Sample diluent for chemiluminescence immunoassay as well as preparation method and application of sample diluent

Abstract

The invention provides a sample diluent for chemiluminescence immunoassay as well as a preparation method and application thereof, and relates to the field of chemiluminescence immunoassay. The sample diluent for chemiluminescence immunoassay comprises the following components in percentage by mass: 0.1-5% of a protein sealing agent, 0.1% of isodecanol polyoxyethylene ether, 2% of glycerol, 0.05-0.5% of a preservative, 0.001-0.1% of a defoaming agent and the balance of a solvent, wherein the solvent is a buffer solution. By limiting the ratio of the components and using the defoaming agent with lower concentration, a better defoaming effect is achieved, bubbles in the diluted reaction liquid can be eliminated, the sampling accuracy of an instrument is improved, and the influence of the defoaming agent on immune reaction is reduced. The diluent is matched with a chemiluminescence immunoassay kit for use, so that the sensitivity and the accuracy are effectively improved, and a plurality of items can be compatible. The sample diluent effectively reduces non-specific reaction in a sample, reduces the cost value, and has good stability.

Description

Technical Field

[0001] The present invention relates to the field of chemiluminescence immunoassay, and particularly to a sample diluent for chemiluminescence immunoassay, a preparation method thereof, and an application thereof. Background Art

[0002] A sample diluent is a functional buffer solution with a specific pH value and osmotic pressure used to dilute and liquefy a sample to be tested so that it can be detected by in vitro diagnostic reagents or instruments. It is mainly used to detect samples that exceed the detection range in an immune reaction after dilution and obtain accurate and reliable results. An effective sample diluent can help reduce false positives and reduce background interference in the sample through dilution.

[0003] Currently, the commonly used sample diluents still have the following problems: (1) There is a matrix effect problem. There are various types of sample diluents for chemiluminescence immunoassay. Different matrix components, pH selections, etc. will interfere with the sample detection, resulting in inaccurate results or poor repeatability; (2) Poor versatility, the sample diluent is not applicable between different projects. (3) Bubbles are easily generated. For the purpose of reducing non-specificity and matrix effect, a certain concentration of protein and surfactant components are added to the sample diluent on the market, which makes the sample diluent extremely prone to generating bubbles. On the one hand, if not detected in time during operation, it will affect the accuracy of the next detection result; on the other hand, it will trigger an error report or suspension of a fully automatic chemiluminescence immunoassay analyzer with a bubble detection function, thereby delaying the experimental results. Therefore, there is an urgent need for a stable sample diluent with weak matrix effect and strong versatility. Summary of the Invention

[0004] The purpose of the present invention is to provide a sample diluent for chemiluminescence immunoassay, a preparation method thereof, and an application thereof, so as to solve the problems of strong matrix effect, poor versatility, and easy generation of bubbles existing in the existing sample diluent in chemiluminescence immunoassay.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides a sample diluent for chemiluminescence immunoassay, comprising the following components in mass fractions: Protein blocker 0.1 - 5%, 0.1% isomeric decanol polyoxyethylene ether, 2% glycerol, preservative 0.05 - 0.5%, defoaming agent 0.001 - 0.1%, and the balance of solvent; The solvent is a buffer solution.

[0006] Preferably, the protein blocker includes one or more of bovine serum albumin, casein, bovine serum, and goat serum.

[0007] Preferably, the preservative is ProClin950 and / or ProClin 300.

[0008] Preferably, the defoaming agent is a polydimethylsiloxane defoaming agent.

[0009] Preferably, the buffer solution is one of phosphate buffer solution, Tris-HCl buffer solution, and citric acid-disodium hydrogen phosphate buffer solution; The pH value of the buffer solution is 6.0 - 8.0; The mass concentration of the buffer in the buffer solution is 0.6 - 1.5 wt%.

[0010] The present invention also provides a method for preparing the sample diluent for chemiluminescent immunoassay as described above, comprising the following steps: Mix the protein blocking agent, surfactant, preservative, defoaming agent, and buffer solution to obtain the sample diluent for chemiluminescent immunoassay.

[0011] The present invention also provides an application of the sample diluent for chemiluminescent immunoassay as described above in chemiluminescent immunoassay.

[0012] The present invention has at least the following beneficial effects: In the sample diluent provided by the present invention, the protein blocking agent (casein) can reduce non-specific reactivity and the background noise of the sample diluent; glycerol has a good wetting effect, forms hydrogen bonds and van der Waals forces with the amino acid residues on the surface of protein molecules. Adding an appropriate amount of glycerol can improve the stability of the protein and enhance the long-term stability of the sample diluent. The present invention achieves the defoaming effect by selecting the ratio of key components and using a lower concentration of defoaming agent, which not only effectively reduces the influence of adding defoaming agent on the immune reaction, but also eliminates the bubbles in the diluted reaction solution to improve the accuracy of instrument sample addition. At the same time, it is beneficial for the full-automatic chemiluminescent immunoassay analyzer to realize intelligent management and reduce the operation difficulty of experimental personnel.

[0013] The present invention is used in conjunction with the chemiluminescent immunoassay kit to effectively improve sensitivity, accuracy and be compatible with multiple items.

[0014] The sample diluent effectively reduces the non-specific reaction in the sample, reduces the background value; and has good stability and can be stored for a long time without quality change. Specific Embodiments

[0015] The present invention provides a sample diluent for chemiluminescent immunoassay, comprising the following components by mass fraction: Protein blocker 0.1 - 5%, isodecyl polyoxyethylene ether 0.1%, glycerol 2%, preservative 0.05 - 0.5%, defoamer 0.001 - 0.1% and the balance solvent, preferably protein blocker 0.5 - 4%, surfactant 0.05 - 0.4%, isodecyl polyoxyethylene ether 0.1%, glycerol 2%, defoamer 0.01 - 0.09% and the balance solvent, further preferably protein blocker 1 - 3%, isodecyl polyoxyethylene ether 0.1%, glycerol 2%, preservative 0.15 - 0.4%, defoamer 0.02 - 0.08% and the balance solvent, more preferably protein blocker 2%, isodecyl polyoxyethylene ether 0.1%, glycerol 2%, preservative 0.2 - 0.3%, defoamer 0.04 - 0.06% and the balance solvent; The solvent is a buffer solution.

[0016] In the present invention, the protein blocker includes one or more of bovine serum albumin, casein, bovine serum and goat serum.

[0017] In the present invention, the surfactant is preferably isodecyl polyoxyethylene ether. This is because isodecyl polyoxyethylene ether has good dispersibility, solubilization and certain defoaming properties. This enables it to not only meet the requirement of the surfactant to reduce the non - specific adsorption of impurities in the sample and the detection antibody, reducing background noise, but also its dispersibility and solubilization to reduce protein aggregation, making the detection have good repeatability and reproducibility; and when used in combination with a very low concentration of defoamer, it can achieve a defoaming effect for the sample diluent, while other surfactants have foaming properties, which will have an adverse impact on subsequent detections.

[0018] In the present invention, the preservative is ProClin950 and / or ProClin 300.

[0019] In the present invention, the defoamer is a polydimethylsiloxane - based defoamer, and can be one or more of defoamer A, defoamer B and defoamer C; Among them, defoamer A is purchased from SIGMA, and the product number is A5633; Among them, defoamer B is purchased from SIGMA, and the product number is A5757; Among them, defoamer C is purchased from SIGMA, and the product number is A8011.

[0020] In the present invention, the buffer solution is one of phosphate buffer solution, Tris - HCl buffer solution and citric acid - disodium hydrogen phosphate buffer solution.

[0021] In the present invention, the pH value of the buffer solution is 6.0 - 8.0; When the buffer solution is a phosphate buffer solution, the pH value is preferably 6.0 - 7.5, more preferably 6.3 - 7.2, still more preferably 6.5 - 7.0, and even more preferably 6.8; When the buffer solution is a Tris-HCl buffer solution, the pH value is preferably 7.0 - 8.0, more preferably 7.2 - 7.8, still more preferably 7.5; When the buffer solution is a citric acid-disodium hydrogen phosphate buffer solution, the pH value is preferably 6.0 - 7.5, more preferably 6.3 - 7.2, still more preferably 6.5 - 7.0, and even more preferably 6.8.

[0022] In the present invention, the mass concentration of the buffer in the buffer solution is 0.2 - 3.5 wt%, preferably 0.5 - 3 wt%, more preferably 1 - 2.5 wt%, and even more preferably 1.5 - 2 wt%.

[0023] The present invention also provides a method for preparing the sample diluent for chemiluminescent immunoassay as described above, comprising the following steps: Mix a protein blocker, a surfactant, a preservative, an antifoaming agent, and a buffer solution to obtain the sample diluent for chemiluminescent immunoassay.

[0024] The present invention also provides an application of the sample diluent for chemiluminescent immunoassay as described above in chemiluminescent immunoassay.

[0025] The technical solutions provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example 1 By mass percentage, measure 0.5% casein, 0.1% isodecyl polyoxyethylene ether, 2% glycerol, 0.2% ProClin 300 (ProClin 300 preservative is purchased from SIGMA and consists of 3% 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT), dissolved in a salt-free proprietary ethylene glycol stabilizer containing alkyl carboxylates), 0.005% antifoaming agent B (SIGMA, product number A5757), 0.24% citric acid monohydrate, 1.38% disodium hydrogen phosphate dodecahydrate, and the balance water; Mix water, citric acid monohydrate, and disodium hydrogen phosphate dodecahydrate to obtain a citric acid-disodium hydrogen phosphate buffer solution with a pH value of 6.8; Then dissolve casein, isodecyl polyoxyethylene ether, glycerol, isothiazolinone, and antifoaming agent B in the citric acid-disodium hydrogen phosphate buffer solution to obtain the sample diluent.

[0027] Example 2

[0028] Measure 0.5% casein, 0.1% isomeric decyl alcohol polyoxyethylene ether, 2% glycerol, 0.1% ProClin 300, 0.005% antifoaming agent B, 0.40% sodium dihydrogen phosphate dihydrate, 0.88% disodium hydrogen phosphate dodecahydrate and the balance of water by mass percentage; Mix water, sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate to obtain a buffer solution with a pH value of 6.8; Then dissolve casein, isomeric decyl alcohol polyoxyethylene ether, glycerol, isothiazolinone and antifoaming agent B in the buffer solution to obtain a sample diluent.

[0029] Example 3

[0030] Measure 0.5% casein, 0.1% isomeric decyl alcohol polyoxyethylene ether, 2% glycerol, 0.1% ProClin 300, 0.005% antifoaming agent B, 0.61% tris(hydroxymethyl)aminomethane and the balance of water by mass percentage; Mix water and 0.61% tris(hydroxymethyl)aminomethane, and then adjust the pH value of the solution to 7.4 with 10 mM hydrochloric acid to obtain a buffer solution; Then dissolve casein, isomeric decyl alcohol polyoxyethylene ether, glycerol, isothiazolinone and antifoaming agent B in the buffer solution to obtain a sample diluent.

[0031] Comparative Example 1 Measure 0.5% casein, 0.1% isomeric decyl alcohol polyoxyethylene ether, 0.1% ProClin 300, 0.005% antifoaming agent B, 0.24% citric acid monohydrate, 1.10% disodium hydrogen phosphate dodecahydrate and the balance of water by mass percentage; Mix water, citric acid monohydrate and disodium hydrogen phosphate dodecahydrate to obtain a buffer solution with a pH value of 6.8; Then dissolve casein, isomeric decyl alcohol polyoxyethylene ether, isothiazolinone and antifoaming agent B in the buffer solution to obtain a sample diluent.

[0032] Comparative Example 2 Measure 0.5% casein, 0.1% ProClin 300, 0.005% antifoaming agent B, 0.24% citric acid monohydrate, 1.10% disodium hydrogen phosphate dodecahydrate and the balance of water by mass percentage; Mix water, citric acid monohydrate and disodium hydrogen phosphate dodecahydrate to obtain a buffer solution with a pH value of 6.8; Then dissolve casein, isothiazolinone and antifoaming agent B in the buffer solution to obtain a sample diluent.

[0033] Comparative Example 3 Measure 0.1% isomeric decanol polyoxyethylene ether, 2% glycerol, 0.1% ProClin 300, 0.005% antifoaming agent B, 0.24% citric acid monohydrate, 1.1% disodium hydrogen phosphate dodecahydrate and the balance water by mass percentage; Mix water, citric acid monohydrate and disodium hydrogen phosphate dodecahydrate to obtain a buffer solution with a pH value of 6.8; Then dissolve isomeric decanol polyoxyethylene ether, glycerol, isothiazolinone and antifoaming agent B in the buffer solution to obtain a sample diluent.

[0034] Comparative Example 4 The sample diluent Diluent Universal of Roche Diagnostics (article number: 03183971122).

[0035] Comparative Example 5 The difference from Example 1 is only that: the antifoaming agent is not added.

[0036] Comparative Example 6 Add antifoaming agent B to the sample diluent Diluent Universal of Roche Diagnostics, and the mass fraction of antifoaming agent B in the sample diluent Diluent Universal is 0.005%.

[0037] Experimental Example Use the alpha-fetoprotein assay kit (AFP), conventional C-reactive protein detection kit (CRP), thyroglobulin antibody detection kit (Anti-TG), estradiol assay kit (E2) of Hybiome to detect on the AE-240 model (using the supporting reaction cups, chemiluminescent substrate solution and cleaning solution for the automatic immunoassay system), and the test samples are AFP project enterprise reference products (all prepared by Hybiome itself; 10 low, medium and high clinical serum samples of these 4 projects are selected), CRP project enterprise reference products, Anti-TG project enterprise reference products and E2 project enterprise reference products.

[0038] The specific detection method is as follows 1. Detect the AFP project enterprise reference product Dilute the AFP project enterprise reference products with different concentrations 20 times respectively using the sample diluents described in Examples 1 to 3 and Comparative Examples 1 to 4, then detect the diluted reference products with the AFP assay kit, calculate the detection results and the measured concentrations (measured concentration = detection result × 20), and calculate the relative deviation according to the theoretical concentration of the enterprise reference product (the theoretical concentration is the concentration before dilution of the AFP project enterprise reference product, C1).

[0039] Sampling mode of AFP during the test: Mix 10 μL of the sample, 100 μL of reagent 1, and 100 μL of reagent 2. After incubation for 10 min, add 20 μL of magnetic particles, incubate for another 10 min, then perform washing and magnetic separation, and finally add the luminescent substrate solution to read the signal value.

[0040] 2. Detect the enterprise reference samples for the CRP project Dilute the enterprise reference samples with different concentrations of the CRP project 100 times respectively using the sample diluents described in Examples 1 - 3 and Comparative Examples 1 - 4. Then detect the diluted reference samples using the CRP assay kit, calculate the test results and the measured concentration (measured concentration = test result × 100), and calculate the relative deviation based on the theoretical concentration of the enterprise reference sample (the theoretical concentration is the concentration of the enterprise reference sample of the CRP project before dilution, C1).

[0041] Sampling mode of CRP during the test: Mix 10 μL of the sample, 100 μL of reagent 1, and 100 μL of reagent 2. After incubation for 10 min, add 20 μL of magnetic particles, incubate for another 10 min, then perform washing and magnetic separation, and finally add the luminescent substrate solution to read the signal value.

[0042] 3. Detect the enterprise reference samples for the Anti - TG project Dilute the enterprise reference samples with different concentrations of the Anti - TG project 80 times respectively using the sample diluents described in Examples 1 - 3 and Comparative Examples 1 - 4. Then detect the diluted reference samples using the Anti - TG assay kit, calculate the test results and the measured concentration (measured concentration = test result × 80), and calculate the relative deviation based on the theoretical concentration of the enterprise reference sample (the theoretical concentration is the concentration of the enterprise reference sample of the Anti - TG project before dilution, C1).

[0043] Sampling mode of Anti - TG during the test: Mix 50 μL of the sample and 100 μL of reagent 1. After incubation for 10 min, add 20 μL of magnetic particles, incubate for another 10 min, perform washing and magnetic separation, then add 100 μL of reagent 2, incubate for 10 min, perform washing and magnetic separation again, and finally add the luminescent substrate solution to read the signal value.

[0044] 4. Detect the enterprise reference samples for the E2 project Dilute the enterprise reference samples with different concentrations of the E2 project 10 times respectively using the sample diluents described in Examples 1 - 3 and Comparative Examples 1 - 4. Then detect the diluted reference samples using the E2 assay kit, calculate the test results and the measured concentration (measured concentration = test result × 10), and calculate the relative deviation based on the theoretical concentration of the enterprise reference sample (the theoretical concentration is the concentration of the enterprise reference sample of the E2 project before dilution, C1).

[0045] Sampling mode of E2 during the test: Mix 50 μL of the sample with 100 μL of Reagent 1. After incubation for 10 min, add 100 μL of Reagent 2 and 20 μL of magnetic particles, incubate for another 10 min, then perform magnetic separation by washing, and finally add the luminescent substrate solution to read the signal value.

[0046] The theoretical concentration C1, measured concentration C2, and relative deviation RD of the enterprise reference samples for the above-mentioned tests 1-4 are shown in Table 1.

[0047] Table 1 Relative deviations of the enterprise reference samples for detecting AFP, CRP, Anti-TG, and E2 items in Examples 1-3 and Comparative Examples 1-4

[0048] Adding casein on the basis of the selected sodium citrate-disodium hydrogen phosphate buffer can significantly inhibit this low value and reduce non-specific reactions. Adding the surfactant isodecyl polyoxyethylene ether and glycerol can improve the detection accuracy of individual items and individual samples, and reduce the matrix effect of the sample diluent among different items. The sample diluent prepared based on the citrate-disodium hydrogen phosphate buffer has better effects than the phosphate buffer and Tris-HCl buffer solution.

[0049] 5. Test the defoaming effects of Example 1 and Comparative Examples 4-6 Take 10 mL each of the diluents prepared in Example 1 and Comparative Examples 4-6, add them to 20 mL transparent plastic bottles, then place them on a rotary mixer and invert and mix for 60 min, then take them down and observe and record the time required for defoaming. The results are shown in Table 2.

[0050] Table 2 Defoaming times of the diluents prepared in Example 1 and Comparative Examples 4-6 under the same conditions

[0051] The defoaming time of Example 1 is 14 s, while there are still bubbles in Comparative Example 4 after standing for 30 min. The sample diluent of the present invention will not generate bubbles due to operations or shaking of the reagent tray of the immunoassay analyzer, thus affecting the detection accuracy of the instrument or delaying the detection. Even if an antifoaming agent is added to the existing sample diluent, the time required for defoaming is longer than that of the diluent prepared in this application.

[0052] 6. Stability of the sample diluent prepared in Test Example 1 Take 50 mL of the sample diluent prepared in Example 1 and perform thermal shock on it according to the thermal shock test procedure in Table 3. After completing steps 1 to 7, take it out, dilute and test the AFP, CRP, Anti-TG and E2 enterprise reference products respectively, and calculate the relative deviation of the measured concentration from the theoretical concentration. The test results are shown in Table 4.

[0053] Table 3 Thermal shock test procedure

[0054] Table 4 Detection results of different samples after dilution of the sample diluent prepared in Example 1 after thermal shock

[0055] The experimental design of the thermal shock of the sample diluent simulates the temperature, freeze-thaw and other condition stimuli during transportation to evaluate the stability of the sample diluent. The results show that the accuracy of the sample diluent after thermal shock still meets the target requirements.

[0056] The above are only the preferred embodiments of the present invention. It should be pointed out 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. A sample diluent for chemiluminescence immunoassay, characterized in that, Comprising components in the following mass fractions: Protein blocking agent 0.1 - 5%, 0.1% isomeric decyl alcohol polyoxyethylene ether, 2% glycerol, preservative 0.05 - 0.5%, defoaming agent 0.001 - 0.1%, and the balance being solvent; The solvent is a buffer solution.

2. The sample diluent for chemiluminescence immunoassay according to claim 1, wherein The protein blocking agent includes one or more of bovine serum albumin, casein, bovine serum, and goat serum.

3. The sample diluent for chemiluminescent immunoassay according to claim 1 or 2, characterized in that, The preservative is ProClin950 and / or ProClin 300.

4. A sample diluent for chemiluminescence immunoassay according to claim 3, characterized in that, The defoaming agent is a polydimethylsiloxane defoaming agent.

5. A sample diluent for chemiluminescence immunoassay according to claim 4, characterized in that, The buffer solution is one of phosphate buffer solution, Tris-HCl buffer solution, and citric acid-disodium hydrogen phosphate buffer solution; The pH value of the buffer solution is 6.0 - 8.0; The mass concentration of the buffer in the buffer solution is 0.2 - 3.5 wt%.

6. A method for preparing a sample diluent for chemiluminescence immunoassay according to any one of claims 1 to 5, characterized in that, Comprising the following steps: Mixing the protein blocking agent, surfactant, preservative, defoaming agent, and buffer solution to obtain a sample diluent for chemiluminescent immunoassay.

7. Use of the sample diluent for chemiluminescent immunoassay prepared by the preparation method of the sample diluent for chemiluminescent immunoassay according to any one of claims 1 - 5 or the sample diluent for chemiluminescent immunoassay according to claim 6 in chemiluminescent immunoassay.

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