WO2022048350A1 - 基于苯硼酸定向偶联抗体的免疫磁吸附剂及其制备方法 - Google Patents

基于苯硼酸定向偶联抗体的免疫磁吸附剂及其制备方法 Download PDF

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WO2022048350A1
WO2022048350A1 PCT/CN2021/108825 CN2021108825W WO2022048350A1 WO 2022048350 A1 WO2022048350 A1 WO 2022048350A1 CN 2021108825 W CN2021108825 W CN 2021108825W WO 2022048350 A1 WO2022048350 A1 WO 2022048350A1
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immunomagnetic
phenylboronic acid
adsorbent
antibody
magnetic carrier
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PCT/CN2021/108825
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French (fr)
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熊勇华
郭亮
冷远逵
黄小林
林童
李响敏
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南昌大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles

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  • the invention belongs to the technical field of food safety detection and pretreatment, and in particular relates to an immunomagnetic adsorbent based on phenylboronic acid directional conjugated antibody and its preparation.
  • pretreatment steps such as sample separation and purification are indispensable and are the key link affecting the reliability of the analysis results.
  • Traditional sample pretreatment methods such as liquid-phase extraction and solid-phase extraction, are cumbersome to operate and have poor specificity, which affects the detection accuracy.
  • Immunoaffinity chromatography is based on the specific recognition between the ligand and the analyte, and its accuracy is good, but the cost is high and time-consuming.
  • the method based on immunomagnetic adsorbent has developed rapidly in the field of sample pretreatment for food safety testing due to its advantages of good specificity, rapidity and high efficiency.
  • the immunomagnetic adsorbent is composed of a superparamagnetic magnetic carrier and an antibody modified on the magnetic carrier.
  • the target object to be detected is captured on the surface of the magnetic carrier through the specific recognition of the antibody, and then under the action of an external magnetic field, the object to be detected is realized. enrichment and separation.
  • the basic principle of the current immunomagnetic adsorbent preparation method is usually to use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) or glutaraldehyde as a coupling reagent to combine the antibody Modified on the surface of carboxylated or aminated magnetic carriers.
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • glutaraldehyde glutaraldehyde
  • the object of the present invention is to aim at the defects of the prior art, to provide an immunomagnetic adsorbent based on a phenylboronic acid directional conjugated antibody and a preparation method thereof.
  • the surface of the provided immunomagnetic adsorbent is oriented in a fixed orientation, and the Fab end of the antigen recognition region is exposed.
  • the present invention provides an immunomagnetic adsorbent based on phenylboronic acid-directed conjugated antibodies, comprising a magnetic carrier and an antibody coupled to the magnetic carrier.
  • the magnetic carrier is any superparamagnetic micro-nano particle, preferably the magnetic carrier is a polymer microsphere or a silica microsphere wrapped with Fe 3 O 4 nanoparticles.
  • all or part of the surface of the magnetic carrier is phenylboronic acid groups.
  • the present invention also provides a method for preparing an immunomagnetic adsorbent based on phenylboronic acid directional conjugated antibodies.
  • a magnetic carrier with surface-modified phenylboronic acid groups is prepared, and then the phenylboronic acid groups are combined with glycoprotein molecules on the antibody Fc fragment.
  • the specific affinity of the cis-diol structure in the target immobilizes the antibody on the magnetic carrier.
  • the first step, the preparation of phenylboronated magnetic carrier, the scheme can be one of the following two.
  • One of the preparation schemes of the magnetic carrier is to synthesize the magnetic carrier modified with the surface carboxyl group or amino group, and then carry out the phenylboronation modification on its surface.
  • EDC as a coupling agent to modify aminophenylboronic acid on the surface of carboxylated magnetic beads to prepare phenylboronated magnetic carriers
  • EDC as a coupling agent to modify 4-carboxyphenylboronic acid on the surface of aminated magnetic beads to prepare phenylboronic acid Magnetic carrier.
  • the second preparation scheme of magnetic carrier 1) see the existing literature to synthesize oil-soluble magnetic nanoparticles; 2) then pass common polymers such as polystyrene maleic anhydride (PSMA), polymaleic anhydride-octadecene copolymer (PMAO), etc.
  • PSMA polystyrene maleic anhydride
  • PMAO polymaleic anhydride-octadecene copolymer
  • the oil phase is used as the oil phase
  • the aqueous solution containing surfactants such as sodium lauryl sulfate is used as the water phase, and water is prepared by emulsification by ultrasound, stirring, etc.
  • the oil-in-oil emulsion, and then the non-polar solvent in the emulsion droplets is removed by volatilization or rotary evaporation to obtain the phenylboronated magnetic carrier.
  • the second step is the directional coupling of the antibody to the magnetic carrier.
  • the phenylboronated magnetic carrier synthesized in the first step and the antibody were mixed in a buffer (0.02M phosphate buffer, pH 5.5-8.0, and the optimal pH of different antibodies was different) at room temperature and 25 °C for 10-20min to achieve the antibody lysis.
  • a buffer 0.02M phosphate buffer, pH 5.5-8.0, and the optimal pH of different antibodies was different
  • the immunomagnetic adsorbent provided by the invention can be used for pre-detection treatment of various food samples, and can be used for the enrichment of hazardous substances such as mycotoxins, pesticide and veterinary drug residues, heavy metals and pathogenic microorganisms in food samples according to the difference of the conjugated antibodies. and purification.
  • the purified hazardous substances can be quantitatively or qualitatively detected by methods such as high performance liquid chromatography and immunoassay.
  • the immunomagnetic adsorbent based on phenylboronic acid directional conjugated antibody and its preparation provided by the present invention, the Fc end of the antibody is directionally coupled to the magnetic carrier, the Fab end is exposed, Reduce the steric hindrance of antigen and antibody binding, thereby improving the antigen affinity of the immunomagnetic adsorbent, improving the enrichment efficiency of the antigen to be tested, effectively controlling the difference between batches, and reducing the cost due to the reduction of antibody dosage.
  • Fig. 1 is the synthesis schematic diagram of traditional immunomagnetic adsorbent
  • Figure 2 is a schematic diagram of the synthesis of immunomagnetic adsorbents based on phenylboronic acid-directed conjugated antibodies.
  • Fig. 3 is a scheme of modification of phenylboronic acid by polystearyl maleic anhydride.
  • the preparation of the immunomagnetic adsorbent based on phenylboronic acid-directed conjugated antibody Salmonella in this embodiment includes the preparation of phenylboronated magnetic carrier, the coupling of antibody and magnetic carrier, and the specific process steps are as follows:
  • Oleic acid modification of Fe 3 O 4 magnetic beads add the magnetic beads synthesized in the previous step into a three-necked flask, pass N 2 , and add 2.4 mL of oleic acid dropwise while stirring. Since the oleic acidified magnetic beads are hydrophobic, they are adsorbed on the stirrer or the wall of the flask. After 3 hours of reaction at 70 °C, the black solution gradually becomes clear. The reaction is stopped, the solution in the beaker is poured out, and 300 mL of ethanol is added to elute the adsorbed oleic acidified magnetic beads. After 2 min, the ethanol solvent was discarded, and this step was repeated 3-5 times until there was no oleic acid layer floating on the surface of the ethanol.
  • Fe 3 O 4 @SiO 2 @-COOH composite nanospheres were surface-modified with phenylboronic acid, and the carboxylated magnetic microspheres synthesized in the previous step were dispersed in EDC-containing buffer (0.02M phosphate buffer, pH 5.5 ), the activation reaction was carried out at room temperature of 25 °C for 30 min. Subsequently, the pH of the system was adjusted to 8.0, 3-aminophenylboronic acid/4-aminophenylboronic acid was added, and the coupling reaction was carried out at room temperature of 25 °C for 30 min. Then, glucosamine or OVA solution was added and incubated at room temperature for 10-20 min to block the remaining carboxyl groups on the magnetic carrier. After the reaction was completed, magnetic microspheres modified with phenylboronic acid were obtained by magnetic separation.
  • EDC-containing buffer 0.02M phosphate buffer, pH 5.5
  • phenylboronated magnetic carrier (15 mg/mL) synthesized in step 1) with a particle size of 180 nm and disperse it in a buffer (0.02 M phosphate buffer, pH 6) containing 100 ⁇ g of anti-Salmonella antibody (2 mg/mL), After 10 min incubation at room temperature 25°C. Add 10uL of glucose and continue to incubate at room temperature for 10min to block the remaining boronic acid groups on the magnetic carrier.
  • T-line absorbance, C-line absorbance and T/C value take the concentration of different bacteria as the abscissa, and draw the standard curve with the T-line absorbance and T/C value respectively.
  • qualitative analysis was carried out by observing the results with the naked eye. If the T line was colored, it indicated that there was bacteria in the sample, and the detection limit was about 10 4 CFU/mL. 10 4 CFU/mL.
  • the preparation of the OTA immunomagnetic adsorbent based on the phenylboronic acid-directed conjugated antibody in this example includes the preparation of the phenylboronated magnetic carrier, the coupling of the antibody and the magnetic carrier, the enrichment and detection of OTA, and the specific application of the immunomagnetic adsorbent.
  • the process steps are as follows:
  • Steps a-d are the same as in Example 1.
  • Fe 3 O 4 @SiO 2 composite nano-microspheres The surface of Fe 3 O 4 @SiO 2 composite nano-microspheres was modified by amination. 10 mg of Fe 3 O 4 @SiO 2 composite nano-microspheres synthesized in the previous step were added to 10 mL of ethanol, ultrasonicated for 1 h to make the dispersion uniform, and 800 ⁇ L of 3 -Aminopropyl triethoxysilane (APTES) reagent, modified on the surface of the microspheres by ultrasonic for 1h, and washed with ultrapure water for 3-5 times after magnetic separation to remove ethanol and unreacted APTES reagent.
  • APTES 3 -Aminopropyl triethoxysilane
  • Fe 3 O 4 @SiO 2 @-NH2 composite nanospheres were surface-modified with phenylboronic acid, first dissolve 2-carboxyphenylboronic acid/4-carboxyphenylboronic acid in a buffer containing EDC (0.02M phosphate buffer, pH is 5.5),
  • the reaction was activated at room temperature of 25°C for 30 min. Subsequently, the pH of the system was adjusted to 8.0, the aminated magnetic microspheres synthesized in the previous step were added, and the coupling reaction was carried out at room temperature of 25 °C for 30 min. After the reaction is completed, magnetic microspheres modified with phenylboronic acid are obtained by magnetic separation.
  • phenylboronated magnetic adsorbent (15 mg/mL) synthesized in step 1) with a particle size of 180 nm and disperse it in a buffer (0.02 M phosphate buffer, pH 6) containing 50 ⁇ g of anti-OTA antibody (2 mg/mL). , after incubating for 10 min at room temperature 25 °C. Add 10uL of glucose and continue to incubate at room temperature for 10min to block the remaining boronic acid groups on the magnetic carrier.
  • carboxylated magnetic adsorbent (15 mg/mL) with a particle size of 180 nm and disperse it in a buffer (0.02 M phosphate buffer, pH 6) containing 300 ⁇ g anti-OTA antibody (2 mg/mL), and add 50 ⁇ g EDC at room temperature. After 90 min incubation at 25°C. Add 10 mg BSA and continue to incubate at room temperature for 10 min to block the remaining carboxyl groups on the magnetic carrier. After the reaction is over, remove the supernatant by magnetic separation, and redissolve the magnetic beads in 0.1% BSA, 0.05% Tween-20, 0.01% NaN 3 PB7.4 buffer (concentration of 1 mg/mL), stored at 4 °C for later use.
  • the recovery rates of the carboxylated immunomagnetic adsorbents all reached 83-88%; the OTA enriched by the immunomagnetic adsorbents eluting the phenylboronic acid-directed conjugated antibodies with the eluent were 8.81ng, 22.2ng, 45.7, and 66.7ng, respectively; The recovery rates of immunomagnetic adsorbents for boronic acid-directed conjugated antibodies were all 88-92%.
  • the above results show that the OTA enrichment effect of the phenylboronic acid-directed conjugated immunomagnetic adsorbent is better when the amount of antibody is less.
  • HPLC reference conditions are listed below:

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Abstract

一种基于苯硼酸定向偶联抗体的免疫磁吸附剂及其制备方法，其中，免疫磁吸附剂利用抗体Fc片段糖蛋白分子中的顺式二醇结构与苯硼酸基团特异性的亲和作用实现抗体定向偶联在磁载体表面。与传统方法相比，本方法最大程度地提高抗体Fab端的利用率，进而提高免疫磁吸附剂对目标物的亲和力与吸附能力，并能有效控制批间差。所制备的免疫磁吸附剂可用于对多种食品样本的检测前处理，根据所偶联抗体的不同可用于食品样本中危害物质如真菌毒素、农兽药残留、重金属以及病原微生物等的富集和纯化。纯化后的危害物质可用高效液相色谱、免疫分析方法以及酶联免疫技术等方法定量或定性检测。

Description

基于苯硼酸定向偶联抗体的免疫磁吸附剂及其制备方法 技术领域
本发明属于食品安全检测和前处理技术领域，具体地说，涉及基于苯硼酸定向偶联抗体的免疫磁吸附剂及其制备。
背景技术
在食品危害因子的检测中，样品的分离、纯化等前处理步骤是必不可少的，是影响分析结果可靠性的关键环节。传统的样品前处理方法如液相萃取、固相萃取等，操作繁琐且特异性差，影响检测准确度。免疫亲和层析法基于配基与待检物之间的特异性识别，其准确度好，但成本高、耗时长。而基于免疫磁吸附剂的方法由于其特异性好、快速高效的优势，在食品安全检测样品前处理领域得到迅速发展。
免疫磁吸附剂由具有超顺磁性的磁载体和修饰在磁载体上的抗体构成，通过抗体特异性识别作用将目标待检物捕获在磁载体表面，然后在外加磁场的作用下，实现对待检物的富集分离。
目前免疫磁吸附剂制备方法的基本原理通常是通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)或戊二醛等作为偶联试剂将抗体修饰在羧基化或氨基化磁载体表面。但这种方法存在一个明显缺陷，即由于抗体Fab端(抗原识别区)和Fc端都具有大量的氨基，导致抗体在磁载体表面的取向随机不可控，这种随机取向减少了Fab端外露的数量，降低了抗体的有效利用率，导致抗体用量大、成本过高、磁分离效率偏低、磁分离可重复性和稳定性偏低等后果。
因此，发展将抗体Fc端定向偶联在磁载体上的方法，使得抗原识别端Fab端外露，减少抗原与抗体结合的空间位阻，从而提高免疫磁吸附剂的抗原亲和力，提高待检物抗原的富集效率，有效控制批间差，同时降低成本，是本领域亟待解决的技术问题。
发明内容
本发明的目的是针对现有技术缺陷，提供基于苯硼酸定向偶联抗体的免疫磁吸附剂及 其制备方法，所提供的免疫磁吸附剂表面抗体取向固定，抗原识别区Fab端外露。
为实现上述目的，本发明采用的技术方案是：
本发明提供了一种基于苯硼酸定向偶联抗体的免疫磁吸附剂，包括磁载体和偶联在磁载体上的抗体，所述抗体通过磁载体表面苯硼酸基团与抗体Fc片段糖蛋白分子中的顺式二醇结构特异性的亲和作用定向偶联在磁载体表面。
进一步地，所述磁载体为任意具有超顺磁性的微纳米粒子，优选磁载体为包裹Fe 3O 4纳米颗粒的聚合物微球或二氧化硅微球。
进一步地，所述磁载体表面全部或部分为苯硼酸基团。
本发明还提供了一种基于苯硼酸定向偶联抗体的免疫磁吸附剂的制备方法，首先制备表面修饰苯硼酸基团的磁载体，然后通过苯硼酸基团与与抗体Fc片段上糖蛋白分子中的顺式二醇结构特异性的亲和作用将抗体定向固定在磁载体上。具体工艺步骤如下所述：
第一步，苯硼酸化磁载体的制备，方案可以是如下两种其中之一。
磁载体制备方案之一，先合成表面羧基或氨基修饰的磁载体，再在其表面进行苯硼酸化修饰。比如，通过用EDC作为偶联剂将氨基苯硼酸修饰在羧基化磁珠表面制备苯硼酸化磁载体；或者通过EDC作为偶联剂将4-羧基苯硼酸修饰在氨基化磁珠表面制备苯硼酸化磁载体。
磁载体制备方案之二，1)参见现有文献合成油溶性磁纳米颗粒；2)再通过对常见聚合物如聚苯乙烯马来酸酐(PSMA)、聚马来酸酐-十八烯共聚物(PMAO)等进行苯硼酸基团改性制备含苯硼酸基团的聚合物，例如，将3-氨基苯硼酸与聚马来酸酐十八醇酯在4-二甲氨基吡啶的催化下通过酰化反应合成含苯硼酸的聚合物；3)然后通过乳化-乳液溶剂挥发法将磁纳米颗粒封装在含苯硼酸基团的聚合物中制备苯硼酸化磁微球载体，具体地，将含有油溶性磁纳米颗粒、聚合物、某种挥发性非极性溶剂如甲苯、氯仿等作为油相，含表面活性剂如十二烷基硫酸钠的水溶液作为水相，通过超声、搅拌等方式乳化制备水包油乳液，随后将乳液滴中的非极性溶剂通过挥发或旋蒸等方式去除即得到苯硼酸化磁载体。
第二步，抗体与磁载体的定向偶联。将第一步合成的苯硼酸化磁载体和抗体混合在缓 冲液(0.02M磷酸盐缓冲液，pH为5.5-8.0，不同抗体最优pH不同)中于室温25℃孵育10～20min实现抗体的定向偶联，而后加入葡萄糖或OVA溶液继续室温孵育10～20min以封闭磁载体上多余的苯硼酸基团，待反应结束后通过磁分离获得抗体定向偶联的免疫磁吸附剂。
本发明提供的免疫磁吸附剂可用于对多种食品样本进行检测前处理，根据所偶联抗体的不同可用于食品样本中危害物质如真菌毒素、农兽药残留、重金属以及病原微生物等的富集和纯化。纯化后的危害物可用高效液相色谱、免疫分析方法等方法定量或定性检测。
与现有技术相比，本发明的有益效果是：
相对传统抗体非定向偶联的免疫磁吸附剂，本发明提供的基于苯硼酸定向偶联抗体的免疫磁吸附剂及其制备，将抗体的Fc端定向偶联到磁载体上，Fab端外露，减少抗原与抗体结合的空间位阻，从而提高免疫磁吸附剂的抗原亲和力，提高待检物抗原的富集效率，有效控制批间差，同时因可降低抗体用量而降低成本。
附图说明
图1为传统免疫磁吸附剂合成示意图；
图2为基于苯硼酸定向偶联抗体的免疫磁吸附剂合成示意图。
图3为聚马来酸酐十八醇酯修饰苯硼酸方案。
具体实施方式
下面结合具体实施例对本发明作进一步详细说明，仅作为说明本发明具体内容而不用于限制本发明范围。
实施例1
本实施例基于苯硼酸定向偶联抗体沙门氏菌的免疫磁吸附剂的制备包括苯硼酸化磁载体的制备，抗体与磁载体的偶联，具体工艺步骤如下所述：
1)苯硼酸化磁载体的制备，
a.Fe 3O 4磁珠的合成，在500mL三口烧瓶中加入300mL超纯水，通入N 2以除去水中氧 气，同时在50℃预热15min。加入3.2gFeCl 2·H 2O，5.2gFeCl 3磁力搅拌混匀后加入25mL氨水，黄色溶液迅速变为黑色，50℃恒温反应30min。将合成的磁珠通过磁吸分离，用超纯水洗3-5次直至磁吸后溶液pH至中性后复溶于300mL超纯水中。
b.Fe 3O 4磁珠的油酸化修饰，将上步所合磁珠加入三口烧瓶中，通入N 2，边搅拌边逐滴加入2.4mL油酸。由于油酸化的磁珠疏水，吸附于搅拌器或烧瓶壁上，70℃反应3h黑色溶液逐渐清澈，停止反应，倒掉烧杯中溶液，加入300mL乙醇将吸附的油酸化磁珠洗脱，磁吸2min弃去乙醇溶剂，重复此步骤3-5次直至乙醇液面无漂浮油酸层。
c.磁微球的合成，通过乳化-乳液溶剂挥发法合成磁微球，将上步合成的油酸化磁珠分散在30mL环己烷中以形成油相，1.5g十二烷基硫酸钠(SDS)溶解在500mL超纯水中以形成水相，将上述混合物超声处理10min形成水包油微乳液。随后放置于60℃过夜挥发环己烷，得到SDS稳定的磁微球。重新将其分散于60mL Tween-80溶液中，之后通过磁吸获得Tween-80修饰的磁微球。
d.Fe 3O 4@SiO 2复合纳米微球的合成，1000mL乙醇，30mL氢氧化铵，20mL超纯水和6mL TEOS于30℃预反应20min，使TEOS预水解。再将上步合成的60mL磁微球加入混合体系中，通过控制反应时间，得到包被合适厚度SiO 2层的磁微球。
e.Fe 3O 4@SiO 2复合纳米微球表面进行羧基化修饰，取10mg上步合成的Fe 3O 4@SiO 2复合纳米微球加入10mL乙醇中，超声1h使其分散均匀，加入800μL 3-(三乙氧基硅基)丙基琥珀酸酐(TEPSA)试剂，超声1h使其修饰在微球表面。磁分离后用超纯水洗3-5次以除去乙醇和未反应的TEPSA试剂，随后加入pH 9.0的碱性超纯水中过夜震荡以水解酸酐，生成羧基基团。
f.Fe 3O 4@SiO 2@-COOH复合纳米微球表面修饰苯硼酸，将上步合成的羧基化的磁微球分散在含有EDC的缓冲液(0.02M磷酸盐缓冲液，pH为5.5)中，室温25℃活化反应30min。随后将体系pH调至8.0，加入3-氨基苯硼酸/4-氨基苯硼酸，室温25℃偶联反应30min。而后加入氨基葡萄糖或OVA溶液继续室温孵育10～20min以封闭磁载体上剩余的羧基基团，待反应结束后通过磁分离获得修饰苯硼酸的磁微球。
2)抗沙门氏菌抗体与磁载体的偶联：
2.1抗沙门氏菌抗体与苯硼酸磁载体的偶联：
取步骤1)合成的1.5mg粒径180nm苯硼酸化的磁载体(15mg/mL)分散于含100μg抗沙门氏菌抗体(2mg/mL)缓冲液(0.02M磷酸盐缓冲液，pH为6)中，于室温25℃孵育10min钟后。加入10uL葡萄糖继续室温孵育10min以封闭磁载体上剩余的硼酸基团，待反应结束后通过磁分离移除上清，将磁珠复溶于0.1％BSA、0.05％吐温-20、0.01％NaN 3的PB7.4缓冲液中(浓度为1mg/mL)，4℃保存备用。
2.2抗沙门氏菌抗体与羧基化化磁载体的偶联(此为传统方法对照)：
取1.5mg粒径180nm传统羧基化的磁吸附剂(15mg/mL)分散于含220μg抗沙门氏菌抗体(2mg/mL)缓冲液(0.02M磷酸盐缓冲液，pH为6)中，并加入50μgEDC于室温25℃孵育90min钟后。加入10mgBSA继续室温孵育10min以封闭磁载体上剩余的羧基基团，待反应结束后通过磁分离移除上清，将磁珠复溶于0.1％BSA、0.05％吐温-20、0.01％NaN 3的PB7.4缓冲液中(浓度为1mg/mL)，4℃保存备用。
3)上述两种免疫磁吸附剂对沙门氏菌的富集
取20mg的猪肉肉糜加入到200mL培养基中，混匀。接种一定浓度的沙门氏菌，温度在37℃，时间为12h震荡培养。将菌液浓度调整为10 4CFU/mL、10 5CFU/mL、10 6CFU/mL.10 7CFU/mL。取1mL各个浓度的菌液、取1mL待测肉糜样品溶液，分别加入封闭后的两种免疫磁吸附剂10μL，混合孵育60min，温度37℃，转速15rpm。孵育后磁分离35min后，弃上清，用PBS清洗后，复溶于PBS中。
4)利用双抗夹心法对样品进行目测和使用仪器测定结果
将捕获到菌的两种免疫磁吸附剂稀释到浓度50μg/mL，取70μL滴加到商业化检测沙门氏菌免疫层析试纸条的加样孔中，10min用免疫层析分析仪读取,记录T线吸光度、C线吸光度和T/C的值,以不同菌的浓度为横坐标，分别以T线吸光度、T/C值为纵坐标绘制标准曲线。同时用肉眼观察结果进行定性分析，T线若有颜色则说明样品中有菌，检测限约为10 4CFU/mL，T线不显色则说明样品中没有沙门氏菌或是含有沙门氏菌的量低于 10 4CFU/mL。
参考所做的标准曲线图,确定样品中沙门氏菌的数量。结果表明：当标准品量在10 4CFU/mL、10 5CFU/mL、10 6CFU/mL、10 7CFU/mL之间时，EDC共价修饰免疫磁吸附剂分别为富集效率均达到了44～50％，苯硼酸定向偶联免疫磁吸附剂富集率均达到了58～63％，说明在抗体消耗量较少的情况下，苯硼酸定向偶联免疫磁吸附剂富集效果明显更好。
实施例2
本实施例基于苯硼酸定向偶联抗体的OTA免疫磁吸附剂的制备包括苯硼酸化磁载体的制备，抗体与磁载体的偶联，OTA的富集与检测，免疫磁吸附剂的应用的具体工艺步骤如下所述：
1)苯硼酸化磁载体的制备
a-d步骤同实施例1。
e.Fe 3O 4@SiO 2复合纳米微球表面进行氨基化修饰，取10mg上步合成的Fe 3O 4@SiO 2复合纳米微球加入10mL乙醇中，超声1h使其分散均匀，加入800μL3-氨基丙基三乙氧基硅烷(APTES)试剂，超声1h使其修饰在微球表面，磁分离后用超纯水洗3-5次以除去乙醇和未反应的APTES试剂。
f.Fe 3O 4@SiO 2@-NH2复合纳米微球表面修饰苯硼酸，先将2-羧基苯硼酸/4-羧基苯硼酸溶解于含有EDC的缓冲液(0.02M磷酸盐缓冲液，pH为5.5)中，
室温25℃活化反应30min。随后将体系pH调至8.0，加入上步合成的氨基化磁微球，室温25℃偶联反应30min。待反应结束后通过磁分离获得修饰苯硼酸的磁微球。
2)抗OTA抗体与磁载体的偶联：
2.1抗OTA抗体与苯硼酸磁载体的偶联：
取步骤1)合成的1.5mg粒径180nm苯硼酸化的磁吸附剂(15mg/mL)分散于含50μg抗OTA抗体(2mg/mL)缓冲液(0.02M磷酸盐缓冲液，pH为6)中，于室温25℃孵育10min钟后。加入10uL葡萄糖继续室温孵育10min以封闭磁载体上剩余的硼酸基团，待反应结束后通过磁分离移除上清，将磁珠复溶于0.1％BSA、0.05％吐温-20、0.01％NaN 3的PB7.4 缓冲液中(浓度为1mg/mL)，4℃保存备用。
2.2抗OTA抗体与羧基化化磁载体的偶联：
取1.5mg粒径180nm羧基化的磁吸附剂(15mg/mL)分散于含300μg抗OTA抗体(2mg/mL)缓冲液(0.02M磷酸盐缓冲液，pH为6)中，并加入50μgEDC于室温25℃孵育90min钟后。加入10mgBSA继续室温孵育10min以封闭磁载体上剩余的羧基基团，待反应结束后通过磁分离移除上清，将磁珠复溶于0.1％BSA、0.05％吐温-20、0.01％NaN 3的PB7.4缓冲液中(浓度为1mg/mL)，4℃保存备用。
3)两种免疫磁吸附剂对OTA的富集与检测：
取制备的两种免疫磁吸附剂1mL(浓度为1mg/mL)于5mL离心管中，然后加入1mL待测样品将OTA标准品用PBS缓冲液分别配制成浓度为(10ng/mL、25ng/mL、50ng/mL、75ng/mL的OTA溶液作为待测样品，并以PBS缓冲液作为空白待测样品)，混匀，捕获10min,磁分离后移除上清液。最后加入1mL洗脱液洗脱，收集洗脱液，用HPLC法按照“GB 5009.96-2016”检测待测样品中OTA的含量。
结果表明：当标准品量在10ng、25ng、50ng、75ng之间时，用洗脱液洗脱EDC共价修饰免疫磁吸附剂富集的OTA分别为8.60ng、21.4ng、44.0、65.7ng，羧基化免疫磁吸附剂回收率均达到83～88％；用洗脱液洗脱苯硼酸定向偶联抗体的免疫磁吸附剂富集的OTA分别为8.81ng、22.2ng、45.7、66.7ng；苯硼酸定向偶联抗体的免疫磁吸附剂回收率均达到88～92％。以上结果说明，在抗体用量更少的情况下，苯硼酸定向偶联免疫磁吸附剂的OTA富集效果更好。
在空白PBS缓冲液中添加5.0μg/kg OTA，平行添加5个,使用苯硼酸定向偶联抗体的免疫磁吸附剂,经上述步骤处理后，其添加回收率在88.1％、89.7％、89.5％、90.9％、89.8％，说明该免疫磁吸附剂具备良好的再生性。
4)苯硼酸定向偶联抗体免疫磁吸附剂的使用方法：
4.1样品前处理
称取试样10.0g玉米(精确至0.01g)，加人50mL三氯甲烷和5mL 0.1mol/L的磷酸水溶 液,于涡旋。振荡器上振荡提取3min～5min，提取液用定性滤纸过滤，取10mL下层滤液至100mL平底烧瓶中，于40C水浴中用旋转蒸发仪旋转燕发至近干，用20mL石油醚溶解残渣后加入10mL取液，再用涡旋振荡器振荡提取3min～5min，静置分层后取下层溶液，用滤纸过滤。
4.2苯硼酸定向偶联抗体的免疫磁吸附剂捕获
取免疫磁吸附剂2mL于10mL离心管中,用5mL去离子水润洗，每次用磁分离架分离洗液；向润洗好的免疫磁吸附剂中加入处理好的滤液5mL，混匀，室温下捕获10min，用磁分离架分离免疫磁吸附剂,用2mL去离子水润洗免疫磁吸附剂2次
4.3苯硼酸定向偶联抗体的免疫磁吸附剂洗脱
用1mL洗脱液洗苯硼酸定向偶联抗体的免疫磁吸附剂，混匀，用磁分离架分离免疫磁吸附剂,回收洗脱液液，即为富集净化后的OTA样品。
5)测定
高效液相色谱参考条件列出如下:
a)色谱柱:C1:柱,柱长150mm,内径4.6mm,粒径5μm,或等效柱:
b)柱温:30C；
c)进样量:10pL；
d)流速:1mL./min；
e)检测波长:激发波长:333nm,发射波长:460nm；
f)流动相及洗脱条件:
流动相:A:冰乙酸-水(2+100),B:乙腈；
等度洗脱条件:A-B(50+50)
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 基于苯硼酸定向偶联抗体的免疫磁吸附剂，包括磁载体和偶联在磁载体上的抗体，其特征在于，所述抗体通过磁载体表面苯硼酸基团与抗体Fc片段糖蛋白分子中的顺式二醇结构特异性的亲和作用定向偶联在磁载体表面。
  2. 根据权利要求1所述的免疫磁吸附剂，其特征在于，所述磁载体为包裹Fe 3O 4纳米颗粒的聚合物微球或二氧化硅微球。
  3. 根据权利要求2所述的免疫磁吸附剂，其特征在于，所述纳米颗粒为Fe 3O 4纳米颗粒。
  4. 根据权利要求1所述的免疫磁吸附剂，其特征在于所述磁载体表面全部或部分为苯硼酸基团。
  5. 根据权利要求1-4任意一项所述的基于苯硼酸定向偶联抗体的免疫磁吸附剂的制备方法，其特征在于，包括如下步骤：
    1)苯硼酸化磁载体的制备：先合成表面羧基或氨基修饰的磁载体，再在其表面进行苯硼酸化修饰，获得苯硼酸化磁载体；
    或者，先合成油溶性磁纳米颗粒，然后将其封装在含苯硼酸基团聚合物中，获得苯硼酸化磁载体；
    2)抗体的定向偶联：将抗体加入到含苯硼酸化磁载体缓冲液中室温孵育，使之定向偶联到磁载体上；而后加入过量含顺式二醇结构的物质封闭磁载体上多余的苯硼酸位点；磁分离洗涤去除多余的抗体和封闭剂，获得免疫磁吸附剂。
  6. 根据权利要求5所述的免疫磁吸附剂的制备方法，其特征在于，所述苯硼酸化修饰为通过酰胺键将氨基苯硼酸修饰在羧基化磁载体表面。
  7. 根据权利要求5所述的免疫磁吸附剂的制备方法，其特征在于，所述含顺式二醇结构的物质为葡萄糖或卵清蛋白OVA。
  8. 根据权利要求5所述的免疫磁吸附剂的制备方法，其特征在于，所述含苯硼酸基团聚合物为利用聚苯乙烯马来酸酐(PSMA)或聚马来酸酐-十八烯共聚物(PMAO)进行苯硼酸基团改性。
  9. 根据权利要求6所述的免疫磁吸附剂的制备方法，其特征在于，所述封装的方法为乳 化-乳液溶剂挥发法。
  10. 根据权利要求1所述的基于苯硼酸定向偶联抗体的免疫磁吸附剂的应用，其特征在于，用于对食品样本中目标危害物质的富集和纯化。
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