CN111896731A - Cleaning solution for chemiluminescence immunoassay - Google Patents
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- G—PHYSICS
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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Abstract
The invention belongs to the technical field of chemiluminescence immunoassay, and relates to a cleaning solution for chemiluminescence immunoassay. The cleaning solution comprises the following components: phosphate buffer solution: 10-50mmol/L, sodium caseinate: 0.05-1 wt%, disodium ethylenediamine tetraacetate: 0.02-0.2 wt%, sodium deoxycholate: 0.01-0.1 wt%, nonionic surfactant: 0.05-1 wt%, preservative: 0.02-0.08 wt%. According to the invention, sodium caseinate, disodium ethylene diamine tetraacetate and sodium deoxycholate are jointly used as effective components in the cleaning solution, and the interaction among the components is utilized, so that the cleaning effect of the cleaning solution is greatly improved, and an antibody-antigen complex is stabilized, thereby improving the accuracy and the reproducibility of the detection reagent.
Description
Technical Field
The invention belongs to the technical field of chemiluminescence immunoassay, and relates to a cleaning solution for chemiluminescence immunoassay.
Background
The chemiluminescence immunoassay technique is a quantitative analysis technique in which an intermediate is excited by free energy released by a chemical reaction to return from an excited state to a ground state, and photons of an equal energy level are released when the intermediate returns from the excited state to the ground state, and the photons are measured. Depending on the immunolabeling, chemiluminescent assays fall into the following three categories: direct chemiluminescence immunoassay, chemiluminescence enzyme immunoassay and electrochemiluminescence immunoassay. The direct chemiluminescence immunoassay is characterized in that acridinium ester, isoluminol derivative ABEI and the like are used as tracer substances to mark antigens or antibody molecules to form a luminescent marker, magnetic microspheres coated with antibodies or antigens are used as a separation mode of a solid phase carrier, alkaline liquid is used for inducing the bound marker to emit light, and the quantitative analysis of ultramicro substances is realized by measuring the luminous intensity.
In the chemiluminescence immunoassay process, after the immunoreaction is finished, the reactant is generally washed by using a washing solution, so as to remove substances which are not bound to the magnetic microsphere-antibody antigen immune complex, remove components irrelevant to the reaction and free binders in a sample and remove non-specific interfering substances adsorbed on a solid phase carrier in the reaction process. The cleaning effect of the cleaning solution is directly related to the accuracy of the detection result, so that the cleaning is a key step for determining the success or failure of the test in the chemiluminescence immunoassay process.
At present, in the aspect of improving the performance (repeatability, sensitivity, specificity and the like) of a chemiluminescence immunoassay reagent, research and development personnel usually strive to screen good main raw materials (antigens and antibodies) and optimize a process reaction system, and the effect of a cleaning solution is usually ignored. Relatively few studies and reports have been made on cleaning solutions: for example, chinese patent application CN 105754733a discloses a chemiluminescent immunoassay analyzer cleaning solution, which is composed of sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride, potassium chloride, a surfactant, a preservative and deionized water, and the cleaning solution is prepared from cheap components, so that the cost is reduced; further, as disclosed in chinese patent application CN 109517670a, the cleaning solution includes potassium dihydrogen phosphate, sodium hydroxide, propylene glycol, preservative and triton, which provides an alkaline environment to stimulate a chemiluminescent reaction. The existing cleaning solution is mainly composed of inorganic salt, a surfactant and a preservative, has limited action on specificity and sensitivity of a chemiluminescence immunoassay reagent, frequently generates false positive and false negative clinically, and cannot meet the requirements of high sensitivity and specificity of chemiluminescence immunoassay.
Disclosure of Invention
Aiming at the defects of the existing cleaning solution for chemiluminescence immunoassay, the invention provides a novel cleaning solution which has excellent cleaning effect, low cost and larger social effect and economic benefit.
One aspect of the present invention provides a cleaning solution for chemiluminescence immunoassay, comprising the following components:
phosphate buffer solution: 10 to 50mmol/L of the mixture is added,
sodium caseinate: 0.05 to 1 weight percent of the total weight of the mixture,
disodium ethylene diamine tetraacetate: 0.02 to 0.2 weight percent,
sodium deoxycholate: 0.01 to 0.1 weight percent,
nonionic surfactant: 0.05 to 1 weight percent of the total weight of the mixture,
preservative: 0.02-0.08 wt%.
The added phosphate buffer solution is used for maintaining the pH value of the cleaning solution, the pH value of the phosphate buffer solution is preferably 6.8-7.5, so that the pH value of the cleaning solution is maintained at 6.8-7.5, and the phosphate buffer solution is favorable for dispersing the magnetic microspheres, improving the cleaning effect and stabilizing the antibody-antigen complex under the neutral and mild conditions of the cleaning solution. In order to ensure the pH buffering effect of the buffering agent, the concentration of the phosphate buffer solution is selected to be 10-50mmol/L, so that the defects that the buffering capacity is poor and the stable pH value of the cleaning solution cannot be efficiently ensured due to the excessively low concentration of the buffering agent are avoided; on the other hand, the crystallization problem caused by excessively high concentration of the buffer is avoided.
Sodium caseinate is a mixture of some phosphoproteins, with more proline residues in the molecule. The addition concentration of the sodium caseinate is reasonably controlled to be 0.05-1 wt%, so that the hydrophobic effect of the antigen-antibody compound can be improved, and the settlement generated by Brownian motion and gravity is reduced, so that the stability of the antigen-antibody compound is improved; the sodium caseinate has negative charges in the neutral buffer solution and is dispersed in colloid, so that the electrostatic interaction between the magnetic microspheres can be reduced, the adsorption speed of the magnetic microspheres under a magnetic field is facilitated, and the recovery efficiency of the magnetic microspheres is improved; the sodium caseinate can also react with peroxy radicals, so that the premature excitation of acridinium ester or isoluminol derivatives on the antibody is prevented, and the detection accuracy is improved.
Sodium caseinate can increase the stability of the antigen-antibody complex within a certain content range, but when the content of sodium caseinate is too high, the stability of the complex is damaged, so that the detection accuracy is influenced. Therefore, the concentration of sodium caseinate of the present invention is further preferably 0.2 to 0.8 wt%.
The added disodium ethylene diamine tetraacetate is an excellent metal ion chelating agent such as calcium, magnesium and the like, and can effectively eliminate metal ions possibly existing in a sample; the ethylene diamine tetraacetic acid can also promote the decomposition of peroxide and prevent the premature excitation of a chemiluminescent agent; the magnetic microsphere cleaning liquid is used as an effective component of the cleaning liquid, and particularly has an excellent cleaning effect on nonspecific binding of the magnetic microsphere. For the purpose of controlling costs and obtaining excellent cleaning effects, the content of disodium ethylenediaminetetraacetate of the present invention is preferably 0.05 to 0.1 wt%.
The sodium deoxycholate can be added to solubilize the sodium caseinate, so that the effective action content of the sodium caseinate in the cleaning solution is improved, metal ions in a sample can be chelated, and the sodium deoxycholate and the disodium ethylenediamine tetraacetate can generate good compounding and synergistic effects to enhance the cleaning effect, so that the repeatability and the accuracy of the detection reagent are further improved. The preferable addition amount of sodium deoxycholate of the present invention is 0.02 to 0.05 wt%.
The mass ratio of the disodium ethylene diamine tetraacetate to the sodium deoxycholate added in the cleaning solution is preferably (1.5-2): 1. the disodium ethylene diamine tetraacetate and the sodium deoxycholate are in a proper mass ratio to generate excellent synergistic effect.
The nonionic surfactant has excellent wetting, emulsifying, dispersing and washing effects, is beneficial to removing hybrid protein combined on the surface of the magnetic particles, and improves the cleaning effect of the cleaning solution. The non-ionic surfactant is preferably one or more of Tween 20, Tween 80, Triton X-100, polyethylene glycol and polyvinylpyrrolidone.
In addition, in order to ensure the long-term storage stability of the cleaning solution, the cleaning solution disclosed by the invention is also added with an antiseptic, and the antiseptic is preferably one or more of sodium azide, ProClin 300 and antibiotics.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention provides a novel cleaning solution, which greatly improves the cleaning effect of the cleaning solution and stabilizes an antibody-antigen complex by jointly using sodium caseinate, disodium ethylene diamine tetraacetate and sodium deoxycholate as effective components in the cleaning solution and utilizing the interaction among the components, thereby improving the accuracy and the reproducibility of a detection reagent;
(2) the disodium ethylene diamine tetraacetate and the sodium deoxycholate added into the cleaning solution have excellent synergistic effect, and the combined use of the disodium ethylene diamine tetraacetate and the sodium deoxycholate can improve the cleaning effect of the cleaning solution on metal ions;
(3) the cleaning solution provided by the invention contains sodium caseinate, and also has the effect of improving the recovery efficiency of the magnetic microspheres.
Detailed Description
Hereinafter, the technical solution of the present invention will be further described and illustrated by specific examples. However, these embodiments are exemplary, and the present disclosure is not limited thereto. Unless otherwise specified, the raw materials used in the following specific examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art.
Example 1
Respectively weighing the components and preparing a cleaning solution according to the following mixture ratio:
phosphate buffer (pH 7.4): 20 mmol/L;
tween 20: 0.05 wt%;
sodium caseinate: 0.5 wt%;
disodium ethylene diamine tetraacetate: 0.05 wt%;
sodium deoxycholate: 0.03 wt%;
sodium azide: 0.05 wt%.
Example 2
Phosphate buffer (pH 7.4): 30 mmol/L;
tween 20: 0.1 wt%;
sodium caseinate: 0.75 wt%;
disodium ethylene diamine tetraacetate: 0.1 wt%;
sodium deoxycholate: 0.05 wt%;
sodium azide: 0.05 wt%.
Comparative example 1
Respectively weighing the components and preparing a cleaning solution according to the following mixture ratio:
phosphate buffer (pH 7.4): 20 mmol/L;
tween 20: 0.05 wt%;
disodium ethylene diamine tetraacetate: 0.05 wt%;
sodium deoxycholate: 0.03 wt%;
sodium azide: 0.05 wt%.
Comparative example 2
Respectively weighing the components and preparing a cleaning solution according to the following mixture ratio:
phosphate buffer (pH 7.4): 20 mmol/L;
tween 20: 0.05 wt%;
sodium caseinate: 0.5 wt%;
sodium deoxycholate: 0.08 wt%;
sodium azide: 0.05 wt%.
Comparative example 3
Respectively weighing the components and preparing a cleaning solution according to the following mixture ratio:
phosphate buffer (pH 7.4): 20 mmol/L;
tween 20: 0.05 wt%;
sodium caseinate: 0.5 wt%;
disodium ethylene diamine tetraacetate: 0.08 wt%;
sodium azide: 0.05 wt%.
Comparative example 4
Original ADVIA CENTAUR WASH1 cleaning solution.
The cleaning solutions of examples 1-2 and comparative examples 1-4 were used in a Procalcitonin (PCT) assay project based on the chemiluminescence immune principle to analyze the effect of different cleaning solutions on the assay results.
Drawing a PCT calibration curve: the calibration samples with the concentrations of 0, 0.02, 0.3, 1.5, 7.5 and 37.5ng/mL are respectively added with the magnetic particle suspension coated with the procalcitonin monoclonal antibody and mixed, incubated at 37 ℃ for 4 minutes, then added with the acridinium ester marker for reaction, incubated at 37 ℃ for 4 minutes, washed by the washing solutions of the examples 1-2 and the comparative examples 1-4 respectively to remove the unbound antigen, antibody and other substances, the experiment is carried out on an ADVIA CENTAUR CP analyzer, the detection is carried out according to the instructions of the instrument and related reagents, the concentration of the calibration samples is used as an abscissa, the light signal value is used as an ordinate, and a standard curve is drawn, and the result is shown in Table 1.
TABLE 1 PCT calibration curves using cleaning solutions of examples 1-2 and comparative examples 1-4
The cleaning solution of the examples 1-2 and the comparative examples 1-4 is used for cleaning the reaction compound, and the linear correlation of the obtained PCT calibration curve is good, so that the PCT calibration curve can be used for sample determination.
And (3) testing precision: respectively adding the magnetic particle suspension coated with the procalcitonin monoclonal antibody into samples with the concentrations of 0.65ng/mL and 6.25ng/mL, mixing, incubating at 37 ℃ for 4 minutes, adding the acridinium ester marker for reaction, further incubating at 37 ℃ for 4 minutes, then respectively washing by using the cleaning solutions of examples 1-2 and comparative examples 1-4 to remove unbound antigen, antibody and the like, carrying out an experiment on an ADVIA CENTAUR CP analyzer, detecting according to the instructions of the instrument and related reagents, repeatedly testing 5 times for each concentration, and calculating the precision, wherein the results are shown in Table 2.
TABLE 2 PCT precision of Using cleaning solutions of examples 1-2 and comparative examples 1-4
And (3) testing the accuracy: serum samples with the concentrations shown in table 3 were tested, washed with the washing solutions of examples 1-2 and comparative examples 1-4, respectively, tested on an ADVIA centraur CP analyzer, tested according to the instructions of the apparatus and related reagents, and the concentration was repeated 5 times to obtain an average value.
TABLE 3 comparison of blood samples
The cleaning solutions of examples 1-2 and comparative examples 1-4 were used in the items of human cardiac troponin i (ctni) assay based on the chemiluminescence immune principle to analyze the effects of different cleaning solutions on the assay results.
Drawing a cTnI calibration curve: the calibration samples with concentrations of 0, 0.02, 0.1, 0.5, 10, 50ng/mL are respectively added with the magnetic particle suspension coated with the cTnI monoclonal antibody to be mixed, incubated for 4 minutes at 37 ℃, then added with the acridinium ester marker to react, incubated for 4 minutes at 37 ℃, then washed by the washing solutions of examples 1-2 and comparative examples 1-4 respectively to remove the unbound antigen, antibody and other substances, the experiment is carried out on an ADVIA CENTAUR CP analyzer, the detection is carried out according to the instructions of the instrument and related reagents, the concentration of the calibration sample is used as an abscissa, the light signal value is used as an ordinate, and a standard curve is drawn, and the result is shown in Table 4.
TABLE 4 cTnI calibration curves using the cleaning solutions of examples 1-2 and comparative examples 1-4
| Concentration of | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 |
| ng/ml | RLU | RLU | RLU | RLU | RLU | RLU |
| 50 | 2894131 | 2911184 | 2580758 | 2365798 | 2394230 | 2118909 |
| 10 | 643464 | 699244 | 623256 | 603145 | 583028 | 584335 |
| 0.5 | 30612 | 36821 | 32612 | 30635 | 39256 | 30234 |
| 0.1 | 7515 | 7468 | 6215 | 7109 | 7211 | 6518 |
| 0.02 | 1529 | 1553 | 1629 | 1511 | 1698 | 1682 |
| 0 | 335 | 360 | 760 | 666 | 822 | 1084 |
| R2 | 0.9995 | 0.9984 | 0.9983 | 0.9969 | 0.9981 | 0.9942 |
The cleaning solution of the examples 1-2 and the comparative examples 1-4 is used for cleaning the reaction compound, and the obtained cTnI calibration curve has good linear correlation and can be used for sample determination.
And (3) testing precision: respectively adding magnetic particle suspensions coated with cTnI monoclonal antibodies into samples of 0.393ng/mL and 8.937ng/mL, mixing, incubating at 37 ℃ for 4 minutes, adding acridinium ester markers for reaction, further incubating at 37 ℃ for 4 minutes, washing with the washing solutions of examples 1-2 and comparative examples 1-4 to remove unbound antigens, antibodies and the like, performing experiments on an ADVIA CENTAUR CP analyzer, detecting according to the instructions of the instrument and related reagents, repeatedly testing each concentration for 5 times, and calculating the precision, wherein the results are shown in Table 5.
TABLE 5 precision of cTnI Using the cleaning solutions of examples 1-2 and comparative examples 1-4
And (3) testing the accuracy: serum samples with the concentrations shown in Table 6 were tested, washed with the washing solutions of examples 1-2 and comparative examples 1-4, respectively, tested on an ADVIA CENTAUR CP analyzer, tested according to the instructions of the apparatus and related reagents, and the concentration was repeated 5 times to obtain an average value.
Table 6 blood sample comparison test results
With respect to the cleaning solution of example 1, the cleaning solution of comparative example 1 did not include sodium caseinate, the cleaning solution of comparative example 2 did not include disodium edetate, and the added amount of sodium deoxycholate was the sum of the contents of disodium edetate and sodium deoxycholate of example 1, the cleaning solution of comparative example 3 did not include sodium deoxycholate, and the added amount of disodium edetate was the sum of the contents of disodium edetate and sodium deoxycholate of example 1. From the experimental results of the procalcitonin and human cardiac troponin I test items, it can be seen that: compared with the cleaning solution sold in the market, the cleaning solution of the embodiment 1-2 has better cleaning effect and higher test result reproducibility; when the cleaning solution lacks sodium caseinate, disodium ethylene diamine tetraacetate or sodium deoxycholate, the necessary effective components and the synergistic action between the effective components are lost, so that the cleaning effect of the cleaning solution is reduced, and the accuracy and the reproducibility of a test result are reduced.
To further evaluate the effect of the washing solution on the recovery efficiency of the magnetic beads, the following experiments were performed:
respectively adding the magnetic particle suspension coated with the procalcitonin monoclonal antibody into 6 samples with the concentration of 0.65ng/mL, mixing, respectively adding the acridinium ester markers for reaction after incubating for 4 minutes at 37 ℃, continuing to incubate for 4 minutes at 37 ℃, then carrying out magnetic attraction on the reaction solution for 1 minute by using a magnetic plate, and sucking and removing the supernatant. After the reaction, the magnetic beads were washed with the washing solutions of examples 1 to 2 and comparative examples 1 to 4, respectively, and then the washing solutions of examples 1 to 2 and comparative examples 1 to 4 were added to resuspend the magnetic beads, and then the magnetic force frame was used to separate the particles from the solution, and the magnetic efficiency (i.e., the time for completion of magnetic bead adsorption) was measured. And recording the agglutination time of the magnetic beads in each scheme by using a timer, observing the suspension liquid of the magnetic beads by naked eyes to be completely clarified, and adsorbing the magnetic beads on a magnetic frame completely to aggregate into a group as a timing end point. The results are shown in table 7 below:
TABLE 7 magnetic attraction time of magnetic beads washed with the washing liquids of examples 1 to 2 and comparative examples 1 to 4
| Magnetic attraction time s | |
| Example 1 magnetic beads washed with washing solution | 28.6 |
| Example 2 magnetic beads washed with washing solution | 28.0 |
| Comparative example 1 magnetic bead washed with washing liquid | 36.2 |
| Comparative example 2 magnetic bead washed with washing liquid | 29.1 |
| Comparative example 3 magnetic bead washed with washing liquid | 29.3 |
| Comparative example 4 magnetic bead washed with washing liquid | 37.5 |
As can be seen from the data results in Table 7, the cleaning solutions of comparative example 1 and comparative example 4 do not contain sodium caseinate, the magnetic attraction time of the magnetic beads cleaned by the cleaning solutions of comparative example 1 and comparative example 4 is obviously longer than that of other groups, and the sodium caseinate in the cleaning solutions has an effect of improving the magnetic bead recovery efficiency.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (8)
1. A cleaning solution for chemiluminescence immunoassay, which is characterized by comprising the following components:
phosphate buffer solution: 10 to 50mmol/L of the mixture is added,
sodium caseinate: 0.05 to 1 weight percent of the total weight of the mixture,
disodium ethylene diamine tetraacetate: 0.02 to 0.2 weight percent,
sodium deoxycholate: 0.01 to 0.1 weight percent,
nonionic surfactant: 0.05 to 1 weight percent of the total weight of the mixture,
preservative: 0.02-0.08 wt%.
2. The washing solution for chemiluminescent immunoassay according to claim 1, wherein the phosphate buffer has a pH of 6.8 to 7.5.
3. The washing solution for chemiluminescent immunoassay according to claim 1, wherein the sodium caseinate is contained in an amount of 0.2 to 0.8 wt%.
4. The washing solution for chemiluminescent immunoassay according to claim 1 wherein the disodium ethylenediaminetetraacetate ranges from 0.05 to 0.1 wt%.
5. The washing solution for chemiluminescent immunoassay according to claim 1 wherein the sodium deoxycholate is present in an amount of 0.02 to 0.05 wt%.
6. The washing solution for chemiluminescent immunoassay according to claim 1 or 4 or 5, wherein the mass ratio of the disodium ethylenediaminetetraacetate to the sodium deoxycholate is (1.5-2): 1.
7. the washing solution for chemiluminescence immunoassay according to claim 1, wherein the nonionic surfactant is one or more of tween 20, tween 80, triton X-100, polyethylene glycol, and polyvinylpyrrolidone.
8. The washing solution for chemiluminescent immunoassay according to claim 1, wherein the preservative is one or more of sodium azide, ProClin 300 and antibiotics.
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Cited By (4)
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| CN113337348A (en) * | 2021-05-31 | 2021-09-03 | 济南迪曼生物科技有限公司 | Acridinium ester chemiluminescence cleaning fluid |
| CN113484306A (en) * | 2021-07-02 | 2021-10-08 | 山东中鸿特检生物科技有限公司 | Magnetic particle chemiluminescence cleaning solution and preparation method and application thereof |
| CN113671169A (en) * | 2021-07-21 | 2021-11-19 | 上海交通大学 | Non-specific adsorption cleaning solution for chemiluminescence immunoassay AEP |
| CN115368976A (en) * | 2022-08-03 | 2022-11-22 | 吉林基蛋生物科技有限公司 | Immune lotion for electrochemiluminescence immunoassay |
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| CN107118865A (en) * | 2017-06-15 | 2017-09-01 | 迈克生物股份有限公司 | Cleaning fluid |
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