Composition for preparing anti-smooth muscle antibody quality control product, anti-smooth muscle antibody quality control product and kit

Abstract

The present invention relates to a composition, an anti-smooth muscle antibody quality control product, and a kit for preparing an anti-smooth muscle antibody quality control product. The composition comprises a matrix and an additive, wherein the matrix includes at least one of fetal bovine serum and newborn bovine serum, and the additive includes morpholineethanesulfonic acid. In the composition, the mass ratio of bovine serum volume to morpholineethanesulfonic acid is 1L:(6.5g-10.2g); and the pH of the composition is 5.8-6.38. The amino acid sequence of the heavy chain variable region of the anti-smooth muscle antibody used is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2. The composition can be used to prepare an anti-smooth muscle antibody quality control product using human serum, and the resulting anti-smooth muscle antibody quality control product has good stability.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a composition, an anti-smooth muscle antibody quality control product and a kit for preparing an anti-smooth muscle antibody quality control product. Background Art

[0002] Anti-smooth muscle antibodies (ASMA or SMA) are serological markers of autoimmune hepatitis (AIH). They are a type of autoantibody that targets cytoskeletal proteins and are primarily classified into IgG and IgM types. Different antibody types can be seen in different diseases. When IgG is predominant, it is more common in patients with AIH, but it can also be seen in other diseases such as mycoplasma pneumonia, Sjögren's syndrome, rheumatoid arthritis, and syphilis. When both IgG and IgM are present, it is more common in patients with overlapping primary biliary cirrhosis and AIH. When IgM is predominant, it is associated with alcoholic hepatitis. ASMA is a type of autoantibody that targets cytoskeletal proteins. The positive rate in AIH patients is as high as 80%, and the higher the titer, the greater the sensitivity for diagnosing AIH.

[0003] The detection of SMA mainly relies on the detection of medical laboratories. The purpose of quality control in medical laboratories is to detect and control the precision of routine work in the laboratory and detect changes in its accuracy, so as to improve the consistency of specimen testing in routine work, thereby ensuring the accurate issuance of test reports. Therefore, it is necessary to develop a quality control product suitable for the quality evaluation of specimen testing for anti-smooth muscle antibody projects in medical laboratories such as clinical laboratories, third-party physical examination centers, provincial and municipal clinical examination centers, etc., which is important for the reliability and accuracy of the test results of the SMA project. This also requires that the quality control product is a quality control product that is close to the patient sample and has high stability, which helps to unbiasedly evaluate or verify the performance of the test kit and instrument, so as to achieve consistency with specimen testing and reduce detection errors.

[0004] At present, the anti-smooth muscle antibody quality control products on the market are mainly prepared with human serum, but clinical serum is difficult to obtain, which is not conducive to mass production. Summary of the Invention

[0005] Based on this, it is necessary to provide a composition for preparing anti-smooth muscle antibody quality control products, which can replace human serum to prepare anti-smooth muscle antibody quality control products, so as to improve the problem of difficulty in obtaining raw materials caused by the use of human serum, facilitate the mass production of anti-smooth muscle antibody quality control products, and improve the reliability and accuracy of the detection results of the SMA project.

[0006] A composition for preparing an anti-smooth muscle antibody quality control product, the composition consisting of a matrix and an additive, the matrix comprising at least one of fetal bovine serum and newborn bovine serum, the additive comprising morpholineethanesulfonic acid, in the composition, the mass ratio of the bovine serum volume to the morpholineethanesulfonic acid is 1L:(6.5g-10.2g), and the pH of the composition is 5.8-6.38.

[0007] The composition for preparing anti-smooth muscle antibodies uses bovine serum as the primary matrix, with a pH of 5.8 to 6.38. The volume ratio of bovine serum to morpholineethanesulfonic acid is 1 L: (6.5 g to 10.2 g). It has been demonstrated that the composition can replace clinical human serum in the preparation of anti-smooth muscle antibody quality control products, facilitating the mass production of anti-smooth muscle antibody quality control products. Furthermore, the anti-smooth muscle antibody quality control products prepared using this composition exhibit good thermal stability and low post-lyophilization loss.

[0008] In one embodiment, the additive further includes a preservative, and the working concentration of the preservative is 0.01 m / V% to 0.5 m / V%.

[0009] In one embodiment, the additive further comprises at least one of bovine serum albumin, casein and inorganic salts.

[0010] In one embodiment, in the composition, the working concentration of the bovine serum albumin is 13.5 g / L to 16 g / L; the concentration of the inorganic salt is 3.2 g / L to 3.6 g / L.

[0011] In one embodiment, the additive further comprises a lyophilization excipient, and the lyophilization excipient comprises at least one of mannitol and trehalose;

[0012] Furthermore, in the composition, the working concentration of mannitol is 27 g / L to 32 g / L; the working concentration of trehalose is 6.7 g / L to 8 g / L.

[0013] An anti-smooth muscle antibody quality control product, the anti-smooth muscle antibody quality control product is prepared by mixing the above-mentioned composition for preparing the anti-smooth muscle antibody quality control product with an anti-smooth muscle antibody, wherein the concentration of morpholineethanesulfonic acid in the composition is 3.9 g / L to 10.2 g / L;

[0014] Alternatively, the anti-smooth muscle antibody quality control product is prepared by mixing the above-mentioned composition for preparing the anti-smooth muscle antibody quality control product with anti-smooth muscle antibodies and then freeze-drying. In the composition, the matrix includes 60% to 65% bovine serum and 35% to 40% water by volume, and the concentration of the morpholineethanesulfonic acid is 3.3 g / L to 3.9 g / L.

[0015] In one embodiment, the amino acid sequence of the heavy chain variable region of the anti-smooth muscle antibody is shown as SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the anti-smooth muscle antibody is shown as SEQ ID NO: 2.

[0016] A method for preparing an anti-smooth muscle antibody quality control product comprises the following steps:

[0017] The above-mentioned composition for preparing an anti-smooth muscle antibody quality control product is mixed with an anti-smooth muscle antibody to prepare an anti-smooth muscle antibody quality control product, wherein the matrix in the composition is bovine serum and the working concentration of the morpholineethanesulfonic acid is 3.9 g / L to 2 g / L;

[0018] Alternatively, the above-mentioned composition for preparing an anti-smooth muscle antibody quality control product is mixed with an anti-smooth muscle antibody and then lyophilized to prepare an anti-smooth muscle antibody quality control product, wherein the matrix comprises 60% to 65% bovine serum and 35% to 40% water by volume, and the working concentration of the morpholineethanesulfonic acid is 3.3 g / L to 3.9 g / L.

[0019] In one embodiment, the freeze-drying includes pre-freezing, first drying and second drying performed in sequence, wherein the pre-freezing is performed in sequence according to the following conditions: 10 min to 50 min at -8°C to -10°C, 30 min to 120 min at -40°C to -60°C, 30 min to 120 min at -20°C to -25°C, and 120 min to 240 min at -40°C to -60°C;

[0020] The first drying is carried out in the following conditions: 10 min to 30 min at 40°C to -60°C and 0.1 mbar to 0.2 mbar, 15 h to 30 h at -25°C to -40°C and 0.1 mbar to 0.2 mbar, and 1 h to 3 h at -5°C to -10°C and 0.1 mbar to 0.2 mbar.

[0021] The second drying is carried out in the following conditions: 30 min to 60 min at 5° C. to 25° C. and 0.001 mbar to 0.01 mbar, and 2 h to 10 h at 5° C. to 25° C. and 0.001 mbar to 0.01 mbar.

[0022] A kit for detecting anti-smooth muscle antibodies comprises the above-mentioned anti-smooth muscle antibody quality control product. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the purification result of SMA IgG chimeric antibody in Example 1;

[0024] Figure 2 Correlation between the concentrations of SMA IgG in the two solutions containing SMA IgG prepared using solution 1 and solution 2 in Example 2 and the solution containing SMA IgG prepared using clinical serum samples;

[0025] Figure 3 Correlation between the concentrations of SMA IgG in the four solutions containing SMA IgG prepared using formulas 1 to 4 in Example 2 and the solution containing SMA IgG prepared using clinical serum samples;

[0026] Figure 4 The correlation between the concentration of SMA IgG after lyophilization and the concentration of SMA IgG in the solution directly prepared with clinical serum in Example 4;

[0027] Figure 5 This is the quality control chart of the anti-smooth muscle antibody in Example 7. DETAILED DESCRIPTION

[0028] In order to facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0029] It should be noted that the terms "first," "second," etc. are used for descriptive purposes only and should not be understood to indicate or imply relative importance. The term "and / or" includes any and all combinations of one or more of the related listed items. "Optionally" means by way of example.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] One embodiment of the present application provides a composition for preparing an anti-smooth muscle antibody quality control product, which is composed of a matrix and an additive, wherein: the matrix includes at least one of fetal bovine serum and newborn bovine serum, and the additive includes morpholineethanesulfonic acid; in the composition, the ratio of the volume of bovine serum to the mass of morpholineethanesulfonic acid is 1L: (6.5g~10.2g), and the pH of the composition is 5.8~6.38.

[0032] Traditional reagents used to prepare anti-smooth muscle antibody quality control products are generally clinical human serum or a solution formed by adding a small amount of auxiliary reagents (including a small amount of bovine serum or no bovine serum) to water as the main matrix. The above-mentioned composition for preparing anti-smooth muscle antibodies uses bovine serum to completely replace or partially replace water as the main matrix, and sets its pH to 5.8-6.38, and the ratio of the volume of bovine serum to the mass of morpholineethanesulfonic acid is 1L: (6.5g-10.2g). It has been verified that when preparing anti-smooth muscle antibody quality control products, the above-mentioned composition for preparing anti-smooth muscle antibody quality control products can replace clinical human serum, and the anti-smooth muscle antibody quality control products prepared using the composition have good thermal stability and a small loss rate after freeze-drying.

[0033] Furthermore, in the composition, the concentration of morpholineethanesulfonic acid is 3.3 g / L to 10.2 g / L.

[0034] In some embodiments, the additives in the above composition also include a preservative. Optionally, the preservative is selected from at least one of Proclin 300, sodium azide and thimerosal. Further, the working concentration of the preservative is 0.01m / V% to 0.5m / V%. ​​In an optional specific example, the working concentration of the preservative is 0.01m / V%, 0.05m / V%, 0.1m / V%, 0.2m / V%, 0.3m / V%, 0.4m / V% or 0.5m / V%. ​​It will be understood that the preservative is not limited to the above, and other substances can also be used. It should be noted that, in this article, "m / V%" represents mass volume percentage concentration. For example, 0.5m / V% means that the mass of the solute in 100mL of solution is 0.5g. "Working concentration" herein refers to the concentration of the component in the composition when the composition is used to prepare anti-smooth muscle antibody quality control. For example, a preservative working concentration of 0.5 m / V% means that when the above composition is used to formulate an anti-smooth muscle antibody control, the preservative concentration in the composition is 0.5 m / V%. ​​Sometimes, the working concentration of a component is the concentration of that component in the anti-smooth muscle antibody control, for example, when the amount of anti-smooth muscle antibody added is small and negligible to the system. It is understood that the packaging concentration of each component of the above composition is not necessarily the working concentration and can be higher than the working concentration to facilitate transportation.

[0035] Furthermore, the additive in the above composition further comprises at least one of bovine serum albumin and an inorganic salt. The bovine serum albumin acts as a stabilizer; the inorganic salt is used to simulate the ion environment in the serum sample.

[0036] In some embodiments, in the above composition, the working concentration of bovine serum albumin is 13.5 g / L to 16 g / L. Alternatively, the working concentration of bovine serum albumin is 13.5 g / L, 14 g / L, 14.5 g / L, 15 g / L, or 15.5 g / L. Further, the working concentration of bovine serum albumin is 15 g / L to 16 g / L.

[0037] In some embodiments, in the above composition, the concentration of the inorganic salt is 3.2 g / L to 3.6 g / L. Optionally, the inorganic salt comprises at least one of sodium chloride and potassium chloride; the concentration of the inorganic salt is 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, or 3.6 g / L. Furthermore, the inorganic salt is sodium chloride, and the working concentration of the inorganic salt is 3.4 g / L to 3.6 g / L.

[0038] In one embodiment, the additives in the above composition further include bovine serum albumin and sodium chloride. In the above composition, the working concentration of bovine serum albumin is 15g / L to 16g / L, and the working concentration of sodium chloride is 3.4g / L to 3.6g / L.

[0039] In some embodiments, the additives in the above composition further include a lyophilization excipient. Optionally, the lyophilization excipient includes at least one of mannitol and trehalose. Mannitol and trehalose are used as lyophilization excipients to facilitate the lyophilization of quality control products. Furthermore, in the above composition, the working concentration of mannitol is 27g / L to 32g / L; the working concentration of trehalose is 6.7g / L to 8g / L. Furthermore, in the above composition, the working concentration of mannitol is 30g / L to 32g / L; the working concentration of trehalose is 7g / L to 8g / L.

[0040] In some embodiments, the above composition is used directly to prepare a liquid quality control. In this case, the matrix in the composition comprises 60% to 100% bovine serum by volume. In other embodiments, the above composition is used to prepare a lyophilized quality control. In this case, the matrix in the composition comprises 60% to 65% bovine serum and 35% to 40% water by volume.

[0041] In addition, one embodiment of the present application further provides an anti-smooth muscle antibody quality control product, which is prepared by mixing the composition for preparing the anti-smooth muscle antibody quality control product of any of the above embodiments with anti-smooth muscle antibodies.

[0042] In some embodiments, the anti-smooth muscle antibody quality control is in liquid form. In the composition used to prepare the anti-smooth muscle antibody quality control, the matrix is ​​bovine serum, and the concentration of the morpholineethanesulfonic acid is 3.9 g / L to 10.2 g / L. Furthermore, in the composition, the matrix is ​​bovine serum, and the concentration of the morpholineethanesulfonic acid is 6.5 g / L to 10.18 g / L.

[0043] In other embodiments, the anti-smooth muscle antibody quality control product is a freeze-dried product. The anti-smooth muscle antibody quality control product is prepared by mixing the above-mentioned composition for preparing the anti-smooth muscle antibody quality control product with the anti-smooth muscle antibody and then freeze-drying. In the composition, the matrix includes 60% to 65% bovine serum and 35% to 40% water by volume, and the working concentration of morpholineethanesulfonic acid is 3.3g / L to 3.9g / L. Of course, when the anti-smooth muscle antibody quality control product is a freeze-dried product, the above-mentioned composition contains a freeze-dried excipient or an additional freeze-dried excipient (such as mannitol, trehalose, etc.) to make the obtained quality control product a freeze-dried product.

[0044] In this embodiment, the amino acid sequence of the heavy chain variable region of the anti-smooth muscle antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the anti-smooth muscle antibody is shown in SEQ ID NO: 2.

[0045] Optionally, the anti-smooth muscle antibody quality control product includes a negative quality control product and a positive quality control product.

[0046] Specifically, the negative control comprises anti-smooth muscle antibodies and the above-mentioned composition. In the working solution of the negative anti-smooth muscle antibody control, the concentration of anti-smooth muscle antibodies is 0.5 COI (±30%), i.e., 0.35 COI to 0.65 COI. In the working solution of the positive anti-smooth muscle antibody control, the concentration of anti-smooth muscle antibodies is 2.5 COI (±30%), i.e., 1.75 COI to 3.25 COI. In this document, "COI" stands for "cut-off index," which refers to the critical threshold value. Generally, the cut-off value for a control is 1.0 COI. A COI ≥ 1.0 indicates elevated antibody levels, but a definitive diagnosis should be confirmed based on clinical manifestations or other diagnostic methods / indicators. For clinical application, it is recommended that: a COI < 0.9 is considered unreactive; 0.9 ≤ COI < 1.1 is considered suspicious, and a retest is recommended after a reasonable period of time; a COI ≥ 1.1 is considered reactive, indicating elevated antibody levels, but a definitive diagnosis should be confirmed based on clinical manifestations or other diagnostic methods / indicators.

[0047] In some embodiments, the water of any of the above embodiments is purified water.

[0048] The anti-smooth muscle antibody quality control product is prepared using the above-mentioned composition for preparing anti-smooth muscle antibody quality control product, has high stability, and can be produced in batches.

[0049] In addition, one embodiment of the present application also provides a method for preparing an anti-smooth muscle antibody quality control product, comprising the following steps: mixing the composition for preparing an anti-smooth muscle antibody quality control product of any of the above embodiments with an anti-smooth muscle antibody to prepare an anti-smooth muscle antibody quality control product.

[0050] In some embodiments, the anti-smooth muscle antibody control is in liquid form. In this case, the method for preparing the anti-smooth muscle antibody control comprises the steps of: mixing the composition for preparing the anti-smooth muscle antibody control according to any of the above embodiments with anti-smooth muscle antibodies to directly obtain a liquid anti-smooth muscle antibody control.

[0051] In other embodiments, the anti-smooth muscle antibody quality control product is a lyophilized product (e.g., lyophilized powder). In this case, the steps of the above-mentioned method for preparing the anti-smooth muscle antibody quality control product include: mixing the composition for preparing the anti-smooth muscle antibody quality control product of any of the above-mentioned embodiments with the anti-smooth muscle antibody, and then lyophilizing to obtain a lyophilized anti-smooth muscle antibody quality control product. It is understood that the lyophilization excipient is included in the composition for preparing the anti-smooth muscle antibody quality control product or is added before the lyophilization process is performed.

[0052] Furthermore, the freeze-drying comprises pre-freezing, first drying, and second drying, which are performed sequentially. Optionally, the pre-freezing is performed sequentially under the following conditions: 10 to 50 minutes at -8°C to -10°C, 30 to 120 minutes at -40°C to -60°C, 30 to 120 minutes at -20°C to -25°C, and 120 to 240 minutes at -40°C to -60°C. The first drying is performed sequentially under the following conditions: 10 to 30 minutes at 40°C to -60°C and 0.1 to 0.2 mbar, 15 to 30 hours at -25°C to -40°C and 0.1 to 0.2 mbar, and 1 to 3 hours at -5°C to -10°C and 0.1 to 0.2 mbar. The second drying is carried out in the following conditions: 30 min to 60 min at 5° C. to 25° C. and 0.001 mbar to 0.01 mbar, and 2 h to 10 h at 5° C. to 25° C. and 0.001 mbar to 0.01 mbar.

[0053] Compared with conventional freeze-drying procedures, when freeze-drying is performed using the freeze-drying procedure, the freeze-drying loss rate is relatively low, which can improve the stability of the anti-smooth muscle antibody, shorten the freeze-drying time, and save production costs.

[0054] The preparation method of the above-mentioned anti-smooth muscle antibody quality control product is simple and easy to produce on a large scale.

[0055] In addition, one embodiment of the present application further provides a detection kit for anti-smooth muscle antibodies, which includes the anti-smooth muscle antibody quality control product of any of the above embodiments.

[0056] The above-mentioned anti-smooth muscle antibody detection kit includes the above-mentioned anti-smooth muscle antibody quality control product and has its corresponding advantages.

[0057] The following is a detailed description of the present invention in conjunction with specific examples. Unless otherwise specified, the following examples do not include other components other than inevitable impurities. The reagents and instruments used in the examples are conventional in the art unless otherwise specified. Experimental methods without specific conditions in the examples were carried out according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0058] Example 1

[0059] SMA IgG antibody preparation

[0060] The prepared SMA antigen was used to immunize BalB / C mice of appropriate age. During the immunization process, the SMA immunogen was coated and the tail blood of the mice was tested by ELISA until the titer reached 10 4 Immunization was stopped at 4 ℃, and the spleen of the mouse was removed. After processing, it was fused with mouse myeloma cells Sp2 / 0, and then positive hybridoma cell lines were screened by limiting dilution method. The corresponding positive hybridoma cell lines were amplified, and the gene sequences of their variable regions were amplified by RT-PCR after extracting their mRNA. After confirmation by Sanger sequencing, they were constructed into IgG chimeric antibody heavy chain and light chain expression vectors pFUSE-CHIg-hG1 (InvivoGen) and pFUSE2-CLIg-hk (InvivoGen), respectively. The endotoxin-free plasmid was extracted, and the light chain and heavy chain expression plasmids were transiently transfected into 293 cells at appropriate ratios. After about 48 hours, the culture cell supernatant was collected and purified by Protein A affinity chromatography and ion exchange chromatography to obtain SMA IgG chimeric antibodies. Among them, the purification results of SMA IgG chimeric antibodies are as follows: Figure 1 shown.

[0061] The amino acid sequence of the heavy chain variable region of the SMA IgG chimeric antibody is as follows:

[0062] EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVREYGNSFAYWGQGTLVTVSA (SEQ ID NO: 1).

[0063] The amino acid sequence of the light chain variable region of the SMA IgG chimeric antibody:

[0064] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK (SEQ ID NO: 2).

[0065] Human IgG1 heavy chain constant region amino acid sequence:

[0066] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)

[0067] Human Kappa light chain constant region amino acid sequence:

[0068] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).

[0069] Example 2

[0070] Screening of quality control matrix formulations

[0071] According to the reference evaluation method given in YY / T 1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents", a quality control matrix solution and a clinical serum sample that do not contain the substance to be tested are first prepared separately. Then, the SMA IgG prepared in Example 1 is added to the matrix solution and the clinical serum sample in parallel (the added SMA IgG can cover the measurement range), and 8 processed samples are prepared for each. The anti-smooth muscle antibody IgG detection kit (chemiluminescence method) of Shenzhen Yahuilong Biotechnology Co., Ltd. is then used for calibration testing, and the quality control matrix solution and clinical serum sample to which SMA IgG has been added are tested at the same time. Then, the concentration value of the clinical serum sample is used as the X-axis, and the concentration value of the quality control matrix solution is used as the Y-axis. A scatter plot is drawn and an ordinary linear regression analysis is performed.

[0072] Solution 1 consists of: 6.57 g / L TRIS hydrochloride (Trizma hydrochloride), 1.01 g / L tris(hydroxymethyl)hydrogen methane (TRIZMA(R) BASE, Vetec), 0.05 m / V% PC300, 80 g / L D-mannitol, 20 g / L trehalose, and 40 g / L bovine serum albumin. The volume was adjusted to 1 L with purified water. The pH of Solution 1 was 7.4 ± 0.1. It should be noted that, with the exception of some components in Example 6 where the pH was adjusted to verify the effect of pH, the pH values ​​of all other components are those obtained after the raw materials were mixed according to their respective proportions.

[0073] Solution 2: 9.762 g / L morpholineethanesulfonic acid (MES MONOHYDRATE, VETEC), 9 g / L sodium chloride, 0.05% m / VPC300, 80 g / L D-mannitol, 20 g / L trehalose, 40 g / L bovine serum albumin, diluted to 1 L with purified water; pH of solution 2 = 6.0 ± 0.1.

[0074] The results of SMA IgG concentrations measured in the above two solutions are shown in Table 1, and the scatter plots are shown in Table 1. Figure 2 shown.

[0075] Table 1 Matrix effect results of different solutions

[0076] Unit: COI

[0077] Clinical serum samples Solution 1 Solution 2 0.66 1.54 1.82 1.42 2.90 3.96 2.75 6.51 7.13 6.27 14.28 17.28 8.42 24.28 31.18 12.87 31.38 43.08 21.17 50.78 65.78 30.27 69.38 87.78 Slope 2.3181 2.9769 Correlation coefficient (R) 0.9972 0.9951

[0078] Refer to the clinical evaluation indicators. For qualitative analysis, the lower limit of the 95% confidence interval should not be less than 90%. For quantitative analysis, the correlation coefficient should generally be greater than 0.975, and the regression coefficient should generally be between 0.9 and 1.1. Figure 2The results showed that the SMA IgG quality control products prepared from the above two solutions did not meet R≥0.975, 0.9≤slope≤1.1.

[0079] Simplify the formula by directly using 4 mL of serum and adding morpholineethanesulfonic acid (MES MONOHYDRATE, VETEC). Prepare the following four different formulas. The required volume / content of each formula is as follows:

[0080] Formulation 1: Add 4 mg PC300 and 0.0407 g morpholineethanesulfonic acid (MESMONOHYDRATE, VETEC) to 4 mL fetal bovine serum, pH = 6.26 / 20.1°C.

[0081] Formulation 2: Add 4 mg PC300 and 0.0203 g morpholineethanesulfonic acid (MESMONOHYDRATE, VETEC) to 4 mL fetal bovine serum, pH = 7.06 / 20.1°C.

[0082] Formulation 3: Add 4 mg PC300 and 0.0407 g morpholineethanesulfonic acid (MESMONOHYDRATE, VETEC) to 4 mL of newborn calf serum, pH = 6.10 / 19.6°C.

[0083] Formulation 4: Add 4 mg PC300 and 0.0203 g morpholineethanesulfonic acid (MESMONOHYDRATE, VETEC) to 4 mL of newborn calf serum, pH = 6.64 / 19.7°C.

[0084] The results of SMA IgG concentrations measured in clinical serum samples and matrix solutions are shown in Table 2, and the scatter plots are shown in Figure 3 shown.

[0085] Table 2 Matrix effect results of different formulations

[0086] Clinical serum samples Recipe 1 Recipe 2 Recipe 3 Recipe 4 0.18 0.14 0.13 0.16 0.13 0.34 0.30 0.26 0.32 0.26 0.67 0.60 0.51 0.63 0.53 1.40 1.29 0.83 1.52 1.07 1.90 2.74 1.73 2.42 1.98 5.31 5.39 4.01 5.58 4.40 12.99 14.19 8.33 14.20 9.99 36.19 34.19 25.39 34.49 29.09 Slope 0.9499 0.6954 0.9576 0.7995 Correlation coefficient (R) 0.9982 0.9992 0.9986 0.9997

[0087] The results showed that only Formula 1 and Formula 3 of the SMA IgG quality control prepared using the above matrix solutions met the ranges of R ≥ 0.975 and slope ≤ 1.1. Therefore, the SMA IgG quality control prepared using the matrix solutions of Formula 1 and Formula 3 can simulate clinical serum samples.

[0088] Example 3

[0089] Thermal stability study

[0090] Take Example 2 Formula 1 and Formula 3 to prepare a negative SMA IgG quality control product with a concentration of 0.5COI (± 30%) and a positive SMA IgG quality control product with a concentration of 2.5COI (± 30%), both of which are liquid quality control products. The prepared negative SMA IgG quality control product and positive SMA IgG quality control product were divided into 2 parts, one part of which was stored in the dark at 37°C as an experimental sample, and the other part was placed at 4°C as a control sample to carry out thermal stability research. After 6 days and 11 days of acceleration, the experimental samples and control samples were taken out and tested on a calibrated Yahuilong chemiluminescence analyzer. The number of tests was set to 3 times, and the mean of the results was calculated. The relative deviation was calculated according to "relative deviation = (mean value of the test results of the experimental group - mean value of the test results of the control group) / mean value of the test results of the control group × 100%", and the results are shown in Table 3.

[0091] Table 3 Thermal stability study results

[0092]

[0093] As shown in Table 3, the relative deviations of the liquid quality control products prepared by Formula 1 and Formula 3 screened in Example 2 at 37°C for 6 and 11 days were no more than ±10%, indicating that the SMA IgG quality control products prepared under these two formulations were stable at 37°C for 11 days.

[0094] Example 4

[0095] Quality control product formulation research

[0096] After studying various formulations, based on the research on formulation 1 and solution 2 in Example 2, fetal bovine serum was mixed with solution 2 at a volume ratio of 1.5:1 (the content of the preservative in the prepared mixture was 0.05 m / v%) to prepare solution A with the following proportions: 60% (v / v) fetal bovine serum, 3.9 g / L MES, 32 g / L D-mannitol, 8 g / L trehalose, 0.05 m / v% PC300, 16 g / L bovine serum albumin, 3.6 g / L sodium chloride, and water;

[0097] In addition, fetal bovine serum was mixed with solution 2 at a volume ratio of 2:1 (except for the preservative added at 0.05 m / V%) to prepare solution B with the following proportions: 66.67% (v / v) fetal bovine serum, 3.25 g / L MES, 26.67 g / L D-mannitol, 6.67 g / L trehalose, 0.05 m / V% PC300, 13.33 g / L bovine serum albumin, 3 g / L sodium chloride and water; then, according to the method of Example 2, 10 clinical serum samples and matrix solution samples were prepared in parallel to conduct matrix effect studies. The results are shown in FIG. Figure 4 shown.

[0098] The results showed that the slope of solution A was 0.9220, R 2 The slope of solution B is 0.8745, R 2 The slope is 0.9969 (R = 0.9984), which does not satisfy the range of 0.9 ≤ slope ≤ 1.1.

[0099] Example 5

[0100] Effects of different freeze-drying procedures on the stability of quality control products

[0101] In order to improve the stability of SMA IgG and save costs, the solution A that meets the requirements in Example 4 was freeze-dried. A conventional freeze-drying procedure was used as a control. Adjustments were made based on the conventional freeze-drying procedure. During the pre-freezing process, the temperature was first lowered to -10°C and maintained for 40 minutes, then lowered to -50°C and maintained for 1.5 hours, then adjusted to -25°C and maintained for 1.5 hours, and then lowered to -55°C and maintained for 1.5 hours. After two annealing and slow freezing, the ice crystals produced were larger, which was conducive to the discharge of water during the sublimation process. The specific optimized freeze-drying procedure and the conventional freeze-drying procedure are shown in Table 4, and the two freeze-drying results are shown in Table 5. In Table 5, the "\" in the vacuum column means that no vacuum was drawn, which is the same as the standard atmospheric pressure of the environment.

[0102] Table 4 SMA IgG quality control product optimization and conventional lyophilization process

[0103]

[0104]

[0105] Table 5 Loss rates of SMA IgG quality control products after optimization and conventional freeze-drying procedures

[0106]

[0107] Compared with the conventional freeze-drying procedure, the freeze-drying loss rate of the SMA IgG quality control prepared by the optimized freeze-drying procedure is also relatively low, which improves the stability of SMA IgG, saves freeze-drying time, and saves production costs.

[0108] Example 6

[0109] Effects of different pH conditions on stability studies

[0110] The negative SMA IgG quality control product prepared by solution A of Example 4 was adjusted to three values ​​of pH 6.38, 6.80, and 7.50 to study the effect of different pH conditions on the stability of the quality control product. One part of the quality control product was placed at 25°C for 9 days or at 37°C for 3 days, protected from light as an experimental sample; the other part was placed at 4°C as a control sample. After the time node arrived, the quality control product was taken out and tested using a calibrated Yahuilong chemiluminescence analyzer. The number of tests was set to 3, and the mean of the results was calculated. The relative deviation was calculated according to "Relative deviation = (mean value of the test results of the experimental group - mean value of the test results of the control group) / mean value of the test results of the control group × 100%", and the results are shown in Table 6.

[0111] Table 6 Stability study results under different pH conditions

[0112]

[0113] As shown in Table 6, as the pH value increases, the relative deviation of the stability results at 25°C and 37°C exceeds the range of ±10%, indicating that the pH value of the SMA IgG quality control product cannot exceed 6.38.

[0114] Take the negative SMA IgG quality control product of Formula 3 in Example 2, adjust the pH to 5.8, 6.0, and 6.3, and study the effect of low pH on stability. One part of the quality control product was placed at 25°C for 6 days / placed at 37°C for 7 days and kept away from light as an experimental sample; the other part was placed at 4°C as a control sample. After the time node arrived, the quality control product was taken out and tested using a calibrated Yahuilong chemiluminescence analyzer. The number of tests was set to 3, and the mean of the results was calculated. The relative deviation was calculated according to "Relative deviation = (mean value of the test results of the experimental group - mean value of the test results of the control group) / mean value of the test results of the control group × 100%", and the results are shown in Table 7.

[0115] Table 7 Stability study results under low pH conditions

[0116]

[0117] From the results in Table 7, it can be seen that the relative deviations of the stability results at 25°C and 37°C under low pH conditions do not exceed ±10%, indicating that the pH range of the SMA IgG quality control product is 5.8-6.38.

[0118] Example 7

[0119] Conduct indoor quality control application experiments

[0120] Negative SMA IgG quality control products and positive SMA IgG quality control products were prepared using Formulation 3 of Example 2. Excel, one of the Microsoft Office software series, was used as the quality control data statistics and graphing software to process the quality control results and achieve the purpose of Westgard multi-rule quality control.

[0121] The specific method is as follows: start Excel, select worksheet 1 "sheet 1" and name it "Anti-smooth muscle antibody project". This worksheet is the anti-smooth muscle antibody worksheet; enter the project name, quality control product name, instrument model, and the names of the reagents and calibrators used in A1, enter "time times" in A2, enter "negative SMA IgG quality control product measurement value" in B2, enter "positive SMA IgG quality control product measurement value" in C2, enter "sx1" in D2, enter "sx2" in E2, and enter "sx1" in F3. F4 Enter Enter "s1" in F5 and "s2" in F6. sx is the standard deviation multiple of the difference between the current measurement value of the quality control material and the target value. and are the means of the negative SMA IgG quality control and the positive SMA IgG quality control, respectively. s1 and s2 are the standard deviations of the negative SMA IgG quality control and the positive SMA IgG quality control, respectively.

[0122] According to the requirements of the indoor quality control value, one bottle of negative SMA IgG quality control product and one bottle of positive SMA IgG quality control product were opened every day, and the anti-smooth muscle antibody project was measured once on the Yahuilong chemiluminescence instrument for 20 consecutive days. 20 data were obtained and the mean value was preliminarily calculated using EXCEL software. and standard deviation (s) as a temporary quality control and s. The specific calculation is performed by calling the corresponding function.

[0123] The calculation formula for sx is as follows: This value has positive and negative directions. After using the function to calculate the respective sx, select the two columns of data D2 and E2, click "Insert" - "Line Chart" in the toolbar, and the quality control chart of anti-smooth muscle antibody is established ( Figure 5 ).

[0124] from Figure 5 The results show that the quality control chart trend of SMA IgG quality control product is consistent with 1 2s , 1 3s , 2 2s 、R 4s , 4 1s 、 If quality control requirements are met, it can be used as a quality control product to meet market demand.

[0125] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0126] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. Use of the composition in preparing anti-smooth muscle antibody quality control products, characterized in that, The composition is 60% v / v fetal bovine serum, 3.9 g / L MES, 32 g / L D-mannitol, 8 g / L trehalose, 0.05 m / V% PC300, 16 g / L bovine serum albumin, 3.6 g / L sodium chloride and water; The pH of the composition is 5.8 to 6.38; The amino acid sequence of the heavy chain variable region of the anti-smooth muscle antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the anti-smooth muscle antibody is shown in SEQ ID NO:

2.

2. An anti-smooth muscle antibody quality control product, characterized in that: The anti-smooth muscle antibody quality control product is prepared by mixing the composition according to claim 1 with the anti-smooth muscle antibody; The amino acid sequence of the heavy chain variable region of the anti-smooth muscle antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the anti-smooth muscle antibody is shown in SEQ ID NO:

2.

3. A method for preparing an anti-smooth muscle antibody quality control product, characterized in that: The steps include: Mixing the composition of claim 1 with an anti-smooth muscle antibody to prepare an anti-smooth muscle antibody quality control product; Alternatively, the composition of claim 1 is mixed with an anti-smooth muscle antibody and then lyophilized to prepare an anti-smooth muscle antibody quality control product; The amino acid sequence of the heavy chain variable region of the anti-smooth muscle antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the anti-smooth muscle antibody is shown in SEQ ID NO:

2.

4. The preparation method according to claim 3, characterized in that The freeze-drying comprises pre-freezing, first drying and second drying, which are performed sequentially. The pre-freezing is performed sequentially under the following conditions: 10 min to 50 min at -8°C to -10°C, 30 min to 120 min at -40°C to -60°C, 30 min to 120 min at -20°C to -25°C, and 120 min to 240 min at -40°C to -60°C. The first drying is carried out in the following conditions: 10 min to 30 min at 40°C to -60°C and 0.1 mbar to 0.2 mbar, 15 h to 30 h at -25°C to -40°C and 0.1 mbar to 0.2 mbar, and 1 h to 3 h at -5°C to -10°C and 0.1 mbar to 0.2 mbar. The second drying is carried out in the following conditions: 30 min to 60 min at 5° C. to 25° C. and 0.001 mbar to 0.01 mbar, and 2 h to 10 h at 5° C. to 25° C. and 0.001 mbar to 0.01 mbar.

5. A kit for detecting anti-smooth muscle antibodies, characterized in that: Including the anti-smooth muscle antibody quality control product according to claim 2.

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