Total bilirubin test kit resistant to interference by high m-protein

Abstract

The present application relates to the technical field of biological detection, and particularly relates to a total bilirubin detection kit resisting high M protein interference. The total bilirubin detection kit resisting high M protein interference provided by the present application prevents the sample from being interfered in the detection process by adding a solubilizing dispersing agent and removing metal ions, optimizes the reagent components, and effectively avoids and reduces the interference of high M protein on the sample detection process. The solubilizing dispersing agent is added in the determination reagent while the ion concentration is reduced, the phenomenon of aggregation and precipitation in the detection process of the high M protein sample is eliminated, the detection process is no longer interfered, and the accuracy of the detection of the sample is improved.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a total bilirubin detection kit that resists interference from high M protein. Background Technology

[0002] Bilirubin is produced by the metabolism of porphyrin compounds in the human body. After being transported to the liver via the bloodstream, it is processed. Bilirubin mainly includes free bilirubin and conjugated bilirubin. Clinically, it is primarily used to diagnose related liver diseases and biliary obstruction, and is one of the most routine items in clinical laboratory tests, playing a very important role in the diagnosis of hepatobiliary diseases. After free bilirubin reaches the liver via blood circulation, it is bound to carrier proteins on the hepatocyte cell membrane by specific apolipoproteins. Through active transport within the cell, bilirubin in the blood can rapidly transfer from the outer surface of the hepatocyte membrane to the inner surface, and then enter the cytoplasm of the hepatocyte. Once in the hepatocyte cytoplasm, bilirubin binds to carrier proteins present in the cytoplasm, and is then passively transported to the smooth endoplasmic reticulum in the form of a complex. Catalyzed by bilirubinuridine diphosphate glucuronyl transferase on the smooth endoplasmic reticulum of hepatocytes, the propionic acid group of bilirubin in the complex can rapidly react with uridine diphosphate-α-glucuronic acid to generate bilirubin glucuronide monoester and diester, thereby transforming into conjugated bilirubin.

[0003] Conjugated bilirubin, within the cytoplasm of hepatocytes, can be secreted along with bile salts via bile secretory organs (Golgi complex, endoplasmic reticulum, lysosomes, etc.) into the bile capillaries and excreted with bile. The liver, as the largest multifunctional solid organ in the human body, is known as the "metabolism center." It is not only the central organ for the metabolism of the three major substances in the body but also plays a vital role in biotransformation, secretion, and excretion, participating in important functions such as fluid balance, blood volume regulation, and immune phagocytosis. Therefore, detecting bilirubin metabolism is widely used clinically as an important indicator for diagnosing hepatobiliary diseases and is one of the most routine items in clinical laboratory testing.

[0004] M protein (also known as monoclonal immunoglobulin or paraprotein) is an abnormal immunoglobulin molecule or fragment thereof secreted by the abnormal proliferation of monoclonal B lymphocytes or plasma cells. It is highly homogeneous in its amino acid composition and sequence. M protein is commonly found in multiple myeloma, heavy chain disease, light chain disease, hypergammaglobulinemia, primary macroglobulinemia, malignant lymphoma, and monoclonal gammaglobulinemia of undetermined significance, among other clonal proliferative disorders of B lymphocytes or plasma cells, with multiple myeloma being the most common. Multiple myeloma is a common and life-threatening hematologic malignancy caused by the abnormal proliferation of clonal plasma cells in the bone marrow, but its pathogenesis and etiology are not fully understood. It is one of the hematologic malignancies with the highest incidence and mortality rates worldwide, ranking second among hematologic malignancies, accounting for 15% of all malignancies and 1% of all malignancies.

[0005] With the continuous progress of social culture, scholars have increasingly proposed that M protein can interfere with the detection of various biochemical indicators and have analyzed the possible mechanisms of this effect. Literature reports that the presence of M protein can affect various biochemical indicators such as high-density lipoprotein, C-reactive protein, serum creatinine, and bilirubin. The mechanism of this effect is largely attributed to the precipitation of M protein when it mixes with detection reagents, resulting in a significantly higher baseline absorbance reading in patient samples than normal, thus leading to inaccurate test results.

[0006] Interference from M proteins in routine biochemical assays may arise from specific analyses, specific or non-specific binding to specific analyte components in the analytical system leading to precipitation, or various chemical or immunological interferences generated during the analytical process. M proteins can also bind to many preanalyte substances, including cations, anions, enzymes, hormones, and lipoproteins, thus affecting test results. Depending on their affinity for the antigenic determinants of these preanalytes, M proteins may alter the function or metabolism of these substances.

[0007] Currently, most clinical methods for bilirubin detection use chemical oxidation methods, with nitrites and vanadates being the commonly chosen oxidants. Changes in the pH, ionic strength, temperature, or solvent properties of the reaction solution can alter the properties of proteins, reducing their solubility and leading to precipitation. Interference occurs when surfactants are unable to dissolve the precipitate formed by M protein in an acidic environment; therefore, the presence of M protein interferes with bilirubin determination using chemical oxidation methods. Clinical studies have revealed cases where high M protein levels significantly affect test results, and kits for serum bilirubin detection do not address the need to avoid M protein interference, nor do they provide methods to mitigate it.

[0008] Currently, methods or kits for determining total bilirubin on the international market ignore the interference caused by high ion concentrations and the lack of solubilizing and dispersing agents when detecting high M protein samples. As a result, the results of this type of sample are inaccurate and affect clinical diagnosis. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a total bilirubin detection kit that is resistant to interference from high M protein. The total bilirubin detection kit that is resistant to interference from high M protein provided by the present invention can eliminate the interference of high M protein samples on the detection and improve the accuracy of the detection.

[0010] This invention provides a reagent combination, comprising reagent 1 and reagent 2, wherein:

[0011] Reagent 1 comprises citric acid, TAGAT CH40, emulsifier OP-09, Kao 20AB (Japan), and NaN3, wherein the mass ratio of citric acid, TAGAT CH40, emulsifier OP-09, Kao 20AB (Japan), and NaN3 is (19.213–57.639):(10–20):(10–20):(50–100):(0.975–1.95).

[0012] The reagent 2 includes triethanolamine, an oxidant, and Proclin. The oxidant includes sodium nitrite or sodium metavanadate, and the mass ratio of triethanolamine to oxidant is (7.45–14.9):(0.138–1.438).

[0013] Further, reagent 1 comprises: 100–300 mmol / L citric acid, 10–20 g / L TAGAT CH40, 10–20 g / L emulsifier OP-09, 50–100 g / L Kao 20AB (Japan), and 0.015–0.03 mol / L NaN3;

[0014] The reagent 2 comprises: 50–100 mmol / L triethanolamine, 2–20 mmol / L oxidant, and at least 0.2 wt% Proclin, wherein the oxidant comprises sodium nitrite or sodium metavanadate.

[0015] Compared to other reagent combinations, the reagent combination provided by this invention, by adding the solubilizing and dispersing agent TAGAT CH40 to the assay reagent and simultaneously reducing the ion concentration (eliminating the addition of NaCl), prevents the aggregation and precipitation phenomenon during the detection of high M protein samples, thus ensuring that the detection process is no longer interfered with, thereby improving the reactivity of the assay reagent. The added solubilizing and dispersing agent and the reduction in ion concentration can act as a dispersant and charge balancer during the detection of high M protein samples, ensuring that the sample detection process is not interfered with by preventing precipitation, thereby improving the accuracy of the detection of such samples and providing more accurate results for the clinical detection of bilirubin in these patients. Due to the synergistic effect between the components in the reagent combination, a more accurate technical effect is obtained.

[0016] In some embodiments, reagent 1 includes:

[0017] The concentration of the citric acid is 100 mmol / L, 200 mmol / L, or 300 mmol / L;

[0018] The concentration of TAGAT CH40 is 10 g / L, 15 g / L or 20 g / L;

[0019] The concentration of the emulsifier OP-09 is 10 g / L, 15 g / L or 20 g / L;

[0020] The concentration of the Japanese Kao 20AB is 50 g / L, 75 g / L or 100 g / L;

[0021] The concentration of NaN3 is 0.015 mol / L, 0.02 mol / L, or 0.03 mol / L.

[0022] In some embodiments, reagent 2 includes:

[0023] The concentration of the triethanolamine is 50 mmol / L, 75 mmol / L, or 100 mmol / L;

[0024] The antioxidant is selected from 7 mmol / L sodium nitrite, 10 mmol / L sodium metavanadate, or 20 mmol / L sodium metavanadate.

[0025] The concentration of Proclin was 0.2 wt%.

[0026] In some specific embodiments, reagent 1 includes water, 200 mmol / L citric acid, 15 g / L TAGAT CH40, 15 g / L emulsifier OP-09, 75 g / L Kao 20AB, and 0.02 mol / L NaN3;

[0027] Reagent 2 comprises water, 75 mmol / L triethanolamine, 7 mmol / L sodium nitrite, and 0.2 wt% Proclin.

[0028] In some specific embodiments, reagent 1 includes water, 100 mmol / L citric acid, 10 g / L TAGAT CH40, 10 g / L emulsifier OP-09, 50 g / L Kao 20AB, and 0.015 mol / L NaN3;

[0029] Reagent 2 comprises water, 50 mmol / L triethanolamine, 10 mmol / L sodium metavanadate, and 0.2 wt% Proclin.

[0030] In some specific embodiments, reagent 1 includes water, 200 mmol / L citric acid, 20 g / L TAGAT CH40, 10 g / L emulsifier OP-09, 50 g / L Kao 20AB, and 0.015 mol / L NaN3;

[0031] Reagent 2 comprises water, 50 mmol / L triethanolamine, 10 mmol / L sodium metavanadate, and 0.2 wt% Proclin.

[0032] In some specific embodiments, reagent 1 includes water, 300 mmol / L citric acid, 20 g / L TAGAT CH40, 20 g / L emulsifier OP-09, 100 g / L Kao 20AB, and 0.03 mol / L NaN3;

[0033] Reagent 2 comprises water, 100 mmol / L triethanolamine, 20 mmol / L sodium metavanadate, and 0.2 wt% Proclin.

[0034] Experiments show that, in the above embodiments, the reagent combination, due to the synergistic effect between its components and concentrations, can better eliminate interference from high M protein samples and more accurately measure the total bilirubin concentration in the sample, thereby achieving more accurate technical results.

[0035] This invention provides the application of the aforementioned reagent combination in the preparation of reagents or kits for the detection of total bilirubin that are resistant to high M protein interference.

[0036] This invention provides a reagent or kit for detecting total bilirubin against high M protein interference, comprising the aforementioned reagent combination.

[0037] The present invention provides a method for detecting total bilirubin, comprising detecting the sample using the total bilirubin detection reagent or kit that resists high M protein interference.

[0038] This invention provides a total bilirubin assay kit resistant to high M protein interference. By adding solubilizing and dispersing agents and removing metal ions, interference with the sample is prevented during the detection process. The optimized reagent composition effectively avoids and reduces the interference of high M protein on the sample detection process. Adding solubilizing and dispersing agents to the assay reagent while reducing the ion concentration (eliminating the addition of NaCl) prevents the aggregation and precipitation of high M protein samples during detection, thus improving the accuracy of the detection process. The added solubilizing and dispersing agents and the reduced ion concentration act as dispersants and charge balancers during the detection of high M protein samples, preventing precipitation and ensuring that the sample detection process is not interfered with, thereby improving the accuracy of the detection of such samples and providing more accurate results for clinical bilirubin testing in these patients. Attached Figure Description

[0039] Figure 1 The results of a correlation analysis between the existing reagents and the reagents in Example 2 were used to test 40 clinical patient samples in the demonstration case.

[0040] Figure 2 The results show the sample reaction curves of the existing reagents in the example and the reagents in Example 2. Detailed Implementation

[0041] This invention provides a total bilirubin detection kit resistant to high M protein interference. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0042] This invention primarily eliminates interference from high M protein samples by adding solubilizing and dispersing agents and reducing ion concentrations in the reagent, thereby greatly improving the reagent's resistance to M protein interference and providing greater possibilities for the diagnosis of special clinical samples.

[0043] A total bilirubin assay kit for detecting high M protein:

[0044] The main principle is that bilirubin is oxidized to biliverdin by a chemical oxidant under the action of surfactants and solubilizing and dispersing agents. The concentration of total bilirubin in the sample is obtained by detecting the change in absorbance at a specific wavelength.

[0045] This invention uses a dual-reagent kit for total bilirubin concentration determination. Previous experiments have demonstrated that the following reagent ratio provides ideal results for both total bilirubin concentration determination and detection of high-M protein samples. The buffer solution used in this invention is a citric acid and triethanolamine buffer.

[0046]

[0047]

[0048] All test materials used in this invention are commercially available products. Specifically, TAGAT CH40 was purchased from Beijing Apis Biotechnology Co., Ltd., emulsifier OP-09 from Shanghai Titan Technology Co., Ltd., and 20AB from Kao Corporation of Japan. The invention is further illustrated below with reference to specific examples.

[0049] Example 1

[0050] This example describes a dual-reagent total bilirubin assay kit against high-M protein, the components of which are as follows:

[0051] Reagent 1:

[0052]

[0053] The following conditions were set on the fully automated biochemical analyzer: temperature: 37℃, reaction time: 10 minutes, initial absorbance: 0.005±0.005, main wavelength: 450nm, secondary wavelength: 548nm, volume ratio of total bilirubin sample to reagent: 1:30, negative reaction, delay time: 60s, detection time: 120s, theoretical K value: -900.

[0054] After adding the sample and reagents, they are thoroughly mixed and a reaction occurs. Finally, the reactants are placed in a biochemical analyzer, and the decrease in absorbance at the main wavelength of 450 nm is detected to obtain the concentration of total bilirubin in the sample.

[0055] Example 2

[0056] This example describes a two-reagent anti-high M protein total bilirubin, the components of which are as follows: including:

[0057] Reagent 1:

[0058]

[0059] Reagent 2:

[0060] Triethanolamine 50 mmol / L

[0061] Sodium metavanadate 10 mmol / L

[0062] Proclin 0.2%

[0063] The following conditions were set on the fully automated biochemical analyzer: temperature: 37℃, reaction time: 10 minutes, initial absorbance: 0.005±0.005, main wavelength: 450nm, secondary wavelength: 548nm, volume ratio of total bilirubin sample to reagent: 1:30, negative reaction, delay time: 60s, detection time: 120s, theoretical K value: -900.

[0064] After adding the sample and reagents, they are thoroughly mixed and a reaction occurs. Finally, the reactants are placed in a biochemical analyzer, and the decrease in absorbance at the main wavelength of 450 nm is detected to obtain the concentration of total bilirubin in the sample.

[0065] Example 3

[0066] This example describes a two-reagent anti-high M protein total bilirubin, the components of which are as follows: including:

[0067] Reagent 1:

[0068]

[0069] Reagent 2:

[0070] Triethanolamine 50 mmol / L

[0071] Sodium metavanadate 10 mmol / L

[0072] Proclin 0.2%

[0073] The following conditions were set on the fully automated biochemical analyzer: temperature: 37℃, reaction time: 10 minutes, initial absorbance: 0.005±0.005, main wavelength: 450nm, secondary wavelength: 548nm, volume ratio of total bilirubin sample to reagent: 1:30, negative reaction, delay time: 60s, detection time: 120s, theoretical K value: -900.

[0074] After adding the sample and reagents, they are thoroughly mixed and a reaction occurs. Finally, the reactants are placed in a biochemical analyzer, and the decrease in absorbance at the main wavelength of 450 nm is detected to obtain the concentration of total bilirubin in the sample.

[0075] Example 4

[0076] This example describes a two-reagent anti-high M protein total bilirubin, the components of which are as follows: including:

[0077] Reagent 1:

[0078]

[0079] Reagent 2:

[0080] Triethanolamine 100 mmol / L

[0081] Sodium metavanadate 20 mmol / L

[0082] Proclin 0.2%

[0083] The following conditions were set on the fully automated biochemical analyzer: temperature: 37℃, reaction time: 10 minutes, initial absorbance: 0.005±0.005, main wavelength: 450nm, secondary wavelength: 548nm, volume ratio of total bilirubin sample to reagent: 1:30, negative reaction, delay time: 60s, detection time: 120s, theoretical K value: -900.

[0084] After adding the sample and reagents, they are thoroughly mixed and a reaction occurs. Finally, the reactants are placed in a biochemical analyzer, and the decrease in absorbance at the main wavelength of 450 nm is detected to obtain the concentration of total bilirubin in the sample.

[0085] Comparative Example 1

[0086] This example describes a two-reagent anti-high M protein total bilirubin, the components of which are as follows: including:

[0087] Reagent 1:

[0088]

[0089]

[0090] Reagent 2:

[0091] Triethanolamine 50 mmol / L

[0092] Sodium metavanadate 10 mmol / L

[0093] Proclin 0.2%

[0094] The following conditions were set on the fully automated biochemical analyzer: temperature: 37℃, reaction time: 10 minutes, initial absorbance: 0.005±0.005, main wavelength: 450nm, secondary wavelength: 548nm, volume ratio of total bilirubin sample to reagent: 1:30, negative reaction, delay time: 60s, detection time: 120s, theoretical K value: -900.

[0095] After adding the sample and reagents, they are thoroughly mixed and a reaction occurs. Finally, the reactants are placed in a biochemical analyzer, and the decrease in absorbance at the main wavelength of 450 nm is detected to obtain the concentration of total bilirubin in the sample.

[0096] Example of effect

[0097] Experimental data and performance evaluation:

[0098] 1) Analytical sensitivity: Different batches of reagents were prepared using the above method and tested for analytical sensitivity together with existing patented reagents. The test results are shown in Table 1 below.

[0099] Existing patented reagent: A total bilirubin detection reagent, comprising a diluent and a reaction reagent, wherein the diluent is composed of the following components:

[0100] Tris buffer 1 (pH 6.5) 0.5 mol / L;

[0101] Triton 5% (by weight);

[0102] Sodium azide 0.2% (by mass);

[0103] Vitamin C oxidase 500 KU / L;

[0104] The reaction reagents described herein consist of the following components:

[0105] Citrate buffer 2 (pH 3.0): 0.5 mol / L;

[0106] Sodium chloride 45g / L;

[0107] Disodium EDTA 2-10 g / L;

[0108] Tween 80 5-20ml / L;

[0109] Sodium dodecyl sulfate 10-100 g / L;

[0110] Sodium persulfate 100-200 g / L;

[0111] Sulfuric acid 0.1-2 ml / L;

[0112] Sodium azide 0.5 g / L;

[0113] Sucrose 20g / L.

[0114] Table 1

[0115] Batch number 1 2 3 4 5 6 Sensitivity of Existing Invention Analysis 0.1215 0.1218 0.1218 0.1247 0.1227 0.1215 Case Study 2: Sensitivity Analysis 0.1265 0.1270 0.1220 0.1227 0.1215 0.1265

[0116] Experimental data show that the analytical sensitivity of the reagent obtained in the second specific embodiment of the present invention is not much different from that of the reagents of the prior art.

[0117] 2) Correlation detection: Using the reagents obtained above, 40 clinical patient samples were tested together with existing reagents and the correlation was compared (30 cases were normal and 10 cases were abnormal). The results are shown in Table 2 below.

[0118] Table 2

[0119]

[0120]

[0121] Correlation data such as Figure 1 As shown, the results indicate that the detection results obtained using the total bilirubin detection reagent provided by this invention have good correlation data.

[0122] 3) Repeatability testing: Under the same conditions and using reagents from the same batch, clinical precision samples and quality control samples were tested 20 times within one day. The mean and SD were calculated for each sample, and the coefficient of variation (CV) was also calculated. The results are shown in Tables 3 and 4 below.

[0123] Table 3

[0124]

[0125]

[0126] Table 4

[0127]

[0128] The experimental results clearly show that the specific implementation examples of this invention are slightly better than existing inventions in terms of repeatability.

[0129] 4) High M protein sample testing:

[0130] 1. Clinical samples with high M protein levels were tested using three reagents, and the results are shown in Table 5 below.

[0131] Table 5

[0132]

[0133] The experimental results clearly show that existing inventions produce negative values ​​when detecting this type of special sample due to interference. Therefore, the specific implementation of this invention is superior to existing inventions in the detection of high M protein. At the same time, the experimental data of Comparative Example 1 also shows the key role played by the addition of TAGAT CH40.

[0134] 2. Differences in sample response curves

[0135] From the reaction curve of sample 7 ( Figure 2 It can be clearly seen that the anti-M protein interference performance of the present invention is far superior to that of the prior art. In the reagent 1 stage, the reaction curve of the prior art shows a clear upward trend, while the present invention shows no trend of interference, and the difference is significant.

[0136] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A total bilirubin detection reagent resistant to high M protein interference, characterized in that, Including reagent 1 and reagent 2, wherein: Reagent 1 is composed of 100-300 mmol / L citric acid, 10-20 g / L TAGAT CH40, 10-20 g / L emulsifier OP-09, 50-100 g / L Kao 20AB (Japan), and 0.015-0.03 mol / L NaN3. The reagent 2 consists of 50-100 mmol / L triethanolamine, 2-20 mmol / L oxidant and 0.2 wt%-0.5 wt% Proclin, wherein the oxidant is sodium nitrite or sodium metavanadate.

2. The total bilirubin detection reagent against high M protein interference according to claim 1, characterized in that, In reagent 1: The concentration of the citric acid is 100 mmol / L, 200 mmol / L, or 300 mmol / L; The concentration of TAGAT CH40 is 10 g / L, 15 g / L or 20 g / L; The concentration of the emulsifier OP-09 is 10 g / L, 15 g / L or 20 g / L; The concentration of the Japanese Kao 20AB is 50 g / L, 75 g / L or 100 g / L; The concentration of NaN3 is 0.015 mol / L, 0.02 mol / L, or 0.03 mol / L.

3. The total bilirubin detection reagent against high M protein interference according to claim 1, characterized in that, In reagent 2: The concentration of the triethanolamine is 50 mmol / L, 75 mmol / L, or 100 mmol / L; The antioxidant is selected from 7 mmol / L sodium nitrite, 10 mmol / L sodium metavanadate, or 20 mmol / L sodium metavanadate. The concentration of Proclin was 0.2 wt%.

4. The total bilirubin detection reagent against high M protein interference according to any one of claims 1 to 3, characterized in that, The reagent 1 is composed of water, 200 mmol / L citric acid, 15 g / L TAGAT CH40, 15 g / L emulsifier OP-09, 75 g / L Kao 20AB, and 0.02 mol / L NaN3; Reagent 2 consists of water, 75 mmol / L triethanolamine, 7 mmol / L sodium nitrite and 0.2 wt% Proclin.

5. The total bilirubin detection reagent against high M protein interference according to any one of claims 1 to 3, characterized in that, The reagent 1 is composed of water, 100 mmol / L citric acid, 10 g / L TAGAT CH40, 10 g / L emulsifier OP-09, 50 g / L Kao 20AB, and 0.015 mol / L NaN3; Reagent 2 consists of water, 50 mmol / L triethanolamine, 10 mmol / L sodium metavanadate, and 0.2 wt% Proclin.

6. The total bilirubin detection reagent against high M protein interference according to any one of claims 1 to 3, characterized in that, The reagent 1 is composed of water, 200 mmol / L citric acid, 20 g / L TAGAT CH40, 10 g / L emulsifier OP-09, 50 g / L Kao 20AB, and 0.015 mol / L NaN3; Reagent 2 consists of water, 50 mmol / L triethanolamine, 10 mmol / L sodium metavanadate, and 0.2 wt% Proclin.

7. The total bilirubin detection reagent against high M protein interference according to any one of claims 1 to 3, characterized in that, The reagent 1 is composed of water, 300 mmol / L citric acid, 20 g / L TAGAT CH40, 20 g / L emulsifier OP-09, 100 g / L Kao 20AB, and 0.03 mol / L NaN3; Reagent 2 consists of water, 100 mmol / L triethanolamine, 20 mmol / L sodium metavanadate, and 0.2 wt% Proclin.

Images