Method for testing removal capability of medium and macromolecular toxins

Abstract

The invention discloses a method for testing medium and macromolecular toxin scavenging capacity, which comprises the following steps: (1) preparing test simulation liquid, and respectively preparing alpha 1-microglobulin, lambda-FLC, kappa-FLC and YKL-40 into the test simulation liquid by using anticoagulant bovine serum; (2) connecting an experimental loop according to a toxin clearance rate test system, adjusting the flow rates of blood and dialysate to be stable, and collecting a blood inlet and outlet sample; (3) concentrating the sample solution, and respectively detecting the concentrations of alpha1-microglobulin, lambda-FLC, kappa-FLC and YKL-40; and (4) calculating according to a formula to obtain the clearance rate. According to the method, pretreatment such as concentration is performed on the detection liquid, so that the detection liquid can conform to the detection range of a detection kit or equipment, the characteristics of high detection sensitivity, high accuracy, good repeatability and the like on medium and macromolecular toxins are realized, and the method can be used for quantitative analysis on medium and macromolecular substances.

Description

Technical Field

[0001] The invention relates to the technical field of blood purification, and in particular to a method for testing the ability to clear medium and macromolecular toxins. Background Art

[0002] Chronic kidney disease (CKD) is a serious disease in which the patient's renal function progressively declines, marked by a decrease in the eGFR or kidney damage or both, and lasts for more than three months. For patients with end-stage renal disease, dialysis is one of the main life-sustaining treatments, but hemodialysis patients are often accompanied by a high symptom burden: uremic pruritus, restless legs syndrome, longer recovery time after dialysis, and a high incidence of cardiovascular complications. The accumulation of medium-molecular toxins, especially medium- and large-molecular toxins (12-45kDa), directly affects the quality of life. Inflammation (accumulation of medium- and large-molecular toxins) is the core of CKD, leading to protein energy waste, anemia, malnutrition and cardiovascular disease, and sarcopenia (characterized by reduced muscle mass and weakness) also increases cardiovascular risk and total mortality. At present, conventional high-throughput dialyzers and hemodialysis filters can only remove small and medium-molecular toxins, and have limited ability to remove medium and large-molecular toxins.

[0003] Medium- and large-molecular toxins such as α1-microglobulin (α1-MG) are associated with the improvement of restless legs syndrome symptoms in hemodialysis patients. It is a glycoprotein in the human body that can sensitively reflect changes in glomerular filtration function. When the glomerular filtration rate (GFR) or endogenous creatinine clearance changes slightly, serum α1-MG can change significantly. The proinflammatory factor human cartilage glycoprotein 39 (YKL-40) is a glycoprotein produced and secreted by different types of cells. It has a promoting effect on cell adhesion, vascular smooth muscle cell migration, and activation of endothelial cell angiogenesis signaling pathways, and is involved in a variety of inflammatory processes, including atherosclerosis and endothelial dysfunction. Its accumulation is related to the prognosis of hemodialysis patients. Light chain protein is a monoclonal immunoglobulin secreted by plasma cells. It is mainly present in the blood and can bind to albumin in plasma to form light chains. The accumulation of λ or κ free light chains (λ-FLC, κ-FLC) can lead to multiple organ damage and increased risk of death. Therefore, during the dialysis process using an ultra-high-pass dialyzer, the establishment of a method to remove these target substances can better improve the performance of the dialysis membrane, improve patient prognosis, improve dialysis quality, and thereby improve survival rate and quality.

[0004] Chinese patent CN113694280A discloses a method for real-time monitoring of toxin content distribution in an extracorporeal dialyzer, mainly for one or more small molecule toxins in phosphate, oxalate, creatine and uric acid, which is not applicable to macromolecular toxins in the present invention; and for the common method of removing macromolecular toxins, such as chemiluminescence, the detection limit of immunoturbidimetry is relatively high, and it is necessary to add a lot of amounts when used for in vitro simulation test experiments to reach the detection range of existing detection methods, so that the cost of testing a dialyzer is up to more than 100,000, and the corresponding detection equipment is rare, and the equipment procurement cost is hundreds of thousands, and the research and testing cost of these macromolecular toxins by dialyzer manufacturers is greatly increased. There is also an enzyme-linked immunosorbent assay (ELISA) with a relatively low detection limit, and the test repeatability and stability are poor. Therefore, an in vitro circulation test method is established, and the effective pretreatment of the test sample solution can solve the problem of high cost and achieve effective detection. Summary of the invention

[0005] The purpose of the present invention is to provide a method for testing the clearance ability of medium and macromolecular toxins, so as to solve the problems of high cost and poor stability in the in vitro circulation test of medium and macromolecular toxins in the prior art.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for testing the ability of removing medium and macromolecular toxins, comprising the following steps:

[0008] (1) preparing test simulation solutions, respectively preparing α1-microglobulin, λ-FLC, κ-FLC, and YKL-40 using anticoagulant bovine serum to prepare test simulation solutions;

[0009] (2) Connect the experimental circuit according to the toxin clearance test system, adjust the blood and dialysate flow rates to stability, and collect blood inlet and outlet samples;

[0010] (3) After the sample solution is concentrated, the concentrations of α1-microglobulin, λ-FLC, κ-FLC, and YKL-40 are detected respectively;

[0011] (4) The clearance rate was calculated according to the formula.

[0012] Furthermore, the concentrations of α1-microglobulin, λ-FLC, κ-FLC and YKL-40 in (1) are 500-2000 ug / L, 20-800 ng / ml, 2-20 mg / l and 5-30 mg / l respectively.

[0013] Furthermore, the clearance test conditions in the toxin clearance test system in (2) are as follows: temperature 37±1°C, setting Q D =500-800ml / min, QBI =200-400ml / min, Q F =0-60ml / min, the Q D is the dialysate flow rate, the Q BI is the blood flow rate at the product inlet, the Q F is the filtrate flow rate.

[0014] Furthermore, the sample solution in (3) is concentrated by methods including but not limited to nitrogen blowing.

[0015] Furthermore, the concentration of α1-microglobulin in (3) is detected using methods including but not limited to ELISA kits.

[0016] Furthermore, the concentrations of λ-FLC, κ-FLC and YKL-40 in (3) are detected using, but not limited to, a chemiluminescence analyzer and a biochemical analyzer.

[0017] Furthermore, the calculation formula in (4) is as follows:

[0018] Where: C BI The concentration of the solution at the blood inlet; C BO The concentration of the solution at the blood outlet, Q BI is the blood flow rate at the product inlet, ml / min; Q F is the filtrate flow rate, ml / min.

[0019] Furthermore, the concentration unit of each substance tested is ug / L, ng / ml or mg / L. In the formula, the numerator and denominator of the concentration unit can be canceled, and the final unit of the clearance rate K is ml / min.

[0020] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects:

[0021] It is used to establish the evaluation index of medium and large molecular toxins for ultra-high-throughput dialyzers, and solve the current situation of lack of evaluation methods and indicators for medium and large molecules for dialyzers. By pre-treating the test liquid by concentration, it can meet the detection range of the test kit or equipment, and achieve high detection sensitivity, accuracy, good repeatability and other characteristics for α1-microglobulin, λ-FLC, κ-FLC, and YKL-40, which can be used for quantitative analysis of medium and large molecular substances. The establishment of this method can more comprehensively evaluate the ability of ultra-high-throughput dialyzers to remove medium and large molecular toxins, and has a promoting effect on improving the performance of ultra-high-throughput dialyzer membranes.

[0022] (1) The present invention provides a method for testing the ability to remove medium and macromolecular toxins. The sample liquid is pretreated and concentrated to within the detection limit of the instrument by nitrogen purging. The method has the characteristics of low preparation cost, high detection accuracy, good repeatability, and no introduction of impurities.

[0023] (2) The present invention provides a method for testing the ability to clear medium and large molecular toxins. The in vitro simulated hemodialysis treatment model simulates clinical conditions to determine the clearance rate of medium and large molecular toxins. The establishment of this method can more comprehensively reflect the clearance ability of ultra-high-flux dialyzers for medium and large molecules, and can provide guidance for improving the performance of high-flux dialyzer membranes.

[0024] (3) Testing the in vitro clearance of α1-MG, YKL-40, and free light chains (λ-FLC, κ-FLC), focusing on the clearance of higher molecular weight toxins, will be helpful in improving the quality of medical device products;

[0025] (4) Research on detection methods for toxins such as α1-MG, YKL-40, free light chains (λ-FLC, κ-FLC), etc. will help promote the establishment of detection standards for such substances;

[0026] (5) Provide a basis for establishing clearance indicators for substances such as α1-MG, YKL-40, free light chains (λ-FLC, κ-FLC), and provide a reference for product performance standardization and performance improvement in the medical device market. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0028] Figure 1 is a connection circuit diagram of a clearance test according to an embodiment of the present invention;

[0029] Figure 2 The embodiment of the present invention is multiple tests Q BI =Result graph of the clearance rate of medium and large molecules at 200 / min;

[0030] Figure 3 The embodiment of the present invention is multiple tests Q BI =Result graph of the clearance rate of medium and large molecules at 300 ml / min;

[0031] Figure 4 The embodiment of the present invention is multiple tests Q BI=The result graph of the clearance rate of medium and large molecules when 400ml / min. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The present invention provides

[0038] Example 1

[0039] Prepare α1-microglobulin at a concentration of 500-2000ug / L and 2L anticoagulated bovine serum Figure 1 Connect the experimental circuit. The clearance test is performed at 37±1℃, set Q D =500ml / min, Q BI =200-400ml / min, Q F= 0ml / min, adjust the blood flow rate to a stable level (including temperature, flow rate and pressure), collect blood inlet and outlet samples. Concentrate the sample solution 10 times, test the concentrated sample according to the ELISA kit instruction manual, calculate the concentration of α1-microglobulin, and calculate the clearance rate using the formula.

[0040] Example 2

[0041] Prepare chitinase protein-40 (YKL-40) at a concentration of 20-800 ng / ml, 2L anticoagulated bovine serum Figure 1 Connect the experimental circuit. At 37±1℃, set QD=500ml / min, QBI=200-400ml / min, QF=0ml / min, adjust the blood flow rate to stability (including temperature, flow rate and pressure), and collect blood inlet and outlet samples. The sample liquid is concentrated 10 times, and the chemiluminescence analyzer is used to detect it and calculate the YKL-40 concentration, and the clearance rate is calculated using the formula.

[0042] Example 3

[0043] Prepare κ free light chain (κ-FLC) at a concentration of 2-20 mg / l, 2L of anticoagulated bovine serum Figure 1 Connect the experimental circuit. At 37±1℃, set QD=500ml / min, QBI=200-400ml / min, QF=0ml / min, adjust the blood flow rate to stability (including temperature, flow rate and pressure), and collect blood inlet and outlet samples. The sample liquid is concentrated 50 times, and the biochemical analyzer is used to detect it and calculate the κ-FLC concentration, and the clearance rate is calculated using the formula.

[0044] Example 4

[0045] Prepare λ-free light chain (λ-FLC) at a concentration of 5-30 mg / l with 2L anticoagulated bovine serum. Figure 1 Connect the experimental circuit. At 37±1℃, set QD=500ml / min, QBI=200-400min, QF=0ml / min. Adjust the blood and filtrate flow rates to stability (including temperature, flow rate and pressure), and collect blood inlet and outlet samples. Concentrate the sample 50 times, use a biochemical analyzer to detect it and calculate the λ-FLC concentration, and use the formula to calculate the clearance rate.

[0046] like Figure 2-4 As shown, when Q D =500ml / min, Q BI =200-400ml / min, Q F =0ml / min, the clearance ability and repeatability test results for medium and large molecules.

[0047] Finally, it should be noted that:

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for testing the ability of removing medium and macromolecular toxins, characterized in that: The following steps are involved: (1) preparing test simulation solutions, respectively preparing α1-microglobulin, λ-FLC, κ-FLC, and YKL-40 using anticoagulant bovine serum to prepare test simulation solutions; (2) Connect the experimental circuit according to the toxin clearance test system, adjust the blood and dialysate flow rates to stability, and collect blood inlet and outlet samples; (3) After the sample solution is concentrated, the concentrations of α1-microglobulin, λ-FLC, κ-FLC, and YKL-40 are detected respectively; (4) The clearance rate was calculated according to the formula.

2. The method for testing the ability of removing medium and macromolecular toxins according to claim 1, characterized in that: The concentrations of α1-microglobulin, λ-FLC, κ-FLC and YKL-40 in (1) are 500-2000ug / L, 20-800ng / ml, 2-20mg / l and 5-30mg / l respectively.

3. The method for testing the ability of removing medium and macromolecular toxins according to claim 1, characterized in that: The clearance test conditions in the toxin clearance test system (2) are as follows: temperature 37±1°C, setting Q D =500-800ml / min, Q BI =200-400ml / min, Q F =0-60ml / min, the Q D is the dialysate / dialysis simulation fluid flow rate, the Q BI is the blood flow rate at the product inlet, the Q F is the filtrate flow rate.

4. The method for testing the ability of removing medium and macromolecular toxins according to claim 1, characterized in that: The sample solution in (3) is concentrated by methods including but not limited to nitrogen blowing.

5. The method for testing the ability of removing medium and macromolecular toxins according to claim 1, characterized in that: The concentration of α1-microglobulin in (3) is detected using methods including but not limited to ELISA kits.

6. The method for testing the ability of removing medium and macromolecular toxins according to claim 1, characterized in that: The concentrations of λ-FLC, κ-FLC and YKL-40 in (3) are detected by methods including but not limited to chemiluminescence analyzer and biochemical analyzer.

7. The method for testing the ability of removing medium and macromolecular toxins according to claim 1, characterized in that: The calculation formula in (4) is as follows: Where: C BI The concentration of the solution at the blood inlet; C BO The concentration of the solution at the blood outlet, Q BI is the blood flow rate at the product inlet, ml / min; Q F is the filtrate flow rate, ml / min.

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