Non-specific adsorption cleaning solution for chemiluminescence immunoassay AEP
阅读说明：本技术 用于化学发光免疫检测aep的非特异性吸附清洗液 (Non-specific adsorption cleaning solution for chemiluminescence immunoassay AEP ) 是由 郭方 陈峻崧 徐文克 李锐 于 2021-07-21 设计创作，主要内容包括：一种用于化学发光免疫检测AEP的非特异性吸附清洗液,每升包括10-100mMol/L缓冲液、0.01-1wt％表面活性剂、100-300mMol/L无机盐、0.5-0.1g/L防腐剂,余量为水。本发明能够去除蛋白粘附,并使样品中内源脂溶性物质增溶分散,降低了分析结果的偏差,提高了检测的准确度,对内源性干扰物清洗效果极佳,一定程度上保证了临床样本检测的准确性。(A non-specific adsorption cleaning solution for AEP of chemiluminescence immunoassay comprises 10-100mMol/L buffer solution, 0.01-1 wt% of surfactant, 100-300mMol/L inorganic salt, 0.5-0.1g/L preservative and the balance of water per liter. The invention can remove protein adhesion, solubilize and disperse endogenous fat-soluble substances in a sample, reduce the deviation of an analysis result, improve the detection accuracy, has excellent cleaning effect on endogenous interferents and ensures the accuracy of clinical sample detection to a certain extent.)
1. A nonspecific adsorption cleaning solution for AEP of chemiluminescence immunoassay is characterized in that each liter comprises 10-100mMol/L buffer solution, 0.01-1 wt% of surfactant, 100-300mMol/L inorganic salt, 0.5-0.1g/L preservative, and the balance of water;
the non-specific adsorption cleaning is as follows: the HLB value and the CMC value of the cleaning buffer solution are changed by utilizing different surfactant combinations under the existence conditions of specific buffer solution and ionic strength so as to achieve the purposes of removing nonspecific adsorption and adhesion and increasing the water solubility of nonpolar substances, thereby meeting the requirements on specificity and sensitivity in the AEP detection process.
2. The non-specific adsorption cleaning solution for AEP chemiluminescent immunoassay according to claim 1 wherein the surfactant is an anionic surfactant, a cationic surfactant, an amphoteric surfactant, a nonionic surfactant or a combination thereof wherein: the anionic surfactant comprises a sulfate salt type, the cationic surfactant comprises an alkyl quaternary ammonium salt, the amphoteric surfactant comprises a betaine type, and the nonionic surfactant comprises a polyol type surfactant and a polyoxyethylene type surfactant.
3. The non-specific adsorption washing solution for AEP chemiluminescent immunoassay according to claim 1 wherein the buffer comprises tris, piperazine-1, 4-diethylsulfonic acid, 3-morpholinopropanesulfonic acid, phosphate buffer or a combination thereof, at pH 7.0-8.5.
4. The non-specific adsorption wash for AEP chemiluminescent immunoassay according to claim 3 wherein the buffer has a pH of 7.5 and 8.0.
5. The non-specific adsorption cleaning solution for AEP in chemiluminescent immunoassay according to claim 1 wherein the inorganic salt is sodium chloride or potassium chloride.
6. The non-specific adsorption cleaning solution for AEP of chemiluminescence immunoassay of claim 1, wherein the preservative is Proclin300, Proclin 950 or sodium azide.
7. The non-specific adsorption cleaning solution for the chemiluminescent immunoassay AEP according to any one of claims 1 to 6, which is specifically any one of the following:
10-200mMol/L of trihydroxymethyl aminomethane, 0.01-1 wt% of mixed solution of polyoxyethylene dodecyl ether and 3-sulfopropyl tetradecyl betaine, 100mMol/L of sodium chloride and 0.5-0.1g/LProclin300, wherein: mixing polyoxyethylene dodecyl ether and 3-sulfopropyl tetradecyl betaine according to a weight ratio of 4:1 to form a compound surfactant;
10-200mMol/L of trihydroxymethyl aminomethane, 0.01-1 wt% of fatty alcohol-polyoxyethylene ether sodium sulfate, 100-300mMol/L of sodium chloride and 0.5-0.1g/LProclin 300;
③ 10-200mMol/L of trihydroxymethyl aminomethane, 0.01-1 wt% of nonionic surfactant, 100-300mMol/L of sodium chloride and 0.5-0.1g/LProclin300, wherein: the nonionic surfactant is a compound surfactant formed by mixing NP 40, Tween20 and polyoxyethylene dodecyl ether in a weight ratio of 1:4: 5.
Technical Field
The invention relates to a technology in the field of chemiluminescence immunoassay, in particular to a non-specific adsorption cleaning solution for chemiluminescence immunoassay AEP.
Background
The chemiluminescence immunoassay CLIA is a novel immunological technique after radioimmunoassay RIA, enzyme-linked immunosorbent assay (ELISA) and fluorescence immunoassay FFIA, combines a chemiluminescence technique with high sensitivity and an immunoreaction with high specificity, and can be used for detecting trace antibodies or antigens. The principle is as follows: the luminescent substance or enzyme is marked on the antigen/antibody, the antigen/antibody forms immune complex in the incubation process, then the excess antigen/antibody is washed away by the aid of a washing solution, and finally an oxidant or a substrate is added to determine the luminescent signal. The intensity of the signal is correlated with the concentration of the analyte.
AEP belongs to asparaginyl endopeptidase, and when the concentration of AEP in human body fluid is detected by adopting a chemiluminescence immune method, the existing chemiluminescence cleaning buffer solution cannot eliminate result deviation caused by nonspecific adsorption of other proteins, hemoglobin, bilirubin and the like.
Disclosure of Invention
Aiming at the problem that in the AEP detection process in the prior art, the adsorption of non-AEP protein in a complex sample and the interference of hemoglobin and bilirubin cannot be well eliminated, so that deviation is generated in the AEP detection, the invention provides the non-specific adsorption cleaning solution for the chemiluminescence immunoassay AEP, which can remove protein adhesion, solubilize and disperse endogenous fat-soluble substances in a sample, reduce the deviation of an analysis result, improve the detection accuracy, has an excellent cleaning effect on endogenous interferents, and ensures the accuracy of clinical sample detection to a certain extent.
The invention is realized by the following technical scheme:
the invention relates to a non-specific adsorption cleaning solution for AEP (aeP) of chemiluminescence immunoassay, wherein each liter of the cleaning solution comprises 10-100mMol/L buffer solution, 0.01-1 wt% of surfactant, 100-300mMol/L inorganic salt, 0.5-0.1g/L preservative and the balance of water.
The non-specific adsorption cleaning is as follows: the HLB value and the CMC value of the cleaning buffer solution are changed by utilizing different surfactant combinations under the existence conditions of specific buffer solution and ionic strength so as to achieve the purposes of removing nonspecific adsorption and adhesion and increasing the water solubility of nonpolar substances, thereby meeting the requirements on specificity and sensitivity in the AEP detection process.
The surfactant is an anionic surfactant, a cationic surfactant, an amphoteric surfactant, a nonionic surfactant or a combination thereof, wherein: the anionic surfactant comprises a sulfate salt type, the cationic surfactant comprises an alkyl quaternary ammonium salt, the amphoteric surfactant comprises a betaine type, and the nonionic surfactant comprises a polyol type surfactant and a polyoxyethylene type surfactant.
The buffer solution comprises tris, piperazine-1, 4-diethylsulfonic acid, 3-morpholinopropanesulfonic acid, phosphate buffer solution or combination, and has a pH value of 7.0-8.5, preferably 7.5 and 8.0.
The inorganic salt is sodium chloride or potassium chloride.
The preservative is Proclin300, Proclin 950 or sodium azide.
The cleaning solution for the chemiluminescence immunoassay AEP is preferably any one of the following solutions:
10-200mMol/L of trihydroxymethyl aminomethane, 0.01-1 wt% of mixed solution of polyoxyethylene dodecyl ether and 3-sulfopropyl tetradecyl betaine, 100mMol/L of sodium chloride and 0.5-0.1g/LProclin300, wherein: the polyoxyethylene dodecyl ether and the 3-sulfopropyl tetradecyl betaine are mixed according to the weight ratio of 4:1 to form the compound surfactant, the HLB value and the CMC value of the compound surfactant are favorable for protein dispersion, the surface tension of the liquid is reduced, and the fat solubility is enhanced, so that a better cleaning effect is achieved.
10-200mMol/L of trihydroxymethyl aminomethane, 0.01-1 wt% of fatty alcohol-polyoxyethylene ether sodium sulfate, 100-300mMol/L of sodium chloride and 0.5-0.1g/LProclin 300.
③ 10-200mMol/L of trihydroxymethyl aminomethane, 0.01-1 wt% of nonionic surfactant, 100-300mMol/L of sodium chloride and 0.5-0.1g/LProclin300, wherein: the nonionic surfactant is prepared by mixing NP 40, Tween20 and polyoxyethylene dodecyl ether in a weight ratio of 1:4:5 to form a compound surfactant; the HLB value and the CMC value of the compound surfactant are favorable for protein dispersion, the surface tension of the liquid is reduced, and the fat solubility is enhanced, so that a better cleaning effect is achieved.
Technical effects
The invention integrally solves the problems of endogenous interference and nonspecific reaction in the process of determining the AEP, which cannot be applied to a reagent for determining the AEP by a chemiluminescence immunoassay method in the prior art. According to the invention, by changing the components and concentration of the surfactant in the washing buffer solution, the surface hydrophilicity of the protein in the sample is changed, the adhesion and aggregation of the protein are removed, and meanwhile, the solubilizing and dispersing effects on fat-soluble substances such as triglyceride, bilirubin and the like are achieved, so that the surfactant is uniformly dispersed in the washing solution in the washing process, and the deviation of the detection result caused by the non-specific adsorption on the surface of heterogeneous particles (magnetic beads) is reduced, thereby improving the accuracy and precision of actual detection.
Detailed Description
The embodiment relates to a non-specific adsorption cleaning solution for chemiluminescence immunoassay AEP, which comprises the following components: 10-200mMol/L buffer solution, 0.01-1 wt% of surfactant, 100-300mMol/L inorganic salt, 0.5-0.1g/L preservative, and the balance of water.
The surfactant is any one or mixture of anionic surfactant, cationic surfactant, amphoteric surfactant or nonionic surfactant.
The anionic surfactant adopts a sulfate salt type and has a molecular general formula of ROSO3 -X+The number of carbon atoms of the hydrocarbon group R is 12 to 15 and X is Na+，K+，NH4 +The concrete formula of the compound comprises fatty alcohol polyoxyethylene ether sulfate (AES), polyoxyethylene alkyl ether sulfate ester salt and polyoxyethylene polycyclic phenyl ether sulfate ester salt.
The cationic surfactant adopts alkyl quaternary ammonium salt and has a molecular general formula of R4N + X-, wherein the total number of carbon atoms of the hydrocarbon radicals R is between 10 and 16, the 4 hydrocarbon radicals R may be the same or different, and X is a halide anion such as Br-, Cl-. Commercially available alkyl quaternary ammonium salts include dodecyltrimethylammonium bromide (alatin), dodecyltrimethylammonium bromide (Sigma), and tetradecyltrimethylammonium chloride (Sigma).
The amphoteric surfactant adopts betaine type, one is sulfobetaine, and the molecular general formula is RN + (CH)3)2CH2COO-, number of carbon atoms of hydrocarbon group RBetween 12 and 18, another is amine oxide betaine with a molecular formula of R4N + X → O, the number of carbon atoms of the hydrocarbon group R is 10 to 16. As commercially available sulfobetaines, there can be mentioned dodecylhydroxypropylsulfobetaine (Shanghai Shengxuan Biochemical Co., Ltd.), 3-sulfopropyltetradecylbetaine (Aladdin). As a commercially available product of amine oxide betaine, cocamidopropyl betaine (Shanghai-derived leaf Biotech Co., Ltd.) and the like can be mentioned.
The nonionic surfactant is polyalcohol surfactant such as span and Tween or polyoxyethylene surfactant such as fatty alcohol polyoxyethylene ether (AEO) and alkylphenol polyoxyethylene, wherein the molecular formula of AEO is RO (CH2CH2O)nH, R is a hydrocarbon group with 12-18 carbon atoms, and n (namely, the degree of polymerization) is 6-10. The alkylphenol polyoxyethylene adopts TX or OP series, and the polymerization degree is between 8 and 12. All the above nonionic surfactant hydrophilic-Lipophilic Balance (HLB) values are between 8 and 18. Examples of the products available on the market include span 20 (bio-engineering (shanghai) corporation). As commercial products of Tween, Tween20 (Solebao), Tween 40, Tween 60, and Tween 80 (as described above, manufactured by Biotechnology, Shanghai, Ltd.) can be cited. Commercially available fatty alcohol-polyoxyethylene ethers include fatty alcohol-polyoxyethylene ether 7(HLB value 12), fatty alcohol-polyoxyethylene ether 9(HLB value 13), fatty alcohol-polyoxyethylene ether 10(13.5), fatty alcohol-polyoxyethylene ether 15(14.5) (the above is liyilvsen), and the like. Commercially available alkylphenol ethoxylates include EMULGEN 105(HLB value 9.7), EMULGEN 106(HLB value 10.5), EMULGEN 108(HLB value 12.1), EMULGEN 109P (HLB value 13.6), distyrylphenol polyoxyethylene ether (HLB value 10.5) (japanese king, supra), triton X100 (west ribbon biotechnology (shanghai) limited), NP 40(Amresco), and the like.
The buffer solution adopts one or more of trihydroxymethyl aminomethane, piperazine-1, 4-diethylsulfonic acid, 3-morpholine propanesulfonic acid and phosphate buffer solution.
The inorganic salt adopts sodium chloride or potassium chloride, and the preservative is Proclin series or sodium azide, wherein: the Proclin series include Proclin300 and Proclin 950.
The preferred formula of the cleaning solution in the embodiment is as follows: 20mM of trihydroxymethyl aminomethane, 150mM of sodium chloride, 0.01-1 wt% of surfactant and 3001g/L of Proclin.
The components are mixed, the pH value is adjusted to 7.4-7.8, and the combination and the dosage are shown in table 1 according to different tables.
TABLE 1 cleaning solution of different combinations and dosages
In the embodiment, an FC-8 small-sized chemiluminescence immunoassay analyzer produced by Ningbo purple garden is adopted, a magnetic bead reagent, an enzyme-labeled antibody reagent, a cleaning solution, a substrate reagent and a sample to be detected are respectively filled into corresponding hole sites of a detection reagent card, quantitative samples are respectively taken, the enzyme-labeled antibody is added into the magnetic bead reagent hole sites and uniformly mixed, incubation is carried out at 37 ℃ for 5-10 min, and immunomagnetic bead particles after reaction are collected; respectively adding quantitative cleaning solution, mixing uniformly, collecting immunomagnetic beads (repeating for 2-3 times), suspending the cleaned immunomagnetic beads with substrate reagent, measuring the luminous signal value, and calculating the concentration value of the sample to be measured according to the relation between the luminous signal value obtained by measurement and the concentration of the sample.
Example 1
The following formulation was used in this example: 10-200mMol/L of trihydroxymethyl aminomethane; 0.01-1 wt% of mixed solution of polyoxyethylene dodecyl ether and 3-sulfopropyl tetradecyl betaine; 100-300mMol/L sodium chloride; 0.5-0.1g/LProclin 300; wherein: the weight ratio of the polyoxyethylene dodecyl ether to the 3-sulfopropyl tetradecyl betaine is 4: 1.
In the embodiment, the cleaning effect of the cleaning solution is verified: cleaning solutions were prepared according to group 4 of table 1, and were subjected to dispensing and sample testing. Whole blood samples of 10 healthy people were taken, one was washed, the other was a control wash (0.5% tween20 phosphate buffer), the reagents were the same except the wash, and the test was performed with troponin I as the item, and the luminescence intensity was observed and compared.
Sample number
Control group
Example 1
1
668
552
2
589
536
3
1056
573
4
814
610
5
730
442
6
621
648
7
884
646
8
557
332
9
619
605
10
863
647
In this example, the accuracy of the cleaning liquid is evaluated: cleaning solutions were prepared according to group 4 of table 1, and were subjected to dispensing and sample testing. Whole blood samples from 50 patients were taken for testing of AEP concentration.
Table 2 example 1 cleaning solution accuracy results
Name of item
Linear regression equation
Correlation coefficient
AEP
y＝1.218x+0.225
0.991
From experimental results, the correlation coefficient reaches above 0.975, which indicates that the cleaning effect is excellent.
In this example, the precision of the cleaning liquid is evaluated: cleaning solutions were prepared according to group 4 of table 1, and were subjected to dispensing and sample testing. Samples of low, medium, and high concentrations of AEP were taken and assayed 20 times repeatedly, comparing the coefficient of variation with control wash buffer.
Table 3 results on precision of cleaning solution in example 1
From the experimental results, the cleaning solution prepared according to this example has a coefficient of variation of less than 5% after repeated measurement for 20 times. The cleaning effect of the cleaning solution of the present embodiment is good.
In this example, evaluation of the stability of the cleaning liquid: cleaning solutions were prepared according to group 4 of table 1, and were subjected to dispensing and sample testing. Testing after placing for 0 day, 3 months, 6 months, 9 months, 12 months and 14 months at the temperature of 2-8 ℃. Troponin I was selected as an item, and the measurement was repeated 10 times using a sample having a concentration of 2ng/mL, and the coefficient of variation was observed, and the results are shown in Table 4.
Table 4 example 1 stability test results
Example 2
The following formulation was used in this example: 10-200mMol/L of trihydroxymethyl aminomethane; 0.01-1 wt% of sodium fatty alcohol-polyoxyethylene ether sulfate; 100-300mMol/L sodium chloride; 0.5-0.1g/L Proclin 300; wherein: the anionic surfactant adopts sodium fatty alcohol-polyoxyethylene ether sulfate.
In the embodiment, the cleaning effect of the cleaning solution is verified: the cleaning solution was prepared according to group 6 in table 1 and subjected to sub-packaging and sample testing. Whole blood samples of 10 healthy people were taken, one was washed, the other was a control wash (0.5% tween20 phosphate buffer), the reagents were the same except the wash, and the test was performed with troponin I as the item, and the luminescence intensity was observed and compared.
Sample number
Control
Example 2
1
668
760
2
589
633
3
1056
857
4
814
648
5
730
538
6
621
602
7
884
652
8
557
484
9
619
585
10
863
826
In this example, the accuracy of the cleaning liquid is evaluated:
cleaning solutions were prepared as set forth in table 1, group 6 of example 1, and were subjected to dispensing and sample testing. Whole blood samples from 50 patients were taken for test AEP and correlated against control wash buffer.
Table 6 example 2 cleaning solution accuracy results
Name of item
Linear regression equation
Correlation coefficient
AEP
y＝1.136x+0.127
0.988
From experimental results, the correlation coefficient reaches above 0.975, which indicates that the cleaning effect is excellent.
In this example, the precision of the cleaning liquid is evaluated: the cleaning solution was prepared according to group 6 in table 1 and subjected to sub-packaging and sample testing. Samples of low, medium, and high concentrations of AEP were taken and assayed 20 times repeatedly, comparing the coefficient of variation with control wash buffer.
Table 7 example 2 cleaning solution precision results
From the experimental results, the cleaning solution prepared in this example has a coefficient of variation of less than 5% after repeated measurements of low, medium and high concentration samples for 20 times. It is demonstrated that the washing liquid of this example has a good washing effect.
In this example, evaluation of the stability of the cleaning liquid: cleaning solutions were prepared according to table 1, group 6, and were subjected to dispensing and sample testing. Testing after placing for 0 day, 3 months, 6 months, 9 months, 12 months and 14 months at the temperature of 2-8 ℃. The AEP concentration of the sample was measured in duplicate for 10 times at 2ng/mL, and the coefficient of variation was observed, and the results are shown in Table 8.
Table 8 example 2 stability test results
Example 3
The following formulation was used in this example: 10-200mMol/L of trihydroxymethyl aminomethane; 0.01-1 wt% of a nonionic surfactant; 100-300mMol/L sodium chloride; 0.5-0.1g/L Proclin 300; wherein: the nonionic surfactant is a mixed solution of NP 40, Tween20 and polyoxyethylene dodecyl ether in a weight ratio of 1:4: 5.
In the embodiment, the cleaning effect of the cleaning solution is verified: a cleaning solution was prepared according to table 1, group 10, and split charged and tested for samples. Whole blood samples of 10 healthy people were taken, one was washed, the other was a control wash (0.5% tween20 phosphate buffer), the reagents were the same except the wash, and the test was performed with troponin I as the item, and the luminescence intensity was observed and compared.
In this example, the accuracy of the cleaning liquid is evaluated: a cleaning solution was prepared according to table 1, group 10, and split charged and tested for samples. Whole blood samples from 50 patients were taken for test AEP and correlated against control wash buffer. Samples of low, medium and high concentration of AEP were taken, and the assay was repeated 20 times to compare the coefficient of variation with the control reagent.
Table 9 example 3 cleaning solution accuracy results
Name of item
Linear regression equation
Correlation coefficient
AEP
y＝1.537x+0.241
0.987
From experimental results, the correlation coefficient reaches above 0.975, which indicates that the cleaning effect is excellent.
In this example, the precision of the cleaning liquid is evaluated: a cleaning solution was prepared according to table 1, group 10, and split charged and tested for samples. Samples of low, medium and high concentration of AEP were taken, and the assay was repeated 20 times to compare the coefficient of variation with the control reagent.
Table 10 example 3 cleaning solution precision results
From the experimental results, the cleaning solution prepared according to this example has a coefficient of variation of less than 5% after repeated measurements of low, medium and high concentration samples for 20 times. The cleaning solution prepared in this example is good in cleaning effect.
In this example, evaluation of the stability of the cleaning liquid: a cleaning solution was prepared according to table 1, group 10, and split charged and tested for samples. After being placed at 2-8 ℃ for 0 day, 3 months, 6 months, 9 months, 12 months and 14 months, the measurement was repeated 10 times with a sample having an AEP concentration of 2ng/mL, and the coefficient of variation was observed, and the results are shown in Table 11.
Table 11 example 1 stability test results
In summary, the whole blood samples from 10 healthy individuals washed in examples 1-3 all had luminescence intensities less than 1000, and had lower background luminescence effects than the control reagent.
From the experimental data, the CV values of the samples were all within 5% when the cleaning solutions prepared in examples 1-3 were stored at 2-8 ℃ for 14 months. From the reagent state, 3 kinds of cleaning solutions are clear and transparent, and no white flocculent precipitate appears, which shows that the 3 kinds of cleaning solutions have good stability and high precision.
The foregoing embodiments may be modified in many different ways by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.