A matrix for a humanized antibody calibrator and a calibrator thereof

Abstract

The present disclosure relates to a matrix for humanized antibody calibrator and a calibrator thereof. The present disclosure provides a calibrator preparation method using humanized antibodies as raw materials, which comprises humanized anti-phospholipid antibodies and a matrix for calibrator. The matrix for calibrator comprises 0.02M Tris, 0.15M NaCl, 0.1M MgCl2, 0.01M ZnCl2, 0.05% Tween20, 0.2% BSA, 0.5% casein, 3% mannitol, 1% PEG20000 and 0.1% PC300. Compared with the human serum calibrator in the existing kit, the calibrator provided by the present disclosure has no pollution risk, high stability, good uniformity, controllable batch difference and can improve the accuracy of the detection result.

Description

Technical Field

[0001] This disclosure belongs to the technical field of immunoassay, specifically to a matrix for humanized antibody calibrators and a chemiluminescent calibrator. Background Technology

[0002] Antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease. Clinically, it is characterized by arterial and venous thrombosis, pathological pregnancy (early miscarriage and mid-to-late-term stillbirth), and thrombocytopenia. Antiphospholipid antibodies (aPL) are present in the serum. These symptoms may occur alone or in combination.

[0003] Antiphospholipid antibodies (aPLs) are a group of autoantibodies that target phospholipids and / or phospholipid-binding proteins. aPLs are primarily found in patients with autoimmune diseases such as antiphospholipid syndrome (APS) and are the most characteristic laboratory marker of APS. aPLs are also risk factors for thrombosis and pathological pregnancy. Simultaneously, aPLs can be found in malignant tumors, infectious diseases, after the use of certain drugs, and even in some healthy individuals. Among them, lupus anticoagulant (LA), anticardiolipin (aCL) antibody, and anti-β2 glycoprotein I (β2GPI) antibody are laboratory markers in the APS classification criteria. They are currently widely used clinically and have become one of the most common autoantibody detection items in clinical laboratories.

[0004] Taking an anti-β2 glycoprotein I antibody kit as an example, the detection substance in its design and development is an antibody against β2 glycoprotein I. Using chemiluminescence as an example, the detection process requires the β2 glycoprotein I antigen to identify and capture the anti-β2 glycoprotein I antibody in the serum to be tested. Then, a secondary antibody labeled with a luminescent marker is used to identify and bind to the captured antibody, ultimately quantifying the anti-β2 glycoprotein I antibody in the serum.

[0005] In clinical testing, calibrators and quality control samples play a crucial role in the accurate quantification of test results and are essential components of reagents. In the development of kits for detecting anti-β2 glycoprotein I antibodies, the calibrators and quality control samples need to contain a certain amount of anti-β2 glycoprotein I antibody.

[0006] In the preparation of calibrators, human serum is the optimal source of raw material, as it better simulates the complexity of human blood and can eliminate the influence of matrix effects. However, due to relevant regulatory restrictions, the preparation and industrialization of human serum are severely limited. Human serum cannot be supplied for the industrial production of calibrators, and it also carries the risk of infection, resulting in a low safety profile. Furthermore, batch-to-batch variation and stability are also key challenges that are difficult to control.

[0007] Currently, most antiphospholipid antibody test kits on the market use human serum as their calibrator. However, human serum calibrators have drawbacks such as difficulty in controlling batch-to-batch variations and the risk of infection, which makes the preparation of calibrators challenging. Summary of the Invention

[0008] To address the aforementioned problems in the existing technology, this disclosure provides a calibrator matrix and chemiluminescence calibrator suitable for humanized antibodies (especially antiphospholipid antibodies).

[0009] Calibration matrix

[0010] A calibrator is a substance used to calibrate clinical laboratory instruments or methods. It possesses a known value and its primary purpose is to establish or verify the accuracy of a testing system (including instruments, reagents, and procedures). In clinical testing, with the continuous development of testing technologies and the increasing demands for accuracy, the use of calibrators has become increasingly standardized. The International Organization for Standardization (ISO) has published a series of standards for in vitro diagnostic medical devices (including calibrators), such as ISO 17511, "In vitro diagnostic medical devices—Measurement of quantities in biological samples—Metrological traceability of values ​​assigned to calibrators and control substances."

[0011] The calibrator matrix refers to the combination of all other components in a calibrator besides the analyte (i.e., the target substance to be calibrated). It is analogous to the environment in which the analyte exists, and is a complex mixture. For example, in blood test calibrators, the matrix includes components such as proteins, salts, lipids, and carbohydrates.

[0012] Interactions may occur between the calibrator matrix and the detection instrument or reagents. For example, in immunoassay, certain components of the calibrator matrix may non-specifically bind to detection antibodies, leading to false positive or false negative results. Matrix effects are also a crucial factor. When the calibrator matrix differs from the patient sample matrix (such as serum or plasma), the detection system may react differently to the calibrator and patient samples. For instance, in chemiluminescent immunoassay, if the calibrator matrix differs from the patient serum matrix, it may cause deviations in the calibration curve when applied to patient samples, affecting the accuracy of the test results.

[0013] Substrate types include aqueous substrates and complex substrates:

[0014] Aqueous matrices are the most common type, with water as their main component. For example, some calibrators used to detect water-soluble small molecules (such as electrolytes) have a water-based matrix, while also containing small amounts of buffer salts and stabilizers to maintain the stability and homogeneity of the analyte.

[0015] Calibrators with complex matrices, such as serum or plasma matrices, are used for blood component analysis. They contain a variety of components, including proteins, lipids, carbohydrates, hormones, and vitamins. The complex interactions between these components mimic the human blood environment, which is beneficial for accurately calibrating instruments and methods related to blood testing.

[0016] Therefore, this disclosure provides a calibrator matrix, particularly suitable for calibrators containing antibodies (specifically antiphospholipid antibodies). The calibrator matrix of this disclosure comprises:

[0017]

[0018] The preservative is selected from: sodium azide, PC150 (Proclin 150), PC300 (Proclin 300), PC950 (Proclin 950), BND (Bronidox), with PC300 being preferred.

[0019] In specific implementations, the calibrator matrix of this disclosure comprises:

[0020]

[0021]

[0022] This disclosure also provides a calibrator comprising the aforementioned calibrator matrix.

[0023] In a specific implementation, the calibrator of this disclosure comprises: 0.2 to 1 mg / ml humanized antiphospholipid antibody; and a calibrator matrix of this disclosure; preferably, the antiphospholipid antibody is selected from: lupus anticoagulant, anticardiolipin antibody, and anti-β2 glycoprotein I antibody.

[0024] In specific implementation schemes, the humanized antiphospholipid antibodies described in the calibrators disclosed herein are selected from:

[0025] Humanized anti-β2 glycoprotein I antibody IgA type

[0026] Humanized anti-β2 glycoprotein I antibody IgG type

[0027] Humanized anti-β2 glycoprotein I antibody IgM type

[0028] Lupus anticoagulant IgG type

[0029] Lupus anticoagulant IgM type

[0030] Humanized anticardiolipin antibody IgA type

[0031] Humanized anticardiolipin antibody IgG type

[0032] Humanized anticardiolipin antibody IgM type.

[0033] In the specific implementation plan, the concentrations of humanized anti-β2 glycoprotein I antibody IgA, humanized anti-β2 glycoprotein I antibody IgG, and humanized anti-β2 glycoprotein I antibody IgM are 0.5 μg / ml, 0.159 μg / ml, and 0.097 μg / ml, respectively.

[0034] This disclosure also provides the use of the aforementioned calibrator matrix in the preparation of humanized antiphospholipid antibody calibrators; preferably, the antiphospholipid antibody is selected from: lupus anticoagulant, anticardiolipin antibody, and anti-β2 glycoprotein I antibody. In a specific embodiment, the humanized antibody is selected from: humanized anti-β2 glycoprotein I antibody IgA, humanized anti-β2 glycoprotein I antibody IgG, humanized anti-β2 glycoprotein I antibody IgM, lupus anticoagulant IgG, lupus anticoagulant IgM, humanized anticardiolipin antibody IgA, humanized anticardiolipin antibody IgG, and humanized anticardiolipin antibody IgM.

[0035] This disclosure also provides a method for preparing a calibrator, comprising dissolving a humanized antiphospholipid antibody into the calibrator matrix of this disclosure.

[0036] This disclosure also provides a method for preparing calibrators, comprising dissolving humanized anti-β2 glycoprotein I antibody IgA, humanized anti-β2 glycoprotein I antibody IgG, and humanized anti-β2 glycoprotein I antibody IgM into the calibrator matrix of this disclosure, respectively. In a specific embodiment, the calibrator matrix comprises 0.02M Tris, 0.15M NaCl, 0.1M MgCl2, 0.01M ZnCl2, 0.05% Tween 20, 0.2% BSA, 0.5% casein, 3% mannitol, 1% PEG20000, and 0.1% PC300; the concentrations of humanized anti-β2 glycoprotein I antibody IgA, humanized anti-β2 glycoprotein I antibody IgG, and humanized anti-β2 glycoprotein I antibody IgM are 0.5 μg / ml, 0.159 μg / ml, and 0.097 μg / ml, respectively.

[0037] The humanized anti-β2 glycoprotein I antibody used in this disclosure is produced using recombinant technology in CHO cells, with a purity of over 90%, and appears as a clear solution. The buffer in the antibody stock solution is 20 mM PBS buffer at pH 7.4. In specific embodiments, the humanized anti-β2 glycoprotein I antibody is commercially available. Detailed Implementation

[0038] Example 1. Screening of calibrator matrix (using β2GPI as an example)

[0039] Currently, there are no humanized antibody calibrators for β2GPI kits.

[0040] Humanized antibodies were formulated using different matrix systems, including:

[0041] Table 1A

[0042]

[0043] Humanized antibody calibrators were prepared at concentrations of 10, 20, 80, and 200 (RU / ml) to examine their reactivity and stability.

[0044] Table 1B. β2GPI IgA calibrator matrix screening

[0045]

[0046] Table 1C

[0047]

[0048] Table 2A. β2GPI IgG calibrator matrix screening

[0049]

[0050]

[0051] Table 2B

[0052]

[0053]

[0054] Table 3A. β2GPI IgM calibrator matrix screening

[0055]

[0056]

[0057] Table 3B

[0058]

[0059]

[0060] As shown in the table above, the accelerated stability of humanized antibodies is difficult to control. In Table 1C, solution 3 maintains a deviation of less than 15% for β2GPI-IgA after 10 days of accelerated heating, demonstrating greater stability compared to other solutions. Table 2B clearly shows the better stabilizing effect of solution 3. In Table 3B, solutions 1, 3, and 5 can all stabilize humanized β2GPI IgM antibody within 10% after 7 days of accelerated heating. Solution 3 can solve the problem of poor accelerated heating stability of humanized antibodies.

[0061] Example 2. Stability verification (using anticardiolipin antibody as an example)

[0062] Humanized anticardiolipin antibody IgA, humanized anticardiolipin antibody IgG, and humanized anticardiolipin antibody IgM were dissolved in solution 3 respectively, and four calibration quality control points of 5, 20, 100, and 200 were prepared for thermal accelerated stability testing.

[0063] Table 4A. Stability Verification of Humanized Anticardiolipin Antibody Calibration Quality Control

[0064]

[0065]

[0066] Table 4B

[0067]

[0068] As can be seen from the table above, after dissolving the humanized anticardiolipin antibody with solution 3, the deviation of the three antibodies remained within 10% after 10 days of thermal acceleration. Solution 3 can also solve the problem of poor thermal acceleration stability of humanized anticardiolipin antibody.

[0069] Example 3. Evaluation of inter-batch variation of calibrators

[0070] Humanized anti-β2 glycoprotein I antibody IgA, humanized anti-β2 glycoprotein I antibody IgG, and humanized anti-β2 glycoprotein I antibody IgM from two different batches were prepared to the same theoretical concentration using a calibrator matrix to assess the deviation of the detection luminescence values ​​of the calibrators from different batches of raw materials.

[0071] As shown in Table 5, there is a difference in the labeled concentration of the two batches of humanized antibody raw materials. The two batches of raw materials were prepared to the same theoretical concentration using a calibrator matrix and then verified.

[0072] Table 5. Labeled concentrations of humanized antibody raw materials from different batches

[0073]

[0074]

[0075] Table 6. Inter-batch variation verification of calibrators prepared from different batches of humanized antibodies

[0076]

[0077] The results in Tables 5 and 6 show that the deviation of the calibrators prepared using different batches of humanized antibody raw materials is less than 5%, indicating that the batch-to-batch difference of humanized antibody calibrators is controllable and meets the needs of clinical application.

[0078] Compared to the batch-to-batch differences of matrix solutions 1-2 and 4-7, solution 3 provided a significant improvement.

[0079] Example 4. Calibrator homogeneity assessment

[0080] Using the calibrator matrix (solution 3 of Example 1), 50 calibrators were prepared for each of humanized anti-β2 glycoprotein I antibody IgA, humanized anti-β2 glycoprotein I antibody IgG, and humanized anti-β2 glycoprotein I antibody IgM. Eight of these calibrators were randomly selected for testing to examine their uniformity.

[0081] Table 7. Calibration homogeneity testing

[0082]

[0083]

[0084] As shown in Table 7, the CV of the luminescence values ​​of 8 randomly selected calibrators out of 50 calibrators were within 5%, indicating that the three humanized antibody calibrators have uniformity.

[0085] Compared to the homogeneity of the control matrix solutions 1-2 and 4-7, solution 3 provided a significant improvement.

[0086] Example 5. Evaluation of freeze-thaw stability of calibrators

[0087] Using the calibrator matrix (solution 3), prepare calibrators for β2 glycoprotein I antibody IgA and β2 glycoprotein I antibody IgG. Divide the prepared calibrators into four equal parts, numbered 1, 2, 3, and 4.

[0088] Number 1 was placed at 4℃;

[0089] Number 2 was placed at -80℃ and completely frozen, then thawed at room temperature, i.e., one freeze-thaw cycle;

[0090] Number 3 was placed at -80℃ and completely frozen, then thawed naturally at room temperature and the freeze-thaw cycle was repeated once, i.e., 2 freeze-thaw cycles.

[0091] Number 4 was placed at -80℃ and completely frozen, then thawed naturally at room temperature, and then the freeze-thaw cycle was repeated twice, for a total of 3 freeze-thaw cycles.

[0092] Finally, the temperatures of numbers 1, 2, 3, and 4 were balanced simultaneously before testing.

[0093] Table 8. Freeze-thaw stability verification of β2GPI IgA calibrators

[0094]

[0095]

[0096] Table 9. Freeze-thaw stability verification of β2GPI IgG calibrators

[0097]

[0098] As shown in Tables 8 and 9, the deviation of the calibrator was within 10% after one freeze-thaw cycle, within 10% after two freeze-thaw cycles, and within 15% after three freeze-thaw cycles. This humanized antibody calibrator maintained stable reactivity after freeze-thaw cycles. Compared to the control matrix solutions 1-2 and 4-7, solution 3 provided a significant improvement.

[0099] This disclosure, through screening different matrix components for calibrators, obtained calibrators derived from humanized antibodies and optimized the matrix for the calibrators, reducing the risks associated with calibrator instability. Data shows that the humanized antibody calibrators described in this disclosure exhibit good stability and batch-to-batch variation within a controllable range. Furthermore, the humanized antibody calibrators of this disclosure do not pose an infection risk compared to serum, and the raw material is obtained through legal and controllable means, making them highly significant for clinical application.

Claims

1. A calibrator matrix, characterized in that it comprises the following: 0.02 mol / L Tris 0.15 mol / L NaCl 0.1 mol / L MgCl2 0.01 mol / L ZnCl2 0.05% v / v Tween20 0.2% w / v BSA 0.5% w / v casein 3% w / v mannitol 1% v / v PEG20000 0.1% v / v preservative.

2. A calibrator, characterized in that it comprises the calibrator matrix as described in claim 1.

3. The calibrator according to claim 2, characterized in that it comprises: 0.2 mg / ml to 1 mg / ml humanized antibodies; and The calibrator matrix as described in claim 1; The humanized antibody is an antiphospholipid antibody.

4. The calibrator according to claim 3, characterized in that the antiphospholipid antibody is selected from: lupus anticoagulant, anticardiolipin antibody, and anti-β2 glycoprotein I antibody.

5. The calibrator according to claim 4, characterized in that the humanized antibody is selected from: Humanized anti-β2 glycoprotein I antibody IgA type Humanized anti-β2 glycoprotein I antibody IgG type Humanized anti-β2 glycoprotein I antibody IgM type Lupus anticoagulant IgG type Lupus anticoagulant IgM type Humanized anticardiolipin antibody IgA type Humanized anticardiolipin antibody IgG type Humanized anticardiolipin antibody IgM type.

6. The use of the calibrator matrix of claim 1 in the preparation of humanized antibody calibrators, characterized in that: The humanized antibody is an antiphospholipid antibody.

7. The use according to claim 6, characterized in that the antiphospholipid antibody is selected from: lupus anticoagulant, anticardiolipin antibody, and anti-β2 glycoprotein I antibody.

8. The use according to claim 7, characterized in that the humanized antibody is selected from: Humanized anti-β2 glycoprotein I antibody IgA type Humanized anti-β2 glycoprotein I antibody IgG type Humanized anti-β2 glycoprotein I antibody IgM type Lupus anticoagulant IgG type Lupus anticoagulant IgM type Humanized anticardiolipin antibody IgA type Humanized anticardiolipin antibody IgG type Humanized anticardiolipin antibody IgM type.

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