Whole blood processing solution for reducing interference in immuno luminescence detection of whole blood samples

Abstract

The present application relates to the field of immune detection technology, especially to a whole blood processing fluid capable of reducing interference in whole blood sample detection, which comprises the following components: a surfactant and an anti-red blood cell antibody; the surfactant is a non-hemolytic surfactant; the concentration ratio of the surfactant to the anti-red blood cell antibody is 20-5000. In the present application, the surfactant and the anti-red blood cell antibody are used together to realize interference-free detection, and the surfactant is preferably an amphoteric surfactant or a non-ionic surfactant, both of which have certain dispersing ability and anti-static effect, can improve the stability of magnetic bead suspension, and thus improve the sensitivity of the reaction system.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more particularly to a whole blood processing solution that reduces interference from immunoluminescence detection of whole blood samples. Background Technology

[0002] In clinical practice, for some tests involving inflammation and cardiac markers, rapid and effective results can facilitate doctors in determining treatment directions and reduce patient suffering. In practice, chemiluminescence immunoassay typically requires serum or plasma samples, the preparation of which necessitates centrifugation of whole blood samples. However, whole blood centrifugation is cumbersome and time-consuming, requires a large sample volume, and results in a slow testing speed. It cannot be performed simultaneously with other conventional whole blood methods, failing to meet the rapid diagnostic needs of emergency and critically ill patients in hospitals. Another testing method is whole blood testing. Whole blood testing requires a smaller sample volume, shortens the initial sample processing time, and allows for testing immediately after blood collection. It is simple and convenient to operate, making it particularly suitable for clinical departments that lack the resources for sample centrifugation and require rapid results.

[0003] Currently, although whole blood testing has the advantages of instant detection and rapid diagnostic results, the complex composition of whole blood and factors such as blood cell aggregation, rupture, and adhesion of blood cells to magnetic beads can significantly interfere with the immune response, resulting in inaccurate test results and poor repeatability.

[0004] Therefore, to address the interference caused by blood cells, whole blood samples need to be processed to reduce the interference of blood cells on the immune response. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a whole blood treatment solution that can reduce interference from blood cells.

[0006] To solve the above-mentioned technical problems, the objective of this invention is achieved through the following technical solution: providing a whole blood processing solution that can reduce interference in whole blood sample detection, the whole blood processing solution comprising the following components: surfactant and anti-erythrocyte antibody;

[0007] The surfactant is a non-hemolytic surfactant;

[0008] Non-hemolytic surfactants are surfactants that do not have hemolytic effects.

[0009] The concentration ratio of the surfactant to the anti-erythrocyte antibody is 20 to 5000.

[0010] The surfactants selected in this invention are those that are stable in dispersion, do not dissolve in blood, can reduce the binding of magnetic bead antibodies to non-specific substances in whole blood, and can improve the sensitivity of reagent reactions.

[0011] The term "non-hemolytic" refers to being gentle on red blood cells and not causing them to rupture, thereby releasing substances within the red blood cells that could interfere with experimental results.

[0012] The "anti-erythrocyte antibody" of this invention has a high affinity for erythrocytes in the sample and can effectively fix / capture erythrocytes in human whole blood without interfering with normal detection.

[0013] This invention is a groundbreaking discovery that the use of anti-erythrocyte antibodies and non-hemolytic surfactants in whole blood testing can achieve highly sensitive and accurate detection of whole blood.

[0014] Further, the concentration of the surfactant is 1% to 5%, and the concentration of the anti-erythrocyte antibody is 0.01 mg / mL to 0.5 mg / mL.

[0015] Furthermore, the surfactant is selected from: TETRONIC 1307, dodecyl hydroxypropyl sulfonyl betaine, sulfopropyl tetradecyl betaine, cocamidopropyl betaine, Surfynol(tm)465, Surfynol(tm)485 or SILWETL7600.

[0016] Furthermore, the whole blood treatment solution also includes: buffer solution, osmotic pressure maintainer, stabilizer, protectant, polymer, and preservative.

[0017] Furthermore, the buffer solution is selected from PBS, Tris-HCl, HEPES, or MES systems, and has a concentration of 10-50 mM;

[0018] The buffer system can provide suitable ion concentration and pH, maintain a constant pH value in the environment, and prevent red blood cell rupture.

[0019] Furthermore, the buffer system is a Tris-HCl system. It maintains the pH of the environment at around 7.0, exhibiting strong buffering capacity and resisting the disruption of the reagent reaction pH environment by whole blood.

[0020] The osmotic pressure maintaining agent is selected from NaCl or KCl.

[0021] The final concentration of the osmotic pressure maintainer is approximately 0.9%, which is comparable to the concentration in the human body. It maintains the osmotic pressure of blood cells, preserves the water balance and morphology inside and outside the cells, and prevents cell lysis.

[0022] The stabilizer is selected from BSA, HAS or casein, and the concentration is 0.5% to 5%.

[0023] The stabilizer can protect proteins in blood samples and maintain blood stability.

[0024] The protective agent is selected from sucrose, mesotriose, lactose, trehalose, hyaluronic acid, or dextran, with a concentration of 1%-5%;

[0025] The protective agent can effectively protect protein molecules from denaturation and inactivation, thus maintaining cell viability.

[0026] The polymer is selected from dextran (5000, 10000, 20000, 40000), sodium dextran sulfate, or PEG (8000, 20000);

[0027] The polymers selected are mild polymers that can disperse red blood cells, prevent red blood cell aggregation, and eliminate interference from red blood cells in the system. Examples include sodium dextran sulfate and PEG. Sodium dextran sulfate can prevent red blood cells from agglomerating, thus improving cell viability and reducing red blood cell rupture; PEG has a red blood cell dispersing effect.

[0028] The preservative is selected from sodium azide, BND or proclin 300, with a concentration of 0.02% to 0.05%; it can prevent bacterial growth and extend the shelf life of the reagent.

[0029] On the other hand, the present invention also discloses an immune detection kit, wherein the kit comprises the whole blood processing solution.

[0030] Furthermore, the immunoassay kit is a chemiluminescent immunoassay kit.

[0031] On the other hand, this invention discloses the application of the whole blood processing solution in chemiluminescence detection. The "concentration" referred to in this invention is the final concentration of each component added to the solution.

[0032] On the other hand, the present invention discloses a method for reducing detection interference, characterized by comprising the following steps:

[0033] S1: Prepare the whole blood treatment solution as described above;

[0034] S2: Mix whole blood samples with whole blood processing solution, incubate, and then test.

[0035] Furthermore, the incubated sample can be subjected to ultrasonic treatment after incubation and before detection in step S2.

[0036] Ultrasonic treatment during magnetic microparticle chemiluminescence can prevent blood cells in some abnormal samples from easily aggregating with magnetic beads in the reagent. The encapsulation or non-specific adsorption of magnetic beads with blood cells can lead to poor repeatability and false positive results.

[0037] The beneficial effects of this invention are as follows: The whole blood processing solution utilizes the combination of surfactants and anti-erythrocyte antibodies to achieve interference-resistant detection. The surfactants are preferably amphoteric surfactants or nonionic surfactants, both of which possess certain dispersing abilities and antistatic properties, improving the stability of the magnetic bead suspension and thus enhancing the sensitivity of the reaction system. The whole blood processing solution in this invention can stabilize cell morphology, disperse blood cells, and reduce the adhesion of blood cells to magnetic beads, thereby reducing interference from non-specific substances in the blood sample. Furthermore, this invention employs an ultrasonic treatment method, performing ultrasonic dispersion after the system reaction is complete before adding the substrate for further reaction, thus solving the problem of inaccurate results caused by the aggregation of blood cells and magnetic beads. In short, this invention achieves rapid, accurate, and highly repeatable whole blood detection through the above methods. Attached Figure Description

[0038] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is a test result diagram of Formula 1 provided in Embodiment 1 of the present invention;

[0040] Figure 2 This is a diagram showing how some abnormal blood cell samples readily aggregate with magnetic beads in the reagent, as provided in Example 4 of the present invention.

[0041] Figure 3 This invention provides a diagram illustrating the PCT and IL-6 treatment process in Embodiment 4.

[0042] Figure 4 This is a diagram showing the processing of CTnI and MYO provided in Embodiment 4 of the present invention;

[0043] Figure 5 A comparison diagram of PCT and IL-6 ultrasound treatment is provided for Embodiment 4 of the present invention;

[0044] Figure 6 A comparison image of CTnI and MYO ultrasound processing is provided for Embodiment 4 of the present invention. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0047] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0048] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0049] Furthermore, terms like "roughly" and "basically" are used to indicate that the content does not require absolute precision, but rather allows for a certain degree of deviation. For example, "roughly equal" does not simply mean absolute equality; in actual production and operation, achieving absolute "equality" is difficult, and a certain degree of deviation is generally present. Therefore, besides absolute equality, "roughly equal to" also includes the aforementioned situation where a certain degree of deviation exists. Using this as an example, in other cases, unless otherwise specified, terms like "roughly" and "basically" have similar meanings.

[0050] Whole blood testing requires a smaller sample volume, shortening the initial sample processing time. Testing can be performed immediately after blood collection, making it simple and convenient to operate. It is especially suitable for clinical departments that lack the facilities to perform specimen centrifugation and require rapid results.

[0051] In current whole blood testing, whole blood processing solutions include both hemolyzing and non-hemolyzing solutions. Hemolyzing solutions typically contain components that dissolve blood cells. When added to a whole blood sample, these cells dissolve, reducing interference with magnetic beads. For example, adding saponins can dissolve red blood cells, while adding a high-molecular-weight polymer (PVP10000) can promote the dissolution of blood cell lysates and adsorb fragments produced after lysis. Alternatively, adding imidazole can eliminate blood cells, preventing them from engulfing the magnetic beads. However, in practical applications, blood cells may not be completely eliminated, still causing some interference with the magnetic beads and resulting in poor repeatability. Furthermore, the lysis of blood cells releases more substances, causing further uncontrollable interference to the reaction system and leading to inaccurate test results.

[0052] Non-hemolytic processing solutions typically contain non-hemolytic surfactants that stabilize blood cells and prevent aggregation. Whole blood samples are more viscous than serum or plasma, making them prone to magnetic bead aggregation. Furthermore, blood cells in whole blood samples may interfere with the binding of antigens and antibodies. Therefore, non-hemolytic processing solutions serve to dilute the sample and prevent blood cell aggregation. Adding a dispersant (C16PB) to the sample diluent can ensure sufficient antigen-antibody reaction with minimal impact on colorimetric assays (CV). However, in practical applications, blood cells easily adhere to magnetic beads, agglomerating and leading to poor reaction system stability, affecting detection sensitivity. Whole blood also significantly affects the pH of the reaction environment, potentially influencing the stability of magnetic bead antibodies and labeled antibodies. Additionally, the complex composition of blood contains more non-specific substances than serum or plasma, which can easily bind non-specifically to magnetic bead antibodies, resulting in inaccurate test results.

[0053] In summary, although whole blood testing has the advantages of instant detection and rapid diagnostic results, the complex composition of whole blood and factors such as blood cell aggregation, rupture, and adhesion of blood cells to magnetic beads can significantly interfere with the immune response, resulting in inaccurate test results and poor repeatability.

[0054] The following detailed description is provided in conjunction with specific embodiments.

[0055] Example 1

[0056] In the examples, chemiluminescence immunoassay was selected to verify the effectiveness of the whole blood treatment solution of the present invention.

[0057] The detection process is as follows: Add the sample to be tested to the reaction vessel, add an equal volume of processing solution to the sample and mix well, then add other components according to the kit instructions (the kit is the IL-6 detection kit from Zhongyuan Huiji Co., Ltd.) and incubate the reaction.

[0058] The formulations in this embodiment all contain 0.03 mg / mL of anti-erythrocyte antibody and 5% S11 (Surfynol(tm)485) surfactant.

[0059] Table 1. Effects of each component on whole blood processing solution

[0060]

[0061]

[0062] This embodiment detects IL-6 in the blood, and the results in Formula 1 are as follows: Figure 1 The given R² = 0.923

[0063] Formulas 2-7 have R2 values ​​of 0.927, 0.915, 0.925, 0.933, 0.916, and 0.919, respectively.

[0064] It can be seen that the lack or replacement of buffer solution, osmotic pressure maintainer, stabilizer, protectant, polymer, and preservative has little impact on the effectiveness of whole blood treatment solution.

[0065] Example 2: Effect of anti-erythrocyte antibody (RBC pAb) on whole blood processing solution

[0066] A. Reagent Preparation

[0067] The whole blood processing solution in this embodiment is formulated as follows: 50 mmol / L Tris-HCl buffer, 1% BSA, 5% sucrose, 1% S9 (TETRONIC 1307), 1% sodium dextran sulfate, 0.05% proclin300 and anti-erythrocyte antibody.

[0068] The concentrations of anti-erythrocyte antibodies were prepared at the following levels: 0.01, 0.05, 0.1, 0.2, 0.5, 1, and 2 (unit: mg / mL).

[0069] The solution has a pH of 7.4. After preparation, the solution is filtered through a 0.22µm filter membrane and stored at 2-8℃.

[0070] B. Clinical validation

[0071] This embodiment detects IL-6 in whole blood.

[0072] The test results are as follows: R was 0.01 mg / mL. 2 =0.912, 0.05 mg / mL R 2 =0.922, 0.1 mg / mL R 2 =0.933, 0.2 mg / mL R 2 =0.925, 0.5 mg / mL R 2 =0.932, 1 mg / mL R 2 =0.451, 2 mg / mL R 2 =0.533.

[0073] It can be seen that the detection effect is good when the concentration of anti-erythrocyte antibody is 0.01mg / mL-0.5mg / mL. If the concentration of anti-erythrocyte antibody is too high (≥1mg / mL), it will cause erythrocyte aggregation, increase system interference, and affect the detection effect.

[0074] Example 3: Effect of surfactants on whole blood treatment solution

[0075] A. Effect of surfactant concentration

[0076] (1) Reagent preparation

[0077] The whole blood processing solution formulation in this embodiment is as follows: 50 mmol / L Tris-HCl buffer, 1% BSA, 5% trehalose, surfactant, 1% sodium dextran sulfate, 0.05% proclin 300 and 0.5% anti-erythrocyte antibody.

[0078] In this embodiment, surfactant S11 (Surfynol(tm)485) was selected, with concentrations of 0.01, 0.1, 1, 2, 5, 10, and 20 (%).

[0079] The test results are as follows: 0.01% of R... 2 =0.654, 0.1% of R 2 =0.735, 1% of R 2 =0.927, 2% R 2 =0.935, 5% of R 2 =0.944, 10% of R 2 =0.641, 20% of R 2 =0.653.

[0080] It can be seen that the detection effect is best when the concentration of surfactant is in the range of 1-5%.

[0081] (2) The influence of the type of surfactant

[0082] In this embodiment, a concentration of 2% is selected, as detailed below:

[0083] Table 2. Effect of Surfactant Type

[0084]

[0085] It can be seen that amphoteric and nonionic surfactants have better detection effects, while cationic and anionic surfactants have poorer effects. Therefore, amphoteric and nonionic surfactants are preferred in this invention.

[0086] Example 4: Increasing the Influence of Ultrasound

[0087] The operation is as follows:

[0088] S1: Prepare the whole blood treatment solution as described in the claim;

[0089] S2: Mix whole blood samples with whole blood processing solution, incubate, and then test.

[0090] S3: The incubated sample can also be subjected to ultrasonic treatment after incubation in step S2 and before detection.

[0091] This processing solution can be used as a standalone component in various existing kits. This example uses a chemiluminescence immunoassay kit (from Zhongyuan Huiji Biotechnology Co., Ltd.) for validation, and requires a chemiluminescence analyzer with a hematocrit (HCT) module. The procedure is as follows: a whole blood sample is divided into two parts. One part undergoes in-vitro HCT detection to calculate the percentage of bleeding cells; the other part is used for testing, with an equal volume of processing solution added, mixed, and then the other components of the kit added for reaction.

[0092] The specific ultrasound operation steps are as follows: 100 μL whole blood sample + 180 μL R2 solution + 100 μL R1 solution + 100 μL M solution, mix well, incubate at 37°C for 5 min, and magnetically separate and wash 3 times with the provided washing solution to remove the supernatant and retain the solid-phase immune complex of the magnetic beads. Then add 1000 μL of whole blood processing solution, vortex to mix well, and use a Xinzhi ultrasonic cell disruptor, select Φ6 amplitude rod, set the power to 30%, sample volume > 500 μL (2 mL centrifuge tube, liquid level above 1.5 cm), sonicate for 1 second each time, with an interval of 3 seconds, for a total of 3 sonications. After sonication, magnetically separate and wash 3 times with the provided washing solution to remove the supernatant and retain the solid-phase immune complex of the magnetic beads. Then add 300 μL of whole blood processing solution to disperse the complex, add AMPPD substrate, and measure light.

[0093] In clinical whole blood sample testing, some abnormal blood cells are prone to aggregation with magnetic beads in the reagent. Figure 2 The magnetic beads may be trapped or non-specifically adsorbed by blood cells, leading to poor repeatability and false positive results.

[0094] To address the aggregation of magnetic beads and blood cells, this invention uses an ultrasonic disruptor to process the aggregated samples, while simultaneously comparing them with plasma samples. By comparing the changes in signal values ​​of the aggregated samples before and after ultrasound, and the differences in signal values ​​between the aggregated samples and plasma samples after ultrasound, the feasibility of this method can be determined.

[0095] Application of whole blood processing solution

[0096] (1) Clinical sample assessment

[0097] Whole blood processing solutions were applied to tests for inflammation and myocardial diseases. Clinical whole blood samples were collected, and each sample was divided into two parts: one part was centrifuged to obtain a plasma sample, and the other part was left unprocessed as a whole blood sample. All samples were tested using either the processing solution or not, with one of the above-mentioned formulations used as the processing solution. The correlation between the plasma and whole blood backtesting values ​​obtained using the two methods was compared.

[0098] Test results as follows Figure 3 , Figure 4 As shown:

[0099] Depend on Figures 3-4 It can be seen that without the treatment solution, the comparison points of low-value whole blood samples are scattered and the correlation is poor when the test items are combined with whole blood treatment solution; after the test items are combined with whole blood treatment solution, the comparison points of low-value whole blood samples are more concentrated and the correlation improves. This indicates that whole blood treatment solution has a good convergence effect on low-value samples, reducing the number of samples with jump values.

[0100] (2) Repeatability assessment

[0101] The clinical samples were reproducible using a treatment solution, and the results are shown in Tables 3 and 4. The CV values ​​were all within 5%, indicating good reproducibility.

[0102] Table 3 IL-6 Projects

[0103]

[0104] Table 4 cTnI Project

[0105]

[0106] In summary, the application of this whole blood processing solution can reduce the relative deviation between plasma and whole blood samples, reduce interference during whole blood sample testing, and improve the accuracy of whole blood testing.

[0107] Results of ultrasound + whole blood processing

[0108] Depend on Figure 3 , 4 As can be seen, while the use of treatment solutions for each item has a good convergence effect on low-value samples and reduces the number of samples with jump values, a few abnormal samples with jump values ​​still exist. To address these abnormal samples, this invention employs ultrasonic treatment. Abnormal samples are screened, and then treated with a treatment solution combined with ultrasonic dispersion. The detection results before and after treatment are compared as follows: Figure 5 , Figure 6 .

[0109] Depend on Figure 5 , Figure 6 It can be seen that processing samples with whole blood solution and ultrasonic dispersion greatly improves the correlation between whole blood and plasma sample measurements and reduces the measurement deviation between whole blood and plasma samples, indicating that the method is feasible.

[0110] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A whole blood processing solution that can reduce interference in whole blood sample detection, characterized in that, The whole blood treatment solution comprises the following components: a surfactant and an anti-erythrocyte antibody; the surfactant is a non-hemolytic surfactant. The concentration ratio of the surfactant to the anti-erythrocyte antibody is 20-5000; The concentration of the surfactant is 1% to 5%, and the concentration of the anti-erythrocyte antibody is 0.01 mg / mL to 0.5 mg / mL; the surfactant is a nonionic surfactant or an amphoteric surfactant.

2. The whole blood treatment solution as described in claim 1, characterized in that, The surfactant is selected from: TETRONIC 1307, dodecyl hydroxypropyl sulfonyl betaine, sulfopropyl tetradecyl betaine, cocamidopropyl betaine, Surfynol(tm) 465, Surfynol(tm) 485 or SILWET L7600.

3. The whole blood treatment solution as described in any one of claims 1-2, characterized in that, The whole blood treatment solution also includes: buffer solution, osmotic pressure maintainer, stabilizer, protectant, polymer, and preservative.

4. The whole blood treatment solution as described in claim 3, characterized in that: The buffer solution is selected from PBS, Tris-HCl, HEPES or MES systems, and the concentration is 10-50 mM. The osmotic pressure maintaining agent is selected from NaCl and KCl; The stabilizer is selected from BSA, HSA, or casein, and the concentration is 0.5%~5%; The protective agent is selected from sucrose, mesotriose, lactose, trehalose, and hyaluronic acid, with a concentration of 1%-5%; The polymer is selected from dextran, sodium dextran sulfate, or PEG; The preservative is selected from sodium azide, BND or proclin 300, with a concentration of 0.02% to 0.05%.

5. An immunoassay kit, characterized in that, The kit contains whole blood processing solution according to any one of claims 1-4.

6. The application of the whole blood processing solution as described in any one of claims 1-4 in chemiluminescence detection.

7. A method for reducing interference from chemiluminescence detection of magnetic particles, characterized in that, Includes the following steps: S1: Prepare the whole blood treatment solution as described in any one of claims 1-4; S2: Mix whole blood samples with whole blood processing solution, incubate, and then test.

8. The method for reducing interference from chemiluminescence detection of magnetic particles as described in claim 7, characterized in that, The incubated sample can also be subjected to ultrasonic treatment after incubation and before detection in step S2.

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