Biological diluent and composite quality control containing the same

Abstract

The application discloses a biological diluent and a composite quality control product containing the same. In the application, the biological diluent can be used for preparing a composite quality control product for urine multi-item detection, improving the stability of the composite quality control product, so that the composite quality control product can be applied to simultaneous detection of 13 common urine detection items, and the time cost and economic cost of multiple quality controls and multiple detections in clinical use can be reduced. The composite quality control product for urine multi-item detection containing the biological diluent provided by the application has a very stable mixed system. After 24 months of stability observation at 2-8 DEG C, the content value drop is within 5%, the quality control measurement value deviation caused by the uncertainty of the quality control itself is extremely small, and the stability of the quality control product meets the requirements of the field.

Description

Technical Field

[0001] This invention relates to the field of medical testing, and particularly to biological diluents and composite quality control products containing them. Background Technology

[0002] Quality control materials are used to detect and control the accuracy and precision of clinical laboratory testing methods, and are an indispensable part of laboratory quality management. Clinically, urine tests can assess kidney damage early, allowing for intervention and treatment. Currently, common urine tests for kidney function include urinary microalbumin, urinary α1-microglobulin, urinary β2-microglobulin, urinary transferrin, urinary immunoglobulin g, urinary retinol-binding protein, and urinary creatinine. However, due to the differences in the chemical properties of the analytes for these tests, the suitable storage conditions for their quality control materials differ, making it unsuitable to formulate a composite quality control material for a single test. For example, the quality control materials for two enzymatic tests, creatinine (CR) and N-acetyl-β-D-glucosidase (NAG), have poor stability and are difficult to prepare into a composite quality control material for joint testing. Multiple tests also suffer from high costs in terms of manpower and resources, high testing prices, and long testing cycles. If a stable composite quality control material could be developed, it would be possible to obtain results for multiple key indicators from a single urine sample test. Therefore, there is an urgent need in this field to develop a stable composite quality control system for multiple urine tests. Summary of the Invention

[0003] The purpose of this invention is to provide a biological diluent.

[0004] Another object of the present invention is to provide a composite quality control product for multiple urine tests.

[0005] To address the aforementioned technical problems, the first aspect of the present invention provides a bio-diluent, the bio-diluent comprising:

[0006] Buffer solution, sodium chloride, trehalose, sucrose, surfactant, glycerol, sarcosine, arginine, metal chelating agent, protectant, preservative and deionized water.

[0007] In some preferred embodiments, the content of the buffer solution is 4-6 parts by weight; more preferably 5-5.5 parts by weight.

[0008] In some preferred embodiments, the sodium chloride content is 20-30 parts by weight; more preferably, it is 25-30 parts by weight.

[0009] In some preferred embodiments, the trehalose content is 15-25 parts by weight; more preferably, it is 17-23 parts by weight.

[0010] In some preferred embodiments, the sucrose content is 15-25 parts by weight; more preferably, it is 17-23 parts by weight.

[0011] In some preferred embodiments, the surfactant content is 1-3 parts by weight; more preferably 1.5-2.5 parts by weight.

[0012] In some preferred embodiments, the glycerol content is 180-210 parts by weight; more preferably, it is 190-200 parts by weight.

[0013] In some preferred embodiments, the content of the protective agent is 5-15 parts by weight; more preferably 7-13 parts by weight.

[0014] In some preferred embodiments, the content of the preservative is 0.5-2 parts by weight; more preferably, it is 0.7-1.5 parts by weight.

[0015] In some preferred embodiments, the content of creatine is 0.5-2 parts by weight; more preferably, it is 0.7-1.5 parts by weight.

[0016] In some preferred embodiments, the content of arginine is 0.5-2 parts by weight; more preferably, it is 0.7-1.5 parts by weight.

[0017] In some preferred embodiments, the content of the metal chelating agent is 10-20 parts by weight; more preferably, it is 13-17 parts by weight.

[0018] In some preferred embodiments, the content of deionized water is 680-720 parts by weight; more preferably, it is 690-710 parts by weight.

[0019] In some preferred embodiments, the surfactant is selected from at least one of Tween-20, Tween-60, Tween-80, and Triton-100.

[0020] In some preferred embodiments, the buffer is phosphate-buffered saline (PBS) or Tris-HCl buffer; more preferably, it is phosphate-buffered saline (PBS).

[0021] In some preferred embodiments, the protective agent is selected from at least one of sodium caseinate, gelatin, peptone, ovalbumin, dextran, and PEG derivatives; more preferably, ovalbumin.

[0022] In some preferred embodiments, the preservative is selected from at least one of sodium azide and P300.

[0023] In some preferred embodiments, the metal chelating agent is selected from at least one of ethylenediaminetetraacetic acid (EDTA), iminodiacetic acid (IDA), tetraethylenepentamine (TEPA), ethylenediamine N,N-diacetic acid (EDDA), and urea.

[0024] In some preferred embodiments, the bio-diluent comprises:

[0025] 4-6 parts by weight of buffer solution;

[0026] Sodium chloride 20-30 parts by weight;

[0027] Trehalose 15-25 parts by weight;

[0028] 15-25 parts by weight of sucrose;

[0029] 1-3 parts by weight of surfactant;

[0030] 180-210 parts by weight of glycerin;

[0031] 13-17 parts by weight of metal chelating agent;

[0032] 0.5-2 parts by weight of creatine;

[0033] Arginine 0.5-2 parts by weight;

[0034] Protectant 5-15 parts by weight;

[0035] Preservative 0.5-2 parts by weight; and

[0036] 680-720 parts by weight of deionized water.

[0037] In some preferred embodiments, the bio-diluent comprises:

[0038] Phosphate buffer, 5-5.5 parts by weight;

[0039] Sodium chloride 25-30 parts by weight;

[0040] Trehalose 17-23 parts by weight;

[0041] 17-23 parts by weight of sucrose;

[0042] Tween-20 1.5-2.5 parts by weight;

[0043] 190-200 parts by weight of glycerin;

[0044] EDTA 10-20 parts by weight;

[0045] Creatine 0.7-1.5 parts by weight;

[0046] Arginine 0.7-1.5 parts by weight;

[0047] 7-13 parts by weight of ovalbumin;

[0048] Sodium azide 0.7-1.5 parts by weight; and

[0049] 690-710 parts by weight of deionized water.

[0050] A second aspect of the present invention provides a composite quality control for multiple urine tests, the composite quality control comprising the bio-diluent described in the first aspect of the present invention and a plurality of urine test quality control samples selected from a group of at least two (preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six, more preferably at least seven, more preferably at least eight, more preferably at least nine, more preferably at least ten, more preferably at least eleven, more preferably at least twelve, and most preferably all thirteen) quality control samples: β2-MG, RBP, CYC, α1-MG, α2-MG, IgG, TRF, CR, κ, λ, NAG, NGAL, and ALB.

[0051] In some preferred embodiments, the plurality of urine test quality control samples include CR and NAG.

[0052] In some preferred embodiments, the plurality of urine test quality controls include CR and NAG, and a combination of at least two (preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six, more preferably at least seven, more preferably at least eight, more preferably at least nine, more preferably at least ten, more preferably at least eleven) quality controls selected from the group consisting of: β2-MG, RBP, CYC, α1-MG, α2-MG, IgG, TRF, κ, λ, NGAL, and ALB.

[0053] In some preferred embodiments, each of the plurality of urine test quality control samples includes a low-level quality control sample and a high-water quality control sample;

[0054] The low-level quality control products include:

[0055] β2-MG 0.31 mg / L;

[0056] RBP 0.39 mg / L;

[0057] CYC 0.21 mg / L;

[0058] α1-MG 14.2 mg / L;

[0059] α2-MG 6.2 mg / L;

[0060] IgG 20.51 mg / L;

[0061] TRF 3.1 mg / L;

[0062] CR 4615umol / L;

[0063] κ 13.8 mg / L;

[0064] λ 7.9 mg / L;

[0065] NAG 13.2 mg / L;

[0066] NGAL 201ug / L; and / or

[0067] ALB 20.3 mg / L;

[0068] The high-level quality control products include:

[0069] β2-MG 11.62 mg / L;

[0070] RBP 1.96 mg / L;

[0071] CYC 1.78 mg / L;

[0072] α1-MG 37.3 mg / L;

[0073] α2-MG 35.1 mg / L;

[0074] IgG 48.22 mg / L;

[0075] TRF 14.1 mg / L;

[0076] CR 9201umol / L;

[0077] κ 60.3 mg / L;

[0078] λ 30.2 mg / L;

[0079] NAG 47.9 mg / L;

[0080] NGAL 512ug / L; and / or

[0081] ALB 160.5mg / L.

[0082] Compared with the prior art, the present invention has at least the following advantages:

[0083] (1) The bio-diluent provided by the present invention can be used to prepare composite quality control products for multiple urine tests. The biosafety and batch-to-batch variation can be effectively controlled, the stability of the composite quality control products can be improved, and it is suitable for large-scale preparation.

[0084] (2) The composite quality control product for multiple urine tests containing the bio-diluent provided by the present invention has a mixture of 13 quality control products that are extremely stable. After 24 months of stability observation at 2-8℃, the content values ​​dropped by less than 5%. The quality control measurement deviation caused by the uncertainty of the quality control itself is very small, which meets the requirements of the field for the stability of quality control products.

[0085] (3) The composite quality control product for multiple urine tests provided by this invention can be used to simultaneously test 13 common urine test items, which can reduce the time and economic costs of multiple tests using multiple quality control products in clinical practice.

[0086] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0087] Through extensive and in-depth research, the inventors have developed a bio-diluent that can be used to dilute biological samples, especially quality control biological samples for urine testing. Using the bio-diluent of this invention to dilute a combination of 13 common urine testing quality control samples, the content stability of the 13 quality control samples is excellent, and a composite quality control for multiple urine tests can be prepared, which can simultaneously test up to 13 items, improving testing efficiency and reducing testing costs.

[0088] the term

[0089] As used in this article, the terms "β2-MG" and "β2-microglobulin" are used interchangeably, referring to a low molecular weight protein with a molecular weight of 11.8 kDa. Serum β2-MG readily passes through the glomerular filtration membrane, but 99.9% is reabsorbed and degraded by proximal tubular cells, resulting in very low levels in urine. Glomerular and tubular dysfunction can lead to changes in serum and urinary β2-MG concentrations.

[0090] As used in this article, the term "RBP" refers to a blood vitamin transporter protein synthesized by the liver and widely distributed in the blood, cerebrospinal fluid, urine, and other body fluids. Measuring retinol-binding protein (RBP) can detect early damage to renal tubule function and sensitively reflect the degree of damage to the proximal convoluted tubules of the kidney.

[0091] As used in this article, the terms "CYC" and "cystatin C" are used interchangeably, referring to a low molecular weight cystine protease inhibitor. All nucleated cells stably produce CYC. Almost all CYC is filtered by the glomerulus and then reabsorbed by the renal tubules. The renal tubules do not secrete or excrete CYC. CYC is not affected by inflammatory responses, sex, muscle mass, or age. Therefore, CYC is a very stable indicator of glomerular filtration rate and is used to evaluate early renal function impairment and assess the recovery of kidney transplant patients.

[0092] As used in this article, the terms “α1-MG” and “α1-microglobulin” are used interchangeably, referring to a glycoprotein with a molecular weight of approximately 33 kDa. Under normal circumstances, the amount of α1-MG in urine is very small, but when the renal tubules are damaged, the excretion of α1-MG in urine increases.

[0093] As used in this article, the terms "α2-MG" and "α2 macroglobulin" are used interchangeably. They refer to the largest molecular weight protein in blood plasma, synthesized by hepatocytes and the mononuclear-macrophage system. It is a protease inhibitor that inhibits endopeptidase activity. Currently, urinary α2MG is considered to have some significance in the diagnosis of diabetes, nephrotic syndrome, kidney transplantation, and glomerular hematuria.

[0094] As used in this article, the term "IgG" refers to the most abundant large molecule protein in blood plasma, with a molecular weight of 160 kDa. Under normal circumstances, it does not easily pass through the glomerular basement membrane due to its selective permeability. When a large amount of large molecules such as IgG appear in the urine, it indicates that the glomerular basement membrane has lost its selective permeability function. This type of proteinuria is called non-selective proteinuria. UigG is mainly used as an indicator of renal function deterioration and prognosis.

[0095] As used in this article, the term “TRF” or “UTRF” is one of the markers of early glomerular injury, primarily reflecting damage to the charge-selective barrier of the glomerular filtration membrane.

[0096] As used in this article, the term "CR" refers to creatinine. 90% of creatine in the human body is found in muscles, mostly in the form of creatine phosphate. Creatine phosphate is dephosphated to form creatinine, which is then excreted in urine as urinary creatinine. The daily excretion of urinary creatinine in normal individuals is quite stable and is not affected by dietary protein content or urine volume. When kidney function is abnormal, creatinine levels can sensitively reflect the problem.

[0097] As used in this article, the term "κ" refers to one of the peptide chains on an immunoglobulin. When the synthesis of κ or κ-light chains in serum is abnormal, the amount of free light chains in the serum increases, exceeding the reabsorption capacity of the glomeruli, and will be excreted in the urine, which can indicate kidney damage.

[0098] As used in this article, the term "λ" refers to one of the peptide chains on an immunoglobulin. When the synthesis of λ or λ-light chains in serum is abnormal, the amount of free light chains in the serum increases, exceeding the reabsorption capacity of the glomeruli, and will be excreted in the urine, which may indicate kidney damage.

[0099] As used in this article, the term "NAG" refers to N-acetyl-β-D-glucosidase, a lysosomal hydrolase. Urinary NAG mainly originates from the kidney, especially the proximal convoluted tubule epithelial cells. It is a sensitive renal tubular marker enzyme, and changes in its urinary enzyme activity can reflect early kidney damage. In cases of renal tubular damage, serum NAG increases. Serum and urinary NAG can be used for early diagnosis, disease detection, or screening.

[0100] As used in this article, the term "NGAL" refers to neutrophil gelatinase-associated lipid transport protein, a novel secretory protein with powerful functions. It is closely related to inflammation, immune response, cell differentiation, apoptosis, tissue remodeling, and the occurrence and development of various tumors. As a novel marker of acute kidney injury, its elevated levels are seen in patients with acute kidney injury, kidney transplantation, and cardiovascular surgery.

[0101] As used in this article, the term "ALB" refers to the most abundant protein in blood plasma, with very low concentrations found in the urine of healthy individuals. When the glomerular basement membrane is damaged (even in the early, minor way), altering its permeability, the concentration of urinary microalbumin (MALB) increases persistently, and mALB can be an early indicator of kidney abnormalities.

[0102] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. The sources of high values ​​used in the following embodiments are shown in Table 1 below; other experimental materials and reagents used are available from commercially available sources unless otherwise specified.

[0103] Table 1

[0104]

[0105] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0106] Example 1

[0107] In this embodiment, a composite quality control diluent was prepared, and its formulation is as follows:

[0108] (1) Preparation of diluent

[0109] The formula for 1L of diluent is as follows:

[0110] Na2HPO4·12H2O 4.65g

[0111] NaH2PO4·2H2O 0.56g

[0112] NaCl 27g

[0113] 20g trehalose

[0114] 20g of sucrose

[0115] Tween-20 2ml (2.2g)

[0116] 150ml (194.55g) of glycerin

[0117] EDTA 15g

[0118] 1g of creatine

[0119] 1g of arginine

[0120] 10g of egg white protein

[0121] 1g of sodium azide

[0122] The specific preparation procedure is as follows: Taking 1L of quality control diluent as an example, first add 700ml of deionized water, then add phosphate and NaCl, stir for 20 minutes until fully dissolved, then add trehalose, sucrose, Tween-20, and glycerol, stir for 30 minutes until fully dissolved, then add sarcosine, arginine, EDTA, and ovalbumin, stir for 30 minutes, finally add preservatives to bring the volume to 1000ml, stir for 30 minutes, filter through a 0.22nm filter membrane, and store at 2-8℃.

[0123] (2) Preparation of composite quality control products

[0124] Two quality control levels were configured according to the target concentration: one level was a negative (or weakly positive) control, and the other was a strong positive control. The reference values ​​for the quality control materials were set based on a comprehensive consideration of clinical needs and the performance of instruments and reagents. In a Class 100,000 cleanroom, the highest values ​​of each item were added to the quality control material diluent according to the target concentration. After stirring for 30 minutes to ensure thorough mixing, the solution was filtered through a 0.22nm filter membrane, and the concentration was measured on a biochemical analyzer. Once the concentration requirements were met, the solution was automatically dispensed into 1ml vials using a Class 100,000 cleanroom and stored at 2-8℃ after assembly.

[0125] Low-level and high-level quality control materials are shown in Table 2.

[0126] Table 2

[0127]

[0128] The quality control samples obtained in the above examples were subjected to performance verification according to the following method, the specific method of which is as follows:

[0129] [Thermal Stability Verification]

[0130] Three sets of quality control samples were taken. Two sets of quality control samples were placed in a 37℃ oven for 14 days and 7 days, respectively, and one set was used as a 0-day control. After mixing, samples were taken and measured using a fully automated biochemical analyzer. The accelerated stability at 37℃ was statistically calculated. The results are recorded in Table 3-1.

[0131] Table 3-1

[0132]

[0133] [Long-term storage stability verification]

[0134] After the quality control system was configured, an unopened set of quality control samples was taken and measured using a fully automated biochemical analyzer, and the results were recorded. Unopened quality control samples were then taken again at 6 months, 12 months, 18 months, 24 months, and 26 months to statistically calculate long-term storage stability. The results were recorded in Tables 3-2-1 and 3-2-2.

[0135] Table 3-2-1

[0136]

[0137] Table 3-2-2

[0138]

[0139] [On-device stability verification]

[0140] Take an unopened quality control sample and perform measurements on a fully automated biochemical analyzer. Measure the sample at 1, 2, 3, 4, 5, and 6 hours, record and calculate the in-system stability data. Record the results in Tables 3-3-1 and 3-3-2.

[0141] Table 3-3-1

[0142]

[0143] Table 3-3-2

[0144]

[0145] [Stability verification after repeated freeze-thaw cycles]

[0146] Four sets of quality control samples were taken. Three sets were subjected to freeze-thaw cycles at -20℃ once, twice, and three times, respectively, while one set served as a zero-cycle control. After thorough mixing, each set was tested using a fully automated biochemical analyzer. The in-system stability data were recorded and calculated. The results are recorded in Table 3-4.

[0147] Table 3-4

[0148]

[0149] [Stability Verification After Opening]

[0150] Take an unopened quality control sample and test it on a fully automated biochemical analyzer. Measure the values ​​after 7, 14, 21, 28, and 35 days, record and calculate the in-system stability data. Record the results in Tables 3-5-1 and 3-5-2.

[0151] Table 3-5-1

[0152]

[0153] Table 3-5-2

[0154]

[0155] [Precision Verification]

[0156] Take a set of quality control samples and perform measurements on a fully automated biochemical analyzer. Perform 20 measurements at each level and calculate the precision. Record the results in Table 3-6-1.

[0157] Table 3-6-1

[0158]

[0159] Table 3-6-1 (continued)

[0160]

[0161] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A biological diluent, characterized in that, The bio-diluent consists of the following components: 4-6 parts by weight of phosphate buffer; Sodium chloride 20-30 parts by weight; Trehalose 15-25 parts by weight; 15-25 parts by weight of sucrose; Tween-20 1-3 parts by weight; 180-210 parts by weight of glycerin; 5-15 parts by weight of ovalbumin; Sodium azide 0.5-2 parts by weight; 0.5-2 parts by weight of creatine; Arginine 0.5-2 parts by weight; EDTA 10-20 parts by weight; and 680-720 parts by weight of deionized water.

2. The bio-diluent according to claim 1, characterized in that, The bio-diluent consists of the following components: Phosphate buffer, 5-5.5 parts by weight; Sodium chloride 25-30 parts by weight; Trehalose 17-23 parts by weight; 17-23 parts by weight of sucrose; Tween-20 1.5-2.5 parts by weight; 190-200 parts by weight of glycerin; EDTA 10-20 parts by weight; Creatine 0.7-1.5 parts by weight; Arginine 0.7-1.5 parts by weight; 7-13 parts by weight of ovalbumin; Sodium azide 0.7-1.5 parts by weight; and 690-710 parts by weight of deionized water.

3. A composite quality control product for multiple urine tests, characterized in that, The composite quality control includes the bio-diluent as described in claim 1 or 2 and multiple urine test quality control products; the urine test quality control products include creatinine and N-acetyl-β-D-glucosidase, and other quality control products selected from at least four of the following groups: β2-microglobulin, retinol-binding protein, cystatin C, α1-microglobulin, α2-macroglobulin, immunoglobulin G, transferrin, κ light chain, λ light chain, neutrophil gelatinase-associated lipotransferase, and albumin.

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