Alkaline phosphatase detection reagent

Abstract

The invention discloses an alkaline phosphatase detection reagent which comprises the following components: a buffer solution, 2-amino-2-methyl-propanol, disodium p-nitrophenyl phosphate, ethylenediamine tetraacetic acid disodium salt, magnesium chloride, zinc sulfate, a surfactant, a preservative, an excipient and a stabilizer. According to the invention, zinc sulfate and magnesium chloride are used as enzyme activators and play a synergistic role, and meanwhile, an effective stabilizer component is added, so that the reaction activity of alkaline phosphatase in the determination reagent is improved, high activity and good stability are ensured when the concentration of alkaline phosphatase is detected, and the accuracy of a detection result is improved.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and particularly to an alkaline phosphatase detection reagent. Background Art

[0002] Alkaline phosphatase (ALP) is an enzyme present in many tissues of the human body, especially in relatively high amounts in the liver, bone, kidney, and small intestine. Therefore, the level of alkaline phosphatase in the blood can reflect the health status of these organs. For example, cirrhosis, hepatitis, liver cancer, or biliary obstruction, osteoporosis, fractures, bone tumors, certain nutritional deficiencies such as vitamin D deficiency, certain kidney diseases, malignant tumors, heart diseases, etc. may all cause an increase in the level of alkaline phosphatase in the blood. Therefore, detecting the level of alkaline phosphatase can help doctors diagnose and monitor the progression of the above diseases. However, due to the wide source of alkaline phosphatase, if the test result is abnormal, further examinations may be required to determine the specific cause.

[0003] The enzyme kinetics method is one of the most commonly used methods for detecting alkaline phosphatase. It is based on the catalytic action of alkaline phosphatase on the substrate, and determines the enzyme activity by measuring the production rate of the metabolite of the substrate or the concentration of the product at the end point of the reaction. Commonly used substrates include p-nitrophenyl phosphate (p-NPP) and p-nitrophenyl phosphate (PNPP). These substrates are catalytically hydrolyzed by alkaline phosphatase under alkaline conditions to produce p-nitrophenol or p-nitrobenzene, and their absorbance can be measured at a specific wavelength.

[0004] The enzyme kinetics method can provide a highly sensitive measurement of the activity of alkaline phosphatase. This enables the enzyme kinetics method to detect low concentrations of alkaline phosphatase and provide accurate results even at the early stages of the disease. The enzyme kinetics method has relatively simple experimental procedures and lower costs compared to other complex techniques such as immunoassays. The substrates it uses are also relatively inexpensive and easily accessible.

[0005] However, during the detection process of alkaline phosphatase, the detection reagent may be affected by various substances, such as certain drugs, antibiotics, metal ions, etc. These substances may interfere with the detection activity of the detection reagent, resulting in an underestimation or overestimation of the alkaline phosphatase activity; or, there may be the activity of other enzymes in the sample, and these enzymes may act on the substrate together with alkaline phosphatase, reducing the detection activity of the detection reagent and resulting in a decrease in the accuracy of the measurement result of the alkaline phosphatase activity. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an alkaline phosphatase detection reagent to solve the problem of reduced accuracy of detection results in the prior art.

[0007] To solve the above problems, the present invention adopts the following solutions:

[0008] An alkaline phosphatase detection reagent, comprising the following components: buffer, 2-amino-2-methyl-1-propanol, disodium 4-nitrophenyl phosphate, disodium ethylenediaminetetraacetate, magnesium chloride, zinc sulfate, surfactant, preservative, excipient, stabilizer.

[0009] Further, the concentration of the buffer is 0.2 - 2 mol / L;

[0010] The concentration of the 2-amino-2-methyl-1-propanol is 10 - 500 mmol / L;

[0011] The concentration of the disodium 4-nitrophenyl phosphate is 50 - 450 mmol / L;

[0012] The concentration of the disodium ethylenediaminetetraacetate is 50 - 450 mmol / L;

[0013] The concentration of the magnesium chloride is 50 - 500 mmol / L;

[0014] The concentration of the zinc sulfate is 50 - 500 mmol / L;

[0015] The volume percentage of the surfactant is 0.01% - 8% (v / v);

[0016] The volume percentage of the preservative is 0.01% - 8% (v / v);

[0017] The concentration of the excipient is 10 - 100 g / L;

[0018] The concentration of the stabilizer is 1 - 10 g / L.

[0019] Further, the buffer is one or a combination of more than one of tris(hydroxymethyl)aminomethane buffer, phosphate buffer, imidazole buffer, ethanesulfonic acid buffer, glycine buffer.

[0020] Further, the surfactant is one or a combination of more than one of Tween - 40, Tween - 80, Tween - 20, Triton X - 100.

[0021] Further, the preservative is one or a combination of more than one of PC300, potassium sorbate, parabens.

[0022] Further, the excipient is one or a combination of more than one of mannitol, PEG - 6K (polyethylene glycol 600), xylitol, PVP, dextran 10,000, dextran 40,000, dextran 50,000.

[0023] Further, the stabilizer is one or a combination of more than one of BSA, D - sorbitol, fructose, xylitol, glycerol.

[0024] The invention adopts the above technical solutions and has the following advantages:

[0025] The present invention uses zinc sulfate and magnesium chloride as enzyme activators, which work synergistically. At the same time, effective stabilizer components are added to avoid the interference of other substances in the sample on the activity of alkaline phosphatase, ensuring good effect and high stability when detecting the concentration of alkaline phosphatase, and improving the accuracy of detecting alkaline phosphatase. Brief Description of the Drawings

[0026] Figure 1 This is the linear regression equation for sample detection of the present invention. Detailed Embodiments

[0027] The following further illustrates the present invention through examples, but it is not a limitation of the present invention. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, as long as it does not depart from the technical solution content of the present invention, any modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

[0028] Example 1

[0029] An alkaline phosphatase detection reagent, comprising the following components: Tris-HCl (Tris-hydroxymethylaminomethane hydrochloride buffer solution), 0.2 mol / L; 2-amino-2-methyl-propanol, 10 mmol / L; p-nitrophenyl phosphate disodium, 50 mmol / L; disodium ethylenediaminetetraacetate, 50 mmol / L; magnesium chloride, 50 mmol / L; zinc sulfate, 50 mmol / L; Triton X-100 (Triton-100), 0.01%; PC 300, 0.01%; mannitol, 5 g / L; PEG-6K, 5 g / L; fructose, 1 g / L.

[0030] Example 2

[0031] An alkaline phosphatase detection reagent, comprising the following components: ethane sulfonic acid buffer solution, 0.5 mol / L; 2-amino-2-methyl-propanol, 30 mmol / L; p-nitrophenyl phosphate disodium, 80 mmol / L; disodium ethylenediaminetetraacetate, 80 mmol / L; magnesium chloride, 80 mmol / L; zinc sulfate, 80 mmol / L; Tween-20, 2%; nipagin ester, 1%; xylitol, 20 g / L; PVP (polyvinylpyrrolidone K23-27) 10 g / L, 5%; D-sorbitol, 3 g / L; glycerol, 2 g / L.

[0032] Example 3

[0033] An alkaline phosphatase detection reagent, comprising the following components: glycine buffer solution, 2 mol / L; 2-amino-2-methyl-1-propanol, 500 mmol / L; disodium p-nitrophenyl phosphate, 450 mmol / L; disodium ethylenediaminetetraacetate, 450 mmol / L; magnesium chloride, 500 mmol / L; zinc sulfate, 500 mmol / L; Tween-80, 6%; Triton X-100, 2%; potassium sorbate, 8%; dextran 10,000, 100 g / L; BSA (bovine serum albumin), 10 g / L.

[0034] Example 4

[0035] The performance of the detection reagent prepared in the examples of the present invention was evaluated by detecting alkaline phosphatase, including linear range, repeatability, accuracy and stability evaluation.

[0036] The detection reagent of the present invention is prepared in the form of microspheres and placed in a chip to form a detection chip. The alkaline phosphatase is detected by the detection chip. The preparation method of the detection reagent in the form of microspheres includes:

[0037] Add the buffer solution to a preset amount of water. After the buffer solution is completely dissolved in water, add 2-amino-2-methyl-1-propanol and adjust the pH value to 10.5 - 11.5. Add excipients, stabilizers, surfactants and preservatives to the solution in turn, and then add disodium p-nitrophenyl phosphate, disodium ethylenediaminetetraacetate, magnesium chloride, and zinc sulfate to obtain the detection reagent; drop the detection reagent into liquid nitrogen through a bead dropping device to make the detection reagent form ice balls, and freeze-dry the ice balls to obtain the detection reagent in the form of microspheres. The microspheres are spherical and the volume is between 3.0 μL and 4.0 μL.

[0038] During detection, place the detection reagent in the form of microspheres in the chip body to prepare a detection chip for alkaline phosphatase detection. The test temperature is 37 °C, the main detection wavelength is 410 nm, and the secondary wavelength is 520 nm.

[0039] The microspheres prepared from the detection reagents of Example 1, Example 2 and Example 3 of the present invention were used as the experimental group to illustrate the relevant performance.

[0040] 1. Linear range

[0041] The test method is as follows: use a low-concentration sample close to the lower limit of the linear range (0 - 20 U / L) and a high-concentration sample close to the upper limit of the linear range (900 - 1000 U / L). Mix the low-concentration sample and the high-concentration sample in proportion to form 5 samples. The mixing ratios of each sample are shown in Table 1:

[0042] Table 1 shows the mixing ratios of the high-concentration sample and the low-concentration sample in 5 samples

[0043] Sample number 1 2 3 4 5 High-concentration sample 0 20％ 40％ 60％ 100％ Low-concentration sample 100％ 80％ 60％ 40％ 0

[0044] The microspheres prepared with the detection reagent of the present invention were used to test the alkaline phosphatase concentrations of 5 plasma samples, and each plasma sample was tested 3 times. The average value (y) of the measured alkaline phosphatase concentration values in the 5 plasma samples was calculated respectively. Taking the diluted concentration (x) of each sample as the independent variable and the average value (y) of the measured concentration value of each sample as the dependent variable, a linear regression equation was obtained. As Figure 1 shown, it can be found that R2 > 0.99 in the concentration range of 0 - 1000 U / L, indicating that the detection reagent of the present invention has a good linear range.

[0045] 2. Repeatability

[0046] The detection chips prepared with the detection reagent of the same batch were used to perform 10 repeated measurements on plasma samples with different concentrations, and the total average value of the measured values and the within - batch standard deviation (S) were calculated respectively, and the within - batch coefficient of variation (CV) was calculated. The results are shown in Table 2.

[0047] Table 2: Repeatability test results of samples with different concentrations

[0048]

[0049] It can be found from Table 2 that for the low - and medium - value samples (concentration less than or equal to 300 U / L) tested with the detection reagent of Examples 1 - 3 of the present invention, CV < 3.5%, and for the high - value samples (concentration greater than 300 U / L), CV < 3%. The detection reagent of the present invention has good repeatability.

[0050] 3. Accuracy

[0051] The alkaline phosphatase concentration detected by the detection reagent of Examples 1 - 3 of the present invention was compared with the target concentration, and the deviation between the two was calculated. The accuracy of the detection reagent of the present invention was evaluated through the deviation.

[0052] Table 3: Deviation between the detected concentration and the target concentration of different examples of the present invention

[0053]

[0054] It can be found from Table 3 that for the low - value samples (concentration less than or equal to 100 U / L), the accuracy deviation of the detection reagent of Examples 1 - 3 of the present invention < ±10%, and for the medium - and high - value samples (concentration greater than 100 U / L), the accuracy deviation < ±5%. It is proved that the detection reagent of the examples of the present invention has high accuracy when detecting samples.

[0055] 4. Stability

[0056] Comparative Example 1: The difference in the preparation raw materials from Example 1 is that it does not contain magnesium chloride and zinc sulfate, and other conditions are the same.

[0057] Comparative Example 2: The difference in the preparation raw materials from Example 1 is that it does not contain a stabilizer, and other conditions are the same.

[0058] Comparative Example 3: The difference in the preparation raw materials from Example 1 is that it does not contain an excipient, and other conditions are the same.

[0059] The lyophilized balls prepared from the detection reagents of Examples 1 - 3 of the present invention and the lyophilized balls prepared from Comparative Examples 1 - 3 were subjected to an accelerated test at 37°C. The change in the concentration of the above lyophilized balls relative to the original value was detected every day to measure their stability. The concentration changes are shown in Table 4.

[0060] Table 4: Concentration changes of different lyophilized balls

[0061]

[0062]

[0063] As can be seen from Table 4, the detection reagents of Examples 1 - 3 of the present invention can be stored stably at 37°C for at least 14 days, and their stability is significantly better than that of the comparative examples. Comparative Example 1 lacks zinc sulfate and magnesium chloride, and its stability deteriorates slightly; Comparative Examples 2 and 3 lack a stabilizer and an excipient respectively, and their stability is greatly affected. The data show that adding a stabilizer and an excipient to the reagent can promote the stability of the lyophilized balls, and this promoting effect is superimposed. Substances such as excipients and stabilizers are the key factors for maintaining the stability of the detection reagent of the present invention. The lack of them will significantly reduce the stability.

[0064] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An alkaline phosphatase detection reagent, characterized in that, It comprises the following components: buffer solution, 2-amino-2-methyl-1-propanol, disodium 4-nitrophenyl phosphate, disodium ethylenediaminetetraacetate, magnesium chloride, zinc sulfate, surfactant, preservative, excipient, stabilizer.

2. The alkaline phosphatase detection reagent according to claim 1, characterized in that, The concentration of the buffer solution is 0.2 - 2 mol / L; The concentration of the 2-amino-2-methyl-1-propanol is 10 - 500 mmol / L; The concentration of the disodium 4-nitrophenyl phosphate is 50 - 450 mmol / L; The concentration of the disodium ethylenediaminetetraacetate is 50 - 450 mmol / L; The concentration of the magnesium chloride is 50 - 500 mmol / L; The concentration of the zinc sulfate is 50 - 500 mmol / L; The volume percentage of the surfactant is 0.01% - 8% (v / v); The volume percentage of the preservative is 0.01% - 8% (v / v); The concentration of the excipient is 10 - 100 g / L; The concentration of the stabilizer is 1 - 10 g / L.

3. The alkaline phosphatase detection reagent according to claim 1, characterized in that, The buffer solution is one or a combination of more than one of tris(hydroxymethyl)aminomethane buffer solution, phosphate buffer solution, imidazole buffer solution, ethanesulfonic acid buffer solution, glycine buffer solution.

4. The alkaline phosphatase detection reagent according to claim 1, characterized in that, The surfactant is one or a combination of more than one of Tween-40, Tween-80, Tween-20, Triton X-100.

5. The alkaline phosphatase detection reagent according to claim 1, characterized in that, The preservative is one or a combination of more than one of PC300, potassium sorbate, parabens.

6. The alkaline phosphatase detection reagent according to claim 1, characterized in that, The excipient is one or a combination of more than one of mannitol, PEG-6K, xylitol, PVP, dextran 10,000, dextran 40,000, dextran 50,000.

7. The alkaline phosphatase detection reagent according to claim 1, characterized in that, The stabilizer is one or a combination of more than one of BSA, D-sorbitol, fructose, xylitol, glycerol.

Images