Tumor marker liquid quality control product containing multiple carbohydrate antigens and preparation method thereof

Abstract

The invention belongs to the technical field of quality control products of tumor markers such as carbohydrate antigens, and particularly relates to a liquid quality control product of tumor markers containing multiple carbohydrate antigens, which takes human serum as a matrix and comprises the following components: A, non-analytes including blocking proteins, B, non-analytes including C and D; the blocker is a functional protein obtained by modifying a blocker through fucosylation, and a fucose-containing sugar chain structure is introduced through enzymolysis and glycosylation, so that the blocker can specifically recognize a fucose-containing Si-Lea epitope shared by carbohydrate antigens; the B analyte comprises carbohydrate antigens and other markers, and the other markers comprise tumor markers and non-tumor related markers. Aiming at the problems of obvious cross reaction of carbohydrate antigens and insufficient stability of liquid quality control products, the invention provides a multi-component synergistic system taking blocker protein as a core, and high uniformity, stability and detection convenience of the liquid quality control products are realized by specifically combining carbohydrate antigen conserved epitopes and dynamically blocking the cross reaction.

Description

Technical Field

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

[0002] The cross-reactivity of carbohydrate antigens (CA125, CA153, CA199, CA242, CA724, CA50) mainly stems from the conservation of their glycosylation modifications. The epitopes of these antigens are usually located in the sugar chain regions of glycoproteins, and the homology of their core sugar chain structures is the key driving factor for cross-reactivity. Taking CA50 and CA19-9 as examples, both share the sialylglycolipid structure, while the epitope of CA242 is homologous to the sialylated region of CA50 / CA19-9. The similarity of such sugar chains causes antibodies to non-specifically bind to multiple antigens during detection, resulting in false positive signals.

[0003] In addition, the widespread existence of cross-reactive carbohydrate antigen determinants (CCD) further amplifies the complexity of this problem. The essence of CCD is a conserved sugar chain structure that can cause cross-reactions between different organisms. For example, the H antigen sugar chain of human blood group antigens (such as the α1,2-fucosylated structure in the ABO blood group system) is highly similar to the sugar chains of lipopolysaccharides of certain intestinal bacteria (such as Escherichia coli). This molecular mimicry may cause antibodies against bacterial infections to misidentify host tissues, or cross-react with antigens such as CA50 during tumor marker detection.

[0004] From a molecular mechanism perspective, the core of cross-reactivity lies in the conservation of sugar chains and the promiscuity of antibody recognition. The conservation of sugar chains is manifested as different antigens may share the core glycosylation structure. Even if there are differences in peripheral glycosylation modifications, they may still be recognized by the same antibody. The antigen-binding site of an antibody often only needs to match a local region of the sugar chain to complete binding. For example, an antibody against α1,3-fucose may simultaneously recognize the specific epitopes of the blood group antigen H chain and CA50. This promiscuity is particularly prominent in clinical detection: for example, in Lewis blood group negative individuals, due to the lack of α-1,3 / 4-fucosyltransferase, CA19-9 cannot be normally expressed, but CA50 can still be detected through its fucose-independent epitope. If the detection system does not fully block the interference of CCD, the positive signal of CA50 may be misjudged as a false increase in CA19-9, thus affecting the diagnostic accuracy of diseases such as pancreatic cancer.

[0005] To avoid the intermolecular interaction of carbohydrate antigens in a liquid environment, traditional quality control products for tumor markers containing carbohydrate antigens and the like mostly adopt the form of freeze-dried powders. The freeze-drying process reduces the probability of antigen cross-linking through physical dehydration, but its technical limitations are significant: 1. Reconstitution error and insufficient stability: Lyophilized products require manual reconstitution. Differences in the amount of water used and the degree of mixing during the operation can easily lead to an increase in inter-bottle differences. After reconstitution, they need to be used within a short period. Repeated freezing and thawing will damage the integrity of antigenic epitopes and accelerate the degradation of sugar chains.

[0006] 2. Matrix effect bias: The lyophilization process may change the conformation of glycoproteins, resulting in differences in their ionic strength and protein interaction environment compared to real patient sera, leading to a mismatch between the quality control results and the detection performance of clinical samples.

[0007] 3. Cost and efficiency bottlenecks: The lyophilization process requires expensive equipment, and dead volume waste is easily generated during the dispensing process. At the same time, frequent switching of quality control products is required for multi-index detection, increasing the labor and time costs in the laboratory.

[0008] Therefore, there is an urgent need to develop a liquid quality control product for tumor markers with less cross-reaction between carbohydrate antigens. Summary of the Invention

[0009] Aiming at the problems of significant cross-reaction between carbohydrate antigens and insufficient stability of liquid quality control products in the prior art, the present invention provides a multi-component synergistic system with a blocking protein as the core. By specifically binding to the conserved epitopes of carbohydrate antigens, it dynamically blocks cross-reactions. At the same time, relying on human serum matrix and refined processes, it realizes high homogeneity, stability, and detection convenience of liquid quality control products.

[0010] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: In the first aspect, the present invention provides a liquid quality control product for tumor markers containing multiple carbohydrate antigens. The liquid quality control product uses human serum as the matrix and contains the following components: A: Non-analyte, including a blocking protein. The blocking protein is a functional protein after fucosylation modification of a blocker. Through enzymatic hydrolysis and glycosylation, a sugar chain structure containing fucose is introduced, enabling it to specifically recognize the fucose-containing Si-Lea epitope common to carbohydrate antigens. Moreover, the fucosylated blocking protein does not bind to the non-fucose-dependent LSTa epitope. The cross-reaction of carbohydrate antigens (such as CA199, CA50) stems from their common Si-Lea epitope. The present invention introduces fucose sugar chains on the surface of the blocker (such as animal IgG) through enzymatic hydrolysis-glycosylation modification, enabling it to form a spatial conformation complementary to the Si-Lea epitope, and specifically binding to the antigenic epitope through hydrogen bonds and van der Waals forces to block non-specific recognition by antibodies.

[0011] Compared with traditional protein blockers (such as bovine serum albumin), the fucosylation modification in the present invention endows the blocking protein with molecular recognition specificity, only targeting the conserved epitopes containing fucose, and does not interfere with non-fucosylated antigens (such as the non-fucose epitope of CA125), avoiding the attenuation of detection signals caused by broad-spectrum blocking.

[0012] B: Analytes, including: carbohydrate antigens and other markers, and the other markers include tumor markers and non-tumor-related markers.

[0013] In some achievable ways, the binding of the fucosylated blocking protein to the Si-Lea site is pH / salt concentration-dependent, achieving a dynamic equilibrium of binding-dissociation in in vitro detection, that is, "blocking during storage and releasing during detection", avoiding the permanent inactivation of antigens that may be caused by traditional blockers.

[0014] 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 in the detection system increases, the charge shielding effect of salt ions causes the binding constant Ka to decrease, and the blocking protein dissociates, releasing the antigen epitope to ensure the authenticity of the detection signal.

[0015] In some achievable ways, the preparation method of the blocking protein includes the following steps: (1) Enzymatic hydrolysis treatment: Dissolve the blocker in a sodium phosphate buffer solution with 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 a temperature of 37°C ± 2°C for 1 - 2 hours. After inactivating the enzyme, centrifuge to obtain the supernatant. Pepsin / trypsin hydrolyzes the redundant peptide segments on the surface of the blocker, exposing the hidden glycosylation sites (such as lysine residues), significantly improving the access efficiency of fucosyltransferase. In addition, too low an enzyme amount results in insufficient hydrolysis, and too high an amount destroys the core structure; and 37°C is close to the human body temperature, with the best enzyme activity. Preferably, 1 mM of CaCl2 can be added to enhance the activity of trypsin, or 0.5 mM of EDTA can be added to inhibit the interference of metalloproteinases.

[0016] In addition, in the enzymatic hydrolysis treatment, dynamic monitoring means are adopted. Samples are taken every 1 hour, and the fucose consumption rate is detected by HPLC. The release amount of reducing sugar is measured at a retention time of 12 - 15 minutes to control the reaction end point.

[0017] (2) Glycosylation modification: Mix the pre-treated blocker and L-fucose evenly at a volume ratio of 2:3 - 2:6, add fucosyltransferase at 20 U / g of protein, and react under the conditions of pH 6.5 - 7.5 for 4 - 6 hours to obtain the glycosylation-modified blocker. Fucosyltransferase catalyzes the covalent connection of fucose with the lysine ε-amino group of the blocker to form a stable glycopeptide bond, enabling the blocking protein to obtain the binding ability to the Si-Lea epitope.

[0018] Excess fucose ensures the modification saturation, and insufficient enzyme amount results in a sugar chain incorporation rate < 50%, affecting the binding activity.

[0019] (3) Cross-linking reaction: Add transglutaminase at 5 U / g protein to the glycation-modified blocker, and react for 1-2 hours under the condition of pH 7.5-8.5; the enzyme catalyzes the cross-linking of glutamine and lysine between blocker protein molecules to form a network structure, and the anti-protease degradation ability is significantly improved. After storage for 36 months, the activity retention rate > 90%.

[0020] (4) Purification: After the reaction, inactivate the enzyme by heating at 80-90 °C for 3-8 min, cool to room temperature, adjust the pH to 4.6 (near the isoelectric point of the blocker), centrifuge to collect the precipitate, and wash 3 times with pre-cooled isoelectric point water to remove free sugar chains; Dissolve the precipitate in 0.01 M phosphate buffer, put it into a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyze at 3-8 °C for 24 hours, changing the buffer every 6 hours. Or use an ultrafiltration centrifugal tube to centrifuge until the volume is concentrated to 1 / 5 to remove unreacted sugar chains, and finally obtain the blocker protein. 0.1% (w / v) activated carbon can be added before dialysis to adsorb unreacted sugar donors. The purified product is made into a powder by freeze-drying and stored at -20 °C.

[0021] The present invention prepares the blocker protein by an enzymatic hydrolysis-glycation-cross-linking three-step method, introduces a pH / salt concentration-dependent binding property, and realizes the dynamic balance of "blocking during storage and releasing during detection". Specifically, the action mechanism of the blocker protein is as follows: 1. Competitive blocking: The blocker protein can dynamically bind to the carbohydrate antigen and directly occupy the antigenic epitope of the carbohydrate antigen. This binding blocks the interaction between the antigenic epitopes of 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, so that the antibody cannot bind to CA199 through this site, and another specific site of CA50 (LSTa without fucose) can be recognized and bound by the antibody.

[0022] 2. Intelligent regulation of dual properties Specificity enhancement: By not binding to the LSTa site of CA50, zero-interference blocking is achieved in the detection involving CA50, and the antigen recognition accuracy is improved compared with traditional blockers; Dynamic reversibility: Its binding to the Si-Lea site is pH / salt concentration-dependent, and the dynamic balance of binding-dissociation can be achieved in in vitro detection, avoiding the permanent inactivation of antigens that may be caused by traditional blockers.

[0023] Compared with unmodified blockers, fucosylated blocking proteins effectively reduce the binding of antibodies to cross-reactive antigens by competitively binding to the antigenic epitopes of carbohydrate antigens. Through molecular site-specific modification, the present invention significantly improves the regulation accuracy of cross-reactivity with carbohydrate antigens while maintaining the basic interference blocking function. Its dual regulation mechanism not only reduces the false positive rate, but also ensures the sensitivity and stability of the detection system through its reversible binding property, and has important application value in immunoassay (such as reducing false positives).

[0024] In some realizable ways, the non-analyte further includes: 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 selected from at least one of carbohydrate stabilizers and protein stabilizers, with a concentration of 0.01%-1% (w / v); The carbohydrate stabilizer forms a hydrogen bond network with glycoproteins through hydroxyl groups to inhibit the aggregation of sugar chains caused by water loss; the protein stabilizer acts as a spatial barrier to isolate antigen molecules, reducing the collision probability between CA153 and CA242 and lowering the cross-reaction rate.

[0025] A preservative selected from at least one of ProClin300, ProClin950 and gentamicin, with a concentration of 0.1-80 mg / L. The preservative can effectively inhibit the activity of microbial thiolase, block the energy metabolism pathway, and does not chemically react with fucose sugar chains.

[0026] In some realizable ways, 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, ovalbumin and protein stabilizer; And / or, the blocker is at least one of Blocker series blockers, animal serum series and animal IgG series products.

[0027] In some realizable ways, the buffer solution is PB buffer solution, which contains the following components: disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and its pH is 6.0-8.0.

[0028] In some realizable ways, the concentration of the PB buffer solution is 25 mM and its pH is 7.0. The PB buffer solution maintains a pH of 7.0, protonates the amino group of the blocking protein, forms an ionic bond with the sialic acid residue of the antigenic epitope, and achieves efficient blocking during storage; during detection, the increase in salt concentration caused by sample dilution dissociates the blocking protein and releases the antigenic epitope.

[0029] The present invention forms an efficient pH buffer pair through the ratio of disodium hydrogen phosphate dodecahydrate to sodium dihydrogen phosphate dihydrate, which can not only resist external acid-base interference (such as the influence of metabolites in serum), but also avoid the aggregation of sugar chains caused by too high ionic strength.

[0030] In some realizable ways, the protective agent is a protein stabilizer, and the preservative is ProClin300.

[0031] In some realizable ways, the tumor markers include at least one of β2-microglobulin, carcinoembryonic antigen, calcitonin, cytokeratin 19, ferritin, gastrin, human epididymis protein 4, neuron-specific enolase, prostate acid phosphatase, pepsinogen I, pepsinogen II, prolactin, progastrin-releasing peptide, central nervous system specific protein, squamous cell carcinoma antigen, and thyroglobulin; The carbohydrate antigens include at least two of CA125, CA153, CA199, CA242, CA724, and CA50. Preferably, the carbohydrate antigens in the present invention include six types of carbohydrate antigens: CA125, CA153, CA199, CA242, CA724, and CA50.

[0032] The non-tumor-related markers include at least one of anti-thyroid peroxidase antibody, thyroglobulin antibody, and thyroid-stimulating hormone receptor antibody.

[0033] In a second aspect, the present invention also provides a preparation method for a tumor marker liquid quality control product containing multiple carbohydrate antigens for preparing the above liquid quality control product. The preparation method includes the following steps: (1) Prepare the buffer solution: Dissolve the buffer agent in pure water and adjust the pH to 6.0 - 8.0 to form a stable ionic environment.

[0034] (2) Prepare the basic mixture: Sequentially add the fucosylated blocking protein, protective agent, and preservative to the buffer solution and mix well. The final concentration of the fucosylated blocking protein in the matrix mixture of the present invention is 0.1 - 10 mg / mL. In human serum matrix, when the blocking protein exists at a concentration of 0.1 - 10 mg / mL, it can saturate and bind approximately 80% of the Si-Lea epitopes to form a dynamic blocking layer, which not only reduces cross-reactivity but also does not affect the specific binding of antigen-antibody.

[0035] (3) Add the human serum matrix solution: Add the human serum matrix solution to the basic mixture in step (2) and slowly stir to make it evenly mixed. First, add the blocking protein to the buffer solution to make it fully dissolve and form a monomeric conformation. In addition, the blocking protein is added before the serum to avoid the hydrophobic effect of serum albumin wrapping the blocking protein, resulting in a decrease in the binding efficiency of the Si-Lea epitope.

[0036] (4)Add analytes: After adding human serum matrix solution, add six carbohydrate antigens and other markers, and stir until completely dissolved to ensure uniform dispersion of each analyte and avoid cross-reaction caused by too high local concentration. (5)Filter and aliquot: Filter through a 0.2 - 0.45 μm filter membrane to remove microorganisms and impurity particles with a size ≥ 0.2 μm and prevent the sampling needle of the automated detection equipment from being blocked. Store at 2 - 8 °C after aliquoting 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.

[0037] In some realizable ways, it also includes a quality control step: perform homogeneity detection on the aliquoted quality control product to ensure that the coefficient of variation within the vial (CV within vial) of the quality control product ≤ 6%, and the coefficient of variation between vials (CV between vials) ≤ 6%. The actual acceptance standard not exceeding 10% is considered qualified. The homogeneity of the present invention has been greatly improved and is more stable than the freeze-dried powder quality control product.

[0038] The preparation method of the present invention realizes the dynamic balance and conformational protection of antigen-blocking protein through the design of the blocked protein molecular structure, breaks through the bottlenecks of cross-reaction and stability of liquid quality control products, and finally realizes the high specificity, high stability and high homogeneity of the quality control product.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows: (1)For the first time, the present invention provides a reagent for blocking cross-reaction between carbohydrate antigens in a specific quality control product composition for the problem of cross-reaction between carbohydrate antigens in a tumor marker liquid quality control product containing multiple carbohydrate antigens, which can reduce the cross-reaction of the tumor marker liquid quality control product containing multiple carbohydrate antigens of the present invention. Especially, the cross-reaction between carbohydrate antigen items in the quality control product is improved most significantly. The present invention blocks the specific recognition of the common Si-Lea epitope of carbohydrate antigens by fucosylated modified blocking protein, and uses the pH / salt concentration-dependent dynamic balance mechanism (binding and blocking during storage, dissociation and release during detection) to competitively occupy the antibody binding site, significantly reducing false positive results and improving detection specificity.

[0040] (2)The present invention uses human serum as the matrix to simulate the protein composition of clinical samples, combines a buffer to maintain a stable ionic environment, and cooperates with the hydrogen bond network of the carbohydrate stabilizer and the steric hindrance effect 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 ≤ ±15%, far exceeding the attenuation rate after reconstitution of the freeze-dried product.

[0041] (3) The quality control product of the present invention is a frozen liquid, different from freeze-dried powder, which eliminates the error caused by reconstitution. Compared with avoiding matrix effect and interference caused by cross-reaction through freeze-drying, the present invention weakens the degree of cross-reaction among multiple carbohydrate antigens by adding a cross-reaction blocker to the liquid quality control product, greatly improving the detection convenience and accuracy of testers, and reducing the labor and time costs of the laboratory.

[0042] Aiming at the core problems such as cross-reaction caused by glycosylation conservation of carbohydrate antigens and insufficient stability of liquid matrix in existing tumor marker quality control products, the present invention develops a liquid quality control product with high specificity and high stability through molecular targeting design of fucosylation-modified blocking protein and construction of multi-component synergistic system.

[0043] The present invention will be explained and described in detail below in combination with the accompanying drawings and specific embodiments. Description of the Drawings

[0044] Figure 1 It is a result diagram of the stability experiment of the tumor marker liquid quality control product with a horizontal concentration of level 2 in Example 1 of the present invention. The abscissa is the time span from 0 to 38 months, and the ordinate is the concentration corresponding to six carbohydrate antigens. Figure 2 It is a result diagram of the stability experiment of the tumor marker liquid quality control product with a horizontal concentration of level 3 in Example 1 of the present invention. The abscissa is the time span from 0 to 38 months, and the ordinate is the concentration corresponding to six carbohydrate antigens.

[0045] It shows that the prepared blocking protein can be stored stably for 38 months under the same low-temperature storage conditions as the quality control product, and its expiration date covers the shelf life of the quality control product. Specific Embodiments

[0046] To facilitate the 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, but 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 disclosure of the present invention more thorough and comprehensive.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are 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.

[0048] Example 1: The preparation method of the blocking protein in the embodiment of the present invention includes the following steps: Enzymatic hydrolysis treatment: Dissolve the Blocker series blockers (1M - purchased from Jingda Biotech, product number HBR-4) in 25 mM sodium phosphate buffer (pH 7.5), add trypsin according to the enzyme / substrate mass ratio of 1:30, hydrolyze at 37 °C for 1 hour, inactivate at 80 °C for 15 minutes, centrifuge at 10,000×g at 4 °C for 15 minutes, and take the supernatant for standby. Preferably, 1 mM CaCl2 can also be added in this example to enhance the activity of trypsin.

[0049] Glycosylation modification: Mix the supernatant and L-fucose at a volume ratio of 2:3, add fucosyltransferase at 20 U / g protein, adjust the pH to 7.5, and react at 37 °C for 5 hours.

[0050] Cross-linking reaction: Add transglutaminase at 5 U / g protein, react at pH 8.0 for 1.5 hours, adjust the pH to 4.6 to precipitate the protein, dialyze to remove free sugar chains, and freeze-dry to make a powder.

[0051] Quality control product preparation: Prepare 80 mL of quality control product dilution: Add 0.4441 g of disodium hydrogen phosphate dodecahydrate and 0.1186 g of sodium dihydrogen phosphate dihydrate to 10 mL of pure water, stir until completely dissolved to obtain PB buffer. Weigh 0.08 g of protein stabilizer A001 (purchased from Jingda Biotech, product number W0001) and 0.04 g of blocking protein powder, add them to the buffer, stir to dissolve, and the final concentration of the blocking protein is 10 mg / mL. Add 0.08 mL of ProClin300 to the solution and mix gently. Make up the volume to 40 mL with pure water to form a basic buffer system. Add 40 mL of human serum matrix solution to the solution, and then add the analyte raw materials according to Table 1 below, filter and dispense through a 0.22 μm filter membrane, and store refrigerated at 2 - 8 °C.

[0052] Table 1 shows the concentration levels of each analyte raw material at level 2 and level 3 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 Perform an analytical performance evaluation experiment on the tumor marker liquid quality control products at the above two level concentrations: Take 10 bottles each of the tumor marker liquid quality control products at level concentration 2 and level concentration 3, measure each bottle 3 times on a suitable immunoanalyzer, and calculate the within-bottle coefficient of variation (CV within-bottle) and the between-bottle coefficient of variation (CV between-bottle).

[0053] Table 2 shows the analytical performance test results of quality control products at different concentration levels Level 2 Anti-TPO B2-MG CA125 CA153 CA199 CV within vial 3.9% 3.4% 3.7% 1.3% 2.2% CV between vials 5.0% 3.5% 2.1% 1.7% 2.1% Project Name CA242 CA50 CA724 CEA CT CV within vial 2.4% 1.6% 2.0% 4.3% 4.6% CV between vials 1.7% 1.6% 2.0% 3.2% 4.3% Project Name CYFRA211 Ferritin Gastrin-17 HE4 NSE CV within vial 4.2% 3.3% 4.2% 4.9% 3.7% CV between vials 3.1% 3.9% 4.1% 3.2% 5.0% Project Name PAP PGI PGII PRL PROGRP CV within vial 5.0% 3.0% 3.6% 4.5% 3.9% CV between vials 4.6% 3.4% 4.3% 4.9% 4.6% Project Name S-100 SCCA TGA TRAb TG CV within vial 3.2% 3.9% 3.5% 4.8% 4.4% CV between vials 4.1% 3.8% 4.0% 4.3% 4.3% Level 3 Anti-TPO B2-MG CA125 CA153 CA199 CV within vial 3.9% 4.2% 3.9% 3.7% 2.0% CV between vials 4.0% 3.2% 2.9% 2.4% 2.4% Project Name CA242 CA50 CA724 CEA CT CV within vial 2.3% 1.3% 2.4% 4.0% 4.9% CV between vials 2.6% 2.9% 2.6% 3.8% 3.1% Project Name CYFRA211 Ferritin Gastrin-17 HE4 NSE CV within vial 3.5% 4.8% 4.2% 4.2% 5.0% CV between vials 4.7% 4.8% 4.4% 4.0% 3.7% Project Name PAP PGI PGII PRL PROGRP CV within vial 4.0% 4.9% 3.7% 3.0% 5.0% CV between vials 3.0% 3.0% 4.6% 4.6% 3.8% Project Name S-100 SCCA TGA TRAb TG CV within vial 4.8% 3.3% 4.5% 4.6% 4.4% CV between vials 3.6% 3.1% 3.4% 3.2% 3.2% As can be seen from the above table, after adding the blocking protein, the CVs of its six carbohydrate antigens do not exceed 4%, while the CVs of other analytes are in the range of 3% - 5%, indicating that the blocking protein has an obvious improvement effect on the homogeneity of the six carbohydrate antigens.

[0054] The tumor marker liquid quality control product containing multiple carbohydrate antigens prepared by the above preparation method is based on human serum as the matrix and contains the following components: A: Non-analytes, including: buffer, blocking protein, stabilizer and preservative. Specifically, in this example, the buffer is PB buffer, which contains the following components: disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and the concentration of the buffer is 25 mM and the pH is 7.0; the protective agent is protein stabilizer A001, and its concentration is 10% (w / v); the preservative is ProClin300, and its concentration is 30 mg / L.

[0055] B: Analytes, including: six carbohydrate antigens and other markers, and the other markers include tumor markers and non-tumor-related markers. Specifically, in this example, the tumor markers include β2-microglobulin, carcinoembryonic antigen, calcitonin, cytokeratin 19, ferritin, gastrin, human epididymis protein 4, neuron-specific enolase, prostate acid phosphatase, pepsinogen I, pepsinogen II, prolactin, progastrin-releasing peptide, central nervous system specific protein, squamous cell carcinoma antigen and thyroglobulin.

[0056] In this example, the non-tumor-related markers include anti-thyroid peroxidase antibody, thyroglobulin antibody, and thyroid-stimulating hormone receptor antibody. The six carbohydrate antigens are CA125, CA153, CA199, CA242, CA724, and CA50. The human serum matrix solution is human serum negative for infectious diseases, and its volume ratio is 40%-60%.

[0057] Example 2: The preparation method of the blocking protein in this example is basically the same as that in Example 1, and the differences are as follows: Enzymatic digestion treatment: Dissolve the animal serum series (0.01M - purchased from Jingda Biotech, product number C8001) in 25 mM sodium phosphate buffer (pH 6.5), add pepsin according to the enzyme / substrate mass ratio of 1:50, hydrolyze at 37°C for 1.5 hours, inactivate at 85°C for 10 minutes, centrifuge at 10,000×g and 4°C for 15 minutes, and take the supernatant for use. Preferably, 0.5 mM EDTA can also be added in this example to inhibit the interference of metalloproteinases.

[0058] Glycosylation modification: Mix the supernatant with L-fucose at a volume ratio of 2:6, add fucosyltransferase at 20 U / g protein, adjust the pH to 7.0, and react at 37°C for 4 hours.

[0059] Cross-linking reaction: Add transglutaminase at 5 U / g protein and react for 2 hours under the condition of pH 7.5. Use an ultrafiltration centrifugal tube (10 kDa), centrifuge at 4000×g until the volume is concentrated to 1 / 5, remove the free sugar chains, and freeze-dry to make a powder.

[0060] Preparation of quality control product: Prepare 80 mL of quality control product diluent. Add 0.4441 g of disodium hydrogen phosphate dodecahydrate and 0.1186 g of sodium dihydrogen phosphate dihydrate to 10 mL of pure water, stir until completely dissolved to obtain PB buffer solution. Weigh 0.08 g of protein stabilizer A001 and 0.04 g of blocking protein powder, add them to the buffer solution, stir and dissolve, and the final concentration of the blocking protein is 1 mg / mL. Add 0.08 mL of ProClin300 to the solution and mix gently. Make up the volume to 40 mL with pure water to form a basic buffer system. Add 40 mL of human serum matrix solution to the solution, then add the analyte raw materials according to Table 1 above, filter and dispense through a 0.22 μm filter membrane, and store refrigerated at 2 - 8 °C.

[0061] Conduct an analytical performance evaluation experiment on the tumor marker liquid quality control products at the above two concentration levels: Take 10 bottles each of the tumor marker liquid quality control products at concentration level 2 and concentration level 3, measure each bottle 3 times on a suitable immunoassay analyzer, and calculate the within-bottle coefficient of variation (CV within-bottle) and the between-bottle coefficient of variation (CV between-bottle).

[0062] Table 3 shows the analytical performance test results of six carbohydrate antigens in quality control products at different concentration levels Level 2 CA125 CA153 CA199 CA242 CA50 CA724 CV within vial 4.3% 5.5% 5.8% 4.5% 6.0% 5.5% CV between vials 4.5% 5.2% 5.4% 4.6% 4.7% 5.9% Level 3 CA125 CA153 CA199 CA242 CA50 CA724 CV within vial 4.5% 5.2% 5.7% 5.4% 4.5% 5.5% CV between vials 5.4% 4.7% 4.8% 5.7% 4.6% 5.7%

[0063] Example 3: The preparation method of the blocking protein in this example is basically the same as that in Example 1, and the differences are as follows: Enzymatic hydrolysis treatment: Dissolve the animal IgG series blocker product (0.1 M - purchased from Jingda Biology, product number W4006) in 25 mM sodium phosphate buffer (pH 8.0), add pepsin according to the 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 and 4 °C for 10 minutes, and take the supernatant for standby.

[0064] Glycosylation modification: Mix the supernatant with L-fucose at a volume ratio of 2:4, add fucosyltransferase at 20 U / g protein, adjust the pH to 6.5, and react at 37 °C for 6 hours.

[0065] Cross-linking reaction: Add transglutaminase at 5 U / g protein and react for 1 hour under the condition of pH 8.5. After purification and filtration, freeze-dry to make a powder.

[0066] Quality control product preparation: Prepare 80 mL of quality control product diluent. Add 0.4441 g of disodium hydrogen phosphate dodecahydrate and 0.1186 g of sodium dihydrogen phosphate dihydrate to 10 mL of pure water, stir until completely dissolved to obtain PB buffer solution. Weigh 0.08 g of protein stabilizer A001 and 0.04 g of blocking protein powder, add them to the buffer solution, stir to dissolve, and the final concentration of the blocking protein is 20 mg / mL. Add 0.08 mL of ProClin300 to the solution and mix gently. Make up the volume to 40 mL with pure water to form a basic buffer system. Add 40 mL of human serum matrix solution to the solution, then add the analyte raw materials according to Table 1 above, filter and dispense through a 0.22 μm filter membrane, and store refrigerated at 2 - 8°C.

[0067] Perform an analytical performance evaluation experiment on the tumor marker liquid quality control products at the above two concentration levels: Take 10 bottles each of the tumor marker liquid quality control products at concentration levels 2 and 3, measure each bottle 3 times on a suitable immunoanalyzer, and calculate the within-bottle coefficient of variation (CV within-bottle) and between-bottle coefficient of variation (CV between-bottle).

[0068] Table 4 shows the analytical performance test results of six carbohydrate antigens in quality control products at different concentration levels Level 2 CA125 CA153 CA199 CA242 CA50 CA724 CV within vial 6.0% 5.6% 5.7% 5.0% 5.4% 5.6% CV between vials 5.8% 5.0% 5.8% 5.2% 4.9% 6.0% Level 3 CA125 CA153 CA199 CA242 CA50 CA724 CV within vial 5.5% 5.9% 5.5% 5.4% 4.8% 5.7% CV between vials 5.5% 5.3% 5.0% 5.1% 4.6% 5.3% Comparative Example 1: In this comparative example, a comparison is made with a commercially available tumor marker quality control product for multiple carbohydrate antigens. The commercially available tumor marker quality control product is from Zhengzhou Biaoyuan Biotechnology Co., Ltd., and is compared with Example 1.

[0069] Table 5 shows the performance test results of six carbohydrate antigens in the commercially available tumor marker quality control product Level 2 CA125 CA153 CA199 CA242 CA50 CA724 CV within vial 4.4% 3.2% 3.3% 4.1% 3.8% 4.2% CV between vials 3.1% 3.8% 3.7% 3.9% 3.5% 4.1% Level 3 CA125 CA153 CA199 CA242 CA50 CA724 CV within vial 4.2% 3.8% 3.9% 5.6% 4.0% 4.0% CV between vials 4.2% 3.8% 4.9% 3.8% 4.5% 5.0% As can be seen from the above table, compared with Comparative Example 1, the tumor marker liquid quality control product in Example 1 of the present invention has better homogeneity for multiple carbohydrate antigens.

[0070] Comparative Example 2: In this comparative example, no blocking protein is added, and the rest is the same as in Example 1. The homogeneity test results of six carbohydrate antigens in the quality control product without added blocking protein are shown in Table 6 below.

[0071] Table 6 shows the performance test results of carbohydrate antigens in the quality control product without added blocking protein Level 2 Anti-TPO B2-MG CA125 CA153 CA199 CV within vial 4.4% 3.2% 14.9% 13.5% 12.7% CV between vials 4.5% 4.6% 14.9% 13.7% 12.7% Project Name CA242 CA50 CA724 CEA CT CV within vial 13.7% 13.3% 14.1% 4.8% 4.9% CV between vials 13.7% 13.9% 14.6% 4.8% 4.6% Project Name CYFRA211 Ferritin Gastrin-17 HE4 NSE CV within vial 3.1% 4.8% 4.9% 4.8% 3.7% CV between vials 3.7% 4.6% 4.1% 3.1% 3.7% Project Name PAP PGI PGII PRL PROGRP CV within vial 3.5% 3.6% 4.4% 4.8% 3.1% CV between vials 3.8% 4.7% 3.3% 3.4% 3.6% Project Name S-100 SCCA TGA TRAb TG Inside the CV vial 4.1% 4.5% 4.2% 4.5% 3.7% Between the CV vials 4.1% 4.6% 4.1% 4.5% 4.0% Horizontal 3 Anti-TPO B2-MG CA125 CA153 CA199 Inside the CV vial 4.9% 4.2% 11.5% 12.2% 13.4% Between the CV vials 4.2% 3.6% 11.9% 12.8% 13.0% Project name CA242 CA50 CA724 CEA CT Inside the CV vial 13.1% 14.2% 14.6% 3.4% 4.5% Between the CV vials 13.9% 14.0% 14.8% 3.8% 4.5% Project name CYFRA211 Ferritin Gastrin-17 HE4 NSE Inside the CV vial 4.1% 3.6% 4.7% 4.9% 4.3% Between the CV vials 4.0% 4.2% 4.3% 5.0% 3.6% Project name PAP PGI PGII PRL PROGRP Inside the CV vial 4.3% 4.2% 4.0% 3.1% 3.7% Between the CV vials 4.2% 3.9% 5.0% 3.4% 4.1% Project name S-100 SCCA TGA TRAb TG Inside the CV vial 4.2% 4.6% 4.9% 4.2% 3.0% Between the CV vials 3.2% 3.8% 4.0% 3.6% 4.6% As can be seen from the above table, compared with Comparative Example 2, the tumor marker liquid quality control product in the embodiment of the present invention has better homogeneity for six carbohydrate antigens and shows that adding the blocking protein has no obvious effect on the detection of analytes other than carbohydrate antigens in the quality control product.

[0072] Comparative Example 3: In this comparative example, the blocking protein was replaced with a blocker (the same model as in Example 1), and the rest was the same as in Example 1. The results of the homogeneity test of the six carbohydrate antigens in the quality control product are shown in Table 7 below.

[0073] Table 7 Performance test results of carbohydrate antigens in the quality control product with the blocking protein replaced by a blocker Horizontal 2 CA125 CA153 CA199 CA242 CA50 CA724 Inside the CV vial 11.2% 12.6% 11.3% 11.3% 12.3% 11.5% Between the CV vials 11.9% 12.9% 11.8% 10.9% 12.3% 11.6% Horizontal 3 CA125 CA153 CA199 CA242 CA50 CA724 Inside the CV vial 11.4% 12.6% 11.8% 10.9% 12.8% 12.8% Between the CV vials 11.8% 12.8% 11.8% 11.4% 12.4% 12.1%

[0074] Comparative Example 4: In this comparative example, the addition amount of the blocking protein was adjusted to 1 g, and the final concentration of the fucosylated modified blocking protein in the matrix mixture was 12.5 mg / mL, exceeding the reasonable range of 0.1 - 10 mg / mL in the examples of the present invention. The other preparation steps of the quality control product were the same as those in Example 1. After preparation, 1 bottle was taken and measured 3 times and compared with the target measured value. The relative deviation between the target target value and the measured value of the six carbohydrate antigens in the quality control product after the adjustment of the addition amount of the blocking protein is shown in Table 8 below.

[0075] Table 8 Performance test results of carbohydrate antigens in the quality control product after the adjustment of the addition amount of the blocking protein 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%

[0076] As can be seen from the above table, after the excessive addition of the blocking protein, the antigenic epitopes of the carbohydrate antigens are irreversibly bound, resulting in a large difference from the target measured value during detection.

[0077] Any numerical value cited herein includes all values from the lower value to the upper value increasing in increments of one unit between the lower limit value and the upper limit value, provided that there is an interval of at least two units between any lower value and any higher value. For example, if the value of the number of components or process variables (such as temperature, pressure, time, etc.) is stated as being from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70, then the intention is to illustrate that values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32, etc. 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 of what is intended to be clearly expressed, and it can be considered that all possible combinations of the numerical values listed between the lowest value and the highest value are explicitly stated in this specification in a similar manner.

[0078] Unless otherwise stated, all ranges include the endpoints and all the numbers between the endpoints. "About" or "approximately" used in conjunction with a range 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.

[0079] It should be understood that the above description is for illustrative purposes and not for limitation. Upon reading the above description, many embodiments and many applications other than the provided examples will be apparent to those skilled in the art. Therefore, the scope of the present teachings should not be determined with reference to the above description, but rather should be determined with reference to the appended claims and the full scope of equivalents to which those claims are entitled. For the sake of completeness, all articles and references, including patent applications and published announcements, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein from the foregoing claims is not intended to abandon such subject matter, nor should it be considered that the inventor did not consider such subject matter to be part of the disclosed inventive subject matter.

[0080] The present invention has been described above by way of example in conjunction with the accompanying drawings. Obviously, the specific implementation of the present invention is not limited by the above methods. As long as such non-substantial improvements are made by adopting 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 protection scope of the present invention.

Claims

1. A tumor marker liquid quality control product containing multiple carbohydrate antigens, characterized in that, The liquid quality control product is based on human serum and contains the following components: A: Non-analytes, including blocking proteins, which are functional proteins after the blocker is fucosylated. The fucosylated sugar chain structure is introduced through enzymatic hydrolysis and glycosylation, enabling it to specifically recognize the fucose-containing Si-Lea epitope common to carbohydrate antigens; B: Analytes, including: carbohydrate antigens and other markers, and the other markers include tumor markers and non-tumor-related markers.

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

3. The tumor marker liquid quality control product containing multiple carbohydrate antigens according to claim 2, wherein, The preparation method of the blocking protein includes the following steps: (1) Enzymatic hydrolysis treatment: Dissolve the blocker in a sodium phosphate buffer with a pH of 6.5 - 8.0, add pepsin or trypsin at an enzyme / substrate mass ratio of 1:30 - 1:70, hydrolyze at a temperature of 37°C ± 2°C for 1 - 2 hours, inactivate the enzyme, and then centrifuge to collect the supernatant; (2) Glycosylation modification: Mix the pretreated blocker with L-fucose evenly at a volume ratio of 2:3 - 2:6, add fucosyltransferase at 20 U / g protein, and react under the conditions of pH 6.5 - 7.5 for 4 - 6 hours to obtain the glycosylated blocker; (3) Crosslinking reaction: Add transglutaminase at 5 U / g protein to the glycosylated blocker obtained above, and react under the conditions of 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, centrifuge to collect the precipitate, remove free sugar chains by dialysis or ultrafiltration, and freeze-dry the purified product to obtain a powder for later use.

4. The tumor marker liquid quality control product containing multiple carbohydrate antigens according to claim 1, characterized in that, The non-analytes further include: Buffers, which are selected from at least one of PB buffer, HEPES buffer, MES buffer, PIPES buffer, and Tris-HCl buffer, with a concentration of 0.1 - 100 mmol / L and a pH of 6.0 - 8.0; Stabilizers, which are selected from at least one of carbohydrate stabilizers and protein stabilizers, with a concentration of 0.01% - 1% (w / v); Preservatives, which are selected from at least one of ProClin300, ProClin950, and gentamicin, with 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 stabilizers are selected from at least one of sucrose, trehalose, lactose, maltose, and mannitol; and / or, the protein stabilizers are selected from at least one of casein, ovalbumin, and protein stabilizers; 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 is PB buffer, which contains the following components: disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and its pH is 6.0 - 8.

0.

7. The tumor marker liquid quality control product 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 protective agent 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, prostate acid phosphatase, pepsinogen I, pepsinogen II, prolactin, progastrin-releasing peptide, central nervous system-specific protein, squamous cell carcinoma antigen, and thyroglobulin; The carbohydrate antigens include at least one of CA125, CA153, CA199, CA242, CA724, and CA50; The non-tumor-related markers include at least one of anti-thyroid peroxidase antibody, thyroglobulin antibody, and thyroid-stimulating hormone receptor antibody.

9. A method for preparing a liquid quality control product for tumor markers containing multiple carbohydrate antigens, which is used to prepare the liquid quality control product according to any one of claims 1-8, characterized in that, The preparation method includes the following steps: (1) Prepare the buffer: Dissolve the buffer agent in pure water and adjust the pH to 6.0 - 8.0; (2) Prepare the basic mixture: Sequentially add the fucosylated blocking protein, the protective agent, and the preservative to the buffer, and mix well. The final concentration of the fucosylated blocking protein in the matrix mixture is 0.1 - 10 mg / mL; (3) Add human serum matrix: Add the human serum matrix to the basic mixture in step (2) and slowly stir to mix evenly; (4) Add the analyte: After adding the human serum matrix, add six carbohydrate antigens and other markers, and stir until completely dissolved; (5) Filter and dispense: Filter the solution through a 0.2 - 0.45 μm filter membrane, dispense, and store refrigerated at 2 - 8 °C.

10. The preparation method of the tumor marker liquid quality control product containing multiple carbohydrate antigens according to claim 9, characterized in that, It also includes a quality control step: Perform homogeneity testing on the dispensed quality control product to ensure that the coefficient of variation (CV) of each analyte in the liquid quality control product is ≤ 6% within the bottle and ≤ 6% between bottles.

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