A marker diluent

Abstract

The present invention relates to the field of in vitro diagnostic products, and in particular, provides a labeling diluent. The labeling diluent provided by the present invention comprises a buffer, a divalent metal ion compound, an inert protein, betaine and sucrose, which can effectively reduce background noise, improve the signal-to-noise ratio, and significantly improve the sensitivity and precision of NSE detection products. At the same time, the clinical relevance of NSE detection products is also improved.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic products, and in particular to a labeled diluent. Background Art

[0002] Chemiluminescence uses enzymes as labels, and the enzyme used is mostly horseradish peroxidase. The main reason is that the enzyme is cheap and the labeling reagent is relatively stable in storage, but the sensitivity of the horseradish peroxidase labeling reagent is lower than that of alkaline phosphatase. The reason why alkaline phosphatase is not widely used is that the luminescent substrate of alkaline phosphatase is a highly sensitive luminescent substrate. High sensitivity also means that the background value noise is also very large. How to improve the signal-to-noise ratio of the alkaline phosphatase system is a difficult problem in the application of the alkaline phosphatase system.

[0003] Alkaline phosphatase (ALP) is an enzyme widely found in animals, plants, and microorganisms. Alkaline phosphatase commonly used in clinical in vitro diagnosis is mainly obtained from calf intestinal mucosa and Escherichia coli. Both sources of enzymes can be purchased on the market, such as Roche, Sigma, etc. Alkaline phosphatase can catalyze the color development of a variety of substrates, so it is widely used in the clinical immunological in vitro diagnostic industry. Antigen-antibody reagents labeled with alkaline phosphatase are an important part of clinical immunological in vitro diagnostic kits. The principle of immunological in vitro diagnostic reagents is mainly through the specific binding of antigens and antibodies, and then the enzyme catalyzes the color development of substrates to achieve the effect of quantitative diagnosis, so it is crucial to ensure the activity and specificity of the antigen-antibody labeled with stable alkaline phosphatase.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first object of the present invention is to provide a labeled diluent.

[0006] The second object of the present invention is to provide the use of the above-mentioned labeling diluent, which can improve the signal-to-noise ratio of the alkaline phosphatase labeling system, improve the precision and clinical relevance.

[0007] The third object of the present invention is to provide a NSE detection reagent.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] A labeling diluent comprises a buffer, a divalent metal ion compound, an inert protein, betaine and sucrose.

[0010] Further, the divalent metal ion compound is a magnesium ion compound and a zinc ion compound;

[0011] Optionally, the magnesium ion compound is MgCl2 or MgSO4;

[0012] Optionally, the zinc ion compound is ZnCl2 or ZnSO4;

[0013] Optionally, the buffer comprises Tris, HEPES, MOPS, PB or MES;

[0014] Optionally, the pH of the buffer is 5.5-6.5;

[0015] Optionally, the inert protein includes 0.5 w / v%-2 w / v% BSA or casein.

[0016] Further, the final concentration of betaine in the labeled diluent is 0.01 w / v%-0.5 w / v%;

[0017] Optionally, the final concentration of betaine in the labeled diluent is 0.05 w / v%-0.3 w / v%;

[0018] Optionally, the final concentration of betaine in the labeled diluent is 0.05 w / v%-0.2 w / v%.

[0019] Further, the final concentration of ZnCl2 in the labeling diluent is 0.05mM-0.1mM, the final concentration of MgCl2 in the labeling diluent is 0.5mM-50mM, and the final concentration of the buffer in the labeling diluent is 25mM-75mM;

[0020] Optionally, the final concentration of MgCl2 in the labeling diluent is 1 mM-30 mM;

[0021] Optionally, the final concentration of MgCl2 in the labeling diluent is 1.5mM-25mM;

[0022] Optionally, the final concentration of MgCl2 in the labeling diluent is 2mM-20mM;

[0023] Optionally, the final concentration of sucrose in the labeled diluent is 0.5 w / v%-4 w / v%.

[0024] Furthermore, the labeling diluent further comprises glycerol;

[0025] Optionally, the final concentration of glycerol in the labeled diluent is 1 w / v%-10 w / v%;

[0026] Optionally, the final concentration of glycerol in the labeled diluent is 3 w / v%-7 w / v%.

[0027] Furthermore, the labeled diluent further comprises 100 mM-300 mM NaCl and 0.05 v / v%-0.2 v / v% preservative;

[0028] Optionally, preservatives include Proclin 300 or sodium azide.

[0029] Furthermore, the labeling diluent is an alkaline phosphatase labeling diluent.

[0030] Application of the above-mentioned labeled diluent in the NSE detection kit.

[0031] An NSE detection reagent comprises the above-mentioned labeled diluent.

[0032] Furthermore, the reagents also include biotin labeling molecules and alkaline phosphatase labeling molecules;

[0033] Labeling diluent is used to dilute biotin-labeled molecules and alkaline phosphatase-labeled molecules.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The labeling diluent provided by the present invention comprises a buffer, a divalent metal ion compound, an inert protein, betaine and sucrose. The labeling diluent can significantly improve the signal-to-noise ratio of the alkaline phosphatase detection system, expand the application range of the alkaline phosphatase system, and in particular, the NSE detection product prepared by the labeling diluent is significantly improved in thermal stability compared with the NSE detection product in the prior art, thereby extending the product shelf life.

[0036] Since the NSE detection reagent provided by the present invention adopts the labeled diluent provided by the present invention as the working solution, the background noise of the kit is reduced, the precision and sensitivity thereof are significantly improved, and the clinical correlation is good, thereby improving the detection accuracy of the kit and extending the shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 This is the clinical relevance result of the unoptimized magnesium ion formula in Test Example 3;

[0039] Figure 2 These are the clinically relevant results of the optimized magnesium ion formula in Experimental Example 3. DETAILED DESCRIPTION

[0040] The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0041] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0042] The invention provides a labeling diluent, which comprises a buffer, a divalent metal ion compound, an inert protein, betaine and sucrose.

[0043] The labeled diluent can significantly improve the signal-to-noise ratio of the alkaline phosphatase detection system and expand the scope of application of the alkaline phosphatase system. In particular, the NSE detection product prepared by the labeled diluent has significantly improved thermal stability compared to the NSE detection product in the prior art, and the product shelf life is extended. When optimizing the composition formula, the inventors effectively reduced the background noise and improved the signal-to-noise ratio through the combined action of components such as betaine and divalent metal ion compounds, which can significantly improve the sensitivity and precision of the NSE detection product. At the same time, the clinical relevance of the NSE detection product has also been improved.

[0044] It should be noted that, in the present invention, "v / v" refers to the milliliters of a substance in 100 ml of solution; "w / v" refers to the grams of a substance in every 100 ml of solution.

[0045] In a preferred embodiment, the divalent metal ion compound is a magnesium ion compound and a zinc ion compound, wherein the magnesium ion compound is preferably MgCl2, and the zinc ion compound is preferably ZnCl2. The final concentration of ZnCl2 in the labeling diluent is preferably 0.05mM-0.1mM, and the final concentration of MgCl2 in the labeling diluent is preferably 0.5mM-50mM, more preferably 1mM-30mM, more preferably 1.5mM-25mM, and more preferably 2mM-20mM. The concentration of ZnCl2 can be, but is not limited to, 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM or 0.1mM; the concentration of MgCl2 can be, but is not limited to, 0.5mM, 5mM, 10mM, 20mM, 30mM, 40mM or 50mM.

[0046] In a preferred embodiment, the buffer comprises Tris, HEPES, MOPS, PB or MES at a pH of 5.5-6.5, wherein the final concentration of the buffer in the labeled diluent is preferably 25 mM-75 mM. The concentration of the buffer may be, but is not limited to, 25 mM, 35 mM, 45 mM, 55 mM, 65 mM or 75 mM.

[0047] In a preferred embodiment, the inert protein includes 0.5 w / v%-2 w / v% BSA. The concentration of BSA can be, but is not limited to, 0.5 w / v%, 1 w / v%, 1.5 w / v% or 2 w / v%.

[0048] In a preferred embodiment, the final concentration of betaine in the labeled diluent is 0.01 w / v%-0.5 w / v%, preferably 0.05 w / v%-0.3 w / v%, and more preferably 0.05 w / v%-0.2 w / v%. The concentration of betaine can be, but is not limited to, 0.05 w / v%, 0.1 w / v%, 0.15 w / v% or 0.2 w / v%.

[0049] In a preferred embodiment, the final concentration of sucrose in the labeled diluent is 0.5 w / v%-4 w / v%. The sucrose concentration may be, but is not limited to, 0.5 w / v%, 1 w / v%, 2 w / v%, 3 w / v% or 4 w / v%.

[0050] In a preferred embodiment, the labeling diluent further comprises glycerol, and the final concentration of glycerol in the labeling diluent is preferably 1 w / v%-10 w / v%, and more preferably 3 w / v%-7 w / v%. The concentration of glycerol can be, but is not limited to, 1 v / v%, 3 v / v%, 4 v / v%, 5 v / v%, 6 v / v%, 7 v / v%, 9 v / v% or 10 v / v%.

[0051] In a preferred embodiment, the labeled diluent further comprises 100mM-300mM NaCl and 0.05v / v%-0.2v / v% preservative, wherein the preservative may be Proclin300 or sodium azide. The concentration of NaCl may be, but is not limited to, 100mM, 130mM, 150mM, 170mM, 200mM, 230mM, 250mM, 270mM or 300mM.

[0052] The labeling diluent provided by the present invention can be used as an alkaline phosphatase labeling diluent.

[0053] The present invention also provides application of the above-mentioned labeled diluent in an NSE detection kit.

[0054] A NSE detection reagent contains the above-mentioned labeling diluent of the present invention. Specifically, the reagent also includes biotin labeling molecules and alkaline phosphatase labeling molecules; the labeling diluent is used to dilute the biotin labeling molecules and alkaline phosphatase labeling molecules.

[0055] Since the labeled diluent provided by the present invention is used as the working solution, the background noise of the reagent is reduced, the precision and sensitivity thereof are significantly improved, and the clinical relevance is good, thereby improving the detection accuracy of the reagent and extending the validity period.

[0056] In a preferred embodiment, the reagent uses a double antibody sandwich method to detect NSE, including a biotin-labeled antibody and an alkaline phosphatase-labeled antibody, wherein both the antibody in the biotin-labeled antibody and the antibody in the alkaline phosphatase-labeled antibody can bind to NSE, but the binding sites are different. The labeling diluent is used to dilute the biotin-labeled antibody and the alkaline phosphatase-labeled antibody.

[0057] In some embodiments, the kit is implemented as follows: a biotin-labeled antibody reacts with NSE in a sample to be detected, and then combines with avidin-coated magnetic beads through a biotin and avidin reaction system, and then an alkaline phosphatase-labeled antibody is used to achieve a double antibody sandwich, and finally an alkaline phosphatase substrate is added to achieve the detection of NSE.

[0058] The substrate of alkaline phosphatase can be CDP-star, AMPPD, CSPD or ADP-Star, etc. In order to improve the sensitivity of the test kit, the substrate of general alkaline phosphatase can select high-sensitivity luminescent substrates such as CDP-star, however, this type of substrate has high luminous efficiency and is also sensitive to background noise. When conventional marker diluents in the prior art are used for this type of substrate, the detection background noise of the product is high, and the interference to sensitivity and precision is relatively large. The marker diluent provided by the present invention is particularly suitable for the detection system of high-sensitivity luminescent substrates, which effectively improves the above problems, improves the overall performance of the test kit, and the product validity period is also improved.

[0059] Methods not described in detail in the specific implementation manner may refer to conventional methods in the art or manufacturer's instructions; reagents not described in the specific implementation manner may be obtained through commercial channels.

[0060] In the present invention, the description of the concentration unit "mM" is expressed as "mmol / L", that is, for example, 10mM is expressed as 10mmol / L, and the description of the concentration unit "M" is expressed as "mol / L", that is, for example, 10M is expressed as 10mol / L.

[0061] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0062] Example 1 Preparation of the Optimized Labeling Dilution of the Present Invention

[0063] According to the conventional preparation method of the reagent, prepare the following dilution solutions of different concentrations:

[0064]

[0065] Example 2 Preparation of biotin-labeled NSE antibody:

[0066] a) 2 mg of NSE monoclonal antibody was dialyzed into 20 mM pH 7.4 PB diluent at 4 °C for 2 hours, with the solution changed every 30 minutes; 10 mmol / L activated biotin (biotin for short) was added, and the reaction was rotated at room temperature for 1 hour; the monoclonal antibody was dialyzed into 20 mM pH 7.4 PB diluent again at 4 °C for 2 hours, with the solution changed every 30 minutes;

[0067] Purify the conjugate: Use size exclusion chromatography to purify the conjugate and measure the concentration

[0068] b) Prepare biotin marker working solution

[0069] Use the diluent in Example 1 to dilute to 1-5 μg / ml to prepare a biotin labeling reagent working solution, store at 4° C., and use directly.

[0070] Example 3 Preparation of alkaline phosphatase labeled NSE antibody:

[0071] a) Preparation of alkaline phosphatase (ALP) marker

[0072] Take 5 mg of NSE antibody, concentrate to 5 mg / ml, add 10 μl of 13.76 mg / ml activator 2-Iminothiolane·HCl (2IT) solution, leave at room temperature for 20 minutes, add glycine to terminate the activation reaction, leave at room temperature for 5 minutes. Use Sephadex G25 to desalt and collect the target protein.

[0073] Take 5mg ALP solution, add 50μl 6.69mg / ml Succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution, leave at room temperature for 30 minutes, add glycine to terminate the reaction, and leave at room temperature for 5 minutes. Use Sephadex G25 to desalt and collect the target protein. Mix the activated NSE antibody with the activated ALP, leave at room temperature for 10 hours, then use Supperdex200 gel chromatography column to separate and purify, remove the unconnected free NSE antibody and ALP, and store the connection at 4°C.

[0074] b) Prepare alkaline phosphatase marker working solution

[0075] The diluent in Example 1 was used to prepare an alkaline phosphatase marker working solution at a concentration of 1-5 μg / ml, which was stored at 4° C. and used directly.

[0076] Comparative Example 1

[0077] This comparative example provides the biotin marker dilution solution and the alkaline phosphatase marker dilution solution as follows (P1):

[0078] PBS 50mM Valine 2w / v% sucrose 2w / v% <![CDATA[ZnCl2]]> 0.1mM BSA 1w / v% <![CDATA[MgCl2]]> 1mM TweenX-100 0.05v / v% ProClin-300 0.1v / v% pH 7.4

[0079] Comparative Example 2

[0080] This comparative example provides the biotin marker dilution solution and the alkaline phosphatase marker dilution solution as follows (P2):

[0081] MES 50mM NaCl 150mM Betaine 0.2w / v% BSA 1w / v% <![CDATA[MgCl2]]> 100mM <![CDATA[CaCl2]]> 0.05mM glycerin 5v / v% ProClin-300 0.1v / v% pH 6

[0082] Test Example 1 Signal-to-Noise Ratio

[0083] The diluent in Example 1 was used as the experimental group, and the diluents in Comparative Examples 1 and 2 replaced the diluent in Example 1 as the control group, and the NSE standard was used as the test sample for detection.

[0084] Experimental process: Add 20μl 0ng / ml or 0.1ng / ml sample to the reaction cup, then add 50μl biotin-labeled antibody, incubate at 42℃ for 2.5mins, then add 30μl avidin-coated magnetic beads and incubate at 42℃ for 2mins. Magnetic separation, wash with TBST. Then add 50μl alkaline phosphatase-labeled antibody and incubate at 42℃ for 3.5mins. Magnetic separation, wash with TBST, add 100μl CDP-star substrate and mix, incubate at 42℃ for 5mins. Read using chemiluminescence analyzer

[0085] The results are shown in the following table. The diluent in Example 1 effectively reduced the noise background and improved the signal-to-noise ratio:

[0086]

[0087]

[0088] Test Example 2 Precision

[0089] The diluent in Example 1 was used as the experimental group, and the diluents in Comparative Examples 1 and 2 replaced the diluent in Example 1 as the control group for testing.

[0090] Experimental plan: Measure the calibration curve, repeat each calibration point 3 times, take the average luminescence value of the calibration point for calibration. Repeat the low, medium and high value NSE quality control products 10 times respectively, measure their concentration values, and calculate CV.

[0091] Experimental process: Add 20μl low, medium or high value NSE quality control to the reaction cup, then add 50μl biotin-labeled antibody, incubate at 42℃ for 2.5mins, then add 30μl avidin-coated magnetic beads and incubate at 42℃ for 2mins. Magnetic separation, wash with TBST. Then add 50μl alkaline phosphatase-labeled antibody and incubate at 42℃ for 3.5mins. Magnetic separation, wash with TBST, add 100μl CDP-star substrate and mix, incubate at 42℃ for 5mins. Read the results using a chemiluminescence analyzer and calculate the measured concentration value.

[0092] The results are shown in the following table. The diluent in Example 1 significantly improves the low-value detection precision while ensuring the detection precision of medium and high values:

[0093]

[0094] At the same time, the background noise (background) was measured 20 times repeatedly, and the CV of the luminescence value was calculated. The results are as follows. It can be seen that the kit containing Example 2 and Example 3 effectively reduces the background and improves product performance:

[0095]

[0096] In addition, the experimental group and the control group were subjected to accelerated stability test at 52°C. Precipitation appeared in the S1 and S5 groups after 3 days, while no precipitation appeared in the S2-S4 and S6 groups, which were clear and transparent.

[0097] Test Example 3 Betaine Optimization Experiment

[0098] Prepare betaine marker dilutions of different concentrations according to the following formula:

[0099]

[0100] The effect of different concentrations of betaine on the signal-to-noise ratio was evaluated according to the method of Experimental Example 1. The results are as follows:

[0101] 0ng / ml 0.1ng / ml Signal-to-Noise Ratio B1 3215 4967 1.54 B2 2746 4781 1.74 B3 2067 5169 2.5 B4 2135 4816 2.26 B5 2279 4597 2.02 B6 2615 4196 1.6 B7 2861 4026 1.41

[0102] Test Example 4 MgCl2 Optimization Experiment

[0103] Prepare MgCl2 labeling dilutions of different concentrations according to the following formula:

[0104]

[0105]

[0106] The effect of different concentrations of MgCl2 on precision was evaluated according to the method of Experimental Example 2. The results are as follows:

[0107] Low value quality control Q1 Median quality control Q2 High value quality control Q3 M1 3.84％ 3.19％ 2.94％ M2 3.64％ 2.85％ 2.51％ M3 3.07％ 2.63％ 2.01％ M4 2.51％ 2.59％ 1.83％ M5 2.35％ 2.21％ 1.63％ M6 2.32％ 2.27％ 1.53％ M7 2.43％ 2.32％ 1.73％ M8 2.86％ 2.49％ 1.79％ M9 2.81％ 2.72％ 1.83％ M10 3.16％ 2.83％ 2.51％

[0108] Test Example 5 Clinical Relevance

[0109] Under the premise of ensuring sensitivity and precision, the clinical relevance was tested. Linear regression analysis was performed with the Elecsys NSE value assigned by the Roche Neuron-Specific Enolase Assay Kit (Electrochemiluminescence) as the X-axis and the optimized value of Experimental Example 4 as the Y-axis.

[0110] Clinically relevant results of non-optimized magnesium formulations are Figure 1 As shown, the clinical relevance of optimizing magnesium ions is as follows Figure 2 As shown, it can be seen that the kit provided by the present invention can significantly improve the clinical relevance of NSE detection.

[0111] Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, it is intended to include all such changes and modifications within the scope of the present invention in the appended claims.

Claims

1. An alkaline phosphatase labeling diluent, characterized in that: including a buffer, a divalent metal ion compound, an inert protein, betaine, sucrose and glycerol; The divalent metal ion compounds are magnesium ion compounds and zinc ion compounds; the final concentration of betaine in the labeled diluent is 0.01 w / v%-0.5 w / v%; the final concentration of glycerol in the labeled diluent is 3 w / v%-7 w / v%.

2. The alkaline phosphatase labeled diluent according to claim 1, characterized in that The magnesium ion compound is MgCl2 or MgSO4.

3. The alkaline phosphatase labeled diluent according to claim 1, characterized in that: The zinc ion compound is ZnCl2 or ZnSO4.

4. The alkaline phosphatase labeled diluent according to claim 1, characterized in that: The buffer comprises Tris, HEPES, MOPS, PB or MES, and the pH of the buffer is 5.5-6.

5.

5. The alkaline phosphatase labeled diluent according to claim 1, characterized in that: The inert protein includes BSA or casein.

6. The alkaline phosphatase labeled diluent according to claim 1, characterized in that The concentration of BSA in the alkaline phosphatase labeling diluent is 0.5 w / v%-2 w / v%, and the concentration of casein is 0.5 w / v%-1 w / v%.

7. The alkaline phosphatase labeled diluent according to any one of claims 1 to 6, characterized in that: The final concentration of betaine in the labeled diluent is 0.05 w / v%-0.3 w / v%.

8. The alkaline phosphatase labeled diluent according to claim 7, characterized in that The final concentration of betaine in the labeled diluent is 0.05 w / v%-0.2 w / v%.

9. The alkaline phosphatase labeled diluent according to claim 8, characterized in that The final concentration of sucrose in the labeled diluent is 0.5 w / v%-4 w / v%.

10. The alkaline phosphatase labeled diluent according to claim 9, characterized in that: The final concentration of ZnCl2 in the labeling diluent is 0.05 mM-0.1 mM, and the final concentration of MgCl2 in the labeling diluent is 0.5 mM-50 mM.

11. The alkaline phosphatase labeled diluent according to claim 10, characterized in that The final concentration of MgCl2 in the labeling diluent is 1 mM-30 mM.

12. The alkaline phosphatase labeled diluent according to claim 11, characterized in that The final concentration of MgCl2 in the labeling diluent is 1.5 mM -25 mM.

13. The alkaline phosphatase labeled diluent according to claim 12, characterized in that The final concentration of MgCl2 in the labeling diluent is 2 mM-20 mM.

14. The alkaline phosphatase labeled diluent according to claim 13, characterized in that The final concentration of the buffer in the labeled diluent is 25 mM-75 mM.

15. The alkaline phosphatase labeled diluent according to claim 14, characterized in that The labeled diluent further comprises 100 mM-300 mM NaCl and 0.05 v / v%-0.2 v / v% preservative.

16. The alkaline phosphatase labeled diluent according to claim 15, characterized in that: Preservatives include Proclin 300 or sodium azide.

17. Use of the alkaline phosphatase labeled diluent according to any one of claims 1 to 16 in a NSE detection kit.

18. A NSE detection reagent, characterized in that: The invention comprises the alkaline phosphatase labeling dilution solution as described in any one of claims 1 to 16.

19. The reagent according to claim 18, characterized in that The reagents also include a biotin-labeled molecule and an alkaline phosphatase-labeled molecule; Labeling diluent is used to dilute biotin-labeled molecules and alkaline phosphatase-labeled molecules.

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