridinium ester antibody conjugates
Wherein brij35 represents surfactant polyoxyethylene lauryl ether brij35; DMF represents N, N-dimethylformamide; the preservative Proclin 300 was purchased from sigma-aldrich (cat. No. 48914-U).
The preparation method of the dilution of the acridinium ester antibody conjugate comprises the following specific steps:
1) Adding a protein stabilizer, a chelating agent, a surfactant, a preservative, an additive and a defoaming agent into a buffer solution according to the formula shown in Table 1, and stirring until the mixture is completely dissolved to obtain a mixed solution;
2) Stirring and uniformly mixing the mixed solution obtained in the step 1), and adding HCl or NaOH to adjust the pH value to 7.2 +/-0.2 to obtain the diluent.
Example 2 dilution of acridinium ester antibody conjugate
(1) Diluent 1-5 was prepared according to the formulation of Table 2, and the diluent 1-5 was a defoamer group.
TABLE 2 content of each component in dilutions 1-5 of acridinium ester antibody conjugates
| Diluent solution | 1 | 2 | 3 | 4 | 5 |
| Bovine IgG | 2g/L | ||||
| Bovine serum albumin | 5g/L | 30g/L | 10g/L | 20g/L | 20g/L |
| Casein sodium salt | 1g/L | ||||
| EDTA-Na 2 | 10mg/L | 10mg/L | 10mg/L | 10mg/L | 10mg/L |
| brij35 | 0.2g/L | 2g/L | 0.5g/L | 0.1g/L | |
| Lauroyl amino acid sodium salt | 1g/L | 0.5g/L | 0.1g/L | ||
| Proclin 300 | 0.050％ | 0.5％ | 0.50％ | 0.50％ | 0.050％ |
| Sodium azide | 0.10％ | ||||
| Sodium chloride | 9g/L | 9g/L | 9g/L | 9g/L | 9g/L |
| Tris-HCl buffer | 10mM | 100mM | 50mM | 50mM | 50mM |
| DMF | 0.10％ | 0.20％ | 0.20％ | 0.20％ | |
| Ethanol | 0.10％ | 0.20％ |
(2) A control diluent 6-10 was prepared according to the formulation in Table 3, the diluent 6-10 containing no antifoaming agent.
TABLE 3 content of respective Components in dilutions 6 to 10 of acridinium ester antibody conjugates
(3) Referring to example 1 of patent application CN201610081312.9, a control diluent 11 was prepared according to the formulation of table 4, and a defoamer DMF was added in an amount of 0.20% by mass based on the formulation to prepare a diluent 12.
TABLE 4 content of each component in the dilution 11 and the dilution 12 of the acridinium ester antibody conjugates (each component content is mass percent)
The specific preparation method of the diluents 11, 12 is as follows:
1) According to the formula shown in Table 4, sucrose, trehalose, glycerol, glycine, bovine Serum Albumin (BSA), DTPA, tween-20, sodium azide, PEG6000, sodium chloride and DMF (added only to the diluent 12) are added into 0.05mol/L citrate buffer solution and stirred until the mixture is completely dissolved to obtain a mixed solution;
2) Stirring and uniformly mixing the mixed solution obtained in the step 1), adding NaOH to adjust the pH value to 6.4 +/-0.1, and obtaining a diluent 11 and a diluent 12.
Example 3 measurement of Effect of diluted solution of acridinium ester antibody conjugate
The acridinium ester-S100 antibody was diluted to a working concentration using the dilutions 1 to 12 prepared in example 2, respectively, to obtain dilutions of the acridinium ester-S100 antibody conjugate, which were designated as dilutions 1 to 12 of the acridinium ester-S100 antibody conjugate, respectively.
From each of the dilutions 1-12 of the acridinium ester-S100 antibody conjugate, 19 bottles of 2ml each were dispensed. Wherein 1 bottle is used for the test on the preparation day, 4 bottles are placed in a 37 ℃ oven for accelerated experiment, and the other 7 bottles are placed in a 2-8 ℃ refrigerator for storage, and the other 7 bottles are placed in room temperature for storage.
(1) Reproducibility test of diluted acridinium ester-S100 antibody conjugates:
the above dilution of acridinium ester-S100 antibody conjugate was tested 30 times, 50. Mu.l of the diluted conjugate was added to the reaction cup, 100. Mu.l of pre-excitation solution (acidic solution containing hydrogen peroxide, pH < 2.0) and 100. Mu.l of excitation solution (alkaline solution containing sodium hydroxide, pH > 12.0) were automatically added to the luminometer, the conjugate immediately emitted light after the addition of the excitation solution, and the luminometer showed the measured Relative Light Units (RLUs). Respectively calculating the average value (M) and the Standard Deviation (SD) of the 30 detection results, obtaining the Coefficient of Variation (CV) according to the formula (1),
CV＝SD/M×100％................(1)
in the formula: CV — coefficient of variation; SD-standard deviation of detection results; m represents the average value of the detection results. The results are shown in Table 5.
TABLE 5 repeatability test results for dilution of acridinium ester antibody conjugates
As can be seen from the results of the repetitive tests in Table 5, the coefficient of variation of the diluted solution of acridinium ester-S100 antibody conjugate after being tested for 30 times in 1-5 times is less than 1%, while the coefficient of variation of the diluted solution of acridinium ester-S100 antibody conjugate after being tested for 30 times in 6-10 times in 6.639%, 4.105%, 3.202%, 1.536%, 10.622%, respectively, is greater than 1%. The individual deviation values appear in the detection results of the dilution liquid 6-10 of the acridinium ester-S100 antibody conjugate repeatedly tested for 30 times, so that the standard deviation of the detection results is increased, the variation coefficient of the detection results is increased, and the repeatability is poor.
(2) Stability testing of diluted acridinium ester-S100 antibody conjugates:
the diluted acridinium ester antibody conjugates were tested for stability at 37 deg.C, 2-8 deg.C and room temperature, respectively, according to the test times set forth in tables 6-8, 50. Mu.l of the diluted conjugate was added to the reaction cup, 100. Mu.l of a pre-excitation solution (acidic solution containing hydrogen peroxide, pH < 2.0) and 100. Mu.l of an excitation solution (alkaline solution containing sodium hydroxide, pH > 12.0) were automatically added to the reaction cup by a luminometer, the conjugate immediately emitted light upon addition of the excitation solution, and the luminometer displayed the measured Relative Light Units (RLUs), with the results shown in tables 6-8.
TABLE 6 stability test results of the diluted conjugates at 37 deg.C
As shown in Table 6, after being left at 37 ℃ for 10 days, the luminous efficiency of the diluted solution 1-5 of the acridinium ester-S100 antibody conjugate decreased by 4.186-6.021%, while that of the diluted solution 11 of the acridinium ester-S100 antibody conjugate decreased by 19.225% and that of the diluted solution 12 of the acridinium ester-S100 antibody conjugate decreased by 20.294%.
TABLE 7 stability test results of the diluted conjugates at 2-8 deg.C
As shown in Table 7, after being placed at 2-8 ℃ for 21 months, the luminous efficiency of the dilution of acridinium ester-S100 antibody conjugate 1-5 decreased by 3.520-5.900%, the luminous efficiency of the dilution of acridinium ester-S100 antibody conjugate 11 decreased by 22.060%, and the luminous efficiency of the dilution of acridinium ester-S100 antibody conjugate 12 decreased by 23.017%.
TABLE 8 stability test results of the diluted conjugates at room temperature
As shown in Table 8, after being left at room temperature for 7 weeks, the luminous efficiency of the diluted solution 1-5 of the acridinium ester-S100 antibody conjugate decreased by 3.970-6.293%, the luminous efficiency of the diluted solution 11 of the acridinium ester-S100 antibody conjugate decreased by 21.541%, and the luminous efficiency of the diluted solution 12 of the acridinium ester-S100 antibody conjugate decreased by 21.302%.
According to the test, the diluted solution of the acridinium ester antibody conjugate provided by the invention is used for diluting the acridinium ester antibody conjugate, and the storage life of the diluted reagent is as follows: can be stored at 2-8 deg.C for more than 18 months, at 37 deg.C for more than 10 days, and can be stored at room temperature for at least 6 weeks.
Claims (4)
1. A diluent of an acridinium ester antibody conjugate, which is characterized by consisting of a protein stabilizer, a chelating agent, a surfactant, a preservative, an additive, a buffer solution and an antifoaming agent;
wherein the protein stabilizer is selected from one or more of bovine IgG, bovine serum albumin, casein sodium salt and mouse IgG, and the content of the protein stabilizer is 5-30g/L;
the chelating agent is EDTA with the content of 10mg/L;
the surfactant is selected from brij35 and/or sodium lauroyl amide, and the content is 0.2-2g/L;
the preservative is selected from one or a mixture of two of Proclin 300 and sodium azide, and the mass percentage content of the preservative is 0.05-0.5%;
the additive is sodium chloride, and the content of the additive is 9g/L;
the buffer solution is Tris-HCl buffer solution, and the final concentration of the buffer solution in the diluent is 10-100mM;
the defoaming agent is selected from DMF, absolute ethyl alcohol or a mixture of the DMF and the absolute ethyl alcohol, and the mass percent content of the defoaming agent is 0.1-0.3%;
the pH value of the diluent is 7.2 +/-0.2.
2. The diluent of claim 1, wherein the pH of the diluent is adjusted to 7.2 ± 0.2 using HCl or NaOH.
3. The dilution according to claim 1 or 2, wherein the protein stabilizer, chelating agent, surfactant, preservative, additive and antifoaming agent are added to the buffer solution, stirred until completely dissolved, and the pH value is adjusted to obtain the dilution.
4. The dilution according to claim 1 or 2, wherein the acridinium ester antibody conjugate is an acridinium ester-S100 antibody conjugate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211201418.XA CN115436621A (en) | 2022-09-29 | 2022-09-29 | Diluent of acridinium ester antibody conjugate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211201418.XA CN115436621A (en) | 2022-09-29 | 2022-09-29 | Diluent of acridinium ester antibody conjugate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115436621A true CN115436621A (en) | 2022-12-06 |
Family
ID=84251257
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211201418.XA Pending CN115436621A (en) | 2022-09-29 | 2022-09-29 | Diluent of acridinium ester antibody conjugate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115436621A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116027019A (en) * | 2022-12-23 | 2023-04-28 | 宁波瑞源生物科技有限公司 | Additive for chemiluminescence immunoassay and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0650054A2 (en) * | 1993-10-20 | 1995-04-26 | Roche Diagnostics GmbH | Process and device for obtaining and detecting immunologically reactive substances from the gas phase |
| CN105158235A (en) * | 2014-06-06 | 2015-12-16 | 厦门万泰凯瑞生物技术有限公司 | Enhanced acridinium ester luminescence system |
| CN105628914A (en) * | 2016-02-04 | 2016-06-01 | 广州科方生物技术有限公司 | Diluent enabling stability for acridinium ester antigen-antibody conjugate and preparation method of diluent |
| CN108680735A (en) * | 2018-05-21 | 2018-10-19 | 苏州佑君环境科技有限公司 | A kind of acridinium ester labeled antibody dilution and preparation method thereof |
| CN111855996A (en) * | 2019-09-19 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Diluent capable of improving stability of acridinium ester antigen-antibody conjugate and reducing background and preparation method thereof |
| CN112578128A (en) * | 2020-12-02 | 2021-03-30 | 苏州海狸生物医学工程有限公司 | Direct chemiluminescence kit for detecting content of beta-hCG and use method thereof |
-
2022
- 2022-09-29 CN CN202211201418.XA patent/CN115436621A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0650054A2 (en) * | 1993-10-20 | 1995-04-26 | Roche Diagnostics GmbH | Process and device for obtaining and detecting immunologically reactive substances from the gas phase |
| CN105158235A (en) * | 2014-06-06 | 2015-12-16 | 厦门万泰凯瑞生物技术有限公司 | Enhanced acridinium ester luminescence system |
| CN105628914A (en) * | 2016-02-04 | 2016-06-01 | 广州科方生物技术有限公司 | Diluent enabling stability for acridinium ester antigen-antibody conjugate and preparation method of diluent |
| CN108680735A (en) * | 2018-05-21 | 2018-10-19 | 苏州佑君环境科技有限公司 | A kind of acridinium ester labeled antibody dilution and preparation method thereof |
| CN111855996A (en) * | 2019-09-19 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Diluent capable of improving stability of acridinium ester antigen-antibody conjugate and reducing background and preparation method thereof |
| CN112578128A (en) * | 2020-12-02 | 2021-03-30 | 苏州海狸生物医学工程有限公司 | Direct chemiluminescence kit for detecting content of beta-hCG and use method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116027019A (en) * | 2022-12-23 | 2023-04-28 | 宁波瑞源生物科技有限公司 | Additive for chemiluminescence immunoassay and application thereof |
| CN116027019B (en) * | 2022-12-23 | 2023-10-03 | 宁波瑞源生物科技有限公司 | Additive for chemiluminescence immunoassay and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105628914B (en) | A kind of dilution and preparation method thereof that acridinium ester antigen-antibody conjugate can be made stable | |
| CN104990912B (en) | A kind of enzyme-catalyzed chemical luminescence substrate of alkaline phosphatase | |
| CN115436621A (en) | Diluent of acridinium ester antibody conjugate | |
| Borëus et al. | Tissue mast cells, histamine and ‘slow reacting substance’in anaphylactic reaction in guinea pig | |
| Peehl et al. | Growth responses of normal, benign hyperplastic, and malignant human prostatic epithelial cells in vitro to cholera toxin, pituitary extract, and hydrocortisone | |
| CN107727587B (en) | Antithrombin III determination kit and detection method thereof | |
| CN109521003A (en) | A kind of anti-Miao Leguan hormone magnetic microparticle chemiluminescence detection kit | |
| Racowsky | Effect of forskolin on the spontaneous maturation and cyclic AMP content of hamster oocyte‐cumulus complexes | |
| CN112051354B (en) | Lipase determination kit and preparation method thereof | |
| CN107449918A (en) | A kind of protein chip stabilizer and its preparation method and application | |
| CN111855996A (en) | Diluent capable of improving stability of acridinium ester antigen-antibody conjugate and reducing background and preparation method thereof | |
| CN111505274B (en) | Preparation method of single-component TMB color developing solution for enzyme-linked immune reaction | |
| CN105850978A (en) | Boar semen room-temperature preservation dilution powder and diluent, and application of dilution powder and diluent | |
| CN108409852A (en) | Sex hormone binding globulin preserves liquid and its application | |
| CN115754299A (en) | Homocysteine detection kit | |
| CN111117623A (en) | Acidic etching auxiliary agent and preparation method thereof | |
| CN103792347A (en) | Special test box for enzyme-linked immunosorbent assay of class-B trichothecenes | |
| CN113504379A (en) | Sex hormone dissociation agent and detection kit | |
| CN109254145A (en) | For improving the dilution of matrix effect between fresh serum and third party's Quality Control | |
| CN108680735A (en) | A kind of acridinium ester labeled antibody dilution and preparation method thereof | |
| CN114428170A (en) | Reagent box for detecting chemicals in sewage and detection method thereof | |
| CN108342359B (en) | Preparation method of digestive juice | |
| CN114324882A (en) | Protein stabilizer and application thereof | |
| Veldhuis et al. | Regulation of ornithine decarboxylase in isolated granulosa cells in vitro by constituents of follicular fluid | |
| CN115327137B (en) | Human immunoglobulin G4 subtype chemiluminescence immunoassay kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |