A tumor marker liquid quality control product containing multiple carbohydrate antigens and its preparation method

Abstract

The present invention belongs to the technical field of quality control products containing tumor markers such as carbohydrate antigens, and specifically relates to a liquid quality control product for tumor markers containing multiple carbohydrate antigens. The liquid quality control product uses human serum as a matrix and contains the following components: A non-analyte includes blocking protein, which is a functional protein modified by fucosylation of the blocking agent. The sugar chain structure containing fucose is introduced through enzymatic hydrolysis and glycosylation, so that it can specifically recognize the fucose-containing Si-Lea epitope shared by carbohydrate antigens; B analytes include carbohydrate antigens and other markers, and other markers include tumor markers and non-tumor-related markers. In response to the problems of significant cross-reaction of carbohydrate antigens and insufficient stability of liquid quality control products, the present invention provides a multi-component synergistic system with blocking protein as the core. By specifically binding to the conserved epitopes of carbohydrate antigens and dynamically blocking cross-reactions, high uniformity, stability and convenient detection of liquid quality control products are achieved.

Description

Technical Field

[0001] The present invention mainly relates to the technical field of quality control products containing tumor markers such as carbohydrate antigens, and specifically relates to a tumor marker liquid quality control product containing multiple carbohydrate antigens and a preparation method thereof. Background Art

[0002] The cross-reactivity of carbohydrate antigens (CA125, CA153, CA199, CA242, CA724, and CA50) stems primarily from the conserved nature of their glycosylation. The epitopes of these antigens are often located in the glycan regions of glycoproteins, and homology in their core glycan structures is a key driver of cross-reactivity. For example, CA50 and CA19-9 share a sialic acid glycosphingolipid structure, while the epitope of CA242 shares homology with the sialylated regions of CA50 / CA19-9. This similarity in glycans can lead to nonspecific binding of antibodies to multiple antigens during testing, resulting in false-positive signals.

[0003] Furthermore, the widespread presence of cross-reactive carbohydrate determinants (CCDs) further complicates this issue. CCDs are essentially conserved sugar chain structures that can elicit cross-reactivity between different organisms. For example, the sugar chains of the H antigen of blood group antigens in humans (such as the α1,2-fucosylated structure in the ABO blood group system) are highly similar to those of lipopolysaccharides of certain intestinal bacteria, such as Escherichia coli. This molecular mimicry can cause antibodies against bacterial infections to mistakenly recognize the host's own tissues or cross-react with antigens such as CA50 in tumor marker assays.

[0004] From a molecular perspective, the core of cross-reactivity lies in the conservation of sugar chains and the promiscuity of antibody recognition. This conserved nature of sugar chains is reflected in the fact that different antigens may share a core glycosylation structure, allowing them to be recognized by the same antibody even if the peripheral sugar modifications differ. The antibody's antigen-binding site often only needs to match a localized region of the sugar chain to complete binding. For example, an antibody targeting α1,3-fucose may recognize specific epitopes on both the H chain of the blood group antigen and CA50. This promiscuity is particularly prominent in clinical testing: for example, in Lewis-negative individuals, CA19-9 cannot be expressed normally due to a lack of α-1,3 / 4-fucosyltransferase, but CA50 can still be detected through its non-fucose-dependent epitope. If the detection system does not adequately block CCD interference, a positive CA50 signal may be misinterpreted as a false increase in CA19-9, thereby affecting the diagnostic accuracy of diseases such as pancreatic cancer.

[0005] To avoid intermolecular interactions between carbohydrate antigens in a liquid environment, traditional quality control products for tumor markers such as carbohydrate antigens are often delivered in freeze-dried powder form. The freeze-drying process reduces the probability of cross-linking between antigens through physical dehydration, but its technical limitations are significant:

[0006] 1. Reconstitution error and insufficient stability: Lyophilized products need to be manually reconstituted. Differences in water usage and mixing degree during the operation can easily lead to increased inter-bottle differences. After reconstitution, they must be used within a short period of time. Repeated freeze-thaw cycles will destroy the integrity of the antigen epitope and accelerate the degradation of the sugar chain.

[0007] 2. Matrix effect deviation: The freeze-drying process may change the conformation of glycoproteins, causing differences in the ionic strength and protein interaction environment between them and the actual patient serum, resulting in a mismatch between the quality control results and the detection performance of clinical samples.

[0008] 3. Cost and efficiency bottlenecks: The freeze-drying process requires expensive equipment, and dead volume waste is easily generated during the packaging process. At the same time, multi-index testing requires frequent switching of quality control products, which increases laboratory manpower and time costs.

[0009] Therefore, there is an urgent need to develop a liquid quality control product for tumor markers with less cross-reactivity between carbohydrate antigens. Summary of the Invention

[0010] In response to the problems of significant cross-reaction of carbohydrate antigens and insufficient stability of liquid quality control products in the existing technology, the present invention provides a multi-component synergistic system with blocking protein as the core. It dynamically blocks cross-reactions by specifically binding to conserved epitopes of carbohydrate antigens. At the same time, relying on the human serum matrix and refined technology, it achieves high uniformity, stability and convenient detection of liquid quality control products.

[0011] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0012] In a first aspect, the present invention provides a liquid quality control product for tumor markers containing multiple carbohydrate antigens. The liquid quality control product is based on human serum and contains the following components:

[0013] A: Non-analytes include blocking proteins. The blocking proteins are functional proteins modified with fucosylation. Enzymatic hydrolysis and glycosylation introduce fucose-containing sugar chains, enabling them to specifically recognize the common fucose-containing Si-Lea epitope of carbohydrate antigens. Furthermore, the fucosylated blocking proteins do not bind to the non-fucose-dependent LSTa epitope. The cross-reactivity of carbohydrate antigens (such as CA199 and CA50) stems from their shared Si-Lea epitope. The present invention introduces fucose sugar chains onto the surface of a blocking agent (such as animal IgG) through enzymatic hydrolysis and glycosylation, creating a spatial conformation complementary to the Si-Lea epitope. The blocking protein specifically binds to the antigen epitope through hydrogen bonds and van der Waals forces, thereby blocking nonspecific antibody recognition.

[0014] Compared with traditional protein blockers (such as bovine serum albumin), the fucosylation modification in the present invention gives the blocking protein molecule recognition specificity, only targeting the conserved epitope containing fucose, without interfering with non-fucosylated antigens (such as the non-fucose epitope of CA125), avoiding the attenuation of detection signals caused by broad-spectrum blocking.

[0015] B: Analytes, including carbohydrate antigens and other markers, including tumor markers and non-tumor related markers.

[0016] In some feasible embodiments, the binding of the fucosylated-modified arrestin to the Si-Lea site is pH / salt concentration dependent, achieving a dynamic equilibrium of binding-dissociation in in vitro detection, i.e., "blocking during storage and releasing during detection", avoiding permanent inactivation of the antigen that may be caused by traditional blockers.

[0017] The main reason is that the binding of the blocking protein to the Si-Lea epitope depends on electrostatic interaction: at pH 7.0, the amino groups on the surface of the blocking protein are protonated and positively charged, forming ionic bonds with the sialic acid residues of the antigen epitope sugar chain to achieve efficient blocking; when the salt concentration of the detection system increases, the salt ions shield the charge, causing the binding constant Ka to decrease, the blocking protein dissociates, and the antigen epitope is released, ensuring the authenticity of the detection signal.

[0018] In some achievable embodiments, the method for preparing the inhibitory protein comprises the following steps:

[0019] (1) Enzymatic hydrolysis: Dissolve the blocker in sodium phosphate buffer at a pH of 6.5-8.0, add pepsin or trypsin at an enzyme / substrate mass ratio of 1:30-1:70, and hydrolyze at 37°C ± 2°C for 1-2 hours. After inactivating the enzyme, centrifuge and collect the supernatant. Pepsin / trypsin hydrolyzes redundant peptides on the surface of the blocker, exposing hidden glycosylation sites (such as lysine residues), which significantly improves the access efficiency of fucosyltransferase. In addition, too low an enzyme amount leads to insufficient hydrolysis, while too high an enzyme amount destroys the core structure; and 37°C is close to human body temperature, and the enzyme activity is optimal. Preferably, 1mM CaCl2 can be added to enhance trypsin activity, or 0.5mM EDTA can be added to inhibit metalloproteinase interference.

[0020] In addition, dynamic monitoring measures were adopted during the enzymatic hydrolysis treatment. Samples were taken every hour, and the fucose consumption rate was detected by HPLC. The amount of reducing sugar released was measured at a retention time of 12-15 minutes to control the reaction endpoint.

[0021] (2) Glycosylation modification: The pretreated blocking agent is mixed evenly with L-fucose at a volume ratio of 2:3-2:6, and 20 U / g protein of fucosyltransferase is added. The reaction is carried out at pH 6.5-7.5 for 4-6 hours to obtain the glycosylated blocking agent. Fucosyltransferase catalyzes the covalent linkage of fucose to the ε-amino group of lysine in the blocking agent to form a stable glycopeptide bond, which enables the blocking protein to acquire the Si-Lea epitope binding ability.

[0022] Excessive fucose ensures modification saturation, while insufficient enzyme leads to a sugar chain incorporation rate of less than 50%, affecting binding activity.

[0023] (3) Cross-linking reaction: Add 5 U / g protein of transglutaminase to the glycosylated blocker and react at pH 7.5-8.5 for 1-2 hours; the enzyme catalyzes the cross-linking of glutamine and lysine between the blocking protein molecules to form a network structure, which significantly improves the ability to resist protease degradation. After 36 months of storage, the activity retention rate is >90%.

[0024] (4) Purification: After the reaction is completed, heat at 80-90°C for 3-8 minutes to inactivate the enzyme, cool to room temperature, adjust the pH to 4.6 (near the isoelectric point of the blocker), collect the precipitate by centrifugation, and wash three times with pre-cooled isoelectric point water to remove free sugar chains;

[0025] Redissolve the precipitate in 0.01 M phosphate buffer, place in a 10 kDa molecular weight cutoff dialysis bag, and dialyze for 24 hours at 3–8°C, changing the buffer every 6 hours. Alternatively, concentrate the volume to 1 / 5 using an ultrafiltration centrifuge tube to remove unreacted glycans and ultimately obtain the arrested protein. Before dialysis, add 0.1% (w / v) activated charcoal to adsorb unreacted sugar donors. The purified product is freeze-dried to a powder and stored at -20°C.

[0026] The present invention prepares the blocking protein through a three-step process of enzymatic hydrolysis-glycosylation-crosslinking, introducing pH / salt concentration-dependent binding properties to achieve a dynamic balance of "blocking during storage and releasing during detection." Specifically, the mechanism of action of the blocking protein is as follows:

[0027] 1. Competitive blocking: Blocker proteins can dynamically bind to carbohydrate antigens and directly occupy their epitopes. This binding blocks the interaction between epitopes on different carbohydrate antigens and the same monoclonal antibody. Specifically, the blocker protein competitively binds to the fucose-containing Si-Lea site shared by CA50 and CA199, preventing the antibody from binding to CA199 through this site. However, CA50 has another specific site (LSTa, which does not contain fucose) that can be recognized and bound by the antibody.

[0028] 2. Dual characteristics of intelligent control

[0029] Enhanced specificity: By not binding to the LSTa site of CA50, it achieves zero-interference blocking in tests involving CA50, improving the accuracy of antigen recognition compared to traditional blockers;

[0030] Dynamic reversibility: Its binding to the Si-Lea site is pH / salt concentration dependent, which can achieve a dynamic equilibrium of binding-dissociation in in vitro assays, avoiding the permanent inactivation of antigens that may be caused by traditional blockers.

[0031] Compared to unmodified blockers, fucosylated retarder proteins competitively bind to carbohydrate antigen epitopes, effectively reducing antibody binding to cross-reactive antigens. Through molecular site-specific modification, this invention significantly improves the precision of regulating carbohydrate antigen cross-reactivity while maintaining the basic interference blocking function. This dual regulatory mechanism not only reduces the false positive rate but also ensures the sensitivity and stability of the detection system through reversible binding properties, thus having important application value in immunodiagnosis (e.g., reducing false positives).

[0032] In some achievable embodiments, the non-analyte further comprises:

[0033] A buffer solution selected from at least one of PB buffer solution, HEPES buffer solution, MES buffer solution, PIPES buffer solution and Tris-HCl buffer solution, with a concentration of 0.1-100 mmol / L and a pH of 6.0-8.0;

[0034] A stabilizer, wherein the stabilizer is selected from at least one of a carbohydrate stabilizer and a protein stabilizer, and the concentration is 0.01%-1% (w / v);

[0035] Carbohydrate stabilizers form hydrogen bond networks with glycoproteins through hydroxyl groups, inhibiting sugar chain aggregation caused by water loss; protein stabilizers act as spatial barriers to isolate antigen molecules, reducing the probability of collision between CA153 and CA242 and lowering the cross-reaction rate.

[0036] A preservative, wherein the preservative is selected from at least one of ProClin 300, ProClin 950, and gentamicin, and has a concentration of 0.1-80 mg / L. The preservative can effectively inhibit the activity of microbial thiols, block the energy metabolism pathway, and does not chemically react with fucose sugar chains.

[0037] In some achievable embodiments, the carbohydrate stabilizer is selected from at least one of sucrose, trehalose, lactose, maltose and mannitol;

[0038] and / or, the protein stabilizer is selected from at least one of casein, ovalbumin, and a protein stabilizer;

[0039] And / or, the blocker is at least one of the Blocker series blockers, animal serum series and animal IgG series products.

[0040] In some achievable embodiments, the buffer is a PB buffer containing the following components: disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and having a pH of 6.0-8.0.

[0041] In some embodiments, the PB buffer has a concentration of 25 mM and a pH of 7.0. Maintaining the pH of 7.0 in the PB buffer protonates the amino groups of the blocking protein, forming ionic bonds with the sialic acid residues of the antigen epitope, achieving efficient blocking during storage. During detection, sample dilution increases the salt concentration, dissociating the blocking protein and releasing the antigen epitope.

[0042] The present invention forms a high-efficiency pH buffer pair through the ratio of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, which can not only resist external acid-base interference (such as the influence of metabolites in serum) but also avoid sugar chain aggregation caused by excessively high ionic strength.

[0043] In some achievable embodiments, the protective agent is a protein stabilizer, and the preservative is ProClin 300.

[0044] In some achievable embodiments, the tumor markers include at least one of β2-microglobulin, carcinoembryonic antigen, calcitonin, cytokeratin 19, ferritin, gastrin, human epididymis protein 4, neuron-specific enolase, prostatic acid phosphatase, pepsinogen I, pepsinogen II, prolactin, progastrin-releasing peptide, central nervous system-specific protein, squamous cell carcinoma antigen, and thyroglobulin;

[0045] The carbohydrate antigens include at least two or more of CA125, CA153, CA199, CA242, CA724 and CA50. Preferably, the carbohydrate antigens in the present invention include six types of carbohydrate antigens, namely CA125, CA153, CA199, CA242, CA724 and CA50.

[0046] The non-tumor-related markers include at least one of anti-thyroid peroxidase antibodies, thyroglobulin antibodies, and thyroid-stimulating hormone receptor antibodies.

[0047] In a second aspect, the present invention further provides a method for preparing a liquid quality control product for tumor markers containing multiple carbohydrate antigens, which is used to prepare the above-mentioned liquid quality control product. The preparation method comprises the following steps:

[0048] (1) Prepare buffer solution: Dissolve the buffer in pure water and adjust the pH to 6.0-8.0 to form a stable ionic environment.

[0049] (2) Prepare a base mixture: add the fucosylated retarder protein, protective agent, and preservative to the buffer solution in sequence and mix thoroughly. The final concentration of the fucosylated retarder protein of the present invention in the matrix mixture is 0.1-10 mg / mL. In the human serum matrix, the retarder protein, when present at a concentration of 0.1-10 mg / mL, can saturate bind to approximately 80% of the Si-Lea epitope, forming a dynamic blocking layer that reduces cross-reactivity without affecting antigen-antibody specific binding.

[0050] (3) Add human serum matrix solution: Add human serum matrix solution to the basic mixture in step (2) and stir slowly to mix evenly. First add the blocking protein to the buffer solution to allow it to fully dissolve and form a monomeric conformation. In addition, the blocking protein is added before the serum to prevent serum albumin from wrapping the blocking protein through hydrophobic interaction, which will lead to a decrease in the Si-Lea epitope binding efficiency.

[0051] (4) Adding analytes: After adding human serum matrix, add six carbohydrate antigens and other markers and stir until completely dissolved to ensure that each analyte is evenly dispersed to avoid cross-reactions caused by excessive local concentrations;

[0052] (5) Filtration and packaging: Filter through a 0.2-0.45 μm filter membrane to remove microorganisms and impurity particles ≥0.2 μm to prevent clogging of the sample needle of the automated detection equipment. After packaging, refrigerate and store at 2-8°C to avoid sugar chain breakage caused by repeated freezing and thawing, ensure long-term stability, and ensure that the performance of the quality control product meets the standards.

[0053] In some feasible methods, a quality control step is also included: a uniformity test is performed on the quality control product after packaging to ensure that the coefficient of variation within the bottle (CV bottle) of the quality control product is ≤6%, and the coefficient of variation between bottles (CV bottle) is ≤6%. The actual acceptance standard is not more than 10%, which is qualified. The uniformity of the present invention is greatly improved, and the quality control product will be more stable compared to the freeze-dried powder.

[0054] The preparation method of the present invention achieves dynamic equilibrium and conformational protection of antigen-arrestin through the molecular structure design of the arrestin, breaks through the cross-reaction and stability bottlenecks of liquid quality control products, and ultimately achieves high specificity, high stability and high uniformity of the quality control products.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The present invention addresses the cross-reaction problem between carbohydrate antigens in a tumor marker liquid quality control product containing multiple carbohydrate antigens for the first time, and provides a reagent for blocking cross-reaction between carbohydrate antigens in this specific quality control product composition, which can reduce the cross-reaction of the tumor marker liquid quality control product containing multiple carbohydrate antigens. In particular, the improvement in cross-reaction between carbohydrate antigen items in the quality control product is most significant. The present invention uses fucosylation to modify the blocking protein to specifically recognize the Si-Lea epitope shared by carbohydrate antigens, and utilizes a pH / salt concentration-dependent dynamic equilibrium mechanism (binding blocking during storage and dissociation and release during detection) to competitively occupy the antibody binding site, significantly reducing false positive results and improving detection specificity.

[0057] (2) The present invention uses human serum as a matrix to simulate the protein composition of clinical samples, combines with a buffer to maintain a stable ionic environment, and cooperates with the hydrogen bond network of the carbohydrate stabilizer and the steric hindrance of the protein stabilizer to inhibit glycoprotein denaturation and aggregation, so that the concentration fluctuation of the liquid quality control product within a 36-month storage period is ≤±15%, which far exceeds the attenuation rate of the lyophilized product after reconstitution.

[0058] (3) The quality control product of the present invention is a frozen liquid, which is different from the freeze-dried powder. It eliminates the error caused by re-dissolution. Compared with freeze-drying to avoid the interference caused by matrix effect and cross-reaction, the present invention adds a cross-reaction blocker to the liquid quality control product to weaken the degree of cross-reaction between multiple carbohydrate antigens, greatly improving the convenience and accuracy of the test for the inspectors and reducing the labor and time costs of the laboratory.

[0059] The present invention addresses the core issues in existing tumor marker control products, such as cross-reactions caused by conserved glycosylation of carbohydrate inhibitors and insufficient stability of liquid matrices. By molecularly targeting the fucosylation-modified inhibitory protein and constructing a multi-component collaborative system, a highly specific and stable liquid quality control product is developed.

[0060] The present invention will be explained in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a graphical representation of the results of a stability experiment of a tumor marker liquid quality control product at level 2 in Example 1 of the present invention, with the abscissa representing the time span of 0-38 months and the ordinate representing the corresponding concentrations of the six carbohydrate antigens;

[0062] Figure 2 This is a graphical representation of the results of a stability experiment of the tumor marker liquid quality control product at level 3 in Example 1 of the present invention. The horizontal axis represents the time span of 0-38 months, and the vertical axis represents the concentrations corresponding to the six carbohydrate antigens.

[0063] This indicates that the prepared blocking protein can be stably stored for 38 months under the same low-temperature storage conditions as the quality control product, and its shelf life covers the shelf life of the quality control product. DETAILED DESCRIPTION

[0064] To facilitate understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in different forms and is not limited to the embodiments described in the text. On the contrary, these embodiments are provided to make the content disclosed in the present invention more thorough and comprehensive.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used by those skilled in the art to which the present invention pertains. The terminology used in the specification of the present invention is for the purpose of describing specific embodiments and is not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0066] Example 1: The method for preparing the inhibitory protein in the embodiment of the present invention comprises the following steps:

[0067] Enzymatic hydrolysis: Dissolve Blocker series blocker (1M, purchased from Jingda Biotechnology, product number HBR-4) in 25mM sodium phosphate buffer (pH 7.5). Add trypsin at an enzyme / substrate ratio of 1:30. Hydrolyze at 37°C for 1 hour, inactivate at 80°C for 15 minutes, and centrifuge at 10,000 × g, 4°C for 15 minutes. The supernatant is then collected for later use. Preferably, 1mM CaCl2 can also be added in this example to enhance trypsin activity.

[0068] Glycosylation modification: The supernatant was mixed with L-fucose at a volume ratio of 2:3, 20 U / g protein of fucosyltransferase was added, the pH was adjusted to 7.5, and the reaction was carried out at 37°C for 5 hours.

[0069] Cross-linking reaction: add 5U / g protein transglutaminase, react at pH 8.0 for 1.5 hours, adjust pH to 4.6 to precipitate protein, dialyze to remove free sugar chains, and freeze-dry to make powder.

[0070] Quality Control Preparation: Prepare 80 mL of quality control diluent: Add 0.4441 g of sodium phosphate dibasic dodecahydrate and 0.1186 g of sodium phosphate dibasic dihydrate to 10 mL of pure water and stir until completely dissolved to prepare PB buffer. Weigh 0.08 g of protein stabilizer A001 (purchased from Jingda Biotechnology, Cat. No. W0001) and 0.04 g of retardin powder into the buffer and stir to dissolve. The final concentration of retardin is 10 mg / mL. Add 0.08 mL of ProClin 300 to the solution and mix gently. Make up to 40 mL with pure water to form the base buffer system. Add 40 mL of human serum matrix to the solution, then add the analyte according to Table 1. Filter through a 0.22 μm filter, aliquot, and store refrigerated at 2-8°C.

[0071] Table 1 shows the concentration levels of each analyte in the raw materials at levels 2 and 3.

[0072] Project Name Concentration unit Level 2 Level 3 Anti-TPO IU / mL 40 95 B2-MG ug / mL 1.8 4 CA125 IU / mL 60 150 CA153 IU / mL 42 115 CA199 IU / mL 40 100 CA242 IU / mL 13 45 CA50 IU / mL 25 70 CA724 IU / mL 20 70 CEA ng / mL 20 59 CT pg / mL 45 660 CYFRA211 ng / mL 4.5 39 Ferritin ng / mL 250 445 Gastrin-17 pmol / L 5.5 50 HE4 pmol / L 65 150 NSE ng / mL 13 50 PAP ng / mL 3 35 PGI ng / mL 30 120 PGII ng / mL 12 30 PRL ng / mL 15 50 PROGRP pg / mL 42 650 S-100 ng / mL 0.35 3.5 SCCA ng / mL 1.8 70 TGA IU / mL 110 300 TRAb IU / L 1.5 18 TG ng / mL 22 60

[0073] The above two levels of tumor marker liquid quality control products were subjected to analytical performance evaluation experiments: 10 bottles of tumor marker liquid quality control products at level 2 and level 3 were taken, and each bottle was measured 3 times on an applicable immunoassay analyzer, and the intra-bottle coefficient of variation (CV intra-bottle) and the inter-bottle coefficient of variation (CV inter-bottle) were calculated.

[0074] Table 2 shows the analytical performance test results of quality control products at different concentration levels

[0075] Level 2 Anti-TPO B2-MG CA125 CA153 CA199 CV bottle 3.9% 3.4% 3.7% 1.3% 2.2% CV Bottle Room 5.0% 3.5% 2.1% 1.7% 2.1% Project Name CA242 CA50 CA724 CEA CT CV bottle 2.4% 1.6% 2.0% 4.3% 4.6% CV Bottle Room 1.7% 1.6% 2.0% 3.2% 4.3% Project Name CYFRA211 Ferritin Gastrin-17 HE4 NSE CV bottle 4.2% 3.3% 4.2% 4.9% 3.7% CV Bottle Room 3.1% 3.9% 4.1% 3.2% 5.0% Project Name PAP PGI PGII PRL PROGRP CV bottle 5.0% 3.0% 3.6% 4.5% 3.9% CV Bottle Room 4.6% 3.4% 4.3% 4.9% 4.6% Project Name S-100 SCCA TGA TRAb TG CV bottle 3.2% 3.9% 3.5% 4.8% 4.4% CV Bottle Room 4.1% 3.8% 4.0% 4.3% 4.3% Level 3 Anti-TPO B2-MG CA125 CA153 CA199 CV bottle 3.9% 4.2% 3.9% 3.7% 2.0% CV Bottle Room 4.0% 3.2% 2.9% 2.4% 2.4% Project Name CA242 CA50 CA724 CEA CT CV bottle 2.3% 1.3% 2.4% 4.0% 4.9% CV Bottle Room 2.6% 2.9% 2.6% 3.8% 3.1% Project Name CYFRA211 Ferritin Gastrin-17 HE4 NSE CV bottle 3.5% 4.8% 4.2% 4.2% 5.0% CV Bottle Room 4.7% 4.8% 4.4% 4.0% 3.7% Project Name PAP PGI PGII PRL PROGRP CV bottle 4.0% 4.9% 3.7% 3.0% 5.0% CV Bottle Room 3.0% 3.0% 4.6% 4.6% 3.8% Project Name S-100 SCCA TGA TRAb TG CV bottle 4.8% 3.3% 4.5% 4.6% 4.4% CV Bottle Room 3.6% 3.1% 3.4% 3.2% 3.2%

[0076] The results can be seen from the table above. After adding the blocking protein, the CVs of the six carbohydrate antigens did not exceed 4%, while the CVs of other analytes were in the range of 3%-5%, indicating that the blocking protein had a significant improvement on the uniformity of the six carbohydrate antigens.

[0077] The liquid quality control product for tumor markers containing multiple carbohydrate antigens prepared by the above preparation method is based on human serum and contains the following components:

[0078] A: Non-analytes, including: buffer, blocking protein, stabilizer, and preservative. Specifically, in this example, the buffer is PB buffer, containing the following components: disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, with a buffer concentration of 25 mM and a pH of 7.0; the protective agent is protein stabilizer A001 at a concentration of 10% (w / v); and the preservative is ProClin 300 at a concentration of 30 mg / L.

[0079] B: Analytes, including six carbohydrate antigens and other markers, including tumor markers and non-tumor-related markers. Specifically, in this embodiment, the tumor markers include β2-microglobulin, carcinoembryonic antigen, calcitonin, cytokeratin 19, ferritin, gastrin, human epididymis protein 4, neuron-specific enolase, prostatic acid phosphatase, pepsinogen I, pepsinogen II, prolactin, progastrin-releasing peptide, central nervous system-specific protein, squamous cell carcinoma antigen, and thyroglobulin.

[0080] In this example, non-tumor-related markers include anti-thyroid peroxidase antibodies, anti-thyroglobulin antibodies, and anti-thyroid stimulating hormone receptor antibodies. The six carbohydrate antigens are CA125, CA153, CA199, CA242, CA724, and CA50. The human serum matrix fluid is infectious-negative human serum, and its volume accounts for 40%-60%.

[0081] Example 2: The method for preparing the arrestin in this example is basically the same as that in Example 1, except that:

[0082] Enzymatic hydrolysis: Animal serum (0.01 M, purchased from Jingda Biotechnology, Cat. No. C8001) was dissolved in 25 mM sodium phosphate buffer (pH 6.5). Pepsin was added at an enzyme / substrate ratio of 1:50. The mixture was hydrolyzed at 37°C for 1.5 hours, inactivated at 85°C for 10 minutes, and centrifuged at 10,000 × g at 4°C for 15 minutes. The supernatant was then collected for later use. Preferably, 0.5 mM EDTA was added in this embodiment to inhibit interference from metalloproteinases.

[0083] Glycosylation modification: The supernatant was mixed with L-fucose at a volume ratio of 2:6, 20 U / g protein of fucosyltransferase was added, the pH was adjusted to 7.0, and the reaction was carried out at 37°C for 4 hours.

[0084] Cross-linking reaction: Add 5 U / g transglutaminase and react for 2 hours at pH 7.5. Use ultrafiltration centrifuge tubes (10 kDa) to concentrate the volume to 1 / 5 by centrifugation at 4000 × g to remove free sugar chains, and lyophilize to obtain a powder.

[0085] Preparation of quality control: Prepare 80 mL of quality control diluent by adding 0.4441 g of sodium phosphate dibasic dodecahydrate and 0.1186 g of sodium phosphate dibasic dihydrate to 10 mL of pure water. Stir until completely dissolved to prepare PB buffer. Weigh 0.08 g of protein stabilizer A001 and 0.04 g of retardin powder into the buffer and stir to dissolve. The final concentration of retardin is 1 mg / mL. Add 0.08 mL of ProClin 300 to the solution and mix gently. Make up to 40 mL with pure water to form the base buffer system. Add 40 mL of human serum matrix to the solution. Then, add the analyte according to Table 1 above. Filter through a 0.22 μm filter, aliquot, and store refrigerated at 2-8°C.

[0086] The above two levels of tumor marker liquid quality control products were subjected to analytical performance evaluation experiments: 10 bottles of tumor marker liquid quality control products at level 2 and level 3 were taken, and each bottle was measured 3 times on an applicable immunoassay analyzer, and the intra-bottle coefficient of variation (CV intra-bottle) and the inter-bottle coefficient of variation (CV inter-bottle) were calculated.

[0087] Table 3 shows the analytical performance test results of six carbohydrate antigens in quality control products at different concentration levels

[0088] Level 2 CA125 CA153 CA199 CA242 CA50 CA724 CV bottle 4.3% 5.5% 5.8% 4.5% 6.0% 5.5% CV Bottle Room 4.5% 5.2% 5.4% 4.6% 4.7% 5.9% Level 3 CA125 CA153 CA199 CA242 CA50 CA724 CV bottle 4.5% 5.2% 5.7% 5.4% 4.5% 5.5% CV Bottle Room 5.4% 4.7% 4.8% 5.7% 4.6% 5.7%

[0089] Example 3: The method for preparing the arrestin in this example is basically the same as that in Example 1, except that:

[0090] Enzymatic hydrolysis: Dissolve the animal IgG series blocker product (0.1M - purchased from Jingda Biotechnology, product number W4006) in 25mM sodium phosphate buffer (pH 8.0), add pepsin at an enzyme / substrate mass ratio of 1:70, hydrolyze at 37°C for 1.5 hours, inactivate at 85°C for 10 minutes, centrifuge at 10,000×g, 4°C for 10 minutes, and remove the supernatant for later use.

[0091] Glycosylation modification: The supernatant was mixed with L-fucose at a volume ratio of 2:4, 20 U / g protein of fucosyltransferase was added, the pH was adjusted to 6.5, and the reaction was carried out at 37°C for 6 hours.

[0092] Cross-linking reaction: Add 5U / g protein of transglutaminase and react at pH 8.5 for 1 hour. Purify, filter, and freeze-dry to obtain a powder.

[0093] Quality Control Preparation: Prepare 80 mL of quality control diluent by adding 0.4441 g of sodium phosphate dibasic dodecahydrate and 0.1186 g of sodium phosphate dibasic dihydrate to 10 mL of pure water. Stir until completely dissolved to prepare PB buffer. Weigh 0.08 g of protein stabilizer A001 and 0.04 g of retardin powder into the buffer and stir to dissolve. The final concentration of retardin is 20 mg / mL. Add 0.08 mL of ProClin 300 to the solution and mix gently. Make up to 40 mL with pure water to form the base buffer system. Add 40 mL of human serum matrix to the solution. Then, add the analyte according to Table 1 above. Filter through a 0.22 μm filter, aliquot, and store refrigerated at 2-8°C.

[0094] The above two levels of tumor marker liquid quality control products were subjected to analytical performance evaluation experiments: 10 bottles of tumor marker liquid quality control products at level 2 and level 3 were taken, and each bottle was measured 3 times on an applicable immunoassay analyzer, and the intra-bottle coefficient of variation (CV intra-bottle) and the inter-bottle coefficient of variation (CV inter-bottle) were calculated.

[0095] Table 4 shows the analytical performance test results of six carbohydrate antigens in quality control products at different concentration levels

[0096] Level 2 CA125 CA153 CA199 CA242 CA50 CA724 CV bottle 6.0% 5.6% 5.7% 5.0% 5.4% 5.6% CV Bottle Room 5.8% 5.0% 5.8% 5.2% 4.9% 6.0% Level 3 CA125 CA153 CA199 CA242 CA50 CA724 CV bottle 5.5% 5.9% 5.5% 5.4% 4.8% 5.7% CV Bottle Room 5.5% 5.3% 5.0% 5.1% 4.6% 5.3%

[0097] Comparative Example 1: This comparative example uses a commercially available tumor marker control product to compare multiple carbohydrate antigens. The commercially available tumor marker control product is from Zhengzhou Biaoyuan Biotechnology Co., Ltd. and is compared with Example 1.

[0098] Table 5 shows the performance test results of six carbohydrate antigens in commercially available tumor marker control products

[0099] Level 2 CA125 CA153 CA199 CA242 CA50 CA724 CV bottle 4.4% 3.2% 3.3% 4.1% 3.8% 4.2% CV Bottle Room 3.1% 3.8% 3.7% 3.9% 3.5% 4.1% Level 3 CA125 CA153 CA199 CA242 CA50 CA724 CV bottle 4.2% 3.8% 3.9% 5.6% 4.0% 4.0% CV Bottle Room 4.2% 3.8% 4.9% 3.8% 4.5% 5.0%

[0100] As can be seen from the above table, compared with Comparative Example 1, the uniformity of multiple carbohydrate antigens in the tumor marker liquid quality control product of Example 1 of the present invention is better.

[0101] Comparative Example 2: In this comparative example, no blocking protein was added, and other aspects were consistent with Example 1. The homogeneity test results of the six carbohydrate antigens in the quality control product without adding blocking protein are shown in Table 6 below.

[0102] Table 6 shows the performance test results of carbohydrate antigens in the quality control product without added blocking protein

[0103] Level 2 Anti-TPO B2-MG CA125 CA153 CA199 CV bottle 4.4% 3.2% 14.9% 13.5% 12.7% CV Bottle Room 4.5% 4.6% 14.9% 13.7% 12.7% Project Name CA242 CA50 CA724 CEA CT CV bottle 13.7% 13.3% 14.1% 4.8% 4.9% CV Bottle Room 13.7% 13.9% 14.6% 4.8% 4.6% Project Name CYFRA211 Ferritin Gastrin-17 HE4 NSE CV bottle 3.1% 4.8% 4.9% 4.8% 3.7% CV Bottle Room 3.7% 4.6% 4.1% 3.1% 3.7% Project Name PAP PGI PGII PRL PROGRP CV bottle 3.5% 3.6% 4.4% 4.8% 3.1% CV Bottle Room 3.8% 4.7% 3.3% 3.4% 3.6% Project Name S-100 SCCA TGA TRAb TG CV bottle 4.1% 4.5% 4.2% 4.5% 3.7% CV Bottle Room 4.1% 4.6% 4.1% 4.5% 4.0% Level 3 Anti-TPO B2-MG CA125 CA153 CA199 CV bottle 4.9% 4.2% 11.5% 12.2% 13.4% CV Bottle Room 4.2% 3.6% 11.9% 12.8% 13.0% Project Name CA242 CA50 CA724 CEA CT CV bottle 13.1% 14.2% 14.6% 3.4% 4.5% CV Bottle Room 13.9% 14.0% 14.8% 3.8% 4.5% Project Name CYFRA211 Ferritin Gastrin-17 HE4 NSE CV bottle 4.1% 3.6% 4.7% 4.9% 4.3% CV Bottle Room 4.0% 4.2% 4.3% 5.0% 3.6% Project Name PAP PGI PGII PRL PROGRP CV bottle 4.3% 4.2% 4.0% 3.1% 3.7% CV Bottle Room 4.2% 3.9% 5.0% 3.4% 4.1% Project Name S-100 SCCA TGA TRAb TG CV bottle 4.2% 4.6% 4.9% 4.2% 3.0% CV Bottle Room 3.2% 3.8% 4.0% 3.6% 4.6%

[0104] As can be seen from the above table, compared with Comparative Example 2, the uniformity of the six carbohydrate antigens in the tumor marker liquid quality control product of the embodiment of the present invention is better, and it is shown that the addition of blocking protein has no obvious effect on the detection of analytes other than carbohydrate antigens in the quality control product.

[0105] Comparative Example 3: In this comparative example, the blocking protein was replaced with a blocking agent (the model was the same as that in Example 1), and the other parameters were consistent with those in Example 1. The homogeneity test results of the six carbohydrate antigens in the quality control product are shown in Table 7 below.

[0106] Table 7 Performance test results of carbohydrate antigens in quality control products where blocking protein was replaced by blocking agent

[0107] Level 2 CA125 CA153 CA199 CA242 CA50 CA724 CV bottle 11.2% 12.6% 11.3% 11.3% 12.3% 11.5% CV Bottle Room 11.9% 12.9% 11.8% 10.9% 12.3% 11.6% Level 3 CA125 CA153 CA199 CA242 CA50 CA724 CV bottle 11.4% 12.6% 11.8% 10.9% 12.8% 12.8% CV Bottle Room 11.8% 12.8% 11.8% 11.4% 12.4% 12.1%

[0108] Comparative Example 4: In this comparative example, the amount of arrestin added was adjusted to 1 g, and the final concentration of the fucosylated arrestin in the matrix mixture was 12.5 mg / mL, which exceeded the reasonable range of 0.1-10 mg / mL in the embodiment of the present invention. The other preparation steps of the quality control product were consistent with those in Example 1. After the preparation was completed, one bottle was measured three times and compared with the target measured value. The relative deviations of the target values and the measured values of the six carbohydrate antigens in the quality control product after the amount of arrestin added was adjusted are shown in Table 8 below.

[0109] Table 8 Performance test results of carbohydrate antigens in quality control products after adjustment of the amount of blocking protein added

[0110] Project Name CA125 CA153 CA199 CA242 CA50 CA724 Relative deviation 14.2% 11.2% 13.2% 14.5% 11.8% 14.4% Relative deviation 13.9% 10.6% 10.8% 13.4% 12.7% 10.8% Relative deviation 13.2% 11.8% 11.2% 14.7% 10.1% 11.1%

[0111] As can be seen from the above table, after excessive addition of blocking protein, the antigenic epitope of the carbohydrate antigen is irreversibly bound, resulting in a large difference between the measured value and the target value during detection.

[0112] Any numerical value cited herein includes all values of the lower and upper values in increments of one unit from the lower limit to the upper limit, and there is an interval of at least two units between any lower value and any higher value. For example, if the value of the quantity of a component or a process variable (such as temperature, pressure, time, etc.) is set forth to be from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70, the purpose is to illustrate that values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32 are also explicitly listed in this specification. For values less than 1, one unit is appropriately considered to be 0.0001, 0.001, 0.01, 0.1. These are merely examples that are intended to be clearly expressed, and it can be considered that all possible combinations of the numerical values listed between the minimum and maximum values are explicitly set forth in this specification in a similar manner.

[0113] Unless otherwise indicated, all ranges include the endpoints and all numbers between the endpoints. When used with a range, "about" or "approximately" applies to both endpoints of the range. Thus, "about 20 to 30" is intended to cover "about 20 to about 30," including at least the specified endpoints.

[0114] It should be understood that the above description is for illustration and not for limitation. Many embodiments and many applications beyond the examples provided will be apparent to those skilled in the art upon reading the above description. Therefore, the scope of the present teachings should not be determined with reference to the above description, but rather with reference to the appended claims and the full scope of equivalents to which such claims are entitled. For the purpose of comprehensiveness, all articles and references, including disclosures of patent applications and publications, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein from the foregoing claims is not intended to be a disclaimer of such subject matter, nor should it be assumed that the inventors did not consider such subject matter to be part of the disclosed inventive subject matter.

[0115] The above description of the present invention is exemplified in conjunction with the accompanying drawings. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned method. As long as such non-substantial improvements are made using the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the scope of protection of the present invention.

Claims

1. A liquid quality control product for tumor markers containing multiple carbohydrate antigens, characterized in that: This liquid control is based on human serum and contains the following components: A: Non-analytes, including blocking proteins, which are functional proteins modified by fucosylation of blocking agents. By enzymatic hydrolysis and glycosylation, fucose-containing sugar chain structures are introduced, enabling the blocking proteins to specifically recognize the fucose-containing Si-Lea epitope common to carbohydrate antigens. B: Analytes, including carbohydrate antigens and other markers, including tumor markers and non-tumor related markers.

2. The liquid quality control product for tumor markers containing multiple carbohydrate antigens according to claim 1, characterized in that: The binding of the fucosylated arrestin to the Si-Lea site is dually dependent on pH and salt concentration, achieving a dynamic equilibrium of binding and dissociation in in vitro detection; And / or, the fucosylated arrestin does not bind to the fucose-independent LSTa epitope.

3. The tumor marker liquid quality control product containing multiple carbohydrate antigens according to claim 2, characterized in that: The preparation method of the inhibitory protein comprises the following steps: (1) Enzymatic hydrolysis: Dissolve the blocking agent in sodium phosphate buffer at pH 6.5-8.0, add pepsin or trypsin at an enzyme / substrate mass ratio of 1:30-1:70, hydrolyze at 37℃±2℃ for 1-2 hours, inactivate the enzyme, and centrifuge to obtain the supernatant; (2) Glycosylation modification: The pretreated blocker was mixed evenly with L-fucose at a volume ratio of 2:3-2:6, 20 U / g protein of fucosyltransferase was added, and the mixture was reacted at pH 6.5-7.5 for 4-6 hours to obtain the glycosylated blocker; (3) Cross-linking reaction: Add 5 U / g protein of transglutaminase to the glycosylated blocker and react at pH 7.5-8.5 for 1-2 hours; (4) Purification and filtration: After inactivating the enzyme, adjust the pH to near the isoelectric point of the blocker, collect the precipitate by centrifugation, remove the free sugar chains by dialysis or ultrafiltration, and freeze-dry the purified product into a powder for future use.

4. The tumor marker liquid quality control product containing multiple carbohydrate antigens according to claim 1, characterized in that: The non-analytes also include: A buffer solution selected from at least one of PB buffer solution, HEPES buffer solution, MES buffer solution, PIPES buffer solution and Tris-HCl buffer solution, with a concentration of 0.1-100 mmol / L and a pH of 6.0-8.0; A stabilizer, wherein the stabilizer is selected from at least one of a carbohydrate stabilizer and a protein stabilizer, and the concentration is 0.01%-1% (w / v); The preservative is selected from at least one of ProClin300, ProClin950 and gentamicin, and has a concentration of 0.1-80 mg / L.

5. The tumor marker liquid quality control product containing multiple carbohydrate antigens according to claim 4, characterized in that: The carbohydrate stabilizer is selected from at least one of sucrose, trehalose, lactose, maltose and mannitol; And / or, the protein stabilizer is selected from at least one of casein and ovalbumin; And / or, the blocker is at least one of the Blocker series blockers, animal serum series and animal IgG series products.

6. The tumor marker liquid quality control product containing multiple carbohydrate antigens according to claim 4 or 5, characterized in that: The buffer solution is a PB buffer solution containing the following components: disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and its pH value is 6.0-8.

0.

7. The liquid quality control product for tumor markers containing multiple carbohydrate antigens according to claim 6, characterized in that: The concentration of the PB buffer is 25 mM and its pH is 7.0; and / or, the stabilizer is a protein stabilizer; And / or, the preservative is ProClin300.

8. The tumor marker liquid quality control product containing multiple carbohydrate antigens according to claim 1, characterized in that: The tumor markers include at least one of β2-microglobulin, carcinoembryonic antigen, calcitonin, cytokeratin 19, ferritin, gastrin, human epididymis protein 4, neuron-specific enolase, prostatic acid phosphatase, pepsinogen I, pepsinogen II, prolactin, progastrin-releasing peptide, central nervous system-specific protein, squamous cell carcinoma antigen, and thyroglobulin; The carbohydrate antigen includes at least one of CA125, CA153, CA199, CA242, CA724 and CA50; The non-tumor-related markers include at least one of anti-thyroid peroxidase antibodies, thyroglobulin antibodies, and thyroid-stimulating hormone receptor antibodies.

9. A method for preparing a liquid quality control product for tumor markers containing multiple carbohydrate antigens, for preparing the liquid quality control product according to any one of claims 1 to 8, characterized in that: The preparation method comprises the following steps: (1) Prepare buffer: dissolve the buffer in pure water and adjust the pH to 6.0-8.0; (2) Prepare a base mixture: add the fucosylated retarder protein, protective agent and preservative to the buffer solution in sequence and mix thoroughly. The final concentration of the fucosylated retarder protein in the base mixture is 0.1-10 mg / mL. (3) Adding human serum matrix solution: add human serum matrix solution to the basic mixture in step (2) and stir slowly to mix evenly; (4) Adding analytes: After adding human serum matrix, add six carbohydrate antigens and other markers and stir until completely dissolved; (5) Filtration and packaging: Filter the solution through a 0.2-0.45 μm filter membrane, and store in a refrigerator at 2-8°C after packaging.

10. The method for preparing a liquid quality control product for tumor markers containing multiple carbohydrate antigens according to claim 9, characterized in that: The quality control step is also included: the quality control product after packaging is subjected to a uniformity test to ensure that the CV of each analyte in the liquid quality control product is ≤6% within the bottle and ≤6% between bottles.

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