Mercaptopyridine derivative and application thereof in hemolytic agent

Abstract

The invention discloses a compound applied to a hemolytic agent and a composition thereof, which are characterized in that a compound which has weak reducibility and can be combined with hemachrome oxygenase to limit enzymatic activity is introduced into a formula of the hemolytic agent, so that the combination ratio of saponin and hemachrome oxygenase is reduced; according to the present invention, the dependence degree of the heme-biliverdin-bilirubin oxidation reduction system on the saponin is limited, the compatibility of the hemolytic agent formula on different batches of commercial saponin products is improved, and the weak reducibility of the compound avoids the oxidation problem of the saponin on the premise of no influence on the disulfide bond of the antibody, such that the heme-biliverdin-bilirubin oxidation reduction system can be easily obtained. Finally, the effects of reducing batch-to-batch clinical deviation of the hemolytic agent and improving the long-term stability of the hemolytic agent are achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical tests, and specifically relates to mercaptopyridine derivatives and their uses in hemolyzing agents. Background Art

[0002] A hemolyzing agent is a reagent used in the analysis by an automatic blood cell analyzer. In the detection reaction, the main functions of the hemolyzing agent include: ① dissolving red blood cells to reduce the interference of red blood cells on the substance to be measured; ② providing a suitable pH and ionic strength for the quantitative detection of the substance to be measured. Currently, most hemolyzing agents on the market use saponins as the main raw material. Compared with surfactants, saponins not only have hemolytic efficacy but also can effectively reduce the influence of the content of red blood cells on the reaction system. Saponins are composed of sapogenins and sugars. According to different sapogenins, they can be divided into steroidal saponins and triterpenoid saponins. Currently, common commercial saponins on the market are all produced by extracting from natural plants. Their essence is a mixture of various saponins, and the hemolytic abilities of saponins with different structures vary. At the same time, due to the difference in the mixing ratio of different types of saponins in different batches of commercial saponins, the hemolytic abilities of saponin products in different batches vary greatly, which will interfere with the test results. Therefore, in the actual use process, it is necessary to verify commercial saponins batch by batch separately.

[0003] At the same time, saponins in the hemolyzing agent may also be oxidized during long-term storage. The possible changes in the structure of oxidized saponins are: ① the hydroxyl groups in the saponin molecule are oxidized to aldehyde groups or carbonyl groups, and the strength of the hemolytic performance of saponins mainly depends on the number of hydroxyl groups at some special positions on the sapogenin. After a large number of hydroxyl groups are oxidized, the hemolytic ability of saponins will be significantly weakened; ② the oxidized saponin molecule further undergoes a reaction to generate hydroxyl free radicals, producing reactive oxygen free radicals, such as peroxy free radicals (ROO - ) or hydroxyl free radicals (-OH), and these reactive oxygen free radicals will react with other surrounding molecules, leading to a series of complex oxidation reactions, ultimately affecting the structure of the saponin molecule. And the change in the molecular structure may lead to a change in the hemolytic performance of saponins, ultimately affecting the detection performance of the reagent.

[0004] At present, in order to solve the impact of saponin oxidation on clinical tests, reducing agents such as tris(2-carboxyethyl)phosphine hydrochloride (Tecp), mercaptoethanol, mercapto glycerol, dithiothreitol (DTT), ascorbic acid, sodium dodecyl sulfate (SDS), urea, etc. are often added to hemolysins on the market. However, reducing agents have certain negative impacts on antibodies in the assay system. They can change the disulfide bonds of antibodies, thereby affecting the conformation and function of antibodies. At the same time, some reducing agents themselves also have certain stability problems. Therefore, when using reducing agents to solve the problem of saponin oxidation, a large amount of time and effort are required to screen the specific types, component combinations, and usage concentrations of the reducing agents used, and the batch-to-batch difference of saponin raw materials cannot be solved. Clinically, it is urgent to improve the stability of the hemolysin formula and its compatibility with saponin raw materials of different batches. Summary of the Invention

[0005] To solve the above problems, the present invention introduces special compounds into the hemolysin formula, improves the compatibility of the hemolysin formula with different batches of commercial saponin products, and the chemical raw material has weak reducibility, which can avoid the oxidation problem of saponin without affecting the disulfide bonds of antibodies, and finally achieves the purpose of reducing the clinical batch-to-batch difference of hemolysins and improving the long-term storage stability.

[0006] To achieve the above object, the present invention adopts the following technical means: a compound having the structure of general formula I:

[0007] (Formula I)

[0008] Wherein

[0009]

[0010] R1 and R2 are each independently selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, mercapto, halogen, carboxyl, ester, trifluoromethyl;

[0011] R3 is selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, mercapto, halogen, trifluoromethyl, sulfonic acid group, polyoxyethylene group;

[0012] R4 is selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, halogen, ester.

[0013] Preferably, R1 is selected from hydrogen, C 1-18 alkyl.

[0014] Preferably, R2 is selected from hydrogen, C 1-18 alkyl, halogen, mercapto, ester, carboxyl.

[0015] Preferably, R3 is selected from hydrogen, halogen, polyoxyethylene group, trifluoromethyl group, and sulfonic acid group.

[0016] Preferably, R4 is selected from hydrogen, C 1-18 alkyl.

[0017] Preferably, the compound is:

[0018]

[0019] A saponin protectant, the protectant comprising the above compound.

[0020] Preferably, the saponin protectant further comprises sodium nitrite, CHAPS, and a reducing agent.

[0021] Preferably, the reducing agent can be selected from at least one of reduced glutathione, mercaptoethanol, dithiothreitol, and TCEP.

[0022] A biological sample lysing agent, the lysing agent comprising saponin and the above compound, or the above saponin protectant.

[0023] Preferably, the lysing agent further comprises a buffer, a preservative, and an ionic strength regulator.

[0024] Preferably, the buffer is selected from at least one of MOPSO buffer, boric acid buffer, MOPS buffer, and PIPES buffer; the preservative is selected from sodium azide, PC300, and BND-10; the ionic strength regulator is selected from at least one of sodium chloride and potassium chloride.

[0025] Use of the above compound, saponin protectant or lysing agent in cell lysis of biological samples.

[0026] The beneficial effects of the present invention are as follows: The lysing agent for blood cell analysis of the present invention is an innovation made on a relatively mature process. While retaining the advantages of saponin as a lysing component, by introducing a saponin protectant containing special components into the reagent, the compatibility of the lysing agent with the batch-to-batch difference of commercial saponin products is improved, and the technical problem of saponin oxidation is overcome, significantly improving the stability of the lysing agent for blood cell analysis. Moreover, the batch-to-batch difference of the introduced new components is controllable, the repeatability is good, and it is easy to scale up, having good clinical application value. Description of the Drawings Specific Embodiments

[0027] The following embodiments are provided to better further understand the present invention. They are not limited to the best mode described, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0028] For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The methods and materials described herein, as well as any methods and materials similar or equivalent thereto, can be used in the practice or testing of the formulations or unit doses herein. Unless otherwise noted, the techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not restrictive.

[0030] As used herein, the terms "comprising", "including", "having", "may", and their variants are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures.

[0031] Definition

[0032] Unless otherwise specified, the terms used herein have the following meanings.

[0033] When defining multiple terms, the "A1", "A2" used herein are general symbols representing various specific substituents. These symbols can be any substituents, not limited to those disclosed herein, and when they are defined as certain substituents in one case, they can be defined as other substituents in other cases.

[0034] The term "alkyl" used herein, whether used alone or in combination with other groups, refers to straight-chain alkyls and branched-chain alkyls containing 1 - 18, preferably 1 - 12, more preferably 1 - 8, and most preferably 1 - 6 carbon atoms. For example, a single alkyl such as "n-propyl" specifically refers only to a straight-chain alkyl, and when referring to a single branched-chain alkyl such as "isopropyl", it specifically refers only to a branched-chain alkyl. For example, "C1-6 alkyl" includes C1-4 alkyl, C1-3 alkyl, methyl, ethyl, n-propyl, isopropyl, and tert-butyl, etc. Similar rules apply to other groups used in this specification.

[0035] As used herein, the term "alkoxy" refers to an alkyl or cycloalkyl group linked by an ether bond; that is to say, "alkoxy" can be defined as -OA1, where A1 is an alkyl or cycloalkyl group as defined above. "Alkoxy" also includes polymers of the alkoxy groups just defined; that is to say, alkoxy can be a polyether, such as -OA1-OA2 or -OA1-(OA2)a-OA3, where "a" is an integer from 1 to 200, and A1, A2 and A3 are alkyl and / or cycloalkyl groups.

[0036] As used herein, the term "mercapto" is represented by the formula -SH.

[0037] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0038] As used herein, the term "carboxyl" is represented by the formula -COOH.

[0039] As used herein, the term "ester group" is represented by the formula -OC(O)A 1 or -C(O)OA 1 where A 1 can be a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein.

[0040] As used herein, the term "trifluoromethyl" is represented by the formula -CF3.

[0041] As used herein, the term "sulfonic acid group" is represented by the formula -SO3H.

[0042] As used herein, the term "polyoxyethylene group" is represented by the formula -(CH2CH2O) n where n can be any integer from 1 to 20, specifically, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0043] As used herein, the term "alkenyl" is a hydrocarbon group having 2 to 24 carbon atoms and containing at least one carbon-carbon double bond in its structural formula.

[0044] As used herein, the term "cycloalkenyl" is a non-aromatic carbocyclic ring composed of at least three carbon atoms and containing at least one carbon-carbon double bond, i.e., C═C. Examples of cycloalkenyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, etc.

[0045] As used herein, the term "alkynyl" is a hydrocarbon group having 2 to 24 carbon atoms and containing at least one carbon-carbon triple bond in its structural formula.

[0046] As used herein, the term "cycloalkynyl" refers to a non-aromatic carbocyclic ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bond. Examples of cycloalkynyl include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, etc.

[0047] As used herein, the term "aryl" refers to a group containing any carbon aromatic group, including but not limited to benzene, naphthalene, phenyl, biphenyl, phenoxyphenyl, etc.

[0048] The term "aryl" also includes "heteroaryl", which is defined as a group in which the aromatic ring of the aromatic group contains at least one heteroatom. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus.

[0049] Compounds of the present invention

[0050] One aspect of the present invention provides a compound having the structure of general formula I:

[0051] (Formula I)

[0052] Wherein

[0053] R1 and R2 are each independently selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, mercapto, halogen, carboxyl, trifluoromethyl, ester group;

[0054] R3 is selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, mercapto, halogen, trifluoromethyl, sulfonic acid group, polyoxyethylene group;

[0055] R4 is selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, halogen, ester group.

[0056] Preferably, R1 is selected from hydrogen, C 1-18 alkyl.

[0057] Preferably, R2 is selected from hydrogen, C 1-18 alkyl, halogen, mercapto, ester group, carboxyl.

[0058]

[0059] Preferably, R3 is selected from hydrogen, C 1-18 alkyl, halogen, trifluoromethyl, sulfonic acid group, polyoxyethylene group.

[0060] Preferably, R4 is selected from hydrogen, C 1-18 alkyl.

[0061] Preferably, the compound of the present invention is:

[0062]

[0063]

[0064] Saponin protectant of the present invention

[0065] One embodiment of the present invention further provides a composition comprising the above-mentioned compound of formula I, which is used as a protective agent for saponins in a hemolytic agent for biological samples. When used together with saponins in the hemolytic agent, it can effectively improve the performance of the hemolytic agent and optimize the detection performance of subsequent detection reagents.

[0066] In addition to the compound of formula I or its conjugate, the saponin protective agent of the present invention may further include sodium nitrite, CHAPS, and a reducing agent. Sodium nitrite and CHAPS are used as hemoglobin protective agents. The former forms nitrosohemoglobin by binding to hemoglobin to slow down the degradation of hemoglobin, and the latter, as a zwitterionic surfactant, forms a film on the surface of hemoglobin and nitrosohemoglobin to prevent damage to hemoglobin by metal ions and the like in the detection system, and can reduce the occurrence of oxidation reactions. The reducing agent can fully react with the oxidant in the system to reduce the damage of the oxidant in the system to hemoglobin. The composition of the present invention may exist in the form of an aqueous solution or in other suitable forms that can be formulated into a solution with water before use.

[0067] Specifically, the reducing agent may be selected from at least one of reduced glutathione, mercaptoethanol, dithiothreitol, TCEP, and 2-mercaptopyridine.

[0068] More specifically, the preferred concentration ranges of the components in the composition are as follows:

[0069]

[0070]

[0071] Hemolytic agent of the present invention

[0072] One embodiment of the present invention further provides a hemolytic agent comprising the above-mentioned compound of formula I or its conjugate and the above-mentioned saponin protective agent, which is mainly used for lysing blood cells in biological samples.

[0073] In addition to the above-mentioned compound of formula I or its conjugate and the above-mentioned saponin protective agent, the hemolytic agent of the present invention may further include other components required for dissolving blood cells in biological samples, such as buffers, preservatives, ionic strength regulators, etc. The hemolytic agent of the present invention may exist in the form of an aqueous solution or in other suitable forms that can be formulated into a solution with water before use.

[0074] Specifically, the buffer can be selected from at least one of MOPSO buffer, boric acid buffer, MOPS buffer, and PIPES buffer, the preservative can be selected from at least one of sodium azide, PC300, and BND-10, and the ionic strength regulator can be selected from at least one of sodium chloride and potassium chloride.

[0075] More specifically, the preferred concentration ranges of the components in the hemolytic agent are as follows:

[0076] Component Concentration Buffer 0.1％-3％ Preservative 0.1％-1％ Ionic strength regulator 0.5％-1.5％ Saponin 0.1％-1％ Sodium nitrite 0.0007％-0.042％ CHAPS 0.003％-0.018％ 2-Mercaptopyridine 0.0002％-0.3％ Compound 0.001％-0.045％

[0077] Use of the compounds, saponin protectant or hemolytic agent of the present invention

[0078] One embodiment of the present invention also provides the use of the above-mentioned compound of formula I or its conjugate, the above-mentioned saponin protectant, or the above-mentioned hemolytic agent in lysing blood cells in a biological sample.

[0079] The features and properties of the present invention will be further described in detail below with reference to specific examples.

[0080] Example 1 Preparation of Compounds

[0081] (1) Preparation of Compound a

[0082] The structural formula of Compound a is as follows:

[0083]

[0084] For its specific synthesis route, refer to US2003 / 055090, 2003, A1.

[0085] (2) Preparation of Compound b

[0086] The structural formula of Compound b is as follows:

[0087]

[0088] For its specific synthesis route, refer to US2003 / 055090, 2003, A1.

[0089] (3) Preparation of Compound c

[0090] The structural formula of Compound c is as follows:

[0091]

[0092] For its specific synthesis route, refer to US2003 / 055090, 2003, A1.

[0093] (4) Preparation of Compound d

[0094] The structural formula of Compound d is as follows:

[0095]

[0096] For its specific synthesis route, refer to US2003 / 055090, 2003, A1.

[0097] (5) Preparation of Compound e

[0098] The structural formula of Compound e is as follows:

[0099]

[0100] For its specific synthesis route, refer to [Journal of the American Chemical Society, 1953, vol. 75, p. 1933, 1934.

[0101] (6) Preparation of Compound f

[0102] The structural formula of Compound f is as follows:

[0103]

[0104] For its specific synthesis route, refer to [Journal of the American Chemical Society, 1953, vol. 75, p. 1933, 1934.

[0105] (7) Preparation of Compound g

[0106] The structural formula of Compound g is as follows:

[0107]

[0108] For its specific synthesis route, refer to Polish Journal of Chemistry, 1991, vol. 65, #2 - 3, p. 377 - 380.

[0109] (8) Preparation of Compound h

[0110] The structural formula of Compound h is as follows:

[0111]

[0112] For its specific synthesis route, refer to US2003 / 055090, 2003, A1.

[0113] (9) Preparation of Compound i

[0114] The structure of Compound i is as follows:

[0115]

[0116] For its specific synthesis route, refer to US2003 / 055090, 2003, A1.

[0117] (10) Preparation of Compound j

[0118] The structural formula of Compound j is as follows:

[0119]

[0120] For its specific synthesis route, refer to US2003 / 055090, 2003, A1.

[0121] (11) Preparation of Compound k

[0122] The structural formula of Compound k is as follows:

[0123]

[0124] For its specific synthesis route, refer to JP2006 / 240999, 2006, A.

[0125] (12) Preparation of Compound l

[0126] The structural formula of Compound l is as follows:

[0127]

[0128] For its specific synthesis route, refer to US2007 / 219276, 2007, A1.

[0129] Preparation of Hemolytic Agent in Example 2

[0130] To verify the effects of the above 12 compounds on the performance of the hemolytic agent, the following 12 groups of tests were set up:

[0131] Prepare the hemolytic agent according to the following method:

[0132] Table 1

[0133] Component Concentration MOPSO buffer 0.8％ Sodium azide 0.9％ Sodium chloride 0.9％ Saponin 0.25％ Compound 0.03％ Sodium nitrite 0.002％ CHAPS 0.004％ 2-Mercaptopyridine 0.0014％

[0134] Among the 12 groups of tests, except for the presence or absence and selection of different compounds, the other components are exactly the same. The specific grouping is shown in the following table:

[0135] Table 2

[0136] A B C D E F G H I J K L M Compound a b c d e f g h i j k l None

[0137] * Among them, each group of hemolytic agents uses the corresponding compound. For example, the hemolytic agent in Group A uses Compound a, and the hemolytic agent in Group M does not add a compound.

[0138] Verification of Hemolytic Agent Performance in Example 3

[0139] To verify the performance differences among the 13 groups of hemolysins in Example 2, the commercially available hemolysin (Mindray LC hemolysin) was set as group N for testing, and the standard batch of hemolysin was set as group O for testing. And the following tests including reactivity test, clinical deviation and long-term stability test were carried out for the 15 groups of hemolysins.

[0140] (1) Verification of C-reactive protein test reactivity

[0141] The standard batch of hemolysin was used in combination with Zhongyuan Huiji C-reactive protein detection reagent (nephelometry) for value determination. Samples with CRP concentrations of 10±2 mg / L, 20±5 mg / L, and 40±10 mg / L were selected. For each concentration range, 3 samples with HCT distributed in the range of 40%-60% were selected as the samples to be tested. The 13 groups of hemolysins in Example 2 were respectively used in combination with Zhongyuan Huiji C-reactive protein detection reagent (nephelometry) and Zhongyuan Huiji specific protein composite calibration product to establish a working curve, and the samples to be tested were tested. Each sample was tested three times repeatedly, and the average reactivity of the three tests was taken as the true reactivity of the sample (the results are shown in Table 3); taking the reactivity of the standard batch of hemolysin as the control, the relative deviation of the clinical test reactivity of the 13 groups of hemolysins in Example 2 was calculated (the results are shown in Table 4). (Both the Mindray instrument and the Zhongyuan Huiji instrument used in the test are closed instruments, and there are differences in their reaction systems. In this experiment, the reactivity of each sample in the Mindray system is only for reference).

[0142] Acceptable standard: The relative deviation of reactivity is less than or equal to ±5%.

[0143] The test results are as follows:

[0144] Table 3

[0145] Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 A 1485 1793 1419 2615 2782 2958 5447 5915 4473 B 1549 1756 1486 2570 2755 2971 5432 5938 4500 C 1540 1837 1444 2567 2804 2990 5488 5880 4582 D 1571 1834 1481 2571 2780 3004 5445 5903 4473 E 1514 1875 1527 2582 2817 2991 5480 5947 4532 F 1590 1896 1524 2634 2817 3037 5496 5924 4505 G 1501 1763 1428 2555 2828 2999 5467 5886 4520 H 1488 1874 1503 2602 2785 3037 5506 5910 4534 I 1590 1829 1442 2554 2795 3048 5552 5901 4562 J 1536 1862 1490 2630 2836 3041 5503 5913 4575 K 1545 1784 1482 2595 2754 2972 5556 5924 4527 L 1507 1789 1435 2554 2759 2963 5532 5914 4526 M 1375 1719 1276 2049 2584 2672 4972 5074 4156 N 2517 3059 2355 4210 4538 4831 8143 8822 6763 O 1540 1828 1473 2595 2791 3004 5486 5897 4539

[0146] Table 4

[0147]

[0148]

[0149] The results showed that the relative deviation of reactivity of 9 samples in each group except group M was within the range of [-5%, 5%]. From the results, it can be seen that the addition of compounds a-l significantly reduced the reactivity deviation of CRP in clinical tests.

[0150] (2) Verification of serum amyloid A test reactivity

[0151] Use the standard batch of hemolysin in combination with Zhongyuan Huiji serum amyloid A detection reagent (nephelometry) for value determination. Select samples with SAA concentrations of 10±2 mg / L, 60±12 mg / L, and 100±20 mg / L. For each concentration range, select 3 samples with HCT distributed in the range of 40%-60% as the samples to be tested. Use the 13 groups of hemolysins in Example 2 in combination with Zhongyuan Huiji serum amyloid A detection reagent (nephelometry) and Zhongyuan Huiji specific protein composite calibration product to establish a working curve, and test the samples to be tested. Each sample is tested three times repeatedly, and the average reactivity of the three tests is taken as the true reactivity of the sample (the results are shown in Table 5); using the reactivity of the standard batch of hemolysin as the control, calculate the relative deviation of the clinical test reactivity of the 13 groups of hemolysins in Example 2 (the results are shown in Table 6). (Both the Mindray instrument and the Zhongyuan Huiji instrument used in the test are closed instruments, and there are differences in their reaction systems. In this experiment, the reactivity of each sample in the Mindray system is only for reference).

[0152] Acceptable standard: The relative deviation of reactivity is less than or equal to ±10%.

[0153] The test results are as follows:

[0154] Table 5

[0155]

[0156]

[0157] Table 6

[0158]

[0159]

[0160] The results show that the relative deviation of reactivity of 9 samples in each group except group M is within the range of [-10%, 10%]. For group M, only the relative deviation of the reactivity of sample 8 reaches the acceptable standard. Considering the reactivity of sample 8, it is considered that the reason for the qualified relative deviation of this sample is that the relatively high reactivity reduces the actually existing deviation during the calculation of the relative deviation. From the results, it can be seen that the addition of compound a-l reduces the reactivity deviation of SAA in clinical tests.

[0161] (3) C-reactive protein clinical test deviation

[0162] According to the formula described in Example 13, hemolyzing agent was prepared using three batches of saponin raw materials (Sigma, batch numbers: MKCS3992, BCBX0632, BCCG2590), and the compounds were added as shown in Table 2. The hemolyzing agent standard batch was used to calibrate the Zhongyuan Huiji C-reactive protein detection reagent (nephelometry) for value determination. Samples with CRP concentrations of 10±2 mg / L, 20±5 mg / L, and 40±10 mg / L were selected, and for each concentration range, 3 samples with HCT distributed in the range of 40%-60% were selected as the samples to be tested. Thirteen groups of hemolyzing agents (each group containing hemolyzing agents prepared from three batches of different saponin raw materials) were used to cooperate with the Zhongyuan Huiji C-reactive protein detection reagent (nephelometry) and the Zhongyuan Huiji specific protein composite calibration product to establish a working curve, and the samples to be tested were tested. Each sample was tested twice repeatedly, and the average value of the two tests was taken as the concentration value of the sample. The relative deviation of the clinical sample test values of the three batches of hemolyzing agents in each group from the clinical test values of the hemolyzing agent standard batch was calculated, and the maximum relative deviation (taking the absolute value) of the thirteen groups of hemolyzing agents was used for comparison. For N groups, three batches of commercially available reagents were selected for comparison of the relative deviation (taking the absolute value) of the clinical test values.

[0163] The test results are as follows:

[0164] Table 7

[0165]

[0166]

[0167] As shown in the results, for the hemolyzing agent added with compounds a-l, the relative deviation of the inter-batch clinical test compared with the standard batch was distributed in the range of [1.09%, 4.99%]; for the hemolyzing agent without added compounds, the relative deviation of the clinical test was distributed in the range of [5.12%, 14.67%]; for the commercially available reagents (N groups), the relative deviation of the inter-batch clinical test was distributed in the range of [5.35%, 9.14%]. It can be seen from the results that the addition of compounds a-l significantly reduced the inter-batch clinical CRP test deviation of the hemolyzing agent.

[0168] (4) Clinical test deviation of serum amyloid A

[0169] According to the formula described in Example 13, hemolyzing agents were prepared using three batches of saponin raw materials (Sigma, batch numbers: MKCS3992, BCBX0632, BCCG2590), and the compounds were added as shown in Table 2. The hemolyzing agent standard batch was used to calibrate the Zhongyuan Huiji serum amyloid A detection reagent (nephelometry), and samples with SAA concentrations of 10±2 mg / L, 60±12 mg / L, and 100±20 mg / L were selected. For each concentration range, 3 samples with HCT distributed in the range of 40%-60% were selected as the samples to be tested. Thirteen groups of hemolyzing agents (each group containing hemolyzing agents prepared from three batches of different saponin raw materials) were used to cooperate with the Zhongyuan Huiji serum amyloid A detection reagent (nephelometry) and the Zhongyuan Huiji specific protein composite calibration product to establish a working curve, and the samples to be tested were tested. Each sample was tested twice, and the average value of the two tests was taken as the concentration value of the sample. The relative deviation of the clinical sample test values of the three batches of hemolyzing agents in each group from the clinical test values of the hemolyzing agent standard batch was calculated, and the maximum relative deviation (taking the absolute value) of the thirteen groups of hemolyzing agents was used for comparison. For N groups, the relative deviation (taking the absolute value) of the clinical test values of three batches of commercially available reagents was compared.

[0170] The test results are as follows:

[0171] Table 8

[0172]

[0173]

[0174] As shown in the results, for the hemolyzing agent added with compounds a-l, the relative deviation of the inter-batch clinical test compared with the standard batch was distributed in the range of [1.88%, 6.84%]; for the hemolyzing agent without added compounds, the relative deviation of the clinical test was distributed in the range of [13.28%, 23.14%]; for the commercially available reagents (N groups), the relative deviation of the inter-batch clinical test was distributed in the range of [7.77%, 9.77%]. It can be seen from the results that the addition of compounds a-l significantly reduced the inter-batch clinical SAA test deviation of the hemolyzing agent.

[0175] (5) Accelerated stability test - C-reactive protein

[0176] The 15 groups of hemolysants described in Example 2 were subjected to thermal acceleration treatment at 37°C for 7 days and 14 days. After the treatment, samples with CRP concentrations of 10±2 mg / L, 20±5 mg / L, and 40±10 mg / L were selected as the samples to be tested. For each concentration range, 3 samples with HCT distributed in the range of 40%-60% were selected. The 15 groups of hemolysants after the acceleration treatment were used in combination with Zhongyuan Huiji C-reactive protein detection reagent (nephelometry) and Zhongyuan Huiji specific protein composite calibration to establish a working curve, and the samples to be tested were tested. Each sample was tested twice, and the mean value of the two tests was taken as the concentration value of the sample. Calculate the mean value of the relative deviation of the CRP test values of 9 clinical samples of each group of hemolysants after the acceleration treatment and the control hemolysant (stored at 2-8°C for 14 days).

[0177] The test results are as follows:

[0178] Table 9

[0179] Thermal acceleration at 37°C for 7 days Thermal acceleration at 37°C for 14 days A 2.90％ 5.85％ B 3.57％ 4.63％ C 3.01％ 4.67％ D 3.36％ 4.92％ E 2.34％ 5.58％ F 2.52％ 4.88％ G 3.69％ 4.71％ H 2.68％ 4.65％ I 3.86％ 4.71％ J 2.63％ 4.68％ K 3.29％ 4.83％ L 2.29％ 5.23％ M 4.34％ 6.65％ N 5.13％ 7.19％ O / /

[0180] As shown in the results, for the hemolysants added with compounds a-l, after thermal acceleration at 37°C for 7 days, the mean value of the relative deviation of the clinical test was distributed in the range of [2.29%, 3.84%]; for the hemolysants without added compounds, the mean value of the relative deviation of the clinical test was 4.34%; for the commercially available reagent (Group N), the mean value of the relative deviation of the clinical test was 5.13%. For the hemolysants added with compounds a-l, after thermal acceleration at 37°C for 14 days, the mean value of the relative deviation of the clinical test was distributed in the range of [4.07%, 5.85%]; for the hemolysants without added compounds, the mean value of the relative deviation of the clinical test was 9.47%; for the commercially available reagent (Group N), the mean value of the relative deviation of the clinical test was 9.19%.

[0181] It can be seen from the results that the addition of compounds a-l significantly reduced the relative deviation of the clinical test after the thermal acceleration of the hemolysant, that is, the addition of compounds a-l significantly improved the acceleration stability of the hemolysant.

[0182] (6) Accelerated stability test - Serum amyloid A

[0183] The 15 groups of hemolyzing agents described in Example 2 were subjected to thermal acceleration treatment at 37°C for 7 days and 14 days. After the treatment, samples with SAA concentrations of 10±2 mg / L, 50±10 mg / L, and 100±20 mg / L were selected as the samples to be tested, and 3 samples with HCT distributed in the range of 40%-60% were selected for each concentration range. The 15 groups of hemolyzing agents after the acceleration treatment were used to establish a working curve in combination with Zhongyuan Huiji serum amyloid A (nephelometry) and Zhongyuan Huiji specific protein composite calibration products, and the samples to be tested were tested. Each sample was tested twice repeatedly, and the mean value of the two tests was taken as the concentration value of the sample. Calculate the mean value of the relative deviation of the SAA test values of 9 clinical samples of each group of hemolyzing agents after the acceleration treatment and the control hemolyzing agent (stored at 2-8°C for 14 days).

[0184] The test results are as follows:

[0185] Table 9

[0186] Thermal acceleration at 37°C for 7 days Thermal acceleration at 37°C for 14 days A 4.18％ 4.53％ B 4.04％ 4.72％ C 4.95％ 5.51％ D 4.96％ 5.95％ E 4.32％ 4.25％ F 4.88％ 5.59％ G 4.39％ 5.75％ H 4.39％ 5.79％ I 4.00％ 5.27％ J 4.50％ 4.67％ K 4.52％ 5.18％ L 4.26％ 4.82％ M 5.85％ 8.08％ N 6.35％ 8.64％ O / /

[0187] As shown in the results, for the hemolyzing agents added with compounds a-l, after thermal acceleration at 37°C for 7 days, the mean value of the relative deviation of the clinical test is distributed in the range of [4.00%, 4.93%]; for the hemolyzing agent without added compounds, the mean value of the relative deviation of the clinical test is 5.85%; for the commercially available reagent (Group N), the mean value of the relative deviation of the clinical test is 6.35%. For the hemolyzing agents added with compounds a-l, after thermal acceleration at 37°C for 14 days, the mean value of the relative deviation of the clinical test is distributed in the range of [4.34%, 5.98%]; for the hemolyzing agent without added compounds, the mean value of the relative deviation of the clinical test is 8.08%; for the commercially available reagent (Group N), the mean value of the relative deviation of the clinical test is 8.64%.

[0188] It can be seen from the results that the addition of compounds a-l significantly reduces the relative deviation of the clinical test of the hemolyzing agent after thermal acceleration, that is, the addition of compounds a-l significantly improves the acceleration stability of the hemolyzing agent.

[0189] As can be seen above, while retaining the advantages of saponin as a hemolytic component, the present invention significantly improves the compatibility of the hemolyzing agent formulation with different batches of commercial saponins by adding saponin protectants (including compounds a-l and their cooperating components) to the hemolyzing agent, and overcomes the technical problem of saponin oxidation, significantly improving the stability of the hemolyzing agent for blood cell analysis. The specific reaction principle is as follows:

[0190] The cell membrane of red blood cells ruptures under the action of a hemolytic agent, releasing cell contents with hemoglobin as the main component. In body fluids, hemoglobin is finally converted into bilirubin under the catalytic action of a series of enzymes such as heme oxygenase. In in vitro diagnostic reagent products developed based on immunoturbidimetry, bilirubin with too high a concentration will interfere with the test results, resulting in clinical test deviations. When saponins are present in the hemolytic agent, the hydrophilic groups on the surface of saponin molecules will bind to the hydrophilic residues (such as hydroxyl groups, amino groups, etc.) on the surface of heme oxygenase molecules through hydrogen bonds; the hydrophobic groups will bind to the amino acid residues on the surface of heme oxygenase molecules through hydrophobic interactions. This binding will affect the conformations of the substrate binding site and catalytic center of heme oxygenase, thereby interfering with the catalytic activity of the enzyme and reducing the bilirubin generated from the conversion of hemoglobin in the sample to be tested. Due to the large batch-to-batch differences in commercial saponins, even at the same usage concentration, the ability of saponins from different batches to bind and inhibit heme oxygenase varies greatly, and further leads to large batch-to-batch differences in the hemolytic agents prepared from them.

[0191] In the present invention, a chemical raw material with weak reducibility and capable of binding to heme oxygenase to limit the enzyme activity is introduced into the hemolytic agent formulation, and a "saponin protective agent component" is formulated with this chemical raw material as the core. Through the synergistic action of the components in the reaction system, the binding ratio of saponin to heme oxygenase is reduced, the dependence of the heme-biliverdin-bilirubin redox system on saponin is limited, and the compatibility of the hemolytic agent formulation with different batches of commercial saponin products is improved. Moreover, the weak reducibility of this raw material avoids the oxidation problem of saponin without affecting the disulfide bonds of antibodies, and finally achieves the effects of reducing the clinical batch-to-batch deviation of the hemolytic agent and improving the long-term stability of the hemolytic agent.

[0192] All data, reagents, and procedures in this article should be understood as illustrative rather than restrictive. Although the present invention has been described in connection with the above specific embodiments, many modifications and other changes are obvious to those skilled in the art. All such modifications and other changes also fall within the scope and protection of the present invention.

Claims

1. Use of a compound having the structure of formula I in cell lysis of biological samples, wherein R1 and R2 are each independently selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, mercapto, halogen, carboxyl, ester, trifluoromethyl; R3 is selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, mercapto, halogen, trifluoromethyl, sulfonic acid group, polyoxyethylene group; R4 is selected from hydrogen, C 1-18 alkyl, C 1-18 alkoxy, halogen, ester group.

2. The compound according to claim 1, wherein R1 is selected from hydrogen, C 1-18 alkyl groups.

3. The compound according to claim 1, wherein R2 is selected from hydrogen, C 1-18 alkyl, halogen, mercapto, ester group, carboxyl group.

4. The compound according to claim 1, wherein R3 is selected from hydrogen, halogen, polyoxyethylene group, trifluoromethyl group, sulfonic acid group.

5. The compound according to claim 1, wherein R4 is selected from hydrogen, C 1-18 alkyl groups.

6. The compound according to claim 1, the compound is:

7. A saponin protectant, the protectant comprising the compound according to any one of claims 1-6.

8. The protectant according to claim 7, the protectant further comprising sodium nitrite, CHAPS, a reducing agent, preferably, the reducing agent is selected from at least one of reduced glutathione, mercaptoethanol, dithiothreitol, TCEP, 2-mercaptopyridine.

9. A biological sample hemolytic agent, the hemolytic agent comprising saponin and the compound according to any one of claims 1-6, or the protectant according to any one of claims 7-8.

10. The hemolytic agent according to claim 9, the hemolytic agent further comprising a buffer, a preservative, an ionic strength regulator; preferably, the buffer is selected from at least one of MOPSO buffer, boric acid buffer, MOPS buffer, PIPES buffer, the preservative is selected from sodium azide, PC300, BND-10, and the ionic strength regulator is selected from at least one of sodium chloride, potassium chloride.

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