An antibody-bsa complex, a method for preparing the same and a magnetic microparticle coated with the same

Abstract

The application belongs to the technical field of in-vitro diagnosis, and relates to an antibody-BSA compound, a preparation method thereof and magnetic microparticles coated by the antibody-BSA compound. The magnetic microparticles coated by the antibody-BSA compound are formed by coupling the antibody-BSA compound with the magnetic microparticles. The antibody-BSA compound is coupled with the magnetic microparticles, and since the hydrophobicity of BSA is stronger than that of the antibody, BSA is more easily combined with the magnetic microparticles than the antibody, so that the coupling rate of the magnetic microparticles and the antibody is improved, meanwhile, the antibody end is exposed outside, the steric hindrance between the antibody and the microspheres is reduced, the specific combination of the antibody and the antigen is more favorable, and the test sensitivity is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of in vitro diagnosis, and relates to an antibody-BSA complex, a preparation method thereof and magnetic microparticles coated with the antibody-BSA complex. BACKGROUND

[0002] Currently, the methods mainly used in in vitro diagnosis include enzyme-linked immunoassay, latex turbidimetry, chemiluminescence and molecular diagnosis. The latex turbidimetry and chemiluminescence involve microsphere-labeled proteins. The methods for coupling microsphere-labeled proteins (antigens, antibodies or avidin) mainly include adsorption and covalent coupling, and the purpose is to coat the corresponding antibodies or antigens of the to-be-detected substance on the microspheres, so as to improve the sensitivity of the test by detecting the characteristics of the reaction between the to-be-detected substance and the microspheres coated with the corresponding antigens or antibodies. The commonly used microsphere surface coupling functional groups include carboxyl, amino and toluenesulfonyl groups.

[0003] So far, the most common reaction strategy for coupling proteins and other amino-containing molecules to the surface of carboxyl microspheres is the water-soluble carbodiimide (EDC, 1-ethyl-(3-dimethylaminopropyl) carbodiimide) mediated method. This method can be carried out in two modes: one-step or two-step. In the one-step method, EDC is directly added to the protein and microsphere mixture, and the carboxyl microsphere particles are activated by water-soluble EDC to generate an intermediate ester, which can directly react with the amino group on the protein. In the two-step method, the carboxyl microsphere particles are first activated with EDC, and then N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (Sulfo-NHS) is further added to generate another secondary intermediate ester (NHS-ester or Sulfo-NHS ester). Compared with the intermediate ester of the one-step method, the NHS-ester or Sulfo-NHS ester is more stable in structure, and therefore, the yield of the two-step method is usually higher. During the coupling process, due to the presence of many amino groups (such as lysine and arginine residues) and carboxyl groups (such as serine and threonine residues) on most proteins, excessive EDC can easily mediate self-polymerization between proteins, and the two-step method can overcome this defect. However, due to the multiple steps, it not only increases the time cost, but also requires enterprises to write more SOP files (operating procedure files) and introduce more quality control links. Obviously, from the perspective of modern large-scale production, the two-step method is not an economical and effective way.

[0004] The method for coupling amino microspheres usually uses the same bifunctional crosslinking agent, such as glutaraldehyde method. Since the same bifunctional crosslinking agent can couple with two identical groups, excessive crosslinking agent is required during the coupling process, and the combination between the microspheres or the proteins cannot be avoided, thereby leading to problems such as aggregation or low coupling efficiency.

[0005] In recent years, tosyl magnetic beads used for in vitro diagnosis have a hydrophilic polymer coating on the surface. Since tosyl groups are introduced into the coating, a carboxyl and agent is not needed, and molecules containing amino groups such as antibodies can be fixed on the surface of the magnetic beads in a chemical binding manner through simple operation. With the chemical binding, tosyl groups are gradually separated, the hydrophilicity of the particle surface is enhanced, so that the biological activity of the conjugate on the surface of the magnetic beads can be maintained, and non-specific reactions can be effectively inhibited. However, due to the complexity of the interaction between proteins and microspheres, ionic bonds and hydrophobic forces will play a role. Individual proteins have their own characteristics, and sometimes it is difficult to predict the corresponding results. Because the titer and isoelectric point of different project antibodies are different, a general process cannot meet the optimal effect of coupling of all projects. SUMMARY

[0006] The present application aims at the deficiencies in the prior art, and provides an antibody-BSA complex, a preparation method thereof, and a magnetic microparticle coated with the antibody-BSA complex.

[0007] One object of the present application is achieved by the following technical solutions.

[0008] An antibody-BSA complex is formed by coupling of an antibody and BSA through thiol groups introduced by the antibody and maleimide groups introduced on the BSA.

[0009] Another object of the present application is achieved by the following technical solutions.

[0010] A preparation method of an antibody-BSA complex comprises the following steps: introducing thiol groups on an antibody, activating BSA with a substance containing maleimide groups, and then mixing and reacting the thiol group-introduced antibody and the activated BSA.

[0011] Thiol groups are introduced on the antibody, maleimide groups are introduced on the BSA, the thiol groups can be coupled and combined with the maleimide groups, and the antibody and the BSA are coupled together through the thiol groups and the maleimide groups, so as to form the antibody-BSA complex.

[0012] As a preferred embodiment, 2-iminothiolane hydrochloride (2IT) is used to react with the antibody, so as to introduce thiol groups on the antibody. 2IT can introduce terminal thiol groups on the antibody through ring-opening reaction with substances (antibodies) containing amino groups. Compared with a disulfide bond reduction method, this method for introducing thiol groups can better maintain the bivalency of the antibody.

[0013] As a preferred embodiment, the mass ratio of 2-iminothiolane hydrochloride and the antibody is 1:10 to 1:100.

[0014] As a preferred embodiment, 2-iminothiolane hydrochloride and the antibody are reacted at room temperature for 10 to 50 min.

[0015] As a preference, the 2-iminothiolane hydrochloride and the antibody are reacted for 10-50 minutes at room temperature, and then a glycine solution having a pH of 7.0-7.5 is added, and the reaction is continued for 5-20 minutes at room temperature.

[0016] As a preference, the substance containing a maleimide group is 4-(N-maleimidomethyl) cyclohexane-1-carboxylate succinimidyl ester (SMCC) and / or a derivative thereof, such as a sulfonated derivative sulfo-SMCC. The SMCC and / or the derivative thereof can activate the BSA, and the BSA can be combined with the antibody having a sulfhydryl group through the activation of the SMCC and / or the derivative thereof.

[0017] As a preference, the mass ratio of the substance containing a maleimide group to the BSA in the activation reaction is 1:10-1:100.

[0018] As a preference, the substance containing a maleimide group and the BSA are activated for 10-50 minutes at room temperature.

[0019] As a preference, the substance containing a maleimide group and the BSA are activated for 10-50 minutes at room temperature, and then a glycine solution having a pH of 7.0-7.5 is added, and the reaction is continued for 10-20 minutes at room temperature.

[0020] As a preference, the antibody having a sulfhydryl group and the activated BSA are reacted for 8-20 hours at 2-8℃.

[0021] As a preference, the molar ratio of the antibody having a sulfhydryl group to the activated BSA is 0.5:1-3:1.

[0022] Another object of the present application is achieved by the following technical solution:

[0023] An antibody-BSA complex coated magnetic microparticle is formed by coupling the above antibody-BSA complex to a magnetic microparticle.

[0024] The antibody-BSA complex is coupled to the magnetic microparticle, and since the hydrophobicity of the BSA is stronger than that of the antibody, the BSA is more easily combined with the magnetic microparticle than the antibody, thereby increasing the coupling rate of the magnetic microparticle to the antibody, i.e., the same antibody can be coupled to more magnetic microparticles. In addition, the coupling of the antibody-BSA complex to the magnetic microparticle exposes the antibody end, reduces the steric hindrance between the antibody and the microparticle, and is more conducive to the specific binding of the antibody and the antigen.

[0025] Another object of the present application is achieved by the following technical solution:

[0026] The application discloses a method for preparing an antibody-BSA complex coated magnetic particle, which comprises the following steps: mixing an antibody-BSA complex and a magnetic particle, then adding a reaction promoter to react, and cleaning and sealing to obtain the antibody-BSA complex coated magnetic particle.

[0027] Preferably, the reaction promoter comprises, but is not limited to, an ammonium sulfate solution and a potassium sulfate solution.

[0028] Preferably, the mass ratio of the magnetic particle to the antibody-BSA complex is 10:0.05-10:0.5.

[0029] Preferably, the reaction promoter is added and the reaction is carried out at 24-42 DEG C for 4-24 hours.

[0030] Preferably, the concentration of the ammonium sulfate solution is 0.5-4 mol / L.

[0031] Preferably, the particle size of the magnetic particle is 0.5-3.0 microns.

[0032] In the above reaction process, the surface of the magnetic particle is coupled with a tosyl functional group. Preferably, the sealing is carried out by using a sealing liquid and the reaction is carried out at 24-42 DEG C for 6-24 hours.

[0033] Compared with the prior art, the application has the following beneficial effects:

[0034] 1. The application introduces a mercapto group on the antibody and a maleimide group on the BSA, and realizes the coupling of the antibody and the BSA through the coupling of the mercapto group and the maleimide group, so as to form the antibody-BSA complex;

[0035] 2. The application introduces a mercapto group on the antibody through the reaction of 2IT and the antibody, and the 2IT can introduce a terminal mercapto group on the antibody through ring-opening reaction with a substance (antibody) with an amino group, and this method for introducing a mercapto group can better maintain the bivalent structure of the antibody;

[0036] 3. The application couples the antibody-BSA complex with the magnetic particle, and since the hydrophobicity of the BSA is stronger than that of the antibody, the BSA is more easily combined with the magnetic particle than the antibody, so that the coupling rate of the magnetic particle and the antibody is improved, and in a conventional method, 1 mg of the antibody is coupled with about 100 mg of the magnetic particle, and after bridging the BSA, 1 mg of the antibody can be coupled with 200 mg of the magnetic particle;

[0037] 4. The coupling of the antibody-BSA complex and the magnetic particle exposes the terminal of the antibody, reduces the steric hindrance between the antibody and the microsphere, and is more beneficial to the specific combination of the antibody and the antigen, so as to improve the test sensitivity;

[0038] 5. This invention uses antibody-BSA complexes to conjugate magnetic microparticles. This method is easier to conjugate and is not affected by the antibody titer or isoelectric point. Different projects can use a common process for conjugation, which reduces production costs and complex procedures, and has high performance. Attached Figure Description

[0039] Figure 1 The correlation graph between the kit test samples and Roche measurements in Comparative Example 1 is shown.

[0040] Figure 2 This is a correlation graph between the test samples from Example 1 and Roche measurements;

[0041] Figure 3 The correlation graph between the kit test samples and Abbott's measured values ​​in Comparative Example 2 is shown.

[0042] Figure 4 This is a correlation graph between the test samples from the kit in Example 2 and the Abbott measurement values. Detailed Implementation

[0043] In the following, embodiments of the preparation method of the antibody-BSA complex and the preparation method of the magnetic microparticles coated with the antibody-BSA complex of the present invention will be described in detail. However, these embodiments are exemplary and the disclosure of the present invention is not limited thereto.

[0044] In some embodiments of the present invention, the method for preparing the antibody-BSA complex includes the following steps: introducing a thiol group onto an antibody, activating BSA with a substance containing a maleimide group, and then mixing and reacting the antibody with the introduced thiol group and the activated BSA.

[0045] In some embodiments of the present invention, 2-iminothione hydrochloride is used to react with the antibody, thereby introducing a thiol group onto the antibody. The specific operation is as follows:

[0046] Replace the antibody with a buffer free of amino and thiol groups, such as borate buffer or HEPES buffer, using dialysis or a PD-10 desalting column, and concentrate it to 1-5 mg / mL using a concentration tube.

[0047] Dissolve 2-iminothionine hydrochloride in a buffer solution free of amino and thiol groups, such as borate buffer or HEPES buffer, at a concentration of 10–15 mg / ml. Mix the antibody solution and the 2IT solution so that the mass ratio of 2-iminothionine hydrochloride to antibody is 1:10 to 1:100. After mixing, react at room temperature for 10–50 min.

[0048] Add a glycine solution (0.2-2 mol / L) with a pH of 7.0-7.5, in the same volume as the 2IT solution, and continue the reaction at room temperature for 5-20 minutes.

[0049] The PD-10 desalting column is replaced with an amino and sulfhydryl-free buffer such as a borate buffer, a HEPES buffer, etc., and concentrated to 1-5 mg / mL for standby, to obtain the sulfhydryl-introduced antibody.

[0050] In some embodiments of the present application, the BSA is activated with a substance containing a maleimide group in the following specific manner:

[0051] The BSA is dissolved in an amino and sulfhydryl-free buffer such as a borate buffer, a HEPES buffer, etc., to 1-5 mg / mL, the substance containing a maleimide group is dissolved in water to 3-10 mg / mL, the BSA solution and the substance containing a maleimide group solution are mixed so that the mass ratio of the substance containing a maleimide group to the BSA is 1:10-1:100, and the activation reaction is carried out at room temperature for 10-50 min.

[0052] A glycine solution (concentration 0.2-2 mol / L) with a pH of 7.0-7.5 is added in the same volume as the amount of the substance containing a maleimide group solution, and the reaction is continued at room temperature for 10-20 min.

[0053] The PD-10 desalting column is replaced with an amino and sulfhydryl-free buffer such as a borate buffer, a HEPES buffer, etc., and concentrated to 1-5 mg / mL for standby, to obtain the activated BSA.

[0054] In some embodiments of the present application, the specific operation steps of the mixed reaction of the sulfhydryl-introduced antibody and the activated BSA are as follows:

[0055] The sulfhydryl-introduced antibody and the activated BSA are mixed in a molar ratio of (0.5-3):1, and reacted at 2-8°C for 8-20 hours to obtain an antibody-BSA complex, which is purified and stored at 2-8°C for standby.

[0056] In some embodiments of the present application, a method for preparing an antibody-BSA complex-coated magnetic microparticle includes the following steps: mixing the above-mentioned antibody-BSA complex and magnetic microparticles, then adding a reaction promoter to react, and obtaining the antibody-BSA complex-coated magnetic microparticle after washing and blocking.

[0057] In some embodiments of the present application, the specific operation steps of the method for preparing an antibody-BSA complex-coated magnetic microparticle are as follows:

[0058] The magnetic microparticles are diluted to 5-20 mg / mL with 0.005-0.05 M phosphate buffer solution at pH 7.0-7.5; the antibody-BSA complex is added in a mass ratio of magnetic microparticles: antibody-BSA complex = 10:0.05-10:0.5; 0.5-4 M ammonium sulfate solution or potassium sulfate solution is added in a volume of 0.5-2 times the total volume of the magnetic microparticles and the antibody-BSA complex; and the mixture is suspended and reacted at 24-42°C for 4-24 hours;

[0059] After magnetic separation and washing, the blocking solution containing BSA is reacted at 24-42°C for 6-24 hours;

[0060] The antibody-BSA complex-coated magnetic microparticles are obtained by diluting the above blocking solution to 5-20 mg / mL, and are stored at 2-8°C until use.

[0061] The magnetic microparticles are diluted to a final concentration of 0.2-1.0 mg / mL when used.

[0062] The technical solutions of the present application are described and explained further below by means of specific examples and drawings. It should be understood that the specific examples described herein are only used to help understand the present application and are not used to limit the present application. The drawings used herein are only used to better illustrate the disclosed content of the present application and do not limit the scope of protection. If not otherwise specified, the raw materials used in the examples of the present application are all commonly used raw materials in the art, and the methods used in the examples are all conventional methods in the art.

[0063] Example 1

[0064] The specific components of the prostate specific antigen (PSA) detection kit are as follows:

[0065] (1) Reagent M: PSA antibody-BSA complex-coated magnetic microparticles 0.5 mg / mL, 0.05 M tris-HCl buffer, 1% BSA, 0.1% Proclin300;

[0066] (2) Enzyme conjugate: PSA labeled antibody-AP complex, 0.05 M tris-HCl buffer, 1% sucrose, 1% BSA, 0.1% Proclin300, 1 mM magnesium chloride, 0.1 mM zinc chloride;

[0067] (3) Calibrators: PSA antigen is diluted into a series of gradients with BSA-containing tris-HCl buffer, 0 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL.

[0068] The preparation method of the PSA antibody-BSA complex coated magnetic microparticles is as follows:

[0069] 1) The PSA antibody is replaced with a borate buffer using a PD-10 desalting column, and concentrated to 2 mg / mL using a concentration tube;

[0070] 2) 2IT is dissolved in a borate buffer to a concentration of 10 mg / ml, and the PSA antibody solution and the 2IT solution are mixed so that the mass ratio of 2-iminothiolane hydrochloride to the antibody is 1:30, and after mixing, the reaction is carried out at room temperature for 30 min;

[0071] 3) 1M glycine solution with a pH of 7.3 is added in the same volume as the addition amount of the 2IT solution, and the reaction is continued at room temperature for 8 min; the buffer is replaced using a PD-10 desalting column, and concentrated to 3 mg / mL for standby;

[0072] 4) BSA is dissolved in a borate buffer to 3 mg / mL, and sulfo-SMCC is dissolved in water to 6 mg / mL, and the BSA solution and the sulfo-SMCC solution are mixed so that the mass ratio of sulfo-SMCC to BSA is 1:30, and the activation reaction is carried out at room temperature for 20 min;

[0073] 5) 1M glycine solution with a pH of 7.3 is added in the same volume as the addition amount of the sulfo-SMCC solution, and the reaction is continued at room temperature for 12 min; the borate buffer is replaced using a PD-10 desalting column, and concentrated to 3 mg / mL for standby;

[0074] 6) The thiol group-introduced antibody of step 3) and the activated BSA of step 5) are mixed in a molar ratio of 1:1, and reacted at 4°C for 12 hours, and the product is purified to obtain an antibody-BSA complex;

[0075] 7) The magnetic microparticles coupled with a tosyl group with an average particle size of 1.5 μm are diluted to 10 mg / mL with a 0.01M phosphate buffer with a pH of 7.4; the antibody-BSA complex is added in a mass ratio of magnetic microparticles: antibody-BSA complex = 10:0.1;

[0076] 8) 1M ammonium sulfate solution is added in an amount of 1 times the total volume of the magnetic microparticles and the antibody-BSA complex; and the reaction is carried out at 25°C for 10 hours;

[0077] 9) After magnetic separation and washing, a blocking solution containing BSA is used, and the reaction is carried out at 25°C for 10 hours;

[0078] 10) The above blocking solution is diluted to a concentration of 10 mg / mL.

[0079] Comparative Example 1

[0080] The prostate specific antigen (PSA) test kit of Comparative Example 1 is different from that of Example 2 in that the PSA antibody-BSA complex-coated magnetic microparticles of Comparative Example 2 are replaced with PSA antibody-coated magnetic microparticles.

[0081] The method for preparing the PSA antibody-coated magnetic microparticles is as follows:

[0082] The PSA antibody is replaced with a borate buffer using a PD-10 desalting column, and concentrated to 2 mg / mL using a concentration tube;

[0083] The magnetic microparticles coupled with tosyl groups having an average particle size of 1.5 μm are diluted to 10 mg / mL using a 0.01 M borate buffer having a pH of 7.4, and the PSA antibody is added in a mass ratio of magnetic microparticles: PSA antibody = 10:0.1;

[0084] 1 M ammonium sulfate solution is added in an amount of 1 times the total volume of the magnetic microparticles and the PSA antibody, and reacted at 25°C for 10 hours. After magnetic separation and washing, a blocking solution containing BSA is added, and reacted at 25°C for 10 hours. The blocking solution is diluted to a concentration of 10 mg / mL.

[0085] Sensitivity test

[0086] The kits of Example 1 and Comparative Example 1 were used to detect the calibrators in parallel, and the luminescence values of the calibrator tests are shown in Table 1.

[0087] Table 1 Luminescence values of calibrator tests

[0088]

[0089]

[0090] The results of Table 1 show that the luminescence values of the PSA antibody-BSA complex-coated magnetic microparticles are nearly 80% higher than those of the ordinary coupling method, and the sensitivity is higher and the raw materials are saved.

[0091] Stability test

[0092] The kit of Example 1 was stored at 37°C and 4°C, respectively, and was subjected to a tracking test.

[0093] The kit stored at 37°C was tested for luminescence values (RLU) twice on day 0 and day 7, the average values were calculated, and the relative deviation from the results of day 0 was calculated. The results are shown in Table 2.

[0094] Table 2 Results of the stability test of the kit at 37°C

[0095]

[0096] The kit stored at 4°C was tested twice for luminescence value (RLU) at the 0th month, 6th month, 9th month, 12th month and 14th month, the average value was calculated, and the relative deviation was calculated with the result of the 0th month. The results are shown in Table 3.

[0097] Table 3 Kit 4°C stability test results

[0098]

[0099]

[0100] The results of Table 2 and Table 3 show that all relative deviations are <10%. It indicates that the kit prepared by the PSA antibody-BSA complex coated magnetic microparticles of the present application has good stability.

[0101] Correlation verification

[0102] The kit of Example 1 of the present application and the kit of Comparative Example 1 simultaneously detect 56 samples, and the correlation with Roche measurement value is as shown in Figure 1 (comparative example 1 kit) and Figure 2 (example 1 kit) as shown.

[0103] As can be seen from the figure, the PSA antibody-BSA complex coated magnetic microparticles of the present application can reduce the steric hindrance of the antibody and the magnetic beads, and the correlation of the test sample can be significantly improved.

[0104] Example 2

[0105] The specific components of the squamous cell carcinoma antigen (SCC) detection kit are as follows:

[0106] (1) Reagent M: SCC antibody-BSA complex coated magnetic microparticles 0.5 mg / ml, 0.05M tris-HCl buffer, 1% BSA, 0.1% Proclin300;

[0107] (2) Enzyme conjugate: SCC labeled antibody-AP complex, 0.05M tris-HCl buffer, 1% sucrose, 1% BSA, 0.1% Proclin300, 1mM magnesium chloride, 0.1mM zinc chloride;

[0108] (3) Calibrator: dilute the SCC antigen into a series of gradients with BSA containing tris-HCl buffer, 0ng / mL, 0.5ng / mL, 1.5ng / mL, 5ng / mL, 20ng / mL, 70ng / mL.

[0109] The preparation method of the above-mentioned SCC antibody-BSA complex coated magnetic microparticles is as follows:

[0110] 1) The SCC antibody is replaced with a PD-10 desalting column with a borate buffer, and concentrated to 4 mg / mL with a concentration tube;

[0111] 2) 2IT is dissolved in a borate buffer to a concentration of 10 mg / ml, and the SCC antibody solution and 2IT solution are mixed so that the mass ratio of 2-iminothiolane hydrochloride to antibody is 1:30, and after mixing, the reaction is carried out at room temperature for 20 min;

[0112] 3) 0.5M glycine solution with a pH of 7.4 is added, and the volume added is the same as the amount of 2IT solution added, and the reaction is continued at room temperature for 12 min; the borate buffer is replaced with a PD-10 desalting column, and concentrated to 4 mg / mL for standby;

[0113] 4) BSA is dissolved in a borate buffer to 2 mg / mL, and sulfo-SMCC is dissolved in water to 8 mg / mL, and the BSA solution and sulfo-SMCC solution are mixed so that the mass ratio of sulfo-SMCC to BSA is 1:30, and the activation reaction is carried out at room temperature for 25 min;

[0114] 5) 0.5M glycine solution with a pH of 7.4 is added, and the volume added is the same as the amount of sulfo-SMCC solution added, and the reaction is continued at room temperature for 15 min; the borate buffer is replaced with a PD-10 desalting column, and concentrated to 3 mg / mL for standby;

[0115] 6) The thiol-introduced antibody of step 3) and the activated BSA of step 5) are mixed according to a molar ratio of 1.5:1, and reacted at 4°C for 15 hours, and the product is purified to obtain an antibody-BSA complex;

[0116] 7) Magnetic microparticles coupled with a tosyl group with an average particle size of 1.5 μm are diluted to 10 mg / mL with a 0.02M phosphate buffer with a pH of 7.4; the antibody-BSA complex is added in a mass ratio of magnetic microparticles: antibody-BSA complex = 10:0.05;

[0117] 8) 2M ammonium sulfate solution is added according to 1 times the total volume of the magnetic microparticles and the antibody-BSA complex; and the reaction is carried out at 25°C for 15 hours;

[0118] 9) After magnetic separation and washing, a blocking solution containing BSA is used, and the reaction is carried out at 25°C for 8 hours;

[0119] 10) The above blocking solution is diluted to a concentration of 10 mg / mL.

[0120] Comparative Example 2

[0121] The SCC detection kit of Comparative Example 2 differs from that of Example 2 in that the SCC antibody-BSA complex-coated magnetic microparticles are replaced with SCC antibody-coated magnetic microparticles.

[0122] The method for preparing the SCC antibody-coated magnetic microparticles is as follows:

[0123] The SCC antibody is replaced with a borate buffer using a PD-10 desalting column, and concentrated to 4 mg / mL using a concentration tube;

[0124] The magnetic microparticles coupled with toluenesulfonyl groups having an average particle size of 1.5 μm are diluted to 10 mg / mL using a 0.02 M phosphate buffer at pH 7.4; the SCC antibody is added in a ratio of magnetic microparticles:SCC antibody = 10:0.05 by mass;

[0125] 2 M ammonium sulfate solution is added in an amount equal to 1 times the total volume of the magnetic microparticles and the SCC antibody; the reaction is carried out at 25°C for 15 hours; after magnetic separation and washing, a blocking solution containing BSA is added, and the reaction is carried out at 25°C for 8 hours; the blocking solution is diluted to a concentration of 10 mg / mL.

[0126] Sensitivity test

[0127] The calibration samples are detected in parallel using the kits of Example 2 and Comparative Example 2, and the luminescence values of the calibration sample tests are shown in Table 4.

[0128] Table 4 Luminescence values of calibration sample tests

[0129]

[0130] The results of Table 4 show that the luminescence values obtained using the SCC antibody-BSA complex-coated magnetic microparticles are nearly 100% higher than those obtained using the ordinary coupling method, and the sensitivity is higher and the raw materials are saved.

[0131] Stability test

[0132] The kit of Example 2 is stored at 37°C and 4°C, respectively, and is subjected to a tracking test.

[0133] The kit stored at 37°C is tested for luminescence values (RLU) twice on day 0 and day 7, the average value is calculated, and the relative deviation from the day 0 result is calculated. The results are shown in Table 5.

[0134] Table 5 Kit stability test results at 37°C

[0135]

[0136] The kit stored at 4°C was tested twice for luminescence value (RLU) at the 0th month, 6th month, 9th month, 12th month and 14th month, the average value was calculated, and the relative deviation was calculated with the result of the 0th month. The results are shown in Table 6.

[0137] Table 6 Kit 4°C stability test results

[0138]

[0139] The results of Table 5 and Table 6 show that all relative deviations are <10%. It indicates that the kit prepared by the magnetic microparticles coated with the SCC antibody-BSA complex of the present application has good stability.

[0140] Correlation verification

[0141] The kit of Example 2 of the present application and the kit of Comparative Example 2 simultaneously detect 80 samples, and the correlation with Abbott measurement is shown in Figure 3 (comparative example 2 kit) and Figure 4 (example 2 kit) as shown.

[0142] As can be seen from the figure, using the magnetic microparticles coated with the SCC antibody-BSA complex of the present application does not affect the specificity of sample testing, achieving improved sensitivity, cost savings without affecting the performance of the reagent.

[0143] Aspects, embodiments, features of the present application should be considered illustrative in all aspects and not limiting the present application, and the scope of the present application is only defined by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed present application.

[0144] In the preparation method of the present application, the order of each step is not limited to the order listed, and for those skilled in the art, the order of each step can be changed without creative labor, which is within the protection scope of the present application. In addition, two or more steps or actions can be performed simultaneously.

[0145] Finally, it should be noted that the specific embodiments described herein are merely illustrative of the present application and do not limit the embodiments of the present application. Those skilled in the art can make various modifications or supplements to the described specific embodiments or use similar ways to replace them, and here it is not necessary or possible to fully exemplify all embodiments. Any obvious changes or variations derived from the essential spirit of the present application still fall within the protection scope of the present application, and any additional limitation is contrary to the spirit of the present application.

Claims

1. A method for preparing magnetic microparticles coated with an antibody-BSA complex, characterized in that, Includes the following steps: The antibody-BSA complex and magnetic microparticles were mixed, and then a reaction promoter was added to react. After washing and blocking, the magnetic microparticles coated with the antibody-BSA complex were obtained. The reaction promoter was an ammonium sulfate solution or a potassium sulfate solution. The preparation method of the antibody-BSA complex includes the following steps: After reacting 2-iminothionane hydrochloride and antibody at room temperature for 10-50 min, a glycine solution with pH 7.0-7.5 is added, and the reaction continues at room temperature for 5-20 min to introduce thiol groups onto the antibody. After activating 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester and / or its derivatives with BSA at room temperature for 10-50 min, a glycine solution with pH 7.0-7.5 is added, and the reaction continues at room temperature for 10-20 min. Then, the thiol-introduced antibody and activated BSA are reacted at 2-8°C for 8-20 hours.

2. The preparation method according to claim 1, characterized in that, The mass ratio of 2-iminothionane hydrochloride to antibody is 1:10 to 1:

100.

3. The preparation method according to claim 1, characterized in that, The mass ratio of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester and / or its derivatives to the activation reaction of BSA is 1:10 to 1:

100.

4. The preparation method according to claim 1, characterized in that, The molar ratio of the antibody with introduced thiol groups to the activated BSA is 0.5:1 to 3:

1.

5. The preparation method according to claim 1, characterized in that, The mass ratio of magnetic microparticles to antibody-BSA complex is 10:0.05 to 10:0.

5.

6. The preparation method according to claim 1, characterized in that, Add a reaction promoter and react at 24~42℃ for 4~24 hours.

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