Method for eliminating lipid interference, immunoturbidimetric kit and application

Abstract

The invention relates to the technical field of biochemical immune in-vitro diagnostic reagents, in particular to a method for eliminating lipid interference, an immunoturbidimetric kit and application of the immunoturbidimetric kit. Surfactants are added into the kit, that is, one or more compound surfactants with low cost and good lipid solubility are added into a reagent I; therefore, the problem of lipid interference can be effectively solved, the lipid interference can be eliminated by adopting a low-cost, high-accuracy and simple-to-operate method for eliminating lipid interference, and the problems that when an existing kit uses enzyme substances to eliminate lipid interference, the reagent cost is greatly increased due to addition of the enzyme substances, and the detection sensitivity is high are solved. The technical problem that the cost pressure of a conventional biochemical immune reagent is increased is solved, and meanwhile, the method can be widely used in cooperation with various biochemical analyzers and has wider universality, so that the method can be widely applied to the lipid blood sample diagnosis process.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical immune in vitro diagnostic reagents, and in particular to a method for eliminating lipidemia interference and an immune turbidimetric kit and application thereof. Background Art

[0002] Lipidemia, also known as chylosing, refers to blood whose plasma color is milky white or turbid. The fat that people consume daily is digested and absorbed in the small intestine, forming tiny chylomicron particles that enter the blood. After the chylomicron particles accumulate, the serum changes from a clear liquid to milky white and viscous lipemia.

[0003] Lipidemia samples can interfere with biochemical immunoassays in vitro, reducing the accuracy of detection. Currently, the methods available to eliminate the interference of lipidemia samples include: sample pretreatment and reagent improvement.

[0004] Common sample pretreatment methods include centrifugation, extraction, and precipitation. Centrifugation is suitable for samples with high lipid content and easy-to-distinguish appearance, but is less suitable for samples with low lipid content and low interference. The extraction method often uses organic solvents such as ether to treat samples, which has a good separation effect on fat-soluble substances in the sample, but organic solvents will also have an uncertain effect on the sample. The precipitation method uses a combination of phosphotungstic acid-magnesium and dextran sulfate-polyethylene glycol to precipitate chylomicrons and low-density lipoproteins in serum to remove interference. With the increasing requirements for result accuracy and detection efficiency, sample pretreatment operations are cumbersome and inefficient, and are gradually being replaced by other methods to eliminate interference.

[0005] In terms of reagent improvement, patents have disclosed the use of enzymes to improve reagents. This method can decompose blood lipids into corresponding fatty acids and propylene glycol. The decomposition products have good water solubility and can reduce interference with test results to a certain extent. However, the addition of enzymes will significantly increase the cost of reagents, increasing the cost pressure of conventional biochemical immunoassay reagents.

[0006] In order to address the deficiencies of the prior art, the present invention intends to use one or more low-cost, lipid-soluble compound surfactants to add to the reagent to solve the problem of lipemia interference. The purpose is to provide a low-cost, high-accuracy, simple-to-operate method for eliminating lipemia interference and its application in immunoturbidimetric kits. Summary of the invention

[0007] The purpose of the present invention is to provide a method for eliminating lipemia interference and an immunoturbidimetric kit and application, which solves the technical problem that when the existing kit uses enzyme substances to eliminate lipemia interference, the addition of enzyme substances will greatly increase the reagent cost and increase the cost pressure of conventional biochemical immunoassay reagents.

[0008] To achieve the above object, in a first aspect, the present invention provides an immunoturbidimetric kit for eliminating lipemia interference, the kit comprising reagent 1 and reagent 2, and the contents of the components in the kit are as follows:

[0009] The reagent 1 includes: 10-100 mmol / L tris(hydroxymethyl)aminomethane (TRIS) buffer or 10-100 mmol / L phosphate buffer, one or more compound surfactants, 0.3-1.0% sodium chloride, 0.3% PC-300, and 0.5-4.0% polyethylene glycol 6000;

[0010] The second reagent includes: 10-100 mmol / L tris(hydroxymethyl)aminomethane buffer or 10-100 mmol / L phosphate buffer, 0.5% trisodium citrate, goat anti-human CRP antiserum, 0.3-1.0% sodium chloride, and 0.3% PC-300.

[0011] Wherein, the compound surfactant is one or more of 0.5-1.0% Tween 20, polyethylene glycol 400, polyvinyl pyrrolidone K30, sodium lauryl sulfate, EMULGEN-108, EMULGEN-707, and EMULGEN-709.

[0012] The reagent 1 is also applicable to other immunoturbidimetric kits, such as immunoturbidimetric kits for complement C3, complement C4 or apolipoprotein A1.

[0013] In a second aspect, the present invention also provides an application of an immunoturbidimetric kit for eliminating lipemia interference, which is an application of a method for detecting specific biochemical indicators in serum samples using immunoturbidimetric method based on the principle of spectrophotometry.

[0014] In a third aspect, the present invention also provides a method for eliminating lipemia interference, using one or more low-cost, lipid-soluble compound surfactants to add to reagent 1 to solve the lipemia interference problem.

[0015] The specific detection steps of using one or more low-cost, fat-soluble composite surfactants to add to reagent 1 to solve the problem of lipemia interference are:

[0016] Adding tris(hydroxymethylaminomethane) buffer or phosphate buffer, one or more compound surfactants, sodium chloride, PC-300 and polyethylene glycol 6000 in a test tube in sequence to prepare reagent 1;

[0017] Mix the reagent 1 and the serum sample, read the absorbance value A1 at the end of 10 seconds, and then read the absorbance value A2 at the end of 5 minutes, and calculate ΔA=A2-A1, where ΔA is the absorbance difference;

[0018] The CRP concentration of the serum sample is calculated based on the obtained absorbance difference of the serum sample.

[0019] The test conditions for mixing the reagent 1 with the serum sample, reading the absorbance value A1 at the end of 10 seconds, and then reading the absorbance value A2 at the end of 5 minutes are: temperature 37° C., wavelength 340 nm.

[0020] Among them, in order to ensure the reliability of the test results, the detection reagent needs to be calibrated with CRP calibrators or standard serum before testing the serum samples.

[0021] The present invention discloses a method for eliminating lipemia interference and an immunoturbidimetric kit and application thereof. By adopting the method for eliminating lipemia interference and the type of surfactant added in the kit, i.e., adding one or more low-cost, lipid-soluble compound surfactants to reagent one, the problem of lipemia interference can be effectively solved, and then the method for eliminating lipemia interference with low cost, high accuracy and simple operation can be adopted to eliminate lipemia interference. The technical problem that when enzyme substances are used to eliminate lipemia interference in the existing kit, the addition of enzyme substances will greatly increase the reagent cost and increase the cost pressure of conventional biochemical immunoassay reagents is solved. At the same time, the method can be widely used in various biochemical analyzers, has wider versatility, and can be widely used in the process of biochemical immunoassay in vitro diagnosis of lipemia samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0023] Figure 1 It is a reaction curve diagram of the test of lipemia sample by the prepared reagent of Example 1 of the present invention.

[0024] Figure 2 This is a reaction curve diagram of the test of lipemia sample by the prepared reagent of Example 2 of the present invention.

[0025] Figure 3 It is a reaction curve diagram of the test of lipemia sample by the prepared reagent of Example 3 of the present invention.

[0026] Figure 4 It is a reaction curve diagram of the test of lipemia sample by the prepared reagent of Example 4 of the present invention.

[0027] Figure 5 It is a reaction curve diagram of the test of lipemia sample by the prepared reagent of Example 5 of the present invention.

[0028] Figure 6 It is a reaction curve diagram of the test of lipemia sample by the prepared reagent of Example 6 of the present invention.

[0029] Figure 7 It is a flowchart of the method for eliminating blood lipid interference of the present invention. DETAILED DESCRIPTION

[0030] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0031] 1. Specific embodiments are as follows:

[0032] Example 1

[0033] Reagent 1

[0034] Table 1

[0035] Drug Name Dosage(1L) Phosphate 30mmol Compound surfactant 1 1.0％ Sodium chloride 0.5％ PC-300 0.3％ Polyethylene glycol 6000 3.0％ pH Adjust pH to 7.60 ± 0.05 with 6M hydrochloric acid

[0036] Reagent 2

[0037] Table 2

[0038] Drug Name Dosage(1L) Phosphate 30mmol Trisodium Citrate 0.5％ Sodium chloride 0.5％ PC-300 0.3％ Sheep anti-human CRP antiserum 30％ pH Adjust pH to 7.40 ± 0.05 with 6M hydrochloric acid

[0039] Preparation of reagent 1: Add phosphate, sodium chloride, PC-300, polyethylene glycol 6000, and one or more compound surfactants selected from Tween 20, polyvinyl pyrrolidone K30, and EMULGEN-108 according to the amount, and finally adjust the pH to 7.60±0.05 with 6M hydrochloric acid.

[0040] Preparation of reagent 2: Add phosphate, trisodium citrate, sodium chloride, PC-300, and goat anti-human CRP antiserum according to the amount, and finally adjust the pH to 7.40±0.05 with 6M hydrochloric acid.

[0041] Example 2

[0042] Reagent 1

[0043] Table 3

[0044]

[0045]

[0046] Reagent 2

[0047] Table 4

[0048] Drug Name Dosage(1L) Phosphate 40mmol Trisodium Citrate 0.5％ Sodium chloride 0.5％ PC-300 0.3％ Sheep anti-human CRP antiserum 30％ pH Adjust pH to 7.40 ± 0.05 with 6M hydrochloric acid

[0049] Preparation of reagent 1: Add phosphate, sodium chloride, PC-300, polyethylene glycol 6000, and one or more compound surfactants selected from Tween 20, polyvinyl pyrrolidone K30, and EMULGEN-707 according to the amount, and finally adjust the pH to 7.60±0.05 with 6M hydrochloric acid.

[0050] Preparation of reagent 2: Add phosphate, trisodium citrate, sodium chloride, PC-300, and goat anti-human CRP antiserum according to the amount, and finally adjust the pH to 7.40±0.05 with 6M hydrochloric acid.

[0051] Example 3

[0052] Reagent 1

[0053] Table 5

[0054] Drug Name Dosage(1L) Phosphate 50mmol Compound surfactant 3 1.0％ Sodium chloride 0.5％ PC-300 0.3％ Polyethylene glycol 6000 3.0％ pH Adjust pH to 7.60 ± 0.05 with 6M hydrochloric acid

[0055] Reagent 2

[0056] Table 6

[0057] Drug Name Dosage(1L) Phosphate 50mmol Trisodium Citrate 0.5％ Sodium chloride 0.5％ PC-300 0.3％ Sheep anti-human CRP antiserum 30％ pH Adjust pH to 7.40 ± 0.05 with 6M hydrochloric acid

[0058] Preparation of reagent 1: Add phosphate, sodium chloride, PC-300, polyethylene glycol 6000, and one or more compound surfactants selected from Tween 20, polyvinyl pyrrolidone K30, and EMULGEN-709 according to the amount, and finally adjust the pH to 7.60±0.05 with 6M hydrochloric acid.

[0059] Preparation of reagent 2: Add phosphate, trisodium citrate, sodium chloride, PC-300, and goat anti-human CRP antiserum according to the amount, and finally adjust the pH to 7.40±0.05 with 6M hydrochloric acid.

[0060] Example 4

[0061] Reagent 1

[0062] Table 7

[0063] Drug Name Dosage(1L) TRIS (Tris(hydroxymethyl)aminomethane) 30mmol Compound surfactant 4 0.5％ Sodium chloride 0.5％ PC-300 0.3％ Polyethylene glycol 6000 4.0％ pH Adjust pH to 7.60 ± 0.05 with 6M hydrochloric acid

[0064] Reagent 2

[0065] Table 8

[0066]

[0067]

[0068] Preparation of reagent 1: Add TRIS, sodium chloride, PC-300, polyethylene glycol 6000, and one or more compound surfactants selected from Tween 20, polyethylene glycol 400, sodium dodecyl sulfate, and EMULGEN-108 according to the amount, and finally adjust the pH to 7.60±0.05 with 6M hydrochloric acid.

[0069] Preparation of reagent 2: Add TRIS, trisodium citrate, sodium chloride, PC-300, and goat anti-human CRP antiserum according to the amount, and finally adjust the pH to 7.40±0.05 with 6M hydrochloric acid.

[0070] Example 5

[0071] Reagent 1

[0072] Table 9

[0073] Drug Name Dosage(1L) TRIS 40mmol Compound surfactant 5 0.5％ Sodium chloride 0.5％ PC-300 0.3％ Polyethylene glycol 6000 4.0％ pH Adjust pH to 7.60 ± 0.05 with 6M hydrochloric acid

[0074] Reagent 2

[0075] Table 10

[0076] Drug Name Dosage(1L) TRIS 40mmol Trisodium Citrate 0.5％ Sodium chloride 0.5％ PC-300 0.3％ Sheep anti-human CRP antiserum 30％ pH Adjust pH to 7.40 ± 0.05 with 6M hydrochloric acid

[0077] Preparation of reagent 1: Add TRIS, sodium chloride, PC-300, polyethylene glycol 6000, and one or more compound surfactants selected from Tween 20, polyethylene glycol 400, sodium dodecyl sulfate, and EMULGEN-707 according to the amount, and finally adjust the pH to 7.60±0.05 with 6M hydrochloric acid.

[0078] Preparation of reagent 2: Add TRIS, trisodium citrate, sodium chloride, PC-300, and goat anti-human CRP antiserum according to the amount, and finally adjust the pH to 7.40±0.05 with 6M hydrochloric acid.

[0079] Example 6

[0080] Reagent 1

[0081] Table 11

[0082] Drug Name Dosage(1L) TRIS 50mmol Compound surfactant 6 0.5％ Sodium chloride 0.5％ PC-300 0.3％ Polyethylene glycol 6000 4.0％ pH Adjust pH to 7.60 ± 0.05 with 6M hydrochloric acid

[0083] Reagent 2

[0084] Table 12

[0085] Drug Name Dosage(1L) TRIS 50mmol Trisodium Citrate 0.5％ Sodium chloride 0.5％ PC-300 0.3％ Sheep anti-human CRP antiserum 30％ pH Adjust pH to 7.40 ± 0.05 with 6M hydrochloric acid

[0086] Preparation of reagent 1: Add TRIS, sodium chloride, PC-300, polyethylene glycol 6000, and one or more compound surfactants selected from Tween 20, polyethylene glycol 400, sodium dodecyl sulfate, and EMULGEN-709 according to the amount, and finally adjust the pH to 7.60±0.05 with 6M hydrochloric acid.

[0087] Preparation of reagent 2: Add TRIS, trisodium citrate, sodium chloride, PC-300, and goat anti-human CRP antiserum according to the amount, and finally adjust the pH to 7.40±0.05 with 6M hydrochloric acid.

[0088] 2. Comparison of test results of different specific embodiments:

[0089] Anti-interference performance:

[0090] 1) Anti-interference performance of the reagent was tested using the chylomicron addition method. A normal serum sample was taken, and its CRP concentration was tested using the reagent prepared in Examples 1-6. The sample was then divided into 4 portions, and each portion was added with 1 / 9 of normal saline, 1 / 9 of 2000 mg / L chylomicron, 1 / 9 of 4000 mg / L chylomicron, and 1 / 9 of 6000 mg / L chylomicron. If the test values ​​of the latter 3 samples were within ±10% of the test values ​​of the sample with normal saline added, it indicated that the reagent had good anti-lipidemia interference performance.

[0091] Table 14

[0092]

[0093] 2) Analysis of the reaction curve of the test sample of lipid blood prepared by the reagent of Example 1-6 (see the specific reaction curve diagram Figure 1-Figure 6 ), after the serum sample containing chylodisruptor was added to the reagent, the absorbance in the reaction curve increased significantly and then decreased rapidly, proving that the reagent had a good dissolving effect on chyle. Combined with the above test results, the deviations were all within 10%, that is, when the reagent for CRP detection of the present invention was used, 6000 mg / L lipemia had basically no interference with the test results of serum CRP.

[0094] 3. Specific steps to eliminate the interference of lipemia:

[0095] The method for eliminating the interference of lipemia is: based on the principle of spectrophotometry, a method of using immunoturbidimetry to detect specific biochemical indicators in serum samples.

[0096] The specific operating steps of the method for eliminating lipemia interference are:

[0097] S101. Add tris(hydroxymethylaminomethane) buffer or phosphate buffer, one or more compound surfactants, sodium chloride, PC-300 and polyethylene glycol 6000 in a test tube in sequence to prepare reagent 1.

[0098] S102, mixing the reagent 1 and the serum sample, reading the absorbance value A1 at the end of 10 seconds, and then reading the absorbance value A2 at the end of 5 minutes, and calculating ΔA=A2-A1, wherein ΔA is the absorbance difference.

[0099] S103. Calculate the CRP concentration of the serum sample according to the obtained absorbance difference of the serum sample.

[0100] Specifically, after obtaining the immunoturbidimetric reagent 1 capable of eliminating lipidemia interference, it is necessary to detect the specific effect of the reagent. The specific detection steps are: mixing the reagent 1 with the serum sample, reading the absorbance value A1 at the end of 10 seconds, and then reading the absorbance value A2 at the end of 5 minutes, and calculating ΔA=A2-A1, wherein ΔA is the absorbance difference. Afterwards, the CRP concentration of the serum sample is calculated according to the absorbance difference of the serum sample obtained, wherein the calculation formula of the CRP concentration is:

[0101] CRP: CRP concentration in the sample;

[0102] ΔA 样本 : absorbance difference of serum samples;

[0103] ΔA 校准品 : Absorbance difference of calibrators;

[0104] C 校准品 : Calibrator concentration.

[0105] In addition, the test conditions for mixing the reagent 1 with the serum sample, reading the absorbance value A1 at the end of 10 seconds, and then reading the absorbance value A2 at the end of 5 minutes are: temperature 37° C., wavelength 340 nm.

[0106] In addition, in order to ensure the reliability of the test results, the detection reagent must be calibrated with CRP calibrators or standard serum before testing the serum samples.

[0107] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. An immunoturbidimetric kit for eliminating lipemia interference, characterized in that: The kit includes reagent 1 and reagent 2, and the contents of each component in the kit are as follows: The reagent 1 includes: 10-100 mmol / L tris(hydroxymethyl)aminomethane buffer or 10-100 mmol / L phosphate buffer, one or more compound surfactants, 0.3-1.0% sodium chloride, 0.3% PC-300, and 0.5-4.0% polyethylene glycol 6000; The second reagent includes: 10-100 mmol / L tris(hydroxymethyl)aminomethane buffer or 10-100 mmol / L phosphate buffer, 0.5% trisodium citrate, goat anti-human CRP antiserum, 0.3-1.0% sodium chloride, and 0.3% PC-300.

2. The immunoturbidimetric kit for eliminating lipemia interference according to claim 1, characterized in that: The compound surfactant is one or more of 0.5-1.0% Tween 20, polyethylene glycol 400, polyvinyl pyrrolidone K30, sodium lauryl sulfate, EMULGEN-108, EMULGEN-707 and EMULGEN-709.

3. The immunoturbidimetric kit for eliminating lipemia interference according to claim 1, characterized in that: The reagent 1 is also applicable to other immunoturbidimetric kits, such as immunoturbidimetric kits for complement C3, complement C4 or apolipoprotein A1.

4. An application of an immunoturbidimetric kit for eliminating lipemia interference, applied to the immunoturbidimetric kit for eliminating lipemia interference according to any one of claims 1 to 3, characterized in that: The application is an application of a method for detecting specific biochemical indicators in serum samples using immunoturbidimetry based on the principle of spectrophotometry.

5. A method for eliminating lipemia interference, applied to the immunoturbidimetric kit for eliminating lipemia interference according to any one of claims 1 to 3, characterized in that: One or more low-cost, lipid-soluble composite surfactants are added to reagent 1 to solve the problem of lipidemia interference.

6. The method for eliminating lipemia interference according to claim 5, characterized in that: The specific detection steps of using one or more low-cost, fat-soluble composite surfactants to add to reagent 1 to solve the problem of lipemia interference are: Adding tris(hydroxymethylaminomethane) buffer or phosphate buffer, one or more compound surfactants, sodium chloride, PC-300 and polyethylene glycol 6000 in a test tube in sequence to prepare reagent 1; Mix the reagent 1 and the serum sample, read the absorbance value A1 at the end of 10 seconds, and then read the absorbance value A2 at the end of 5 minutes, and calculate ΔA=A2-A1, where ΔA is the absorbance difference; The CRP concentration of the serum sample is calculated based on the absorbance difference of the serum sample.

7. The method for eliminating lipemia interference according to claim 6, characterized in that: The test conditions for mixing the reagent 1 with the serum sample, reading the absorbance value A1 at the end of 10 seconds, and then reading the absorbance value A2 at the end of 5 minutes are: temperature 37° C., wavelength 340 nm.

8. The method for eliminating lipidemia interference according to claim 7, characterized in that: In order to ensure the reliability of the test results, the detection reagent must be calibrated with CRP calibrators or standard serum before testing the serum samples.

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