Matrix for humanized antibody calibrator and calibrator thereof

Abstract

The invention relates to a matrix for a humanized antibody calibrator and a calibrator thereof. The invention provides a preparation method of a calibrator taking a humanized antibody as a raw material. The calibrator comprises a humanized anti-phospholipid antibody and a substrate for the calibrator. The matrix for the calibrator comprises 0.02 M of Tris, 0.15 M of NaCl, 0.1 M of MgCl2, 0.01 M of ZnCl2, 0.05% of Tween 20, 0.2% of BSA (Bovine Serum Albumin), 0.5% of casein, 3% of mannitol, 1% of PEG (Polyethylene Glycol) 20000 and 0.1% of PC300. Compared with a human serum calibrator in an existing kit, the calibrator provided by the invention has the advantages of no pollution risk, high stability, good uniformity and controllable batch difference, and can improve the accuracy of a detection result.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and specifically relates to a humanized antibody calibrator matrix and a chemiluminescent calibrator. Background Art

[0002] Antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease. Clinically, it is manifested by arterial and venous thrombosis, morbid pregnancy (early pregnancy miscarriage and late pregnancy stillbirth), and thrombocytopenia. Antiphospholipid antibodies (aPL) are present in the serum. The above symptoms can exist alone or in combination.

[0003] Antiphospholipid antibodies (aPLs) are a group of autoantibodies that use phospholipids and / or phospholipid-binding proteins as target antigens. aPLs are mainly found in patients with autoimmune diseases such as antiphospholipid syndrome (APS) and are the most characteristic laboratory indicators of APS. aPLs are also a risk factor for thrombosis and pathological pregnancy. At the same time, aPLs can be seen in malignant tumors, infectious diseases, after the use of certain drugs, and even in some healthy people. Among them, lupus anticoagulant (LA), anti-cardiolipin (aCL) antibodies, and anti-β2-glycoprotein I (β2GPI) antibodies are laboratory indicators in the APS classification standards. They are currently widely used in clinical practice and have become one of the most common autoantibody detection items in clinical laboratories.

[0004] Taking the anti-β2 glycoprotein I antibody kit as an example, in its design and development, the detection substance is the antibody against β2 glycoprotein I. Taking the chemiluminescence method as an example, in the detection process, the anti-β2 glycoprotein I antibody in the serum to be tested needs to be recognized and captured by the β2 glycoprotein I antigen substance, and then the secondary antibody labeled with a luminescent marker is used to recognize and bind to the captured antibody, and finally the anti-β2 glycoprotein I antibody in the serum is quantitatively obtained.

[0005] In the clinical testing process, calibrators and quality control products play a key role in the accurate quantification of test results and are essential components of reagents. In the development of the anti-β2 glycoprotein I antibody detection kit, calibrators and quality control products need to contain a certain amount of anti-β2 glycoprotein I antibodies.

[0006] In the preparation of calibrators, human serum is the best source of raw materials, which can better simulate the complexity of human blood and eliminate the influencing factors caused by matrix effects. However, due to the restrictions of relevant regulations, the preparation and industrialization of human serum are very limited. Human serum cannot be supplied for the industrial preparation of calibrators, and human serum has the risk of infection and a low safety factor. In addition, batch difference and stability are also key points that are difficult to control.

[0007] Among the antiphospholipid antibody detection kits currently available on the market, the calibrant components of most products are human serum. However, human serum calibrants have disadvantages such as difficulty in controlling batch differences and infectious risks, which makes it difficult to prepare calibrants. Summary of the invention

[0008] In view of the above problems existing in the prior art, the present disclosure provides a calibrator matrix and a chemiluminescent calibrator suitable for humanized antibodies (especially antiphospholipid antibodies).

[0009] Calibrator Matrix

[0010] A calibrator is a substance used to calibrate clinical testing instruments or testing methods. It has the characteristic of known value, and its main purpose is to establish or verify the accuracy of the detection system (including instruments, reagents, operating procedures, etc.). In clinical testing, with the continuous development of testing technology and the increasing requirements for the accuracy of test results, the use of calibrators has gradually become standardized. The International Organization for Standardization (ISO) has issued a series of standards on in vitro diagnostic medical devices (including calibrators), such as ISO17511 "In vitro diagnostic medical devices-Measurement of quantities in biological samples-Metrological traceability of the assignment of calibrators and control materials".

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

[0012] Interactions may occur between the calibrator matrix and the test instrument or test reagents. Taking immunoassay as an example, certain components in the calibrator matrix may non-specifically bind to the detection antibody, resulting in false positive or false negative results. Matrix effect is also a key factor. When there is a difference between the calibrator matrix and the patient sample matrix (such as serum, plasma), the detection system may respond differently to the calibrator and patient samples. For example, in chemiluminescent immunoassay, if the calibrator matrix is ​​different from the patient serum matrix, it may cause deviations in the calibration curve when applied to patient sample testing, affecting the accuracy of the test results.

[0013] Matrix types include aqueous matrices and complex matrices:

[0014] Aqueous matrix is ​​the most common type, with water as the main component. For example, some calibrants used to detect water-soluble small molecules (such as electrolytes) have a matrix that is mainly water, and also contains small amounts of buffer salts and stabilizers to maintain the stability and homogeneity of the analyte.

[0015] Complex matrices such as serum or plasma matrices are used for blood component testing. They contain multiple components such as proteins, lipids, carbohydrates, hormones, vitamins, etc. The complex interactions between these components simulate the human blood environment and are conducive to the accurate calibration of blood testing related instruments and methods.

[0016] Therefore, the present disclosure provides a matrix for calibration, which is particularly suitable for calibration containing antibodies (particularly antiphospholipid antibodies). The matrix for calibration of the present disclosure comprises:

[0017]

[0018] The preservative is selected from the group consisting of sodium azide, PC150 (Proclin150), PC300 (Proclin300), PC950 (Proclin950), BND (Bronidox), preferably PC300.

[0019] In a specific embodiment, the calibrator matrix of the present disclosure comprises:

[0020]

[0021]

[0022] The present disclosure also provides a calibrator, which comprises the aforementioned calibrator matrix.

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

[0024] In a specific embodiment, the humanized antiphospholipid antibody in the calibrator of the present disclosure is 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,

[0029] Lupus anticoagulant IgM

[0030] Humanized antiphospholipid antibody IgA type,

[0031] Humanized anti-cardiolipin antibody IgG type,

[0032] Humanized anti-cardiolipin antibody IgM type.

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

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

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

[0036] The present disclosure also provides a method for preparing a calibrator, comprising dissolving a humanized anti-β2 glycoprotein I antibody IgA type, a humanized anti-β2 glycoprotein I antibody IgG type, and a humanized anti-β2 glycoprotein I antibody IgM type in a calibrator matrix of the present disclosure, respectively. In a specific embodiment, the calibrator matrix comprises 0.02M Tris, 0.15M NaCl, 0.1MMgCl2, 0.01M ZnCl2, 0.05% Tween20, 0.2% BSA, 0.5% casein, 3% mannitol, 1% PEG20000, 0.1% PC300; the concentrations of humanized anti-β2 glycoprotein I antibody IgA type, humanized anti-β2 glycoprotein I antibody IgG type, and humanized anti-β2 glycoprotein I antibody IgM type are 0.5μg / ml, 0.159μg / ml, and 0.097μg / ml, respectively.

[0037] The humanized anti-β2 glycoprotein I antibody used in the present disclosure is produced by recombinant technology in CHO cells, with a purity of more than 90%, and is presented as a transparent solution. The buffer in the antibody storage solution is 20mM PBS buffer pH 7.4. In a specific embodiment, the humanized anti-β2 glycoprotein I antibody can be obtained commercially. DETAILED DESCRIPTION

[0038] Example 1. Screening of Calibrator Matrix (Taking β2GPI as an Example)

[0039] In the prior art, there is no humanized antibody calibrator for β2GPI kit.

[0040] Humanized antibodies are 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 investigate the reactivity and stability of the calibrators.

[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 can be seen from the above table, the accelerated stability of humanized antibodies is difficult to control. Solution 3 in Table 1C keeps β2GPI-IgA within 15% deviation after 10 days of thermal acceleration, which can make humanized antibodies more stable compared with other solutions. It is obvious from Table 2B that solution 3 has a better stabilizing effect. Solution 1, solution 3, and solution 5 in Table 3B can stabilize the humanized β2GPI IgM antibody within 10% after 7 days of thermal acceleration. Solution 3 can solve the problem of poor thermal acceleration stability of humanized antibodies.

[0061] Example 2. Stability verification (taking anti-cardiolipin antibody as an example)

[0062] Solution 3 was used to dissolve humanized antiphospholipid antibody IgA, humanized antiphospholipid antibody IgG, and humanized antiphospholipid antibody IgM, respectively, and four calibration quality control points of 5, 20, 100, and 200 were prepared for thermal acceleration stability investigation.

[0063] Table 4A. Humanized antiphospholipid antibody calibration quality control stability verification

[0064]

[0065]

[0066] Table 4B

[0067]

[0068] It can be seen from the above table that after using solution 3 to dissolve the humanized antiphospholipid antibodies, the three antibodies still maintained a deviation within 10% after 10 days of thermal acceleration. Solution 3 can also solve the problem of poor thermal acceleration stability of humanized antiphospholipid antibodies.

[0069] Example 3. Assessment of inter-batch differences of calibrators

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

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

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

[0073]

[0074]

[0075] Table 6. Verification of batch-to-batch differences of calibrators prepared from different batches of humanized antibodies

[0076]

[0077] It can be seen from the results in Tables 5 and 6 that the deviation of the measurement results of the calibrators prepared using different batches of humanized antibody raw materials is less than 5%, indicating that the batch difference of the humanized antibody calibrators is controllable and meets the needs of clinical application.

[0078] Solution 3 provided a significant improvement compared to the batch-to-batch variation of matrix solutions 1-2, 4-7.

[0079] Example 4. Calibrator homogeneity assessment

[0080] Using the calibration matrix (Solution 3 of Example 1), 50 calibrators of humanized anti-β2 glycoprotein I antibody IgA, humanized anti-β2 glycoprotein I antibody IgG, and humanized anti-β2 glycoprotein I antibody IgM were prepared, and 8 of them were randomly selected for testing to examine the homogeneity of the calibrators.

[0081] Table 7. Calibrator homogeneity test

[0082]

[0083]

[0084] As can be seen from Table 7, the luminescence value CVs of 8 calibrators randomly selected from the 50 calibrators were within 5%, indicating that the three humanized antibody calibrators were homogeneous.

[0085] Solution 3 provided a significant improvement in homogeneity compared to the comparative matrix solutions 1-2, 4-7.

[0086] Example 5. Freeze-thaw stability assessment of calibrators

[0087] Use the calibrator matrix (solution 3) to prepare the β2 glycoprotein I antibody IgA and β2 glycoprotein I antibody IgG calibrators, and divide the prepared calibrators into four equal parts, numbered 1, 2, 3, and 4, where:

[0088] No. 1 was placed at 4°C;

[0089] No. 2 was completely frozen at -80°C and then thawed at room temperature, i.e., frozen and thawed once;

[0090] No. 3 was completely frozen at -80°C, then thawed naturally at room temperature and repeated freezing and thawing once, i.e., freezing and thawing twice;

[0091] No. 4 was completely frozen at -80°C, then naturally thawed at room temperature and repeated freezing and thawing twice, i.e., freezing and thawing three times.

[0092] Finally, the temperatures of No. 1, 2, 3, and 4 are balanced and then tested.

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

[0094]

[0095]

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

[0097]

[0098] As can be seen from Tables 8 and 9, the deviation of the calibrator is within 10% after one freeze-thaw cycle, within 10% after two freeze-thaw cycles, and within 15% after three freeze-thaw cycles. The humanized antibody calibrator still has stable reactivity after freeze-thaw cycles. Solution 3 provides significant improvement over the comparative matrix solutions 1-2 and 4-7.

[0099] The present disclosure obtains calibrators using humanized antibodies as raw materials by screening different matrix components for calibrators, and optimizes the matrix for calibrators, thereby reducing the risk of unstable calibrators. Data show that the humanized antibody calibrators disclosed in the present disclosure have good stability and the batch-to-batch difference is within a controllable range. At the same time, the humanized antibody calibrators disclosed in the present disclosure have no infection risk compared to serum, and the raw material acquisition route is legal and controllable, which has strong clinical application significance.

Claims

1. A matrix for calibration, comprising:

2. The calibrator matrix according to claim 1, comprising:

3. A calibrator comprising the calibrator matrix according to claim 1 or 2.

4. The calibrator according to claim 3, comprising: 0.2 mg / ml to 1 mg / ml humanized antibody; and The calibrator matrix according to claim 1 or 2; The humanized antibody is an antiphospholipid antibody; Preferably, the antiphospholipid antibody is selected from the group consisting of: lupus anticoagulant, anticardiolipin antibody, and anti-β2 glycoprotein I antibody.

5. The calibrator according to claim 4, wherein 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, Lupus anticoagulant IgM Humanized antiphospholipid antibody IgA type, Humanized anti-cardiolipin antibody IgG type, Humanized anti-cardiolipin antibody IgM type.

6. Use of the calibrator matrix according to claim 1 or 2 in the preparation of a humanized antibody calibrator, wherein: The humanized antibody is an antiphospholipid antibody; Preferably, the antiphospholipid antibody is selected from the group consisting of: lupus anticoagulant, anticardiolipin antibody, and anti-β2 glycoprotein I antibody.

7. The use according to claim 6, wherein 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, Lupus anticoagulant IgM Humanized antiphospholipid antibody IgA type, Humanized anti-cardiolipin antibody IgG type, Humanized anti-cardiolipin antibody IgM type.

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