A sample diluent and its preparation method

Abstract

This disclosure relates to a sample diluent and its preparation method. Specifically, this disclosure relates to a sample diluent composed of Tris, Tween-20, BSA, mixed serum, and PC300. A key improvement is that the mixed serum is prepared by mixing goat serum and fetal bovine serum at a volume ratio of 2:1, and the mixed serum accounts for 40% of the sample diluent. The sample diluent of this disclosure is designed for the detection of lambda and kappa type free light chains, significantly reducing sample matrix interference, improving detection specificity and stability, and solving the technical problem of disproportionate dilution after high-value sample dilution. It is suitable for clinical testing, biomedical research, and other scenarios.

Description

Technical Field

[0001] This disclosure relates to the field of biological detection reagent technology, specifically to the preparation of a sample diluent, which is particularly suitable for the in vitro detection of lambda-type and kappa-type free light chains. Background Technology

[0002] Free light chains (FLCs) are an important component of immunoglobulin (Ig) molecules. Ig consists of two light chains (lambda chain and kappa chain) and two heavy chains. Synthesized during B lymphocyte maturation, light chains not bound to heavy chains are released into the bloodstream as free light chains. Abnormal serum concentrations of free light chains are closely associated with multiple myeloma, macroglobulinemia, and kidney disease, and are important biomarkers for clinical diagnosis and prognostic assessment. Typically, the levels of free light chains are abnormally high in the serum of patients with multiple myeloma and related conditions, often exceeding the detection limit. Therefore, sample dilution is a crucial step in free light chain detection, and the effectiveness of the dilution directly determines the accuracy of the test results.

[0003] Currently, there is no specific diluent for free light chains. Samples diluted with physiological saline or pure water exhibit a tendency for the calculated values ​​to increase with increasing dilution factor, severely interfering with physicians' judgment. Therefore, there is an urgent need for a sample diluent specifically designed for free light chains. This diluent should significantly reduce the matrix effect caused by increasing dilution factor, thereby better restoring the true measured values ​​of the sample and reducing errors. Summary of the Invention

[0004] In view of the aforementioned needs in the art, a sample diluent is provided.

[0005] Sample diluent is a buffer solution used to dilute the sample to be tested.

[0006] In some implementations, the sample diluent contains or consists of the following components, based on the final concentration: 8 mmol / L to 12 mmol / L Tris(hydroxymethylaminomethane) 0.08% to 0.12% (v / v) Tween-20, 0.8% to 1.2% (w / v) BSA, 35% to 45% (v / v) mixed serum, 0.05% to 1% (v / v) PC300 Deionized water; The pH of the sample diluent is 6.5 to 8 (preferably 7.2 to 7.6). The mixed serum comprises goat serum and fetal bovine serum in a volume ratio of 1.8:1 to 2.2:1.

[0007] Goat serum is a serum component isolated from goat blood; it is a well-known material in the field and is commercially available. Fetal bovine serum is serum derived from fetal bovine blood; it is a well-known material in the field and is commercially available.

[0008] In some embodiments, the volume ratio of the goat serum to the fetal bovine serum is 2:1.

[0009] In some embodiments, the final concentration of the mixed serum is 40% (v / v).

[0010] In some specific embodiments, the sample diluent, based on its final concentration, comprises or consists of the following components: 10 mmol / L Tris 0.1% (v / v) Tween-20 1.0% (w / v) BSA, 40% (v / v) mixed serum, 0.1% (v / v) PC300; The remainder is deionized water. The pH of the sample diluent was 7.4.

[0011] This disclosure also provides the use of the aforementioned sample diluent in the preparation of the test kit.

[0012] In some implementations, the kits disclosed herein are used for the detection of free light chains.

[0013] In some implementations, the free light chain is selected from lambda-type free light chains and Kappa-type free light chains.

[0014] This disclosure also provides a detection kit for free light chains, which includes the sample diluent described above. Detailed Implementation

[0015] Example 1 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: fetal bovine serum = 2:1) 40% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0016] 2. Preparation method: (1) Take 800 mL of deionized water, add Tris powder and stir to dissolve, then adjust the pH to 7.4; (2) Add Tween-20, BSA and PC300 in sequence and stir until completely dissolved; (3) Mix goat serum and fetal bovine serum at a volume ratio of 2:1 to prepare mixed serum; (4) Add 400 mL of mixed serum to the above solution and stir evenly; (5) Add deionized water to 1000 mL, filter through a 0.22 μm filter membrane for sterilization, seal and store at 4℃. This is designated as dilution 1.

[0017] Example 2 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: fetal bovine serum = 1:1) 40% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0018] 2. Preparation method: Same as in Example 1. This is designated as diluent 2.

[0019] Example 3 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: fetal bovine serum = 1:2) 40% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0020] 2. Preparation method: Same as in Example 1. The solution is designated as diluent 3.

[0021] Example 4 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: fetal bovine serum = 3:1) 40% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0022] 2. Preparation method: Same as in Example 1. The solution is designated as diluent 4.

[0023] Example 5 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: fetal bovine serum = 2:1) 45% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0024] 2. Preparation method: Same as in Example 1. The solution is designated as diluent 5.

[0025] Example 6 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: fetal bovine serum = 2:1) 35% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0026] 2. Method: Same as in Example 1. Diluent 6 is used.

[0027] Example 7 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: calf serum = 2:1) 40% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0028] 2. Method: Same as in Example 1. The solution is designated as diluent 7.

[0029] Example 8 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: horse serum = 2:1) 40% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0030] 2. Method: Same as in Example 1. The solution is designated as diluent 8.

[0031] Example 9 1. Composition: Tris 10 mmol / L Tween-20 0.1% (v / v) BSA 1.0% (w / v) Mixed serum (goat serum: rabbit serum = 2:1) 40% (v / v) PC300 0.1% (v / v) The remainder is deionized water with a pH of 7.4.

[0032] 2. Method: Same as in Example 1. The solution is designated as diluent 9.

[0033] Test Example 1 By comparing the diluents used in Examples 1 to 6, the recovery rates of random high-value myeloma samples were verified, and the recovery rates of different diluents at different dilution factors were compared.

[0034] See Tables 1 and 2 for specific data.

[0035] Table 1. Recovery rates of different diluents It is evident that diluent 1 performs best among all formulations, and its stability also meets the requirements. Through stability testing, after being placed at 37℃ for 7 days, the dilution effect is still no different from that stored at 4℃.

[0036] Table 2. Stability study of dilution 1 Test Example 2 Different serums were matched with goat serum, and the specific results are shown in Table 3 below.

[0037] Table 3. Recovery rates of different sera matched with goat serum Surprisingly, only diluent 1 (containing fetal bovine serum) performed best among all formulations. Replacing the fetal bovine serum with horse serum or rabbit serum commonly used in clinical testing did not show any feasible anti-interference ability.

[0038] This disclosure aims to overcome the shortcomings of the prior art and provide a sample diluent that, by optimizing the formulation of the mixed serum and its proportion in the diluent, solves the technical problems of poor specificity, insufficient stability, and low sensitivity of existing diluents, thereby achieving accurate and efficient detection of free light chains.

[0039] Although not limited to specific theories, goat serum has low background interference and stable composition, while fetal bovine serum is rich in nutritional factors that can maintain protein activity. Surprisingly, the optimal volume ratio of the two was found to be 2:1, which can maximize the balance between interference inhibition and activity maintenance. The volume ratio of the mixed serum in the diluent is 35%-45%, with 40% being optimal. This ratio can ensure the dilution effect while avoiding matrix interference caused by excessive serum content.

Claims

1. A sample diluent, comprising or consisting of the following components based on a final concentration: 8 mmol / L to 12 mmol / L Tris 0.08% to 0.12% (v / v) Tween-20, 0.8% to 1.2% (w / v) BSA, 35% to 45% (v / v) mixed serum, 0.05% to 1% (v / v) PC300 Deionized water; The pH of the sample diluent is 6.5 to 8, preferably 7.2 to 7.6; The mixed serum comprises goat serum and fetal bovine serum in a volume ratio of 1.8:1 to 2.2:

1.

2. The sample diluent according to claim 1, wherein: The volume ratio of the goat serum to the fetal bovine serum is 2:

1.

3. The sample diluent according to claim 1, wherein: The final concentration of the mixed serum was 40% (v / v).

4. The sample diluent according to claim 1, comprising or consisting of the following components based on the final concentration: 10 mmol / L Tris 0.1% (v / v) Tween-20 1.0% (w / v) BSA, 40% (v / v) mixed serum, 0.1% (v / v) PC300; The remainder is deionized water. The pH of the sample diluent was 7.

4.

5. Use of the sample diluent according to any one of claims 1 to 4 in the preparation of a detection kit for the detection of free light chains.

6. The use according to claim 5, wherein: The free light chains are selected from lambda-type free light chains and Kappa-type free light chains.

7. A detection kit for free light chains, comprising: The sample diluent according to any one of claims 1 to 4.

8. The detection kit for free light chains according to claim 7, wherein: The free light chains are selected from lambda-type free light chains and Kappa-type free light chains.

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