CN114778858A - Lupus anticoagulant detection kit - Google Patents
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Abstract
The invention discloses a lupus anticoagulant detection kit, which comprises a LA1 screening reagent and a LA2 confirmation reagent; the LA1 screening reagent comprises recombinant snake venom enzyme, recombinant phospholipid, calcium ions, a first heparin neutralizer, a first buffer solution and a first auxiliary material, and the LA1 screening reagent is in a liquid state; LA2 confirms that the reagent includes recombinate snake venom enzyme, recombinate phosphatide, calcium ion, second heparin neutralizer, second buffer solution and second supplementary product, LA2 confirms that the reagent is the liquid; the content of the recombinant phospholipid in the LA1 screening reagent is lower than that in the LA2 confirmation reagent; according to the invention, the recombinant snake venom enzyme and the recombinant phospholipid are matched for use, so that the sensitivity of the reagent is higher, the change amplitude in unit time is obviously enhanced when a positive or weak positive sample is tested, the detection rate of the positive sample is improved, and the false negative is reduced; the heparin neutralizer is also included, so that the influence of heparin is eliminated, and the accuracy is improved; the reagent is liquid, and has convenient use, low cost and good stability.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a lupus anticoagulant detection kit.
Background
Lupus Anticoagulant (LA) is a group of immunoglobulins that can bind directly to negatively charged phospholipids or to phospholipoprotein complexes, and belongs to the group of antiphospholipid Antibodies (APL). Persistent LA positive patients are considered to have a higher risk of thrombosis and recurrence and are also a risk signal for unexplained recurrent abortion, stillbirth, thrombotic diseases and certain autoimmune diseases; LA also enhances platelet aggregation and inhibits fibrinolytic activity, so patients with positive LA are prone to thrombotic complications.
Currently, the commonly used methods for detecting LA include Activated Partial Thromboplastin Time (APTT), modified diluted viper venom time (dvrvvt) and Silica Coagulation Time (SCT) detection methods, and usually a traditional three-step method of a screening test, a mixing test and a confirmation test is used to judge whether LA exists, and a second detection is performed after 12 weeks, and diagnosis can be made when LA is still positive. LA plays a very important role in the diagnosis and treatment of APS. Compared with the SCT method, the dRVVT method has higher specificity, can accurately reflect the blood coagulation process participated in after the activation of the factor X in the body of a patient, is not influenced by the abnormality or the deletion of the blood coagulation factors VII, VIII, IX, XI and the like, has stronger anti-interference capability, can be suitable for a full-automatic blood coagulation analyzer, and has wide prospect in clinical application.
However, the mainstream lupus anticoagulant detection kits (dRVVT method) in the market are all in the form of freeze-dried powder, the freeze-drying process is too complex, the difference between bottles of the reagent is large, re-dissolving operation is required before use, manual operation steps are added, and the kit is easily influenced by re-dissolving water quality, accuracy of a pipettor and a manipulation of an operator; the sensitivity of a lupus anticoagulant detection kit (dRVVT method) needs to be improved, and particularly, false negative results often occur under the condition that weak LA exists in a sample to be detected; the stability of the lupus anticoagulant detection kit (dRVVT method) needs to be improved, the effective period is not long enough, and the use and the reagent storage of a client are not facilitated, so that the improvement of the stability of the reagent is very necessary; the existing lupus anticoagulant detection kit (dRVVT method) has insufficient anti-heparin interference capability, and when a sample containing heparin is tested, the obtained result is unreliable, so that the anti-heparin interference capability of the existing kit is urgently required to be improved; lupus anticoagulant detection kits (dRVVT method) are mostly imported abroad, are expensive in price setting, long in transportation time and long in arrival period, and have certain influence on hospitals and patients.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a lupus anticoagulant detection kit aiming at the defects of the prior art, which has the advantages of low price, full liquid, high reagent sensitivity, convenience, rapidness, strong anti-interference capability, good stability and good accuracy.
The technical scheme adopted by the invention for solving the technical problems is as follows: a lupus anticoagulant detection kit comprises a LA1 screening reagent and a LA2 confirmation reagent;
the LA1 screening reagent comprises recombinant snake venom enzyme, recombinant phospholipid, calcium ions, a first heparin neutralizer, a first buffer solution and a first auxiliary material, and the LA1 screening reagent is in a liquid state;
the LA2 confirmation reagent comprises recombinant snake venom enzyme, recombinant phospholipid, calcium ions, a second heparin neutralizer, a second buffer solution and a second auxiliary material, and the LA2 confirmation reagent is in a liquid state;
the content of the recombinant phospholipid in the LA1 screening reagent is lower than that in the LA2 confirmation reagent.
Preferably, the contents of the screening reagent for the recombinant snake venom enzyme in LA1 and the confirming reagent for LA2 are both 1-3 IU/mL.
Preferably, the content of the recombinant phospholipid in the LA1 screening reagent is 0.02-0.06%, and the content of the recombinant phospholipid in the LA2 confirmation reagent is 0.4-0.8%.
Further, preferably, the first heparin neutralizing agent comprises polyacrylamide, and the content of the polyacrylamide in the LA1 screening reagent is 1-3 mg/mL.
Further, the second heparin neutralization agent preferably comprises polyacrylamide, and the content of the polyacrylamide in the LA2 confirmation reagent is 1-3 mg/mL.
Preferably, the first buffer solution and the second buffer solution are at least one of a PBS buffer solution, a Tris buffer solution, a Hepes buffer solution, a MES buffer solution, a MOPS buffer solution and a citric acid buffer solution, and the pH of the LA1 screening reagent and the LA2 confirmation reagent is 7.2 to 7.4.
Preferably, the first buffer solution and the second buffer solution are PBS buffer solutions with the concentration of 100-200 mM.
Further, preferably, the first auxiliary material comprises a first stabilizer, and the first stabilizer comprises one or more of glycine, polyethylene glycol 2000, Span-80, BSA, HSA, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin and mannitol.
Further, it is preferred that the first stabilizer comprises the components: glycine, polyethylene glycol 2000 and Span-80, wherein the components in the LA1 screening reagent comprise the following components in percentage by mass: 1-3% of glycine, 20001-5% of polyethylene glycol and-800.1-1% of Span.
Further, preferably, the second auxiliary material comprises a second stabilizer, and the second stabilizer comprises one or more of glycine, polyethylene glycol 2000, Span-80, BSA, HSA, gelatin, Tween-20, trehalose, glucose, beta-cyclodextrin and mannitol.
Further, it is preferred that the second stabilizer comprises the components: glycine, polyethylene glycol 2000 and Span-80, wherein the mass percentages of the components in the LA1 screening reagent are as follows: 1-3% of glycine, 20001-5% of polyethylene glycol and-800.1-1% of Span.
Further, it is preferable that each of the LA1 screening reagent and the LA2 confirmation reagent includes a preservative, and the preservative is at least one of sodium benzoate, sodium azide, Proclin-300, gentamicin, and nitrite.
Further, preferably, the LA1 screening reagent and the LA2 confirmation reagent both comprise Proclin-300 with the mass percent of 0.03-0.05%.
Preferably, the LA1 screening reagent and the LA2 confirmation reagent both comprise protease protective agents, and the mass percentage of the protease protective agents in the LA1 screening reagent and the LA2 confirmation reagent is 2-4%.
Further, preferably, the protease protective agent is bamboo polysaccharide.
Preferably, the contents of the calcium ion in the LA1 screening reagent and the LA2 confirmation reagent are both 10-20 mM.
The invention has the beneficial effects that: according to the lupus anticoagulant detection kit, the screening reagent and the confirmation reagent are both in liquid state, the use is convenient, the cost is low, the sensitivity is high, the anti-interference capability is strong, the accuracy is good, the stability is good, the kit is a domestic pioneer full-liquid lupus anticoagulant detection kit, the substitution of the imported lupus anticoagulant detection kit can be realized, and the requirement of clinical examination is fully met; the cost of purchasing detection reagents in hospitals can be reduced, the detection cost of patients is reduced, and the burden is reduced; the recombinant snake venom enzyme and the recombinant phospholipid are used as raw materials in a matched manner, so that the coagulation time of a LA1 screening reagent for a positive patient (containing weak positive) can be prolonged, the detection rate of a positive sample can be improved, false negative can be reduced, the sensitivity of the reagent is higher, the change amplitude in unit time is remarkably enhanced when the positive sample is tested, the detection capability of the reagent is higher when the weak positive sample is tested, and the reagent is more helpful and superior for clinical diagnosis; the LA1 screening reagent and the LA2 confirmation reagent also comprise a heparin neutralizer, and the accuracy of a detection result is improved except for the influence on the detection result caused by the existence of heparin in a sample to be detected.
Detailed Description
For a more clear understanding of the technical features, objects, and effects of the present invention, specific embodiments of the present invention will now be described in detail.
A lupus anticoagulant detection kit comprises a LA1 screening reagent and a LA2 confirmation reagent; the LA1 screening reagent comprises recombinant snake venom enzyme, recombinant phospholipid, calcium ions, a first heparin neutralizer, a first buffer solution and a first auxiliary material, and the LA1 screening reagent is in a liquid state; LA2 confirms that the reagent includes recombined snake venom enzyme, recombined phospholipid, calcium ion, second heparin neutralizer, second buffer solution and second auxiliary material, LA2 confirms that the reagent is liquid; the content of recombinant phospholipid in the LA1 screening reagent is lower than that in the LA2 confirmation reagent.
The working principle of the kit of the invention is as follows: under the condition of calcium ion, the recombinant snake venom enzyme can directly activate the factor X in a sample to be detected to change the factor X into Xa, the LA1 screening reagent contains low-concentration recombinant phospholipid and is sensitive to LA, and LA2 confirms that the reagent contains excessive recombinant phospholipid and can be neutralized with LA to obtain shorter coagulation time. If LA is present, the clotting time of the LA1 screening reagent is prolonged, and if LA2 is confirmed, the clotting time can be corrected.
According to the lupus anticoagulant detection kit provided by the invention, the LA1 screening reagent and the LA2 confirming reagent are both in liquid state, the use is convenient, the cost is low, the sensitivity is high, the linear range is wide, the anti-interference capability is strong, the stability is good, the kit is a domestic pioneer full-liquid lupus anticoagulant detection kit, the substitution of an imported lupus anticoagulant detection kit can be realized, and the requirement of clinical examination is fully met; the cost of purchasing detection reagents in hospitals can be reduced, the detection cost of patients is reduced, and the burden is reduced; the recombinant snake venom enzyme and the recombinant phospholipid are used as raw materials in a matched manner, so that the coagulation time of a LA1 screening reagent for a positive patient (containing weak positive) can be prolonged, the detection rate of a positive sample can be improved, false negative can be reduced, the sensitivity of the reagent is higher, the change amplitude in unit time is remarkably enhanced when the positive sample is tested, the detection capability of the reagent is higher when the weak positive sample is tested, and the reagent is more helpful and superior for clinical diagnosis; the LA1 screening reagent and the LA2 confirmation reagent also comprise heparin neutralizer, so that the influence of the existence of heparin in a sample to be detected on a detection result is eliminated, and the accuracy of the detection result is improved.
The recombinant snake venom enzyme and the recombinant phospholipid are prepared by adopting genetic engineering, so that the purity is higher, the unit activity is stronger, the use amount is lower, and the cost is saved; the two reagents are used in combination, so that the solidification time of a positive patient (containing weak positive) LA1 screening reagent can be prolonged, the detection rate of a positive sample is favorably improved, false negative is reduced, the sensitivity of the reagent is higher, the change amplitude in unit time is obviously enhanced when the positive sample is tested, the detection capability of the reagent is higher when the weak positive sample is tested, and the reagent is more helpful and superior to clinical diagnosis. In terms of reaction time, reagents containing the recombinant snake venom enzyme and the recombinant phospholipid only need to be incubated for 45s when added into a sample to be detected, and incubation time of existing kits on the market after the sample to be detected is different from 90-200 s.
The contents of the recombinant snake venom enzymes in an LA1 screening reagent and an LA2 confirmation reagent are 1-3 IU/mL, the content of recombinant phospholipid in an LA1 screening reagent is 0.02-0.06%, and the content of recombinant phospholipid in an LA2 confirmation reagent is 0.4-0.8%.
The first heparin neutralizer comprises polyacrylamide, the second heparin neutralizer comprises polyacrylamide, and due to the fact that heparin exists in a sample to be detected and influences of the heparin on a detection result, the polyacrylamide with a certain concentration is added into the LA1 screening reagent and the LA2 confirming reagent, the influences on the detection result due to the existence of the heparin in the sample can be eliminated, the anti-interference capability of the kit is enhanced, and the detection accuracy is further improved; polyacrylamide has positive charge, heparin has negative charge, the positive charge of the polyacrylamide can neutralize the negative charge of the heparin, so that the heparin loses activity and cannot play an anticoagulation role, and the effect of neutralizing the heparin by the polyacrylamide is better than that of the traditional heparin neutralizers such as coagulated polyamine, protamine and the like under the condition of the same use concentration. The content of the polyacrylamide in the LA1 screening reagent is 1-3 mg/mL, the content of the polyacrylamide in the LA2 confirmation reagent is 1-3 mg/mL, and the LA1 screening reagent and the LA2 confirmation reagent have strong anti-interference capability on heparin under the above mixture ratio.
The first buffer solution and the second buffer solution are respectively at least one of a PBS buffer solution, a Tris buffer solution, a Hepes buffer solution, a MES buffer solution, a MOPS buffer solution and a citric acid buffer solution, the types of the buffer solutions are selected randomly, and the PBS buffer solution is preferred; the pH values of the first buffer solution and the second buffer solution are both 7.2-7.4. By using any of the above buffers, the LA1 screening reagent and the LA2 confirmation reagent can be effectively maintained in a predetermined range, namely, the solution is stabilized at a pH of 7.2-7.4, and the first buffer, the second buffer and the third buffer do not cause adverse effects on the detection of lupus anticoagulant in a sample to be detected; furthermore, the first buffer solution, the second buffer solution and the third buffer solution are preferably PBS buffer solutions with the concentration of 100-200 mM, and in the buffer solutions, the recombinant snake venom enzymes and the recombinant phospholipids can exert the maximum efficiency, the sensitivity of the reagent is improved, and the detection efficiency and the accuracy are improved.
The first auxiliary material comprises a first stabilizer, the first stabilizer comprises one or more of glycine, polyethylene glycol 2000, Span-80, BSA, HSA, gelatin, Tween-20, trehalose, glucose, beta-cyclodextrin and mannitol, and the type of the first stabilizer can be selectively added or not added; the addition of the first stabilizer can improve the stability of the reagent, inhibit the nonspecific adsorption in a system on the premise of not influencing the sensitivity of a reaction system, and more importantly, can effectively protect the activity of the recombinant snake venom enzyme in the LA1 screening reagent and improve the stability of the recombinant snake venom enzyme; for example, mannitol, trehalose, glucose, gelatin and the like are added, so that the freeze denaturation of the protein is inhibited, and the condition that the LA1 screening reagent is precipitated and non-specifically aggregated can not be caused by freezing, so that the stability of the reagent in storage, transportation and the like is ensured; the addition of polyhydric alcohols such as polyethylene glycol 2000 and the like can improve the viscosity of the reagent, facilitate the uniform suspension of the recombinant snake venom enzyme and the recombinant phospholipid in the first buffer solution, and prevent the recombinant snake venom enzyme and the recombinant phospholipid from settling easily, thereby playing a good stability role, and can also be used as an antifreeze agent to effectively reduce the freezing point, thereby achieving a good freeze-thaw resistance effect; amino acids such as glycine are added as basic units constituting proteins, and the addition of amino acids is advantageous for improving the stability of the reagent. Tween-20, Span-80 and the like are added to play a role of a surfactant, so that the stability of the reagent is further improved; the protein concentration of the LA1 screening reagent can be increased by adding BSA, HSA and the like, a certain protection effect on protease is achieved, and the stability of the reagent is improved.
Further, it is preferred that the first stabilizer comprises the components: glycine, polyethylene glycol 2000 and Span-80, wherein the components in the LA1 screening reagent comprise the following components in percentage by mass: 1-3% of glycine, 20001-5% of polyethylene glycol and 1-800.1% of Span; the accuracy and the stability of the kit can be improved.
The second auxiliary material comprises a second stabilizer, and the second stabilizer comprises one or more of glycine, polyethylene glycol 2000, Span-80, BSA, HSA, gelatin, Tween-20, trehalose, glucose, beta-cyclodextrin and mannitol; the type of the second stabilizer can be optionally added or not added; the addition of the second stabilizer can improve the stability of the reagent, inhibit the nonspecific adsorption in the system on the premise of not influencing the sensitivity of the reaction system, and more importantly, can also effectively protect the activity of the recombinant snake venom enzyme in the LA2 confirmation reagent and improve the stability of the recombinant snake venom enzyme; for example, mannitol, trehalose, glucose, gelatin and the like are added, so that on one hand, the freezing denaturation of protein can be inhibited, and the freezing condition that LA2 confirms that the reagent is precipitated and non-specifically aggregated can be avoided, so that the stability of storage, transportation and the like of the reagent is ensured, on the other hand, gelatin macromolecules have a net structure, so that a sieve pore effect can be generated, the space limitation can be performed on the protein, the mutual collision among protein molecules is reduced, and the stability of the protein is improved to a certain extent; the addition of polyhydric alcohols such as polyethylene glycol 2000 can improve the viscosity of the reagent, facilitate the uniform suspension of the recombinant snake venom enzyme and the recombinant phospholipid in the second buffer solution, and prevent sedimentation, so that the reagent has a good stability effect, and can be used as an antifreeze to effectively reduce the freezing point, so that a good freeze-thaw resistance effect is achieved; amino acids such as glycine are added as basic units constituting proteins, and the addition of amino acids is advantageous for improving the stability of the reagent. Tween-20, Span-80 and the like are added to play a role of a surfactant, so that the stability of the reagent is further improved; the addition of BSA, HSA, etc. can increase the protein concentration in LA2 confirmation reagent, protect protease to a certain extent, and improve the stability of the reagent.
Further, it is preferred that the second stabilizer comprises the components: glycine, polyethylene glycol 2000 and Span-80, wherein the mass percentages of the components in the LA1 screening reagent are as follows: 1-3% of glycine, 20001-5% of polyethylene glycol and-800.1-1% of Span. The accuracy and the stability of the kit can be improved.
The LA1 screening reagent and the LA2 confirmation reagent both comprise preservatives, and the preservatives are at least one of sodium benzoate, sodium azide, Proclin-300, gentamicin and nitrite; of course, the type of the preservative may be optionally added or not. The preservative can be added to achieve the preservative effect, so that the kit is prevented from losing efficacy due to microbial pollution, and the storage period of the kit is prolonged. Furthermore, the LA1 screening reagent and the LA2 confirming reagent preferably both comprise 0.03-0.05% of Proclin-300 by mass percent, and the LA1 screening reagent and the LA2 confirming reagent have better preservative effect and are beneficial to improving the stability of the reagents in the above proportion.
Further, both the LA1 screening reagent and the LA2 confirmation reagent include a protease inhibitor, and it is needless to say that the protease inhibitor may be added selectively or not. The protease protective agent is used for prolonging the stability of a LA1 screening reagent and a LA2 confirmation reagent in the kit, is beneficial to long-term use and storage of the kit, is beneficial to protecting the activity of the enzyme, prevents the denaturation of the recombinant snake venom enzyme, and plays a role in protecting the recombinant snake venom enzyme; prevent enzyme decomposition and nonspecific adsorption, and reduce the denaturation of some enzymes and adverse environmental factors such as heat, surface tension and chemical factors. Further, the protease protective agent is preferably 2 to 4 mass% of both the LA1 screening reagent and the LA2 confirmation reagent, and the protease protective agent has a good protein protection effect on the LA1 screening reagent and the LA2 confirmation reagent at the mixture ratio. Further, the preferred protease protective agent is bamboo polysaccharide, which is beneficial to protecting the activity of the enzyme and preventing the enzyme from being denatured, hydroxyl groups of the bamboo polysaccharide can replace water molecules to form hydrogen bonds with amide groups on the surface of the enzyme, and can also restrict the change of the biological activity of the wrapped enzyme, play a role in protecting the enzyme, prevent the decomposition and the non-specific adsorption of the enzyme, relieve the denaturation of some enzymes, and relieve the denaturation caused by adverse environmental factors such as heating, surface tension and chemical factors.
The contents of the screening reagent of the calcium ions in LA1 and the confirmation reagent of LA2 are both 10-20 mM; calcium ions can be provided by calcium chloride, under the action of the calcium ions with the concentration, the factor X in a sample to be detected can be quickly activated by the recombinant snake venom enzyme in the LA1 screening reagent and the LA2 confirmation reagent, so that the factor X is activated into Xa, the sensitivity of the reagent is improved, and the accuracy of a detection result is improved.
The present invention is further explained below by means of specific embodiments.
Example 1
A lupus anticoagulant detection kit comprises a LA1 screening reagent and a LA2 confirmation reagent; the specific components are shown in the following table:
TABLE 1 reagent Components of the kit of example 1
Note: the PBS buffer solution can be replaced by any one or more of Tris buffer solution, Hepes buffer solution, MES buffer solution, MOPS buffer solution and citric acid buffer solution; glycine, polyethylene glycol 2000, Span-80 can be replaced by any one or more of tween-20, BSA, HSA, gelatin, trehalose, glucose, beta-cyclodextrin and mannitol; proclin-300 can be replaced by any one or more of sodium benzoate, sodium azide, gentamicin and nitrite; the calcium ions are provided by anhydrous calcium chloride; "/" indicates the absence of this component.
Example 2
A lupus anticoagulant detection kit comprises a LA1 screening reagent and a LA2 confirmation reagent; the specific components are shown in the following table:
TABLE 2 reagent Components of the kit of example 2
Note: the calcium ions are provided by calcium chloride; "/" indicates the absence of this component.
Example 3
A lupus anticoagulant detection kit comprises a LA1 screening reagent and a LA2 confirmation reagent; the specific components are shown in the following table:
TABLE 3 reagent Components of the kit of example 3
Note: the calcium ions are provided by anhydrous calcium chloride; "/" indicates the absence of this component.
Comparative example 1
In comparative example 1, both the LA1 screening reagent and LA2 confirmation reagent contained no polyacrylamide, and the other components and amounts were the same as in example 1.
Comparative example 2
In comparative example 2, neither the LA1 screening reagent nor LA2 confirmation reagent contained bamboo polysaccharide, and the other components and amounts were the same as in example 1.
Comparative example 3
In the LA1 screening reagent and LA2 confirming reagent in comparative example 3, the recombinant snake venom enzyme and the recombinant phospholipid were replaced with the common snake venom enzyme and the common phospholipid, respectively, and the other components and the amounts were the same as in example 1.
The lupus anticoagulant assay kits of examples 1-3 and comparative examples 1-3 were used to test accuracy, reproducibility, stability, and the yin-yang coincidence rate of clinical samples. The LA1 screening and LA2 confirmation assays of the dRVVT method should be used in parallel as a complete assay, and in conjunction with the procedures listed, a Normalized Ratio (NR) can be calculated with a cutoff value of NR > 1.2.
NR was calculated by applying the following formulas (1) (2) (3):
formula (1): normalized Ratio (NR) ═
；
Formula (2): LA1 screening reagent ratio =
；
Formula (A)3): LA2 confirmation reagent ratio =
。
TABLE 4 test results of the lupus anticoagulant assay kit of examples 1 to 3 and comparative examples 1 to 3 on normal human specimens
1. Accuracy test and results thereof
LA negative control products and LA positive control products containing exogenously added heparin are respectively measured by adopting the lupus anticoagulant detection kits in the embodiments 1 to 3 and the comparative example 1, each control product is tested for 3 times repeatedly, relative deviation is calculated according to the formulas (4) and (5), and the test results are shown in tables 5 to 8. The test result is required to be: the relative deviation should be within 7.5%.
Formula (4):
formula (5):
；
in the formula:
t is the target value of the quality control product;
b-relative deviation.
TABLE 5 accuracy test results of the lupus anticoagulant assay kit of example 1
TABLE 6 accuracy test results of the lupus anticoagulant assay kit of example 2
TABLE 7 accuracy test results of the lupus anticoagulant assay kit of example 3
TABLE 8 accuracy test results of the lupus anticoagulant assay kit of comparative example 1
The test results in tables 5-8 show that the relative deviation of the examples 1-3 is relatively small, and the examples 1-3 are proved to have good accuracy. From the test results in table 8, it is found that the relative deviation of comparative example 1 does not meet the requirement that the relative deviation is within the range of ± 7.5%, and from the results of example 1 and comparative example 1, the influence of heparin in the sample to be tested can be reduced by adding a proper amount of polyacrylamide, and the interference of heparin in the sample to lupus anticoagulant determination can be prevented, the LA1 screening reagent and the LA2 confirmation reagent in example 1 contain a certain concentration of polyacrylamide, so that the influence of heparin in the sample on the detection result can be eliminated, and the accuracy of the detection result can be increased, and the interference of free heparin in the sample in comparative example 1 causes the reduction of Xa and thrombin participating in the coagulation process, the prolongation of the coagulation time, and the deviation of the test result from the target value is larger. Thus, comparative example 1 is not as accurate as examples 1 to 3.
2. Repeatability tests and results thereof
The LA negative quality control substance and the LA positive quality control substance were each tested 10 times using the lupus anticoagulant assay kit of examples 1 to 3. Calculating the average value of the test results according to equations (4) and (6) ((
) And Standard Deviation (SD), Coefficient of Variation (CV) was calculated according to equation (4), and the test results are shown in tables 9 to 11. The test result is required to be: the Coefficient of Variation (CV) of LA negative quality control products is less than or equal to 10 percent; the Coefficient of Variation (CV) of the LA positive quality control product is less than or equal to 10 percent.
Formula (6):
in the formula: CV is the coefficient of variation.
TABLE 9 repetitive test results of the lupus anticoagulant assay kit of example 1
TABLE 10 repetitive test results of the lupus anticoagulant assay kit of example 2
TABLE 11 example 3 repeatability tests results of lupus anticoagulant assay kit
From the test results in tables 9 to 11, it was found that examples 1 to 3 all meet the above-mentioned reproducibility requirements.
3. Stability testing and results thereof
1) Bottle opening stability: the screening reagents for LA1 and the confirmation reagents for LA2 in the lupus anticoagulant detection kits of examples 1 to 3 were stored at 2 to 8 ℃ after decapping, and accuracy tests were performed on days 0, 14, 21, 28, 30, and 32, and the test results are shown in tables 12 to 14. The test result is required to be: in the lupus anticoagulant detection kit, LA1 screening reagent and LA2 confirmation reagent are placed at 2-8 ℃ after being unpacked and can be stored stably for 30 days, namely, the relative deviation is within the range of +/-7.5%.
TABLE 12 open bottle stability test results of the lupus anticoagulant assay kit of example 1
TABLE 13 open bottle stability test results of the lupus anticoagulant assay kit of example 2
TABLE 14 open bottle stability test results of the lupus anticoagulant assay kit of example 3
The test results in tables 12 to 14 show that examples 1 to 3 meet the above requirements for the stability of the reagent in the opened state, and that the reagent in the LA1 screening test and LA2 test were stable for at least 32 days when placed at 2 to 8 ℃ after the reagent was opened.
2) Accelerated stability: the LA1 screening reagent and LA2 confirmation reagent in the lupus anticoagulant assay kit of examples 1 to 3 and comparative example 2 were stored at 37 ℃ in an unopened state, and accuracy tests were performed on days 0, 12, 14, and 16, and the test results are shown in tables 15 to 18. The test result meets the requirements: in the lupus anticoagulant detection kit, the LA1 screening reagent and the LA2 confirmation reagent can be stored at 37 ℃ for 14 days in an unopened state, namely, the relative deviation is within the range of +/-7.5%.
TABLE 15 accelerated stability test results of the lupus anticoagulant assay kit of example 1
TABLE 16 accelerated stability test results of the lupus anticoagulant assay kit of example 2
TABLE 17 accelerated stability test results of the lupus anticoagulant assay kit of example 3
TABLE 18 accelerated stability test results of the lupus anticoagulant assay kit of comparative example 2
From the test results in tables 15 to 18, it was found that examples 1 to 3 satisfy the above requirement for accelerated stability, and that the LA1 screening reagent and the LA2 confirmed that the reagent can be stored stably at 37 ℃ for at least 16 days in an unopened state. The relative deviation of comparative example 2 was not met by the requirements of + -7.5% both measured on day 12, i.e., the LA1 screening reagent and LA2 confirmed that the reagents could not be stably stored at 37 ℃ for 12 days without being opened, and did not meet the requirement of accelerated stability, as can be seen from the results of example 1 and comparative example 2, the addition of a proper amount of the enzyme protein protectant, bamboo polysaccharide, facilitates the long-term use and storage of the kit, protects the activity of the enzyme, prevents the denaturation of thrombin, and provides the kit with excellent accelerated stability, and the kits of examples 1-3 have higher accelerated stability than comparative example 2.
4. Clinical sample testing and results thereof
A group of clinical samples (50 in total, 25 in each of negative and positive) were tested simultaneously by using the reference kit, the lupus anticoagulant assay kits of examples 1 to 3 and comparative example 3, and Kappa analysis was performed with the Normalized Ratio (NR) of the reference kit (siemens, lot number 638157) as the X-axis and the Normalized Ratio (NR) of the kits of examples 1 to 3 and comparative example 3 as the Y-axis, and the test results are shown in tables 19 to 20. The test result is required to be: the negative coincidence rate is more than or equal to 90 percent, the positive coincidence rate is more than or equal to 90 percent, the total coincidence rate is more than or equal to 90 percent, and the Kappa value is more than or equal to 0.75.
TABLE 19 statistics of the yin-yang coincidence rate of clinical samples tested in examples 1-2 and reference reagent
TABLE 20 statistics of the yin-yang coincidence rates of clinical specimens tested in example 3, comparative example 3 and reference reagents
The test results in tables 19-20 show that the reagent kit and the reference reagent in examples 1-3 can meet the requirements of both the yin-yang coincidence rate and the Kappa value of the clinical sample, and the comparative example 3 can not meet the requirements. The kit and the reference reagent in the embodiments 1-3 are proved to have higher consistent diagnosis rate, while the consistent diagnosis rate in the comparative example 3 is poorer, so that the condition of inconsistency between yin and yang is easy to occur, and the misdiagnosis probability is higher; further, when the recombinant snake venom enzyme and the recombinant phospholipid are used in a matching manner, the detection rate of a positive sample is improved, false negative is reduced, the sensitivity of the reagent is higher, the change amplitude in unit time is obviously enhanced when the positive sample is tested, and the detection capability of the reagent is higher when the weak positive sample is tested.
The kit can quickly and effectively detect lupus anticoagulant in a patient, is not easily influenced by endogenous and exogenous coagulation factors, has higher specificity compared with APTT and SCT tests, and can qualitatively evaluate whether the patient contains the lupus anticoagulant; the kit is a full-liquid reagent, and has the advantages of convenient use, low cost, high sensitivity, strong specificity, strong anti-interference capability and good stability. Can realize the replacement of an imported lupus anticoagulant detection kit (dRVVT method) and fully meet the requirement of clinical examination. The cost of purchasing detection reagents in hospitals can be reduced, the detection cost of patients is reduced, and the burden is reduced; the raw materials of the recombinant snake venom enzyme and the recombinant phospholipid are used, so that the sensitivity of the reagent can be improved, the solidification time of a positive patient (containing Wenyang) LA1 screening reagent is prolonged, the detection rate of a positive sample is improved, and false negative is reduced; the influence of heparin in a sample is reduced by adding polyacrylamide, and the influence of heparin on Xa and thrombin is prevented, so that the coagulation time is prolonged, and the deviation of a test result is large; the stability of the kit is effectively prolonged by adding a proper amount of bamboo polysaccharide, and the long-term use and storage of the kit are facilitated.
The data show that the results obtained by using the kit of the invention to carry out related performance tests all meet the acceptance standards. The feasibility and rationality of the present invention was demonstrated by the above specific embodiment, which is only illustrative of an alternative embodiment of the present invention, but not limiting thereto. All changes, modifications and substitutions that may be suggested to one skilled in the art and included within the scope of the appended claims are intended to be embraced by the invention.
Claims (16)
1. A lupus anticoagulant detection kit is characterized by comprising a LA1 screening reagent and a LA2 confirming reagent;
the LA1 screening reagent comprises recombinant snake venom enzyme, recombinant phospholipid, calcium ions, a first heparin neutralizer, a first buffer solution and a first auxiliary material, and the LA1 screening reagent is in a liquid state;
the LA2 confirmation reagent comprises recombinant snake venom enzyme, recombinant phospholipid, calcium ions, a second heparin neutralizer, a second buffer solution and a second auxiliary material, and the LA2 confirmation reagent is in a liquid state;
the content of the recombinant phospholipid in the LA1 screening reagent is lower than that in the LA2 confirmation reagent.
2. The kit according to claim 1, wherein the contents of the recombinant snake venom enzyme in the LA1 screening reagent and the LA2 confirmation reagent are both 1-3 IU/mL.
3. The kit according to claim 1, wherein the content of the recombinant phospholipid in the LA1 screening reagent is 0.02-0.06%, and the content of the recombinant phospholipid in the LA2 confirmation reagent is 0.4-0.8%.
4. The kit according to claim 1, wherein the first heparin neutralization agent comprises polyacrylamide, and the content of the polyacrylamide in the LA1 screening reagent is 1-3 mg/mL.
5. The kit according to claim 1, wherein the second heparin neutralization agent comprises polyacrylamide, and the content of the polyacrylamide in the LA2 confirmation reagent is 1-3 mg/mL.
6. The kit according to claim 1, wherein the first buffer solution and the second buffer solution are at least one of a PBS buffer solution, a Tris buffer solution, a Hepes buffer solution, a MES buffer solution, a MOPS buffer solution and a citric acid buffer solution, and the pH of the LA1 screening reagent and the LA2 confirmation reagent is 7.2-7.4.
7. The kit according to claim 6, wherein the first buffer solution and the second buffer solution are PBS buffer solutions with the concentration of 100-200 mM.
8. The kit of claim 1, wherein the first excipient comprises a first stabilizer, and the first stabilizer comprises one or more of glycine, polyethylene glycol 2000, Span-80, BSA, HSA, gelatin, tween-20, trehalose, glucose, β -cyclodextrin, and mannitol.
9. The kit of claim 8, wherein the first stabilizer comprises the components: glycine, polyethylene glycol 2000 and Span-80, wherein the components in the LA1 screening reagent comprise the following components in percentage by mass: 1-3% of glycine, 20001-5% of polyethylene glycol and-800.1-1% of Span.
10. The kit of claim 1, wherein the second excipient comprises a second stabilizer comprising one or more of glycine, polyethylene glycol 2000, Span-80, BSA, HSA, gelatin, tween-20, trehalose, glucose, β -cyclodextrin, and mannitol.
11. The kit according to claim 10, characterized in that said second stabilizer comprises the components: glycine, polyethylene glycol 2000 and Span-80, wherein the components in the LA1 screening reagent comprise the following components in percentage by mass: 1-3% of glycine, 20001-5% of polyethylene glycol and 800.1-1% of Span.
12. The kit of claim 1, wherein each of the LA1 screening reagent and the LA2 confirmation reagent comprises a preservative, and the preservative is at least one of sodium benzoate, sodium azide, Proclin-300, gentamicin, and nitrite.
13. The kit according to claim 12, wherein the LA1 screening reagent and the LA2 confirmation reagent each comprise 0.03-0.05% by mass of Proclin-300.
14. The kit according to claim 1, wherein the LA1 screening reagent and the LA2 confirmation reagent both comprise protease protection agents, and the mass percentages of the protease protection agents in the LA1 screening reagent and the LA2 confirmation reagent are 2-4%.
15. The kit of claim 14, wherein the protease protectant is bamboo polysaccharide.
16. The kit according to claim 1, wherein the content of the calcium ion in the LA1 screening reagent and the LA2 confirmation reagent is 10 to 20 mM.
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| CN117825681A (en) * | 2023-12-26 | 2024-04-05 | 米度医疗科技（中山）有限公司 | Full liquid reagent for lupus anticoagulant and preparation method thereof |
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