Reagent for testing platelet aggregation function and use thereof

Abstract

The present application relates to the technical field of in vitro detection, and particularly relates to a platelet aggregation function detection reagent and application thereof.The platelet aggregation function detection reagent provided by the present application comprises a fibrin activator and an ADP reagent, the fibrin activator adopts a novel high-purity porcine dermal collagen polypeptide component Prionex, and the combination with glycine significantly improves the stability of the fibrin activator, the platelet inhibitor tirofiban is used in combination with PEG6000, the accuracy of the detection result is improved, and the sensitivity to the anti-platelet aggregation drug is improved; the ADP reagent adopts chitosan, the activity and stability are improved, and the ADP reagent can be widely applied to clinical diagnosis of platelet aggregation function.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology, and in particular to reagents for detecting platelet aggregation function and their applications. Background Technology

[0002] Platelets are small, disc-shaped fragments of cytoplasm shed from the cytoplasm of mature megakaryocytes in the bone marrow. Their main functions are blood clotting and hemostasis, and repairing damaged blood vessels. The functional activity of platelets in the physiological hemostasis process can be roughly divided into two stages: the first stage mainly involves platelets rapidly adhering to the wound site after trauma and aggregating to form a relatively soft hemostatic embolus; the second stage mainly involves promoting blood clotting and forming a firm hemostatic embolus. Numerous studies have shown that platelet activation and aggregation are the initiating factors of thrombus formation in the body and one of the most critical components in the thrombus formation process. Therefore, platelet aggregation function testing can not only assess the diagnosis and treatment of thrombotic and hemorrhagic diseases, but also provide a preliminary evaluation of the clinical efficacy of antiplatelet aggregation drugs, offering excellent clinical guidance.

[0003] One of the most important pathways for platelet aggregation is the ADP pathway. ADP is the most important substance in the human body that induces platelet aggregation: on the one hand, after ADP binds to the platelet membrane receptor P2Y1, it couples with the Gq protein to activate phospholipase C, causing Ca2+ to accumulate in the platelet aggregates. 2+ Ca2+ flows from outside the cell into the cell; intracellular Ca2+ 2+ Increased concentration activates protein kinase C, causing platelet deformability and aggregation; on the other hand, ADP interacts with platelet P2Y. 12 It binds to membrane receptors, couples with Gi protein, inhibits the activation of adenylate cyclase AC, reduces cAMP levels in platelets, and induces platelet aggregation. Thromboelastography (TEG) uses whole blood as the test sample and can reflect the dynamic changes in blood coagulation (including the rate of fibrin formation, dissolution state, and coagulation strength and elasticity). It can perform continuous monitoring, accurately describe the patient's coagulation profile, and thus quickly determine whether the patient has coagulation dysfunction and analyze the cause.

[0004] Clopidogrel is a common drug that inhibits platelet aggregation and is used to prevent and treat cardiovascular, cerebrovascular, and other circulatory disorders caused by high platelet aggregation. Its mechanism of action is that clopidogrel can inhibit platelet aggregation via hepatic cytochrome P450. 450 Enzyme metabolism forms active substances, which irreversibly interact with platelet P2Y. 12Membrane receptors bind, block ADP on adenylyl cyclase AC inhibition, promote vasodilator phosphoprotein VASP phosphorylation, inhibit the activation of GPIIb / IIIa membrane receptors, thereby inhibiting platelet aggregation. However, there are variations in the response of platelets of different individuals to clopidogrel, about 5%-10% of patients still have thrombosis after taking the drug, and about 4%-30% of patients have clopidogrel resistance. Therefore, we need a precise and efficient method to detect platelet aggregation function, so as to take individualized anti-platelet therapy.

[0005] However, the platelet aggregation function detection reagent on the domestic market at present generally has poor stability, and the detection results are mostly false positives. In order to solve these problems, most manufacturers add saccharides or polymer lyophilization protectants to the detection reagent. In addition, in order to avoid the phenomenon of platelet activation caused by external factors, some manufacturers add platelet inhibitors to the fibrin activator, but do not fully consider the different reactivity of the diagnostic reagent to anti-platelet drugs, therefore, a platelet aggregation function detection reagent kit for ADP activation pathway with good stability, high accuracy and good sensitivity is still needed to be developed on the market. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide a platelet aggregation function detection reagent and its application. The present application provides a platelet aggregation function detection reagent with good stability of fibrin activator and ADP reagent, and sensitivity to anti-platelet aggregation drugs, and its application in thrombelastography.

[0007] The present application provides a platelet aggregation function detection reagent, which comprises a fibrin activator and an ADP reagent.

[0008] The working concentration of each component in the fibrin activator is: batroxobin 10-100 BU / mL, HFXIIIa 50-500 U / mL, sodium chloride 0.1%-1.5%(w / v), Prionex 0.5%-2.0%(w / v), trehalose 0.1%-1.0%(w / v), amino acids 0.1%-1.5%(w / v), surfactant 1%-10%(w / v), platelet inhibitor 0.001%-10%(w / v), Tris 10-100 mmol / L, and preservative 0.0001%-0.05%(w / v);

[0009] The working concentration of each component in the ADP reagent is: adenosine diphosphate or adenosine diphosphate sodium salt 2-200 μmol / L, sodium chloride 0.1%-1.5% (w / v), trehalose 0.2%-2.0% (w / v), chitosan 0.005%-0.1% (w / v), and preservative 0.005%-0.1% (w / v).

[0010] The w / v in the present application is mass / volume ratio, and the unit is g / mL.

[0011] Specifically, the working concentration in the present application is the optimal concentration of fibrin activator and ADP reagent in actual application, which can be in the form of lyophilized powder or 1-100 times of mother liquor, such as 10 times, 20 times or 50 times of mother liquor, and the present application does not make any limitation.

[0012] The platelet aggregation function detection reagent provided by the present application has good stability, high accuracy of detection results and good sensitivity, and is used in cooperation with a thrombelastograph to detect related coagulation indexes by taking whole blood as a sample.

[0013] In the detection reagent provided by the present application, the amino acid is at least one selected from glycine, lysine, histidine and valine;

[0014] The surfactant is at least one selected from PEG6000, PEG8000 and Tween 80;

[0015] The platelet inhibitor is at least one selected from tirofiban, abciximab, eptifibatide and ticagrelor;

[0016] The adenosine diphosphate sodium salt is at least one selected from adenosine diphosphate sodium, adenosine diphosphate disodium and adenosine diphosphate disodium salt.

[0017] Specifically, the working concentration of each component in the fibrin activator in the detection reagent provided by the present application is: batroxobin 50 BU / mL, HFXIIIa 200 U / mL, sodium chloride 0.6% (w / v), Prionex 1.0% (w / v), trehalose 0.6% (w / v), amino acid 0.5% (w / v), surfactant 1.0% (w / v), platelet inhibitor 0.005% (w / v), Tris 20 mmol / L, and preservative 0.0005% (w / v).

[0018] The working concentration of each component in the ADP reagent is: adenosine diphosphate or adenosine diphosphate sodium salt 150 μmol / L, sodium chloride 0.9% (w / v), trehalose 1.0% (w / v), chitosan 0.02% (w / v), and preservative 0.005% (w / v).

[0019] Specifically, the amino acid is glycine, the surfactant is PEG6000, the platelet inhibitor is tirofiban, and the preservative is gentamicin.

[0020] In some embodiments, the combination of Prionex and glycine can improve the stability of the fibrin activator; the combination of tirofiban and PEG6000 can avoid false positives in test results, improve the sensitivity to anti-platelet aggregation drugs, and the correlation coefficient between the MA value and the drug concentration reaches 0.996; chitosan can improve the activity and stability of the ADP reagent.

[0021] The working concentrations of the components in the fibrin activator provided by the application are as follows: batroxobin 50 BU / mL, HFXIIIa 200 U / mL, sodium chloride 0.6% (w / v), Prionex 1.0% (w / v), trehalose 0.6% (w / v), glycine 0.5% (w / v), PEG6000 1.0% (w / v), tirofiban 0.005% (w / v), Tris 20 mmol / L, and gentamicin 0.0005% (w / v);

[0022] The working concentrations of the components in the ADP reagent are as follows: adenosine diphosphate 150 μmol / L, sodium chloride 0.9% (w / v), trehalose 1.0% (w / v), chitosan 0.02% (w / v), and gentamicin 0.005% (w / v).

[0023] The pH value of the fibrin activator provided by the application is 7.0-8.0.

[0024] In some embodiments, the pH value of the fibrin activator is 7.5.

[0025] The application provides the use of the detection reagent in the preparation of a platelet aggregation function detection kit.

[0026] The application also provides a platelet aggregation function detection kit, which comprises the detection reagent and calcium chloride, sodium citrate, and heparin.

[0027] In some embodiments, the concentration of the calcium chloride is 0.2 mol / L, the concentration of the sodium citrate is 0.109 mol / mL, and the concentration of the heparin is not less than 14.5 IU / mL.

[0028] The application also provides a use method of the platelet aggregation function detection kit, which comprises the use of the detection reagent, and the steps include:

[0029] S1, detecting the blood clotting intensity data of the blood sample added with sodium citrate and calcium chloride by using the reconstituted fibrin activator and ADP reagent;

[0030] S2, detecting the blood clotting intensity data of the blood sample added with the fibrin activator and heparin;

[0031] S3, detecting the blood clotting intensity data of the blood sample added with the fibrin activator, ADP reagent and heparin;

[0032] In some embodiments, the method for using the platelet aggregation function detection kit comprises the following steps:

[0033] 1, re-warming the detection reagent to room temperature, adding 50 μL and 100 μL of distilled water in the fibrin activator and ADP reagent bottles respectively, and shaking to fully mix.

[0034] 2, opening the application software, inputting the patient information, selecting the corresponding test type, and loading the sample cups on the three channels of the thrombelastograph.

[0035] 3, adding 1 mL of citrated blood to the sodium citrate whole blood activator tube, and inverting and mixing for 5 times.

[0036] 4, sucking 20 μL of calcium chloride into the bottom of the sample cup of channel 1, sucking 340 μL of the blood sample activated by the above-mentioned sodium citrate whole blood activator into the cup, and clicking the button to start the test.

[0037] 5, sucking 10 μL of the reconstituted fibrin activator into the bottom of the sample cup of channel 2 and channel 3 respectively, and sucking 10 μL of the reconstituted ADP reagent into the bottom of the sample cup of channel 3.

[0038] 6, adding 360 μL of heparinized blood into the sample cups of channels 2 and 3 respectively, and blowing and sucking for 3 times, and quickly clicking the button to start the test after adding the sample.

[0039] 7, after the test is completed, clicking "stop" on the test interface, selecting the detection results of the three cups on the data interface, clicking "ADP inhibition rate", and obtaining the platelet inhibition rate.

[0040] The application also provides a preparation method of the detection reagent, comprising mixing all components of the fibrin activator and ADP reagent respectively, and vacuum freeze-drying to obtain the freeze-dried powder of the detection reagent.

[0041] The scheme provided by the present application has excellent stable protein characteristics of Prionex, and the combination of glycine improves the stability of fibrin activator; the combination of tirofiban and PEG6000 avoids false positive detection results, and improves the sensitivity of anti-platelet aggregation drugs; chitosan can promote the aggregation of platelets, release active substances, and has a synergistic effect with ADP, which can improve the activity and stability of ADP reagent.

[0042] The platelet aggregation function detection reagent provided by the present application can quantitatively and qualitatively detect the platelet aggregation function of patients taking platelet inhibiting drugs with whole blood as a sample, which has guiding significance for the clinical effect of platelet inhibiting drugs. DETAILED DESCRIPTION

[0043] The present application provides a platelet aggregation function detection reagent and its application. Those skilled in the art can refer to the content of the present application and appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of the present application without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0044] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market.

[0045] The present application will be further described below in combination with examples:

[0046] Preparation of fibrin activator and ADP reagent

[0047] 1. Preparation of fibrin activator

[0048] Ingredients: 10-100 BU / mL batroxobin, 50-500 U / mL HFXIIIa, 0.1%-1.5% (w / v) NaCl, 0.5%-2.0% (w / v) Prionex, 0.1%-1.5% (w / v) Gly, 1%-10% (w / v) PEG6000, 0.1%-1.0% (w / v) trehalose, 0.0001%-0.05% (w / v) gentamicin, 0.001%-10% (w / v) tirofiban, and 10-100 mmol / L Tris.

[0049] Preparation method: (1) Tris and NaCl are taken according to the formula amount, purified water is added to dissolve thoroughly, and the pH value is adjusted to 7.5;

[0050] (2) Take Prionex, Gly, PEG6000, trehalose according to the formula amount, add to the buffer solution in step (1), stir to fully dissolve;

[0051] (3) Take Batroxin and HFXIIIa according to the formula amount, add to the solution in step (2), adjust the pH value to 7.5;

[0052] (4) Take Gentamicin and Tirofiban according to the formula amount, add to the solution in step (3), fully dissolve to obtain fibrin activator;

[0053] (5) The fibrin activator obtained in step (4) is divided into 50 μL per bottle, vacuum freeze-dried to obtain fibrin activator freeze-dried powder.

[0054] 2. Preparation of ADP reagent

[0055] Ingredients: 2-200 μmol / L adenosine diphosphate (ADP), 0.1%-1.5% (w / v) NaCl, 0.2%-2.0% (w / v) trehalose, 0.005%-0.1% (w / v) gentamicin, and 0.005%-0.1% (w / v) chitosan.

[0056] Preparation method: (1) Take NaCl, trehalose, gentamicin, and chitosan according to the formula amount, add to purified water and fully dissolve;

[0057] (2) Take adenosine diphosphate according to the formula amount, add to the buffer solution in step (1), stir to fully dissolve;

[0058] (3) The ADP reagent obtained in step (2) is divided into 100 μL per bottle, vacuum freeze-dried to obtain ADP reagent freeze-dried powder.

[0059] Example 1 Optimization of fibrin activator and ADP reagent

[0060] 1. Optimization of fibrin activator

[0061] 1.1. Different components of fibrin activator

[0062] Component 1: 25 BU / mL Batroxin, 300 U / mL HFXIIIa, 0.9% (w / v) NaCl, 50 mM Tris (pH 7.0).

[0063] Component 2: 40 BU / mL Batroxin, 250 U / mL HFXIIIa, 0.9% (w / v) NaCl, 1.0% (w / v) Prionex, 1.0% (w / v) Gly, 50 mM Tris (pH 7.0).

[0064] Component 3: 50 BU / mL Batroxin, 200 U / mL HFXIIIa, 0.3% (w / v) NaCl, 0.5% (w / v) Prionex, 0.5% (w / v) Gly, 1.0% (w / v) Trehalose, 20 mM Tris (pH 7.0).

[0065] Component 4: 50 BU / mL Batroxin, 100 U / mL HFXIIIa, 0.6% (w / v) NaCl, 2.0% (w / v) Prionex, 0.5% (w / v) Gly, 0.6% (w / v) Trehalose, 0.005% (w / v) Gentamicin, 0.05% (w / v) Tirofiban, 20 mM Tris (pH 7.5).

[0066] Component 5: 50 BU / mL Batroxin, 200 U / mL HFXIIIa, 0.6% (w / v) NaCl, 1.0% (w / v) Prionex, 5.0% (w / v) Gly, 0.3% (w / v) Trehalose, 0.005% (w / v) Gentamicin, 0.005% (w / v) Tirofiban, 20 mM Tris (pH 7.5).

[0067] Component 6: 50 BU / mL Batroxin, 200 U / mL HFXIIIa, 0.6% (w / v) NaCl, 1.0% (w / v) Prionex, 2.0% (w / v) Gly, 0.3% (w / v) Trehalose, 0.0005% (w / v) Gentamicin, 5.0% (w / v) PEG6000, 0.005% (w / v) Tirofiban, 20 mM Tris (pH 7.5).

[0068] Component 7: 50 BU / mL Batroxin, 200 U / mL HFXIIIa, 0.6% (w / v) NaCl, 1.0% (w / v) Prionex, 0.5% (w / v) Gly, 0.6% (w / v) Trehalose, 0.0005% (w / v) Gentamicin, 1.0% (w / v) PEG6000, 0.005% (w / v) Tirofiban, 20 mM Tris (pH 7.5).

[0069] 1.2, Screening of the Optimal Component of Fibrin Activator

[0070] The blood samples (heparin anticoagulation) of the same person were tested by using components 1-7, and a certain imported reagent with the similar detection principle and the present application as the comparison reagent. The results are shown in Table 1.

[0071] Relative deviation (%) = [(average value of the example-average value of the comparison reagent) / average value of the comparison reagent]*100%

[0072] Table 1

[0073]

[0074] The results show that the relative deviation of each parameter of component 7 from the comparison reagent is the smallest, and therefore the fibrin activator of the present application is preferably component 7.

[0075] 2. Optimization of ADP reagent

[0076] 2.1 Different components of ADP reagent

[0077] Component 8: 100 μM ADP, 0.9% (w / v) NaCl, 2.0% (w / v) trehalose.

[0078] Component 9: 200 μM ADP, 0.3% (w / v) NaCl, 2.0% (w / v) trehalose, 0.05% (w / v) gentamicin.

[0079] Component 10: 150 μM ADP, 0.9% (w / v) NaCl, 1.0% (w / v) trehalose, 0.05% (w / v) gentamicin, 0.02% (w / v) chitosan.

[0080] Component 11: 100 μM ADP, 0.6% (w / v) NaCl, 0.5% (w / v) trehalose, 0.005% (w / v) gentamicin, 0.05% (w / v) chitosan.

[0081] Component 12: 150 μM ADP, 0.9% (w / v) NaCl, 1.0% (w / v) trehalose, 0.005% (w / v) gentamicin, 0.02% (w / v) chitosan.

[0082] 2.2 Screening of the optimal component of ADP reagent

[0083] The blood samples (heparin anticoagulation) of the same person were detected by components 8-12, and a certain imported reagent was used as the comparison reagent. The results are shown in Table 2.

[0084] Relative deviation (%) = [(average of examples - average of comparison reagent) / average of comparison reagent] * 100%

[0085] Table 2

[0086]

[0087] The results show that the relative deviation of each parameter of component 12 from the comparison reagent is the smallest, and therefore the ADP reagent of the present application is preferably component 12.

[0088] Example 2 Accuracy detection

[0089] The same blood sample (heparin anticoagulation) of one person was detected for 10 times by component 3 and component 7 respectively. The results are shown in Table 3.

[0090] Table 3

[0091]

[0092]

[0093] The results show that in 10 detections, component 3 has 3 false positive results (No. 3, 5, 8); and component 7 has no false positive, and the CV of each parameter value obtained by detection is within 10%, which proves that the combination of tirofiban and PEG6000 can avoid false positive detection results and improve the accuracy of measurement results.

[0094] Example 3 correlation test

[0095] The same blood sample (heparin anticoagulation and adding different concentrations of dipyridamole) of one person was detected by component 7 and component 12, and an imported reagent was used as a comparison reagent. The results are shown in Table 4 (MA represents blood clotting intensity).

[0096] Table 4

[0097] Dipyridamole concentration Reagent ADP cup MA (mm) Reagent ADP cup MA (mm) 0 μg / mL 58.0 56.0 1 μg / mL 51.1 49.1 2 μg / mL 47.3 48.1 3 μg / mL 43.4 42.6 4 μg / mL 37.4 34.3 5 μg / mL 32.5 30.5 Correlation coefficient (r) 0.996 0.985

[0098] The results show that the concentration of the fibrin activator and ADP reagent of the application and the anti-platelet aggregation drug dipyridamole has good correlation, the correlation coefficient r is 0.996, which is better than the comparison reagent, and can accurately evaluate the effect of the patient taking the drug and guide the clinical drug dosage.

[0099] Example 4 stability test

[0100] The reagent of the application and the comparison reagent were both placed in a 2-8℃ environment for 10 days, and the same blood sample (heparin anticoagulation) of one person was detected at regular intervals. The results are shown in Table 5.

[0101] Table 5

[0102]

[0103] The results show that the fibrin activator and ADP reagent of the application have good stability within 10 days, and the relative range of MA values obtained by detection is within 10%, which is much better than the comparison reagent.

[0104] Example 5 precision test

[0105] The same blood sample (sodium citrate and heparin anticoagulation, and adding a certain amount of dipyridamole) of one person was detected for 10 times by the same batch of platelet aggregation function detection reagent, and the detection results are shown in Table 6.

[0106] The detection method is as follows:

[0107] 1. Warm the detection reagent to room temperature, and add 50 μL and 100 μL of distilled water to the fibrin activator and ADP reagent bottles respectively, and shake to mix thoroughly.

[0108] 2. Open the application software, input the patient information, and select the corresponding test type.

[0109] 3. Load the sample cups on the three channels of the thrombelastograph.

[0110] 4. Add 1 mL of citrated blood to the sodium citrate whole blood activator tube, and invert to mix thoroughly 5 times.

[0111] 5. Take 20 μL of calcium chloride and add to the bottom of the sample cup in channel 1, take 340 μL of the above-mentioned sodium citrate whole blood activator activated blood sample and add to the cup, and click the button to start the test.

[0112] 6. Take 10 μL of the reconstituted fibrin activator and add to the bottom of the sample cups in channels 2 and 3 respectively.

[0113] 7. Take 10 μL of the reconstituted ADP reagent and add to the bottom of the sample cup in channel 3.

[0114] 8. Add 360 μL of heparinized blood to the sample cups in channels 2 and 3 respectively, and blow and suck to mix 3 times. After adding the sample, quickly click the button to start the test.

[0115] 9. After the test is completed, click “stop” on the test interface, remove the sample cups, and dispose of the medical waste.

[0116] 10. Select the detection results of the three cups in the data interface, click “ADP inhibition rate”, and obtain the platelet inhibition rate.

[0117] The platelet inhibition rate calculation formula is as follows:

[0118]

[0119] MA(ADP) is the blood clotting intensity of heparin anticoagulated whole blood added with fibrin activator and platelet activator (ADP); MA(fibrin) is the blood clotting intensity of heparin anticoagulated whole blood added with only fibrin activator; and MA(total) is the blood clotting intensity of sodium citrate anticoagulated whole blood added with sodium citrate whole blood activator.

[0120] Table 6

[0121]

[0122] The results show that the CV of the MA value obtained by the kit is within 5%, and the CV of the platelet inhibition rate is also within 5%, which indicates that the detection precision of the platelet aggregation function detection kit is good.

[0123] The above is only the preferred embodiment of the present application, and it should be pointed out that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A reagent for detecting platelet aggregation function, characterized in that, It includes fibrin activator and ADP reagent; The working concentrations of each component in the fibrin activator are as follows: batroxobin 50 BU / mL, HFXIIIa 200 U / mL, sodium chloride 0.6% (w / v), Prionex 1.0% (w / v), trehalose 0.6% (w / v), glycine 0.5% (w / v), PEG6000 1.0% (w / v), tirofiban 0.005% (w / v), Tris 20 mmol / L, and gentamicin 0.0005% (w / v); The working concentrations of each component in the ADP reagent are: adenosine diphosphate 150 μmol / L, sodium chloride 0.9% (w / v), trehalose 1.0% (w / v), chitosan 0.02% (w / v), and gentamicin 0.005% (w / v).

2. The detection reagent according to claim 1, characterized in that, The pH value of the fibrin activator is 7.0 to 8.

0.

3. The use of the detection reagent according to claim 1 or 2 in the preparation of a platelet aggregation function detection kit.

4. A platelet aggregation function detection kit, characterized in that, It includes the detection reagent as described in claim 1 or 2, as well as calcium chloride, sodium citrate, and heparin.

5. The method of using the platelet aggregation function detection kit according to claim 4, characterized in that, It includes the following steps: S1, reconstituted fibrin activator and ADP reagent, were used to detect the blood coagulation strength data of blood samples with added sodium citrate and calcium chloride; S2. Detect the blood coagulation strength data of blood samples with added fibrin activator and heparin; S3. Detect the blood coagulation strength data of blood samples with added fibrin activator, ADP reagent and heparin.

6. The method for preparing the detection reagent according to claim 1 or 2, characterized in that, It includes mixing all components of the fibrin activator and ADP reagent separately and then freeze-drying them under vacuum to obtain the lyophilized powder of the detection reagent.

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