Method for preparing magnetic microsphere for separation of biological protein and use thereof

Abstract

Disclosed is a method for preparing a magnetic microsphere for the separation of biological proteins. A magnetic microsphere matrix is treated by formulating and using an appropriate emulsified liquid, and modification of the surface of the magnetic microsphere matrix is realized by emulsion polymerization, thereby obtaining a magnetic microsphere coated with a polyacrylate polymer layer. Said emulsified liquid comprises the following components therein: acyclic acid monoester compounds, acyclic acid glycol compounds, initiators and optionally anionic surface active agents and water. The magnetic microsphere significantly reduces the non-specific adsorption of other proteins, without affecting the joining ability for a specific protein, when used in the separation of biological proteins. A new selection is provided to realize the separation engineering of high protein specificity adsorption.

Claims

1. A method for preparing a magnetic microsphere for separation of biological protein, characterized in that it comprises: a. preparing an emulsion; b. dispersing a magnetic microsphere matrix with magnetism into the emulsion to generate a dispersion system; and c. subjecting the dispersion to a polymerization reaction; wherein the magnetic microsphere matrix is a magnetic particle modified with an active group on surface thereof; further wherein the active group is at least one selected from the group consisting of: a carboxyl group and a sulfonic acid group; and wherein the emulsion comprises the following components: an acrylic monoester compound selected from the group consisting of methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, n-propyl methacrylate, hydroxypropyl methacrylate, n-butyl methacrylate, and hydroxybutyl methacrylate; an acrylic diol ester compound selected from the group consisting of glycol dimethacrylate, 1,3-propanediol dimethacrylate, 1,3-butanediol dimethacrylate, 1,4-butanediol dimethacrylate, neopentyl glycol dimethacrylate and 1,6-hexanediol dimethacrylate; an anionic surfactant selected from the group consisting of sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium hexadecyl sulfonate, and sodium n-decyl sulfate; water; and a water-soluble initiator selected from the group consisting of sodium persulfate, potassium persulfate, and ammonium persulfate.

2. The method of claim 1, characterized in that the emulsion comprises 0.5 to 30 wt % of the acrylic monoester compound, 0.05 to 5 wt % of the acrylic diol ester compound, 0.2 to 2 wt % of the water-soluble initiator, 0.1 to 1 wt % of the anionic surfactant, and 62 to 99 wt % of water, based on the total weight of the emulsion.

3. The method of claim 1, characterized in that the weight content of the acrylic diol ester compound is 5 to 15% of the weight content of the acrylic monoester compound.

4. The method of claim 3, characterized in that the weight content of the acrylic diol ester compound is 10% of the weight content of the acrylic monoester compound.

5. The method of claim 1, characterized in that the active group is capable of bonding with a biological protein.

6. The method of claim 1, characterized in that the particle size of the magnetic microsphere matrix is 0.1 to 5 m.

7. The method of claim 1, characterized in that the magnetic microsphere matrix in the dispersion system has a concentration of 10 to 150 mg/m L.

8. The method of claim 7, characterized in that the polymerization reaction in the step c is performed under a temperature of 60 to 90 C. with a reaction time of 10-40 h.

9. The method of claim 1, characterized in that in the step a, the emulsion is prepared by mixing together the components of the emulsion uniformly; and the method further comprises a step d following the step c: performing a solid-liquid separation with the product of the step c and washing solid matter obtained therefrom to generate the magnetic microsphere; wherein the solid-liquid separation in the step d is performed by magnetic separation or centrifugation, the washing is performed for a plurality of times with organic solvent and water in sequence, and the organic solvent is one or more selected from the group consisting of alcohol, ester, and halogenated hydrocarbon.

10. The method of claim 1, wherein the active group is a carboxyl group.

11. The method of claim 1, wherein the acrylic monoester compound is methyl methacrylate.

12. The method of claim 1, wherein the acrylic diol ester is glycol dimethacrylate.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

(1) Further description of the present disclosure will be provided below with specific embodiments. However, it should be understood that the scope of the present disclosure is not limited thereto.

Example 1

1) Preparation of Magnetic Microsphere Matrix

(2) The magnetic microsphere matrix with carboxyl functional group used in the present example was prepared according to Example 1 of the Chinese Patent CN92105584.

2) Preparation of Emulsion

(3) The components of the emulsion and their percentages by weight were as follows:

(4) TABLE-US-00001 Methyl methacrylate .sup.5%; Ethylene glycol dimethacrylate 0.5%; Potassium persulfate 0.3%; Sodium dodecyl sulfate .sup.0.2%; and Purified Water 94%.

(5) The above components were mixed according to their proportions, sonicated to fully dissolve the solid components, and further well mixed.

3) Preparation of Dispersion System

(6) The magnetic microsphere matrix obtained from step 1) was dispersed in the emulsion prepared from step 2) at a concentration of 20 mg/mL, and sonicated for uniform dispersion of the magnetic microsphere matrix to obtain a dispersion system.

4) Polymerization Reaction and Post-Treatment

(7) The dispersion system obtained from step 3) was stirred at 75 C. for 30 hours. The supernatant was removed by magnetic separation. The solids were washed 3 times with methanol and 5 times with water to give the magnetic microspheres.

5) Determination of Binding Performance of Surface Active Groups of Magnetic Microspheres

(8) 5a) Choice of test samples: toxoplasma IgG (TOXO IgG) negative and positive samples (n=10 for each), glutamic acid decarboxylase (GAD65) negative and positive samples (n=10 for each), and cytomegalovirus IgM (CMV IgM) negative and positive samples (n=10 for each). All of the above samples were confirmed by clinical verification.

(9) 5b) Antigen-coated magnetic microspheres: The TOXO antigen, GAD65 antigen, and CMV antigen were used for coating the magnetic microspheres prepared in the present example. The coated magnetic microspheres were diluted with phosphate buffer saline (PBS) by a certain proportion to a working concentration of 0.5 mg/mL.

(10) 5c) Preparation of luminescent markers: ABEI (N-(4-aminobutyl)-N-ethyl isoluminol)-labeled monoclonal mouse anti-human IgG antibody (anti-hIgG-ABEI) (diluted with PBS to 0.02 g/mL), ABEI-labeled staphylococcal protein A (SPA-ABEI) (diluted with PBS to 0.04 g/mL), and ABEI-labeled mouse anti-human IgM monoclonal antibody (anti-hIgM-ABEI) (diluted with PBS to 0.02 g/mL) were used respectively as the luminescent markers for detection of TOXO IgG, GAD65, and CMV IgM.

(11) 5d) Method for sample adding determination: Toxoplasma IgG antibody (TOXO IgG) assay kit, glutamic acid decarboxylase antibody (GAD65) assay kit, and cytomegalovirus IgM antibody (CMV IgM) assay kit from New Industries Biomedical Engineering Co., LTD (Shenzhen, China) were used in sample adding determination according to the corresponding kit instructions. Luminescent signal intensities were determined using MAGLUMI 2000 Plus Automated Chemiluminescence Analyzer (New Industries Biomedical Engineering Co., LTD, Shenzhen, China).

(12) The results are shown in Table 1 to 3.

6) Non-Specific Adsorption Test of Magnetic Microspheres

(13) 6a) Choice of test samples: normal human serum with clinically verified negativity for TOXO IgG, GAD65 and CMV IgM.

(14) 6b) Preparation of luminescent markers: ABEI (N-(4-aminobutyl)-N-ethyl isoluminol)-labeled monoclonal mouse anti-human IgG antibody (anti-hIgG-ABEI) (1:4000 diluted with PBS), ABEI-labeled staphylococcal protein A (SPA-ABEI) (1:3000 diluted with PBS), and ABEI-labeled mouse anti-human monoclonal IgM antibody (anti-hIgM-ABEI) (1:4000 diluted with PBS) were used as luminescent markers for detecting TOXO IgG, GAD65, and CMV IgM.

(15) 6c) Antigen-coated magnetic microspheres: The TOXO antigen, GAD65 antigen, and CMV antigen were used for coating the magnetic microspheres of the present example. The coated magnetic microspheres were diluted with phosphate buffer saline (PBS) by a certain proportion to a working concentration of 0.5 mg/mL.

(16) 6d) Method for sample adding determination: Toxoplasma IgG antibody (TOXO IgG) assay kit, glutamic acid decarboxylase antibody (GAD65) assay kit, and cytomegalovirus IgM antibody (CMV IgM) assay kit from New Industries Biomedical Engineering Co., LTD (Shenzhen, China) were used in sample adding determination according to the corresponding kit instructions. The results are shown in Table 4.

Comparative Example 1

(17) The magnetic microsphere matrix was prepared according to the method disclosed in the Chinese Patent CN92105584, and tested for the binding performance and non-specific adsorption of their surface active groups. The tests were performed in similar process and conditions according to the steps 5) and 6) of Example 1, except that the magnetic microsphere matrix used in the steps 5) and 6) of Example 1 were replaced with the magnetic microspheres prepared in the present comparative example. The results are shown in Table 1 to 4.

(18) TABLE-US-00002 TABLE 1 Luminescent signal Luminescent signal intensity and intensity and negative/positive Se- negative/positive determination Positive rum determination of the magnetic or Sam- of the magnetic microspheres of Test Negative ple microspheres of comparative Item Sample ID Example 1 Example 1 CMV Negative C336 5356 (Negative) 105983 (Positive) IgM C128 1208 (Negative) 62563 (Positive) C500 4211 (Negative) 8359 (Negative) C228 1806 (Negative) 5698 (Negative) C86 2149 (Negative) 7562 (Negative) C471 2350 (Negative) 2359 (Negative) C60 4215 (Negative) 10698 (Negative) C348 1089 (Negative) 2439 (Negative) C502 4356 (Negative) 5326 (Negative) C377 6607 (Negative) 7659 (Negative) Positive CP1 159563 (Positive) 165230 (Positive) CP2 32859 (Positive) 23597 (Positive) CP3 60538 (Positive) 53695 (Positive) CP4 254714 (Positive) 247796 (Positive) CP5 114689 (Positive) 120874 (Positive) CP6 70899 (Positive) 60899 (Positive) CP7 240563 (Positive) 257465 (Positive) CP8 95686 (Positive) 105693 (Positive) CP9 95631 (Positive) 75132 (Positive) CP10 586044 (Positive) 569212 (Positive) Note: Luminescent signal intensity is marked as positive when above 20,000 or negative when below 20,000. The same applies below.

(19) TABLE-US-00003 TABLE 2 Luminescent signal Luminescent signal intensity and intensity and negative/positive Se- negative/positive determination Positive rum determination of the magnetic or Sam- of the magnetic microspheres of Test Negative ple microspheres of comparative Item Sample ID Example 1 Example 1 TOXO Negative C336 7546 (Negative) 1168936 (Positive) IgG C348 5327 (Negative) 5632 (Negative) C389 2358 (Negative) 905326 (Positive) C323 4781 (Negative) 5356 (Negative) C369 5632 (Negative) 153262 (Positive) C350 8461 (Negative) 50329 (Negative) C377 2596 (Negative) 268954 (Positive) C86 5632 (Negative) 7653 (Negative) C60 2579 (Negative) 4698 (Negative) C102 5612 (Negative) 7658 (Negative) Positive TP1 286591 (Positive) 295336 (Positive) TP2 168354 (Positive) 170562 (Positive) TP3 105936 (Positive) 98653 (Positive) TP4 468992 (Positive) 450879 (Positive) TP5 56798 (Positive) 50598 (Positive) TP6 136587 (Positive) 143287 (Positive) TP7 154805 (Positive) 168953 (Positive) TP8 347761 (Positive) 332567 (Positive) TP9 269547 (Positive) 254687 (Positive) TP10 165392 (Positive) 170589 (Positive)

(20) TABLE-US-00004 TABLE 3 Luminescent signal Luminescent signal intensity and intensity and negative/positive Se- negative/positive determination Positive rum determination of the magnetic or Sam- of the magnetic microspheres of Test Negative ple microspheres of comparative Item Sample ID Example 1 Example 1 GAD65 Negative C336 5669 (Negative) 496652 (Positive) C348 3569 (Negative) 3956 (Negative) C369 7452 (Negative) 6724 (Negative) C323 4689 (Negative) 8693 (Negative) C377 3569 (Negative) 2563 (Negative) C321 5689 (Negative) 6395 (Negative) C350 4567 (Negative) 7841 (Negative) C60 5966 (Negative) 3633 (Negative) C471 5368 (Negative) 5689 (Negative) C500 2045 (Negative) 196745 (Positive) Positive GP1 40569 (Positive) 36995 (Positive) GP2 58611 (Positive) 56321 (Positive) GP3 106335 (Positive) 86596 (Positive) GP4 175986 (Positive) 187642 (Positive) GP5 40539 (Positive) 46952 (Positive) GP6 363571 (Positive) 369882 (Positive) GP7 140266 (Positive) 132569 (Positive) GP8 80563 (Positive) 78536 (Positive) GP9 253658 (Positive) 265714 (Positive) GP10 87420 (Positive) 86536 (Positive)

(21) It can be seen from above Tables 1 to 3, TOXO antigen, GAD65 antigen, and CMV antigen coating the magnetic microspheres prepared according to Example 1 of the present disclosure demonstrated good binding ability with the corresponding antibody-positive samples, respectively, while presenting a completely negative signal for corresponding antibody-negative samples. This indicated that the magnetic microspheres prepared according to Example 1 disclosed herein had surface active groups with excellent binding ability with proteins, i.e., antigens. The binding of the surface active groups of the magnetic microspheres to the proteins was not affected at all by the polyacrylate polymers on the surface of the magnetic microspheres.

(22) TABLE-US-00005 TABLE 4 Luminescent signal Luminescent signal intensity and intensity and negative/positive Se- negative/positive determination rum determination of the magnetic Sam- of the magnetic microspheres of Test ple microspheres of comparative Item ID Example 1 Example 1 TOXO C336 4284 (Negative) 1224643 (Positive) IgG C348 3999 (Negative) 3792 (Negative) C389 2736 (Negative) 824763 (Positive) C323 5742 (Negative) 6224 (Negative) C369 4840 (Negative) 123416 (Positive) C350 5631 (Negative) 62498 (Positive) C377 3829 (Negative) 243310 (Positive) C86 8763 (Negative) 8511 (Negative) C60 6012 (Negative) 5491 (Negative) C102 5843 (Negative) 6161 (Negative) GAD65 C336 4464 (Negative) 628768 (Positive) C348 3129 (Negative) 2560 (Negative) C369 5412 (Negative) 3072 (Negative) C323 3408 (Negative) 103748 (Positive) C377 4012 (Negative) 3178 (Negative) C321 3218 (Positive) 3400 (Negative) C350 3520 (Negative) 3078 (Negative) C60 1982 (Negative) 2634 (Negative) C471 2561 (Negative) 22766 (Positive) C500 2973 (Negative) 201428 (Positive) CMV C336 2636 (Negative) 133538 (Positive) IgM C128 2715 (Negative) 139161 (Positive) C500 5012 (Negative) 5843 (Negative) C228 1892 (Negative) 3584 (Negative) C86 2360 (Negative) 4509 (Negative) C471 1743 (Negative) 3764 (Negative) C60 3264 (Negative) 202597 (Positive) C348 3464 (Negative) 2676 (Negative) C502 1525 (Negative) 2127 (Negative) C377 3672 (Negative) 2085 (Negative)

(23) As indicated by Table 4, the magnetic microspheres prepared according to Example 1 disclosed herein had an obviously lower non-specific adsorption of antibodies in the samples than the non-specific adsorption of antibodies of the magnetic microsphere matrix in Comparative Example 1 that was not modified with the emulsion prepared as disclosed herein. In addition, for some special samples, Comparative Example 1 had falsely high test results which were not consistent with the clinical samples. The experiments and their data above has demonstrated that the magnetic microspheres in Example 1 disclosed herein have significant effects in reducing or avoiding non-specific protein adsorption.

Example 2

1) Preparation of Magnetic Microsphere Matrix

(24) The magnetic microsphere matrix used in the present example was prepared according to the Chinese Patent Application Publication CN102746529A, especially Examples 1 to 6 thereof. That is, seed particles of polystyrene polymer were prepared, then modified with emulsion containing polyvinylpyrrolidone, divinylbenzene, styrene, and toluene (porogen), and subjected to emulsion polymerization to obtain porous polystyrene particles. The porous polystyrene particles were nitrified with nitric acid and incorporated with iron using FeSO.sub.4. The resulting particles were coated and carboxyl functionalized to give the magnetic microsphere matrix with carboxyl functional groups used in the present example.

2) Preparation of Emulsion

(25) The components of the emulsion and their percentages by weight were as follows:

(26) TABLE-US-00006 Hydroxyethyl methacrylate 0.5%; Neopentyl glycol dimethacrylate 0.05%; Ammonium persulfate 0.2%; Sodium dodecyl sulfonate, .sup.0.5%; and Purified water 98.75%.

(27) The above components were mixed according to their proportions, sonicated to fully dissolve the solid components, and further well mixed.

3) Preparation of Dispersion System

(28) The magnetic microsphere matrix obtained from step 1) was dispersed in the emulsion prepared from step 2) at a concentration of 40 mg/mL, and sonicated for uniform dispersion of the magnetic microsphere matrix to obtain a dispersion system.

4) Polymerization Reaction and Post-Treatment

(29) The dispersion system obtained from step 3) was stirred at 80 C. for 18 hours. The supernatant was removed by magnetic separation. The solids were washed 3 times with methanol and 5 times with water to give the magnetic microspheres.

5) Determination of Binding Performance of Surface Active Groups of Magnetic Microspheres

(30) The tests were performed similarly to the step 5) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example. The results are shown in Tables 5-7.

6) Non-Specific Adsorption Test of Magnetic Microspheres

(31) The tests were performed by repeating the step 6) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example. The results are shown in Table 8.

Example 3

(32) The experiment was carried out following the procedures of Example 2, except that the emulsion of Example 2 was replaced with the emulsion described below.

(33) The components of the emulsion and their percentages by weight were as follows:

(34) TABLE-US-00007 Hydroxyethyl methacrylate 0.5%; Neopentyl glycol dimethacrylate 0.2%; Ammonium persulfate 0.2%; Sodium dodecyl sulfonate, .sup.0.5%; and Purified water 98.6%.

(35) The results are shown in Tables 5 to 8.

Comparative Example 2

(36) The magnetic microsphere matrix was prepared according to the Chinese Patent Application Publication CN102746529A, especially Examples 1 to 4 thereof, and tested for the binding performance and non-specific adsorption of their surface active groups. The tests were performed in similar process and conditions according to the steps 5) and 6) of Example 2, except that the magnetic microsphere matrix used in the steps 5) and 6) of Example 2 were replaced with the magnetic microspheres prepared in the present example. The results are shown in Tables 5 to 8.

(37) TABLE-US-00008 TABLE 5 Luminescent signal Luminescent signal Luminescent signal intensity and intensity and intensity and negative/positive Se- negative/positive negative/positive determination Positive rum determination determination of the magnetic or Sam- of the magnetic of the magnetic microspheres of Test Negative ple microspheres of microspheres of Comparative Item Sample ID Example 2 Example 3 Example 2 CMV Negative C336 4728 (Negative) 5044 (Negative) 987762 (Positive) IgM C128 5689 (Negative) 5682 (Negative) 156942 (Positive) C500 3529 (Negative) 3768 (Negative) 5782 (Negative) C228 5430 (Negative) 6048Negative) 2634 (Negative) C86 1945 (Negative) 2044 (Negative) 6851 (Negative) C471 2356 (Negative) 3086 (Negative) 3654 (Negative) C60 4879 (Negative) 5047 (Negative) 4810 (Negative) C348 2561 (Negative) 2758 (Negative) 5730 (Negative) C502 5873 (Negative) 6048 (Negative) 6820 (Negative) C377 5126 (Negative) 4985 (Negative) 7873 (Negative) Positive CP1 175642 (Positive) 172484 (Positive) 145637 (Positive) CP2 43215 (Positive) 49387 (Positive) 26548 (Positive) CP3 57863 (Positive) 68240 (Positive) 65487 (Positive) CP4 234561 (Positive) 234578 (Positive) 235611 (Positive) CP5 108431 (Positive) 118524 (Positive) 102453 (Positive) CP6 894321 (Positive) 824554 (Positive) 785421 (Positive) CP7 345210 (Positive) 304117 (Positive) 301245 (Positive) CP8 86510 (Positive) 86584 (Positive) 98703 (Positive) CP9 76431 (Positive) 74438 (Positive) 103241 (Positive) CP10 421302 (Positive) 440378 (Positive) 486310 (Positive)

(38) TABLE-US-00009 TABLE 6 Luminescent signal Luminescent signal Luminescent signal intensity and intensity and intensity and negative/positive Se- negative/positive negative/positive determination Positive rum determination determination of the magnetic or Sam- of the magnetic of the magnetic microspheres of Test Negative ple microspheres of microspheres of Comparative Item Sample ID Example 2 Example 3 Example 2 TOXO Negative C336 5412 (Negative) 6685 (Negative) 1251097 (Positive) IgG C348 5687 (Negative) 5487 (Negative) 6583 (Negative) C389 3654 (Negative) 1568 (Negative) 897621 (Positive) C323 4628 (Negative) 5724 (Negative) 4598 (Negative) C369 5132 (Negative) 5252 (Negative) 168439 (Positive) C350 2356 (Negative) 4435 (Negative) 19874 (Negative) C377 3541 (Negative) 2015 (Negative) 284710 (Positive) C86 6874 (Negative) 7593 (Negative) 8910 (Negative) C60 3559 (Negative) 3554 (Negative) 5431 (Negative) C102 6215 (Negative) 6861 (Negative) 7357 (Negative) Positive TP1 275610 (Positive) 245810 (Positive) 259841 (Positive) TP2 176354 (Positive) 203357 (Positive) 201684 (Positive) TP3 96854 (Positive) 95794 (Positive) 102543 (Positive) TP4 385672 (Positive) 307972 (Positive) 475689 (Positive) TP5 46983 (Positive) 52988 (Positive) 49612 (Positive) TP6 156432 (Positive) 142583 (Positive) 184256 (Positive) TP7 179531 (Positive) 205953 (Positive) 135682 (Positive) TP8 354613 (Positive) 330610 (Positive) 354621 (Positive) TP9 254983 (Positive) 304988 (Positive) 201873 (Positive) TP10 196543 (Positive) 195579 (Positive) 196826 (Positive)

(39) TABLE-US-00010 TABLE 7 Luminescent signal Luminescent signal Luminescent signal intensity and intensity and intensity and negative/positive Se- negative/positive negative/positive determination Positive rum determination determination of the magnetic or Sam- of the magnetic of the magnetic microspheres of Test Negative ple microspheres of microspheres of Comparative Item Sample ID Example 2 Example 3 Example 2 GAD65 Negative C336 5596 (Negative) 4586 (Negative) 516892 (Positive) C348 3854 (Negative) 4573 (Negative) 4195 (Negative) C369 7261 (Negative) 6678 (Negative) 5792 (Negative) C323 4195 (Negative) 4458 (Negative) 7982 (Negative) C377 2943 (Negative) 3044 (Negative) 3018 (Negative) C321 6813 (Negative) 8427 (Negative) 5943 (Negative) C350 5192 (Negative) 6458 (Negative) 8019 (Negative) C60 5713 (Negative) 4397 (Negative) 3529 (Negative) C471 4952 (Negative) 5521 (Negative) 4695 (Negative) C500 2946 (Negative) 3486 (Negative) 186452 (Positive) Positive GP1 50136 (Positive) 82754 (Positive) 49613 (Positive) GP2 69842 (Positive) 75864 (Positive) 47621 (Positive) GP3 163542 (Positive) 182475 (Positive) 98651 (Positive) GP4 132549 (Positive) 124458 (Positive) 175626 (Positive) GP5 50134 (Positive) 66457 (Positive) 56138 (Positive) GP6 431562 (Positive) 337581 (Positive) 416235 (Positive) GP7 150234 (Positive) 204574 (Positive) 153426 (Positive) GP8 90125 (Positive) 100254 (Positive) 86423 (Positive) GP9 176950 (Positive) 186990 (Positive) 214683 (Positive) GP10 65432 (Positive) 66784 (Positive) 95634 (Positive)

(40) It can be seen from the above Table 5-7, TOXO antigen, GAD65 antigen, and CMV antigen coating the magnetic microspheres prepared according to Examples 2 and 3 of the present disclosure demonstrated good binding ability with the corresponding antibody-positive samples, respectively, while presenting a completely negative signal for corresponding antibody-negative samples. This indicated that the magnetic microspheres prepared according to Examples 2 and 3 disclosed herein had surface active groups with excellent binding ability with proteins, i.e., antigens. The binding of the surface active groups of the magnetic microspheres to the proteins was not affected at all by the polyacrylate polymers on the surface of the magnetic microspheres.

(41) TABLE-US-00011 TABLE 8 Luminescent signal Luminescent signal Luminescent signal intensity and intensity and intensity and negative/positive Se- negative/positive negative/positive determination rum determination determination of the magnetic Sam- of the magnetic of the magnetic microspheres of Test ple microspheres of microspheres of comparative Item ID Example 2 Example 3 Example 2 TOXO C336 5613 (Negative) 6717 (Negative) 135649 (Positive) IgG C348 4201 (Negative) 4254 (Negative) 6739 (Negative) C389 3265 (Negative) 3858 (Negative) 984352 (Positive) C323 4952 (Negative) 5554 (Negative) 7762 (Negative) C369 5169 (Negative) 4867 (Negative) 163852 (Positive) C350 4821 (Negative) 2021 (Negative) 59613 (Positive) C377 2956 (Negative) 3467 (Negative) 34162 (Positive) C86 9543 (Negative) 10420 (Negative) 6591 (Negative) C60 6830 (Negative) 7240 (Negative) 6135 (Negative) C102 2465 (Negative) 1867 (Negative) 4261 (Negative) GAD65 C336 5913 (Negative) 6785 (Negative) 694351 (Positive) C348 4256 (Negative) 4572 (Negative) 6521 (Negative) C369 6289 (Negative) 4420 (Negative) 2564 (Negative) C323 6205 (Negative) 5796 (Negative) 4694 (Negative) C377 6031 (Negative) 6457 (Negative) 6191 (Negative) C321 4316 (Negative) 4358 (Negative) 4603 (Negative) C350 2546 (Negative) 2781 (Negative) 6405 (Negative) C60 3194 (Negative) 3368 (Negative) 2019 (Negative) C471 2468 (Negative) 2941 (Negative) 3461 (Negative) C500 4619 (Negative) 5501 (Negative) 235874 (Positive) CMV C336 3491 (Negative) 3934 (Negative) 191635 (Positive) IgM C128 6159 (Negative) 6679 (Negative) 124671 (Positive) C500 3461 (Negative) 3587 (Negative) 3054 (Negative) C228 2016 (Negative) 2847 (Negative) 4916 (Negative) C86 3469 (Negative) 4013 (Negative) 2046 (Negative) C471 1954 (Negative) 2428 (Negative) 4671 (Negative) C60 5068 (Negative) 244618 (Positive) 2749 (Negative) C348 5319 (Negative) 5679 (Negative) 6942 Negative) C502 1605 (Negative) 1785 (Negative) 3491 (Negative) C377 3259 (Negative) 4840 (Negative) 5192 (Negative)

(42) It can be seen from Table 8, the magnetic microspheres prepared according to Examples 2 and 3 disclosed herein had a non-specific adsorption of antibodies in the samples that was obviously lower than that of the magnetic microsphere matrix in Comparative Example 2 which was not modified with the emulsion prepared as disclosed herein. In addition, for some special samples, Comparative Example 2 had falsely high test results which were not consistent with the clinical samples. The experiments and data above had demonstrated that the magnetic microspheres in Examples 2 and 3 disclosed herein had significant effects in reducing or avoiding non-specific protein adsorption.

Example 4

1) Preparation of Magnetic Microsphere Matrix

(43) The magnetic microsphere matrix used in the present example was prepared according to the Chinese Patent Application Publication CN102746529A, especially Examples 1 to 6 thereof.

2) Preparation of Emulsion

(44) The components of the emulsion and their percentages by weight were as follows:

(45) TABLE-US-00012 Butyl methacrylate 10%; 1,6-hexanediol dimethacrylate 2%; Potassium persulfate 1%; Sodium dodecyl sulfate 0.5%; and Water 86.5%.

(46) The above components were mixed according to their proportions, sonicated to fully dissolve the solid components, and emulsified in a high-shear emulsifying machine for 20 minutes.

3) Preparation of Dispersion System

(47) The magnetic microsphere matrix obtained from step 1) was dispersed in the emulsion prepared from step 2) at a concentration of 50 mg/mL, and sonicated for uniform dispersion of the magnetic microsphere matrix to obtain a dispersion system.

4) Polymerization Reaction and Post-Treatment

(48) The dispersion system obtained from step 3) was stirred at 75 C. for 28 hours. The supernatant was removed by magnetic separation. The solids were washed 3 times with methanol and 5 times with water to give the magnetic microspheres.

5) Determination of Binding Performance of Surface Active Groups of Magnetic Microspheres

(49) The tests were performed similarly to the step 5) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example.

(50) It can be seen from the experimental data (not listed here) that the active groups of magnetic microspheres had similar binding capacity with antigen proteins with those prepared in Examples 1 and 2. Antigens coating the magnetic microspheres were excellently binded with the antibodies in the sample, while presenting a completely negative signal in the negative samples.

6) Non-Specific Adsorption Test of Magnetic Microspheres

(51) The tests were performed by repeating the step 6) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example.

(52) It can be seen from the experimental data (not listed here) that, similar as those in Example 1 and 2, the antigen-coated magnetic microspheres prepared according to the present example had little non-specific adsorption in an adsorption test.

Example 5

1) Preparation of Magnetic Microsphere Matrix

(53) The magnetic microsphere matrix used in the present example was prepared according to the Chinese Patent Application Publication CN102746529A, especially Examples 1 to 4 thereof.

2) Preparation of Emulsion

(54) The components of the emulsion and their percentages by weight were as follows:

(55) TABLE-US-00013 Ethyl methacrylate 10%; 1,3-propanediol dimethacrylate 2%; Ammonium persulfate 1%; N-decyl sulfate 0.5%; and Water 86.5%.

(56) The above components were mixed according to their proportions, sonicated to fully dissolve the solid components, and homogenized a high-pressure homogenizer for 20 minutes.

3) Preparation of Dispersion System

(57) The magnetic microsphere matrix obtained from step 1) was dispersed in the emulsion prepared from step 2) at a concentration of 50 mg/mL, and sonicated for uniform dispersion of the magnetic microsphere matrix to obtain a dispersion system.

4) Polymerization Reaction and Post-Treatment

(58) The dispersion system obtained from step 3) was stirred at 70 C. for 24 hours. The supernatant was removed by magnetic separation. The solids were washed 3 times with methanol and 5 times with water to give the magnetic microspheres.

5) Determination of Binding Performance of Surface Active Groups of Magnetic Microspheres

(59) The tests were performed similarly to the step 5) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example.

(60) It can be seen from the experimental data (not listed here) that the active groups of magnetic microspheres had similar binding capacity with antigen proteins with those prepared in Examples 1 and 2. Antigens coating the magnetic microspheres were excellently binded with the antibodies in the sample, while presenting a completely negative signal in the negative samples.

6) Non-Specific Adsorption Test of Magnetic Microspheres

(61) The tests were performed by repeating the step 6) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example.

(62) It can be seen from the experimental data (not listed here) that, similar as those in Example 1 and 2 the antigen-coated magnetic microspheres prepared according to the present example had little non-specific adsorption in an adsorption test.

Example 6

1) Preparation of Magnetic Microsphere Matrix

(63) The magnetic microsphere matrix used in the present example was prepared according to the Chinese Patent Application Publication CN102746529A, especially Examples 1 to 4 thereof.

2) Preparation of Emulsion

(64) The components of the emulsion and their percentages by weight were as follows:

(65) TABLE-US-00014 Hydroxypropyl methacrylate 12%; 1,3-propanediol dimethacrylate 2%; Sodium persulfate 1%; Sodium dodecyl sulfate 0.5%; and Water 84.5%.

(66) The above components were mixed according to their proportions, sonicated to fully dissolve the solid components, and emulsified in a high-shear emulsifying machine for 20 minutes.

3) Preparation of Dispersion System

(67) The magnetic microsphere matrix obtained from step 1) was dispersed in the emulsion prepared from step 2) at a concentration of 100 mg/mL, and sonicated for uniform dispersion of the magnetic microsphere matrix to obtain a dispersion system.

4) Polymerization Reaction and Post-Treatment

(68) The dispersion system obtained from step 3) was stirred at 70 C. for 20 hours. The supernatant was removed by magnetic separation. The solids were washed 3 times with ethanol and 5 times with water to give the magnetic microspheres.

5) Determination of Binding Performance of Surface Active Groups of Magnetic Microspheres

(69) The tests were performed similarly to the step 5) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example.

(70) It can be seen from the experimental data (not listed here) that the active groups of magnetic microspheres have similar binding capacity with antigen proteins as those prepared in Example 1 and 2. Antigens coating the magnetic microspheres were excellently binded with the antibodies in the sample, while presenting a completely negative signal in the negative samples.

6) Non-Specific Adsorption Test of Magnetic Microspheres

(71) The tests were performed by repeating the step 6) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example. It can be seen from the experimental data (not listed here) that, similar as those in Examples 1 and 2, the antigen-coated magnetic microspheres prepared according to the present example had little non-specific adsorption in an adsorption test.

(72) The description above has provided specific details of the present disclosure. However, a person skilled in the art can readily appreciate modifications of the embodiments disclosed herein without deviating from the spirit and scope of the present disclosure. It should also be noted that the respective aspects, various components of different embodiments, and numerous technical features recited herein can be in combined or interchanged in part or in whole. In the embodiment above, each particular embodiment referring to another can be suitably combined with any other embodiments, which could be appreciated by a person skilled in the art. Finally, a person skilled in the art can comprehend that the description above is only for the purpose of illustration by way of example, without limiting the present disclosure in any aspect.