Particles with antibodies bound to their surfaces

Abstract

This application relates to particles with antibodies bound to their surfaces. Specifically, this application relates to a particle with an antibody bound to its surface, and the structure of the particle is shown by the following formula: antibody - bridging protein - particle. This structure is beneficial to maintaining the activity of the antibody, and at the same time, the long arms formed by the bridging protein help to reduce the steric hindrance effect of the antibody and facilitate the binding to the antigen. The particles and reagents prepared in this application have improved accuracy and sensitivity.

Description

[0001] This application is a divisional application of the patent application 2018101332939 (a method for covalently conjugating an antibody or its antigen-binding fragment to the surface of a particle) filed on February 9, 2018. Technical Field

[0002] This application relates to the field of clinical testing. In particular, it relates to a method for covalently conjugating an antibody or its antigen-binding fragment to the surface of a particle. Background Art

[0003] Anti-Mullerian hormone (AMH) is a member of the transforming growth factor-β (TGF-β) superfamily. AMH is a glycoprotein secreted by immature Sertoli cells in the testis and granulosa cells of preantral follicles and small antral follicles in the ovary. The more small antral follicles there are in the ovary, the higher the concentration of AMH; conversely, when follicles are gradually consumed with age and various factors, the AMH concentration will also decrease; when approaching menopause, AMH will gradually tend to 0. In view of this, AMH can be used as a marker for predicting ovarian reserve.

[0004] The clinical significance of AMH testing lies in:

[0005] (1) Evaluation of ovarian reserve function: When a female is born, the serum AMH concentration level is very low. After puberty, the AMH concentration reaches a peak and remains at a high level throughout the reproductive age. After that, with age and various factors, the AMH concentration will decrease until it cannot be detected after menopause. It can be seen that the AMH value fluctuates with the change of ovarian function. The higher the AMH index, the larger the inventory of eggs and the stronger the fertility. When AMH decreases, it represents that the ovary is aging, indicating the decline of female fertility. By detecting AMH, the decline of age-related ovarian reserve function can be reflected earlier and more accurately.

[0006] (2) Polycystic ovary syndrome (PCOS): PCOS is the most common reproductive endocrine disorder in the population, but the current diagnostic methods are not accurate. Calculating antral follicles under traditional ultrasound is difficult and is greatly affected by subjective factors. Clinical studies have found that the serum AMH level in PCOS patients is 2 to 3 times higher than the normal level. Serum AMH can be used to diagnose PCOS and evaluate its curative effect, with a sensitivity of 67% and a specificity of 92%. Therefore, the AMH concentration is an accurate reflection of the number of ovarian antral follicles. Replacing the count of small antral follicles under ultrasound with the serum AMH level can be used as one of the diagnostic criteria for PCOS.

[0007] (3) Premature ovarian failure (POF): The serum AMH level in POF patients is significantly lower than that of normal women of the same age and is comparable to that of menopausal women. Moreover, since AMH is produced not only by small antral follicles but also by small follicles that cannot be detected by color ultrasound, and it is non-cycle-dependent and does not participate in the negative feedback regulation of the hypothalamic-pituitary-gonadal axis. Therefore, AMH is expected to become a good clinical indicator for early prediction of POF onset.

[0008] (4) Assisted reproductive technology: The AMH level can predict ovarian responsiveness, identify women at risk of ovarian hyperstimulation syndrome, and the dosage of ovulation induction drugs can be determined according to the AMH value. Studies have found that the higher the AMH level in the serum and follicular fluid of patients undergoing IVF / ICSI treatment, the higher the fertilization rate, and AMH may become an indicator for predicting the fertilization rate. In addition, AMH predicts OHSS better than age and BMI. The basal AMH in patients with OHSS is 6 times higher than that in normal people, suggesting that AMH may predict OHSS in advance.

[0009] (5) Ovarian granulosa cell tumor: Since AMH is only expressed by granulosa cells and its expression level is determined by the granulosa cells of ovarian primary follicles and preantral follicles, this indicates that AMH can be used as a marker for ovarian granulosa cell tumors. It is reported that 76% to 93% of ovarian granulosa cell tumors are positive for AMH expression, and AMH has an independent correlation with the tumor size and differentiation degree of ovarian granulosa cell tumors and has a certain predictive value for the recurrence of ovarian granulosa cell tumors.

[0010] (6) Predicting the age of menopause: The AMH level is closely related to the age of menopause. The serum AMH level gradually decreases 5 years before menopause. Therefore, it is currently considered that serum AMH can predict the age of menopause.

[0011] (7) Disorders of sex development: It can assist in diagnosing whether children have developmental disorders such as gonadal dysplasia, precocious puberty, and cryptorchidism. If the serum AMH level is very low or undetectable, it highly suggests the absence of testicular tissue. Studies have shown that in patients with cryptorchidism, 50% of patients have normal serum AMH levels, and the serum AMH level in patients with cryptorchidism is significantly lower than that in patients with a single testis.

[0012] (8) Assisting in the diagnosis and treatment of related diseases: The serum AMH level is also related to a series of autoimmune diseases because the ovarian function of such patients is often damaged. Such as autoimmune thyroid diseases, systemic lupus erythematosus, and Crohn's disease. In addition, the detection of AMH can be used to predict and evaluate the impact of treatments such as radiotherapy, chemotherapy, and ovarian surgery on the ovarian function of patients, and to guide the selection of treatment plans.

[0013] Chinese Patent Application CN107192827A discloses a colloidal gold detection device for anti-Müllerian hormone. A nitrocellulose membrane is provided inside the detection device. One end of the nitrocellulose membrane is connected to a sample pad, and the other end is connected to an absorbent pad. A sample hole is provided on the outer shell at the position of the sample pad, and an observation hole is provided at the position of the nitrocellulose membrane. A detection line and a quality control line are provided on the nitrocellulose membrane. An AMH monoclonal antibody labeled with colloidal gold is coated on the sample pad; a second AMH monoclonal antibody is fixed on the detection line. The colloidal gold detection device in CN107192827A is conducive to rapid preliminary interpretation. This method can achieve preliminary qualitative or semi-quantitative detection, but cannot achieve accurate quantification.

[0014] Currently, the quantitative detection method of AMH in this field is mainly based on magnetic particle chemiluminescence analysis, which is a detection method combining sandwich chemiluminescence immunoassay and magnetic particle separation technology. Its detection principle is as follows: A quantitative enzyme-labeled AMH monoclonal antibody is added to a sample (standard, calibrator or quality control product). After incubation at 37 °C with the AMH monoclonal antibody bound to magnetic particles, the AMH monoclonal antibody bound to the magnetic particles and the enzyme-labeled AMH monoclonal antibody respectively bind to different epitopes of the AMH molecule in the sample to form a "sandwich" structure; it can be directly precipitated in an external magnetic field without centrifugation for separation; the supernatant is poured off, and the precipitated complex is washed, and then an enzyme-catalyzed chemiluminescence substrate is added; the substrate is catalytically cleaved under the action of the enzyme to form an unstable excited-state intermediate, and when the excited-state intermediate returns to the ground state, photons are emitted to form a luminescence reaction; the luminescence intensity of the reaction can be detected using a luminometer, and the AMH content in the sample can be calculated according to the standard curve. Within the detection range, the luminescence intensity is proportional to the AMH concentration in the sample.

[0015] Indian Patent Application IN2015CH00633A discloses a method for quantifying AMH in human serum. This method involves chemiluminescence based on graphene (oxide) and / or ELISA diagnostic technology. In this method, graphene oxide has the ability to retain a large amount of capture antibody (anti-AMH polyclonal antibody), thereby improving the accuracy and sensitivity of detection. The detection device prepared according to this method includes: a multi-well plate, graphene oxide conjugated with 3-aminopropyltriethoxysilane; and a capture antibody conjugated with EDC.

[0016] European Patent Application EP2817621 discloses a lateral flow immunoassay device for detecting AMH in whole blood. By conjugating gold particles or fluorescent particles to antibodies, the device can generate a chromogenic or fluorescent signal, and the intensity of this signal is proportional to the AMH concentration.

[0017] At present, there are many methods for coupling antibodies to antibodies. However, these coupling methods have more or less some problems. For example, direct coupling to magnetic beads will lead to steric hindrance, resulting in increased sensitivity and antibody usage; through the biotin avidin system, patients taking vitamin H treatment will have inaccurate test results.

[0018] CN101625366A discloses a method for coupling antibodies to latex particles. The surface-modified latex particles are combined with antibodies by covalently bonding the carboxyl or amino groups on their surfaces to the amino end of the antibodies. There is a bridging chemical arm between the microspheres and the antibodies, which reduces the steric effect. This not only increases the binding rate of the antibodies, but also provides a suitable three-dimensional spatial structure for the antibodies, effectively protecting the active area where the antibodies bind to the antigens.

[0019] CN102161716A provides another method for coupling antibodies to latex particles, wherein the latex is firstly combined with an unrelated protein by chemical bonds, and then the antibody is cross-linked to the unrelated protein using glutaraldehyde, thereby sensitizing the latex. This method can improve the sensitivity of the reagent.

[0020] CN101625366A uses EDC for coupling, so it does not play a role in increasing the arm length, and EDC acts as a simple chemical cross-linking agent. In practical applications, the method described in CN102161716A uses glutaraldehyde, which leads to low cross-linking efficiency and long cross-linking time. This method has been basically eliminated in commercial applications.

[0021] In view of the above problems, there is still a need in the art for a new coupling process, which requires that the original activity of the antibody be maintained after being coupled to the magnetic beads while avoiding interference in the sample. Summary of the invention

[0022] Therefore, according to some embodiments of the present application, a bridging protein is provided, which comprises NHS-PEG and an irrelevant protein; NHS-PEG and the irrelevant protein are covalently linked in a linear manner. Wherein, NHS-PEG is covalently bound to the carboxyl end and the amino end of the irrelevant protein, respectively. The PEG is selected from: PEG500, PEG1000, PEG2000; the NHS is N-hydroxysulfosuccinimide ester, or N-hydroxysuccinimide ester.

[0023] In some embodiments, the total arm length of the bridge protein is to Best to

[0024] The total arm length refers to the distance separating the particle and the antibody (or its antigen-binding fragment). The total arm length can be measured by an amino acid analyzer based on the amino acid structure.

[0025] In some embodiments, the molecular weight of the irrelevant protein is between 8 kDa and 12 kDa, preferably between 9 kDa and 11 kDa. The molecular weight can be determined by any well-known method in the art.

[0026] In some embodiments, the irrelevant protein is selected from: irrelevant antibodies or their fragments, ovalbumin or its fragments, gelatin or its fragments, keyhole limpet hemocyanin or its fragments, bovine serum albumin or its fragments, human serum albumin or its fragments, rabbit serum albumin or its fragments, polylysine.

[0027] The technical solutions involved in this application will be applied to the field of clinical testing, thus involving the detection of analytes in samples. In view of this, in such a context, an irrelevant antibody refers to an antibody that does not significantly affect (or interfere with) the recognition or binding of the antibody (or its antigen-binding fragment) on the coated particle to the analyte in a statistical sense. The technical solutions of this application do not depend on the specific sequence of the irrelevant protein. In fact, the irrelevant protein at least serves to separate the particle and the antibody, thus avoiding steric hindrance. Therefore, in addition to the types of irrelevant proteins mentioned above, even non-functional linear polypeptides can be artificially synthesized. In the context of this application, "non-functional" means that it does not significantly affect (or interfere with) the detection of analytes in samples in a statistical sense and does not affect the preparation of coated particles. Such "non-functionality" can be pre-judged by those skilled in the art through experiments. For example, when those skilled in the art are instructed in this application to prepare a reagent for detecting A, the anti-A antibody needs to be coated onto the particle with the help of a bridging protein. Those skilled in the art synthesize a linear polypeptide in advance and test whether it affects the binding between A and the anti-A antibody. Therefore, there is no need for specific sequence limitation on the artificially synthesized non-functional linear polypeptide. In an exemplary embodiment, the artificially synthesized non-functional linear polypeptide is a fragment of thymopentin, and its sequence is Arg-Lys-Asp-Val-Tyr, and this structure has no effect on the analyte to be detected. In other embodiments, the artificially synthesized non-functional linear polypeptide can also be a fragment of glutathione with antioxidant properties, or a fragment of MDpep9 antimicrobial peptide.

[0028] According to some embodiments of the present application, a reagent is provided, which contains the bridging protein of the present application.

[0029] According to some embodiments of the present application, the use of the bridging protein of the present application in the preparation of coated particles is provided.

[0030] According to some embodiments of the present application, a particle with an antibody bound to its surface is provided.

[0031] In some embodiments, provided is a particle having an antibody bound thereto, comprising or consisting of:

[0032] - a particle;

[0033] - an antibody or an antigen-binding fragment thereof; and

[0034] - a bridging protein.

[0035] In some specific embodiments, the particle is bound to the antibody (or an antigen-binding fragment thereof) via the bridging protein.

[0036] In some specific embodiments, the particle is covalently bound to the antibody (or an antigen-binding fragment thereof) via the bridging protein.

[0037] In some specific embodiments, the antibody is selected from: monoclonal antibody, polyclonal antibody, recombinant antibody (such as chimeric antibody, humanized antibody, camel antibody), or a combination thereof.

[0038] In some specific embodiments, the antigen-binding fragment is selected from: Fv, sc-Fv, Fab, F(ab’)2, Fab’, scFv-Fc fragment, bispecific antibody.

[0039] In some specific embodiments, the bridging protein refers to an irrelevant protein covalently bound to NHS-PEG at the carboxyl terminus and the amino terminus, respectively.

[0040] In some embodiments, the molecular weight of the irrelevant protein is between 8 kDa and 12 kDa. In some specific embodiments, the irrelevant protein does not affect the binding of the antibody (or an antigen-binding fragment thereof) to its antigen.

[0041] In some embodiments, the molecular weight of the irrelevant protein is between 9 kDa and 11 kDa.

[0042] In some embodiments, the PEG is selected from: PEG500, PEG1000, PEG2000.

[0043] In some embodiments, the NHS is N-hydroxythiosuccinimide ester, or N-hydroxysuccinimide ester.

[0044] In some embodiments, the total arm length of the bridging protein is For example to

[0045] In some embodiments, the irrelevant protein is selected from: irrelevant antibodies or fragments thereof (e.g., Fab), ovalbumin or fragments thereof, gelatin or fragments thereof, keyhole limpet hemocyanin or fragments thereof, bovine serum albumin or fragments thereof, human serum albumin or fragments thereof, rabbit serum albumin or fragments thereof, polylysine, synthetic non-functional linear polypeptides.

[0046] In a specific embodiment, the irrelevant protein is BSA or a fragment thereof. BSA has stable physical and chemical properties, is not easily denatured, is inexpensive and readily available, contains many free amino groups in its molecule, and can maintain a large solubility over a wide pH range and different ionic strengths, which is beneficial to the coupling reaction.

[0047] In a specific embodiment, the irrelevant protein is a synthetic non-functional linear polypeptide.

[0048] In the broadest sense, the terms "antibody", "ab" or "immunoglobulin" can be used interchangeably and include monoclonal antibodies, engineered antibodies, chemically synthesized antibodies or recombinant antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies. In one embodiment, the present application relates to a monoclonal or polyclonal antibody.

[0049] As used herein, the term "monoclonal antibody" or "Mab" refers to an antibody obtained from a substantially homogeneous population of antibodies; in other words, the individual antibodies in the population are considered identical except for possible natural mutations that may be present in minimal amounts. Monoclonal antibodies are highly specific for a single epitope. Such monoclonal antibodies can be produced by monoclonal B cells or hybridomas. Monoclonal antibodies can also be recombinant, i.e., produced by protein engineering. Monoclonal antibodies can also be isolated from phage antibody libraries.

[0050] In the present application, "antigen-binding fragment" is intended to mean any peptide, polypeptide or protein that retains the ability of an antibody to bind to its target (generally also referred to as an antigen). In one embodiment, such "antigen-binding fragments" are selected from: Fv, scFv (sc represents single-chain), Fab, F(ab’)2, Fab’, scFv-Fc fragments or bispecific antibodies. The antigen-binding fragment has at least one CDR derived from its intact antibody.

[0051] The term "recombinant antibody" refers to an antibody obtained by the expression of recombinant DNA.

[0052] In some embodiments, the particles are selected from: latex particles, gold particles, magnetic particles, plastic particles. In a preferred embodiment, the particles are magnetic particles.

[0053] In some embodiments, the antibody is an antibody specific for a small molecule analyte, such as, but not limited to, a hormone antibody or a hapten antibody.

[0054] In a preferred embodiment, the hapten is selected from Müllerian inhibiting substance, adrenocorticotropic hormone, aldosterone, angiotensin, renin, osteocalcin, parathyroid hormone, vitamins.

[0055] In some embodiments, the particle size of the particles is 50 to 200 nm; for example, 80 to 150 nm.

[0056] In some embodiments, the particles are particles with surface modification groups, and the modification groups are selected from: amino, carboxyl, hydroxyl, mercapto; preferably, the particles are amino-modified particles.

[0057] According to some embodiments, a method for covalently binding an antibody or an antigen-binding fragment thereof to the surface of a particle is also provided, which includes the steps of:

[0058] 1) Providing the particles;

[0059] 2) Providing the antibody or an antigen-binding fragment thereof;

[0060] 3) Providing a bridging protein;

[0061] 4) Covalently binding the bridging protein and the particles to form a bridging protein-particle complex;

[0062] 5) Covalently binding the bridging protein-particle complex and the antibody or an antigen-binding fragment thereof to form an antibody or an antigen-binding fragment thereof-bridging protein-particle complex;

[0063] 6) Optionally, blocking the antibody or an antigen-binding fragment thereof-bridging protein-particle complex;

[0064] Wherein, steps 1), 2), or 3) can be interchanged with each other.

[0065] In some embodiments, one or more of the following are used for the blocking in step 6): PEG1000, PEG2000, PEG500, casein, BSA.

[0066] In some embodiments, the covalent binding is carried out in a PB buffer solution with a pH of 7 to 9 containing NaCl.

[0067] In some embodiments, the covalent binding is carried out in a 0.01 M PB buffer solution with a pH of 8 containing 0.15 M NaCl.

[0068] In some embodiments, the covalent binding is carried out at 28 to 40 degrees Celsius, for example, but not limited to, 30, 31, 32, 33, 34, 35, 36, 37, 38 degrees Celsius.

[0069] In some embodiments, the covalent binding lasts for 20 to 60 minutes, such as but not limited to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 minutes.

[0070] According to some embodiments, a method for covalently binding an antibody or an antigen-binding fragment thereof to the surface of a particle is provided, which comprises the steps of:

[0071] 1) Providing 100 nm NH4-modified magnetic particles in 0.01 M PB buffer at pH 8 containing 0.15 M NaCl;

[0072] 2) Providing an antibody (or an antigen-binding fragment thereof) in 0.01 M PB buffer at pH 8 containing 0.15 M NaCl;

[0073] 3) Providing a bridging protein in 0.01 M PB buffer at pH 8 containing 0.15 M NaCl;

[0074] 4) Covalently binding the bridging protein and the particle at 37 °C for 30 minutes to form a bridging protein-particle complex;

[0075] 5) Covalently binding the bridging protein-particle complex and the antibody or an antigen-binding fragment thereof at 37 °C for 30 minutes to form an antibody or an antigen-binding fragment thereof-bridging protein-particle complex;

[0076] 6) Optionally, blocking the antibody or an antigen-binding fragment thereof-bridging protein-particle complex; wherein steps 1), 2) or 3) can be interchanged with each other.

[0077] According to some embodiments, a detection reagent is provided, which comprises:

[0078] - A first reagent, which comprises an enzyme-labeled first antibody and a buffer; or which comprises an enzyme-labeled antigen and a buffer;

[0079] - A second reagent, which comprises the coated particles of the present invention;

[0080] - An optional diluent, which comprises BSA and a buffer;

[0081] - Optionally, a calibrator, and

[0082] - Optionally, a quality control product.

[0083] In some embodiments, the diluent comprises BSA and phosphate buffer; or comprises BSA and normal saline;

[0084] In some embodiments, the first antibody labeled with an enzyme is the first antibody labeled with alkaline phosphatase.

[0085] In some embodiments, the first reagent comprises a first antibody (or antigen) labeled with an enzyme and a buffer (Tris, acetate buffer), pH 6 to 8.

[0086] In some embodiments, the second reagent comprises from 0.2 mg / L to 2.0 mg / L of particles having an antibody (or antigen-binding fragment thereof) bound to the surface according to the present application. The concentration of the particles in the second reagent is, for example but not limited to, 0.3 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.1 mg / L, 1.5 mg / L. Detailed embodiments

[0087] Example 1: Method for preparing a bridging protein

[0088] 1. Selection of an irrelevant protein:

[0089] Before proceeding with the formal preparation, an irrelevant protein needs to be selected first.

[0090] Based on the antibody to be covalently bound to the particle (e.g., the type of antibody, the species origin of the antibody), the ultimate use of the antibody (for what kind of detection, what antigen it binds to, what sample is being detected), and the reagent environment in which the particle / antibody is located (other components in the kit where the particle / antibody is located), an irrelevant protein is selected such that in the final detection, this irrelevant protein does not affect the detection function of the antibody. For example, the irrelevant protein does not react with the detection system (sample, analyte, antibody, other components in the reagent) in a way that affects the detection purpose.

[0091] The molecular weight of the irrelevant protein is approximately 10 kDa.

[0092] The irrelevant protein can be a naturally occurring protein, a purified protein, a protein fragment, or a synthetic non-functional linear polypeptide. For example, in the subsequent examples, a non-limiting example of the irrelevant protein is a synthetic linear polypeptide with reactive groups (such as amino groups) at both ends (the carboxyl end is chemically modified to be linked with a reactive group to facilitate connection with the NHS group). In view of this, this reactive group is not contained in the subsequent buffer.

[0093] 2. Preparation of the bridging protein:

[0094] PEG (polyethylene glycol) 2000 with NHS groups is covalently linked to the N-terminus and C-terminus of the irrelevant protein respectively (PEG500 or PEG1000 can also be used, and the results have no significant difference), and it is named the bridging protein.

[0095] Example 2: Particle Coupling Method

[0096] Magnetic beads of 80 nm, 100 nm, and 150 nm were selected, with the surface modified with NH4 groups and purchased from Merck.

[0097] The bridging protein prepared in Example 1 is first covalently linked to magnetic beads; then, it is covalently linked to various monoclonal antibodies (commercially available antibodies, see subsequent examples for details, brand is not limited) to form a magnetic bead-bridging protein-antibody complex;

[0098] The magnetic beads are blocked with PEG1000 (or PEG2000, PEG500, casein).

[0099] The specific steps are as follows:

[0100] (1) Take 0.05 ml of amino magnetic beads and wash them three times with a solution of 0.01 M PB and 0.15 M NaCl at pH 8.0. Finally, mix the magnetic beads with 1 ml of a solution of 0.01 M PB and 0.15 M NaCl at pH 8.0 and set aside;

[0101] (2) 0.2 mg of antibody solution, volume no greater than 100 μl, was added to 2 ml of 0.01 M PB, 0.15 M NaCl solution at pH 8.0, mixed thoroughly, added to a Centricon-10 concentrator tube and then placed in a Sigma 2-16k high-speed refrigerated centrifuge and concentrated at 3000 g for about 30 min until the volume was no greater than 0.5 ml;

[0102] (3) After re-dissolving the bridging protein with a solution of 0.01 M PB and 0.15 M NaCl at pH 8.0, 0.1 mg was taken out, the volume of which was no more than 100 μl, and then added to the magnetic particle suspension of step (1), and rotated and mixed in an oven at 37 degrees for 30 minutes;

[0103] (4) Wash the magnetic beads again three times with a 0.01M PB, 0.15M NaCl solution at pH 8.0, and finally mix the magnetic beads with 1 ml of a 0.01M PB, 0.15M NaCl solution at pH 8.0, add the antibody prepared in step (2), and rotate and mix in a 37°C oven for 30 minutes;

[0104] (5) Wash the magnetic beads again three times with 0.01 M PB, 0.15 M NaCl solution at pH 8.0 to obtain antibody-bridging protein-particle complexes;

[0105] (6) Optionally, add the antibody-bridging protein-particle complex to a 2 ml solution of 0.01 M TRIS, 0.15 M NaCl, 1% PEG1000, 1% PEG2000, 1% PEG500, 0.2% casein, and 0.5% BSA at pH 8.0, and keep it mixed for 6 hours to block the surface of the magnetic beads;

[0106] (7) The particles obtained after blocking can be used directly (e.g., but not limited to, used after dilution 50 times).

[0107] Example 3. Preparation of an AMH kit

[0108] 1. Prepare and assemble the particles obtained in Example 2 into a kit according to the composition components in the following table.

[0109] Table 1. Composition of the AMH kit

[0110]

[0111] Self-prepare calibration products and quality control products, or commercial calibration products and quality control products can also be used.

[0112] 2. Principle of the kit:

[0113] Add a quantitative amount of enzyme-labeled AMH monoclonal antibody (R1), AMH antibody bound to magnetic particles (separation reagent), and R2 to the sample, calibration product, and quality control product.

[0114] After incubation at 37°C, the AMH monoclonal antibody bound to the magnetic particles and the enzyme-labeled monoclonal antibody bind to different epitopes of the AMH molecule respectively, forming a "sandwich" structure.

[0115] Precipitate directly in an external magnetic field and separate without centrifugation. Pour off the supernatant, wash the precipitated complex, and then add an enzyme-catalyzed chemiluminescent substrate.

[0116] The substrate is catalytically cleaved by the enzyme to form an unstable excited-state intermediate. When the excited-state intermediate returns to the ground state, photons are emitted, forming a luminescence reaction. The luminescence intensity of the reaction can be detected using a luminometer, and the AMH content in the sample can be calculated according to the standard curve. Within the detection range, the luminescence intensity is proportional to the AMH concentration in the sample.

[0117] Example 4. Preparation of control particles and their kits

[0118] Adopt the method in CN101625366A to directly couple carboxyl microspheres with antibodies using EDC. The specific coupling method is as follows:

[0119] (1) Take 0.05 ml of carboxyl microspheres (80, 100, or 150 nm), wash them 3 times with 0.05 M MES solution at pH 6.0, and finally mix them evenly with 1 ml of 0.05 M MES solution at pH 6.0;

[0120] (2) Take 10 mg of EDC, dissolve it in 0.05 M MES at pH 6.0, and the concentration is 50 mg / ml;

[0121] (3) Take 0.02 ml of 50 mg / ml EDC solution, add it to (1), and at the same time add 0.2 mg of antibody, mix well and react for 30 minutes; then wash and mix well with 0.01 M TRIS solution at pH 7.4 for at least 30 minutes;

[0122] (4) After washing 2 times with PB at pH 7.4, 0.01 M, 1% BSA, and 0.01% TWEEN-20, add 2 ml of the magnetic beads in step (1), and mix well;

[0123] (5) Dilute and set aside.

[0124] Test Example

[0125] Test Example 1: Stability Verification

[0126] The present inventors further compared the magnetic beads prepared in Example 3 (the particles and their kits of Example 2) and Example 4 (the comparative particles and their kits), and their performance in the kits.

[0127] The specific method is as follows:

[0128] Prepare self-prepared calibration points of AMH (S0, S1, S2, S3, S4, S5), and configure the magnetic beads according to the same formula, namely PB at pH 7.4 0.01 M, 0.5% BSA (1% BSA), and 0.01% TWEEN-20. A total of 10 ml is configured for each magnetic bead at each BSA concentration, and then divided into 5 ml tubes, with a total of 2 tubes. One tube is placed in an oven at 37 °C for 4 days, and the other tube is placed in a refrigerator for cold storage at a temperature of 2 °C to 8 °C.

[0129] After taking them out after 4 days, according to the operation instructions of the manual, compare and measure the luminescence values of calibration points S1, S3, and S5. The results show that the ratio of the luminescence values of the measured points of the magnetic beads labeled with bridging protein is close to 1, indicating that the titer of the whole complex is basically not lost. For the magnetic beads labeled with EDC, the ratio is close to 1 at high protein concentrations; but at low protein concentrations, the attenuation of its titer is significantly greater than that of the magnetic beads labeled with bridging protein.

[0130] Table 2. Comparison of Magnetic Bead Stability

[0131]

[0132] When preparing the reagent, those skilled in the art usually add a certain amount of protein protectant to the reagent components to maintain the biological activity of proteins (such as enzymes, antibodies, etc.). The applicant has found that even if a very low concentration of protein protectant (such as BSA, trehalose, etc.) is added to the reagent, the antibody-bridging protein-particle prepared by the method of Example 2 can still maintain the activity of the protein. However, the particles prepared by the conventional method (Example 4) (antibody directly conjugated to the particle) can only be stable in the presence of a higher concentration of protectant.

[0133] Test Example 2. Sensitivity

[0134] Use the zero-concentration calibrator or sample diluent as the sample for detection, repeat the determination 20 times, obtain the RLU values (relative luminescence values) of the 20 measurement results, calculate their average value (M) and standard deviation (SD), and obtain M - 2SD.

[0135] According to the concentration-RLU value results between the zero-concentration calibrator and the adjacent calibrator, perform two-point regression fitting to obtain a linear equation, substitute the RLU value of M - 2SD into the above equation, and find the corresponding concentration value, which is the lowest detection limit. The results are as follows.

[0136] Table 3. Comparison of reagent sensitivities

[0137] Sensitivity Example 3 0.01 ng / ml Example 4 0.06 ng / ml

[0138] This result shows that the particles obtained by using the preparation method of Example 2 can achieve higher detection sensitivity.

[0139] Test Example 3. Correlation with commercially available mature products

[0140] The inventor compared the test results of the reagents of Example 3 and Example 4 with the Roche AMH kit respectively.

[0141] Table 4. Correlation comparison

[0142]

[0143]

[0144] The comparison was carried out on serum samples in different concentration ranges. The inventor found that: even in the low-concentration sample range of 0 to 1 ng / ml, the reagent prepared by the method of Example 3 still maintains excellent correlation with the commercially available mature kit.

[0145] Test Example 4. Performance indicators of the AMH kit of the present application

[0146] 1. Anti-interference:

[0147] If the test sample contains interfering substances at the following concentrations, it has no significant impact on the test results: hemoglobin ≤ 160 g / l, triglyceride ≤ 150 mg / dl, bilirubin ≤ 17 g / dl, alkaline phosphatase ≤ 150 U / L.

[0148] 2. Detection limit:

[0149] The lowest detection limit: not greater than 0.03 ng / ml.

[0150] 3. Accuracy:

[0151] Recovery experiments were conducted using pure products (purchased from ORIGENE), and the recovery rate was in the range of 85% to 115%.

[0152] 4. Repeatability:

[0153] Two samples with different concentrations were tested, and each was repeatedly tested 10 times. The coefficient of variation CV was not greater than 10%.

[0154] 5. Inter-batch difference:

[0155] The same sample was tested using three batches of kits, and the inter-batch coefficient of variation CV among the three batches of kits was not greater than 15%.

[0156] 6. Linear range:

[0157] In the measurement range of 0.03 ng / ml to 25 ng / ml, the linear correlation coefficient R of the dose-response curve 2 ≥ 0.9900.

[0158] The improvement of this application lies in:

[0159] 1) Using a bridging protein complex It can avoid steric hindrance effects;

[0160] 2) The bridging protein can ensure the stability of the entire conformation;

[0161] 3) Both ends of the bridging protein carry NHS groups, which can control the entire coupling reaction time within 60 minutes;

[0162] 4) Antibody Fab fragments of non-test substances can also be introduced onto the bridging protein to play a role in eliminating specific interference.

Claims

1. A particle with anti - Müllerian hormone antibody bound to its surface, which comprises the following: - A particle; - An antibody or its antigen - binding fragment; and - A bridging protein; Among them, The particle is bound to the antibody or its antigen - binding fragment through the bridging protein; the binding is a covalent binding; Wherein, the bridging protein refers to an irrelevant protein covalently bound with NHS - PEG at the carboxyl - terminal and amino - terminal respectively; The molecular weight of the irrelevant protein is between 8 kDa and 12 kDa, The total arm length of the bridging protein is 80 Å to 200 Å, The irrelevant protein is selected from: irrelevant antibody or its fragment, ovalbumin or its fragment, gelatin or its fragment, keyhole limpet hemocyanin or its fragment, bovine serum albumin or its fragment, human serum albumin or its fragment, rabbit serum albumin or its fragment, polylysine, synthetic non - functional linear polypeptide; The irrelevant protein does not affect the binding of the antibody or its antigen - binding fragment to anti - Müllerian hormone; The antibody is selected from: monoclonal antibody, polyclonal antibody, recombinant antibody, or a combination thereof; The antigen - binding fragment is selected from: Fv, sc - Fv, Fab, F(ab’)2, Fab’, scFv - Fc fragment, bispecific antibody; The particle is selected from: latex particle, gold particle, magnetic particle, plastic particle; the antibody or its antigen - binding fragment is specific to anti - Müllerian hormone.

2. The particle with anti - Müllerian hormone antibody bound to its surface according to claim 1, wherein the particle is a magnetic particle.

3. The particle with anti - Müllerian hormone antibody bound to its surface according to claim 1, wherein the total arm length of the bridging protein is 100 Å to 150 Å.

4. The particle with anti - Müllerian hormone antibody bound to its surface according to claim 1, wherein: The particle has a particle size of 50 nm to 200 nm; The particle is a particle with surface - modifying groups, and the modifying groups are selected from: amino group, carboxyl group, hydroxyl group, mercapto group; The molecular weight of the irrelevant protein is between 8 kDa and 12 kDa; The PEG is selected from: PEG500, PEG1000, PEG2000; The NHS is N - hydroxythiosuccinimide ester, or N - hydroxysuccinimide ester.

5. The particle with anti - Müllerian hormone antibody bound to its surface according to claim 4, wherein the particle has a particle size of 80 nm to 150 nm.

6. The particle with anti - Müllerian hormone antibody bound to its surface according to claim 4, wherein the particle is an amino - modified particle.

7. The particle with anti - Müllerian hormone antibody bound to its surface according to claim 4, wherein the molecular weight of the irrelevant protein is between 9 kDa and 11 kDa.

8. A method for covalently binding an anti - Müllerian hormone antibody or its antigen - binding fragment to the surface of a particle, which comprises the steps of: 1) Providing a particle; 2) Providing an antibody or its antigen - binding fragment; 3) Providing a bridging protein; 4) Covalently binding the bridging protein and the particle to form a bridging protein - particle complex; 5) Covalently bind the bridging protein-particle complex and the antibody or its antigen-binding fragment to form an antibody or its antigen-binding fragment-bridging protein-particle complex; 6) Block the antibody or its antigen-binding fragment-bridging protein-particle complex; Among them, steps 1), 2) or 3) can be interchanged with each other; The antibody or its antigen-binding fragment is specific to anti-Müllerian hormone.

9. The method according to claim 8, wherein: The bridging protein refers to an irrelevant protein covalently bound with NHS-PEG at the carboxyl terminus and amino terminus respectively; The molecular weight of the irrelevant protein is between 8 kDa and 12 kDa, The total arm length of the bridging protein is 80 Å to 200 Å, The irrelevant protein is selected from: irrelevant antibody or its fragment, ovalbumin or its fragment, gelatin or its fragment, keyhole limpet hemocyanin or its fragment, bovine serum albumin or its fragment, human serum albumin or its fragment, rabbit serum albumin or its fragment, polylysine, synthetic non-functional linear polypeptide; The irrelevant protein does not affect the binding of the antibody or its antigen-binding fragment to anti-Müllerian hormone; The antibody is selected from: monoclonal antibody, polyclonal antibody, recombinant antibody, or a combination thereof; The antigen-binding fragment is selected from: Fv, sc-Fv, Fab, F(ab’)2, Fab’, scFv-Fc fragment, bispecific antibody; The particle is selected from: latex particle, gold particle, magnetic particle, plastic particle.

10. The method according to claim 9, wherein the molecular weight of the irrelevant protein is between 9 kDa and 11 kDa.

11. The method according to claim 9, wherein the total arm length of the bridging protein is 100 Å to 150 Å.

12. The method according to claim 9, wherein the particle is a magnetic particle.

13. The method according to claim 9, wherein: The particle has a particle size of 50 nm to 200 nm; The particle is a particle with surface modification groups, and the modification groups are selected from: amino group, carboxyl group, hydroxyl group, mercapto group; The molecular weight of the irrelevant protein is between 8 kDa and 12 kDa; The PEG is selected from: PEG500, PEG1000, PEG2000; The NHS is N-hydroxythiosuccinimide ester, or N-hydroxysuccinimide ester.

14. The method according to claim 13, wherein the particle has a particle size of 80 nm to 150 nm.

15. The method according to claim 13, wherein the particle is an amino-modified particle.

16. The method according to claim 13, wherein the molecular weight of the irrelevant protein is between 9 kDa and 11 kDa.

17. The method according to claim 8, wherein the blocking is carried out using one or a combination selected from the following: PEG500, PEG1000, PEG2000, casein, BSA.

18. The method according to claim 8, wherein the covalent binding is carried out under the following selected conditions: PB buffer with pH 7 to 9 containing NaCl; 28 to 40 degrees Celsius; Lasts for 20 to 60 minutes.

19. The method according to claim 18, wherein the buffer is a 0.01 M PB buffer with a pH of 8 containing 0.15 M NaCl.

20. The method according to claim 18, wherein the covalent binding is carried out under conditions of 30, 31, 32, 33, 34, 35, 36, 37 or 38 degrees Celsius.

21. The method according to claim 18, wherein the covalent binding lasts for 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 minutes.

22. A reagent, comprising: The particles with anti-Müllerian hormone antibody bound to the surface as claimed in claim 1.

23. An anti-Müllerian hormone kit, comprising: - A first reagent, - A second reagent, - A diluent, - A separation reagent, wherein: The first reagent consists of: 0.3 mg / L of the first anti-Müllerian hormone monoclonal antibody labeled with alkaline phosphatase, and a TRIS buffer with a pH of 8.0; The second reagent consists of: sheep, horse or pig IgG, and a TRIS buffer with a pH of 8.0; The diluent consists of: 0.01% BSA, and a phosphate buffer with a pH of 7.4; The separation reagent consists of: 1.1 mg / L of the particles with anti-Müllerian hormone antibody bound to the surface as claimed in claim 1; The anti-Müllerian hormone antibody in the separation reagent is the second anti-Müllerian hormone monoclonal antibody; The first anti-Müllerian hormone monoclonal antibody and the second anti-Müllerian hormone monoclonal antibody recognize different epitopes of anti-Müllerian hormone; The particle size of the particles is 100 nm.

24. The anti-Müllerian hormone kit according to claim 23, further comprising any one or a combination selected from: calibrators, quality control products.

25. The anti-Müllerian hormone kit according to claim 24, wherein: The calibrators contain: 0 ng / ml, 0.2 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 25 ng / ml of anti-Müllerian hormone; The quality control products contain: 1 ng / ml, 10 ng / ml of anti-Müllerian hormone.

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