CN211453649U - Immunochromatography test strip based on quantum dot fluorescent microspheres - Google Patents
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Abstract
Description
技术领域technical field
本实用新型属于免疫分析技术领域,具体涉及一种快速检测盐酸赛庚啶的量子点荧光微球免疫层析试纸条。The utility model belongs to the technical field of immunoassay, in particular to a quantum dot fluorescent microsphere immunochromatographic test strip for rapidly detecting cyproheptadine hydrochloride.
背景技术Background technique
盐酸赛庚啶作为医用药物,被用于皮肤瘙痒、接触性皮炎、过敏性鼻炎等多种过敏病症的治疗,其同时具有抑制下丘脑的饱觉中枢而刺激食欲、增加体重、促进生长的作用。盐酸赛庚啶也被非法添加在动物饲料中,可产生与“瘦肉精”盐酸克仑特罗类似效果,促进畜禽类生长和提高瘦肉率,因此被称为新型“瘦肉精”。人长期食用有盐酸赛庚啶残留的肉类食品,可严重损害人体健康,产生乏力、头晕、瞌睡、呼吸抑制等不良症状,严重者可导致昏迷甚至死亡,尤其以儿童与老人最为敏感。因此,中国农业部于2010年发布1519号公告,严禁在饲料和动物饮水中使用或添加盐酸赛庚啶。Cyproheptadine hydrochloride, as a medical drug, is used for the treatment of various allergic diseases such as skin itching, contact dermatitis, allergic rhinitis, etc. It also inhibits the satiety center of the hypothalamus to stimulate appetite, increase body weight, and promote growth. . Cyproheptadine hydrochloride is also illegally added to animal feed, which can produce a similar effect to "clenbuterol" Clenbuterol hydrochloride, promote the growth of livestock and poultry and improve the lean meat rate, so it is called a new type of "clenbuterol". . Human long-term consumption of meat food with cyproheptadine hydrochloride residues can seriously damage human health, resulting in fatigue, dizziness, drowsiness, respiratory depression and other adverse symptoms, severe cases can lead to coma or even death, especially children and the elderly are the most sensitive. Therefore, the Ministry of Agriculture of China issued Announcement No. 1519 in 2010, prohibiting the use or addition of cyproheptadine hydrochloride in feed and animal drinking water.
目前盐酸赛庚啶的检测方法主要为仪器检测,如高效液相色谱法和液质联用检测方法。这些检测方法检测需要耗费大量时间,所需的检测设备昂贵,而且需要专门的实验人员操作,不利于大量样品的快速检测。酶联免疫分析方法(ELISA)具有灵敏度高、回收率好、特异性强等优点,且可以实现现场快速定性和半定量分析,在药物残留检测中得到广泛的应用,但该方法仍需要操作人员的专业培训。基于ELISA原理的免疫层析试纸条,不需要专业培训的操作人员,具有更加简单快速的特点,且成本更加低廉、灵敏度高且不需要复杂的仪器设备,在生物医药、农兽药残留、食品安全检测等领域得到广泛应用。At present, the detection methods of cyproheptadine hydrochloride are mainly instrument detection, such as high performance liquid chromatography and liquid chromatography-mass spectrometry detection methods. The detection of these detection methods takes a lot of time, the required detection equipment is expensive, and the operation of specialized laboratory personnel is required, which is not conducive to the rapid detection of a large number of samples. Enzyme-linked immunoassay (ELISA) has the advantages of high sensitivity, good recovery and strong specificity, and can achieve rapid on-site qualitative and semi-quantitative analysis, and has been widely used in drug residue detection, but this method still requires operators professional training. Immunochromatographic test strips based on the ELISA principle do not require professionally trained operators, are simpler and faster, have lower costs, are more sensitive, and do not require complex instruments and equipment. It has been widely used in security detection and other fields.
量子点荧光微球免疫层析法是以量子点荧光微球作为标记物而建立的一种免疫层析技术。量子点具有良好的生物相容性,对其表面进行化学修饰后可以与核酸、抗体和DNA等生物分子进行特异性的偶联。量子点荧光微球是一种将量子点荧光染料进行包埋或在高聚物载体上通过吸附作用而形成的一种荧光标记物,可有效提高反应灵敏度,在免疫层析检测技术领域被视为最具发展潜力的荧光标记物,量子点荧光微球偶联抗体过程是整个方法的关键部分,偶联的效果最终影响灵敏度的提高。目前量子点技术主要用来检测蛋白生物标记物以及其他小分子化合物的检测。利用量子点荧光微球免疫层析法对盐酸赛庚啶的检测尚未见报道。Quantum dot fluorescent microsphere immunochromatography is an immunochromatographic technique established with quantum dot fluorescent microspheres as markers. Quantum dots have good biocompatibility and can be specifically coupled with biomolecules such as nucleic acids, antibodies and DNA after chemical modification on their surfaces. Quantum dot fluorescent microspheres are a kind of fluorescent marker formed by embedding quantum dot fluorescent dyes or by adsorption on a polymer carrier, which can effectively improve the sensitivity of the reaction, and is regarded as a kind of fluorescent label in the field of immunochromatographic detection technology. As the most promising fluorescent marker, the process of coupling antibody to quantum dot fluorescent microspheres is a key part of the whole method, and the effect of coupling ultimately affects the improvement of sensitivity. At present, quantum dot technology is mainly used to detect protein biomarkers and other small molecule compounds. The detection of cyproheptadine hydrochloride by quantum dot fluorescent microsphere immunochromatography has not been reported yet.
实用新型内容Utility model content
本实用新型提供了一种基于量子点荧光微球的免疫层析试纸条,可实现对盐酸赛庚啶的简便、快速、高灵敏的定量检测。实用新型是通过如下技术方案实现的:The utility model provides an immunochromatographic test strip based on quantum dot fluorescent microspheres, which can realize simple, fast and highly sensitive quantitative detection of cyproheptadine hydrochloride. The utility model is realized through the following technical solutions:
首先,本实用新型提供了一种基于量子点荧光微球的免疫层析试纸条,包括底板,样品垫、硝酸纤维素膜和吸水垫,样品垫搭接于硝酸纤维素膜的一端上表面,吸水垫接于硝酸纤维素膜的另一端上表面,样品垫端部与吸水垫端部之间外露的硝酸纤维素膜上包被有与底板端面平行的检测线和质控线;First, the utility model provides an immunochromatographic test strip based on quantum dot fluorescent microspheres, comprising a bottom plate, a sample pad, a nitrocellulose membrane and a water-absorbing pad, and the sample pad is overlapped on the upper surface of one end of the nitrocellulose membrane , the absorbent pad is connected to the upper surface of the other end of the nitrocellulose membrane, and the exposed nitrocellulose membrane between the end of the sample pad and the end of the absorbent pad is coated with a detection line and a quality control line parallel to the end face of the bottom plate;
所述检测线表面固定有1 mg/mL盐酸赛庚啶竞争抗原;1 mg/mL cyproheptadine hydrochloride competition antigen is fixed on the surface of the detection line;
所述质控线表面固定有浓度为0.3 mg/mL羊抗鼠IgG;The surface of the quality control line is immobilized with goat anti-mouse IgG at a concentration of 0.3 mg/mL;
具体实施中,样品垫装配前,优选以缓冲液浸泡24 h,所述缓冲液成分为:以质量百分比计,2% BSA,2.5% 蔗糖, 0.02% 叠氮化钠NaN3,以PBS补足余量,pH 7.4;In the specific implementation, before the sample pad is assembled, it is preferably soaked in a buffer solution for 24 hours, and the buffer solution components are: in mass percentage, 2% BSA, 2.5% sucrose, 0.02% sodium azide NaN 3 , and PBS is used to make up the remainder. amount, pH 7.4;
该试纸条对盐酸赛庚啶的检测灵敏度达到0.096 ng/mL。The detection sensitivity of the test strip for cyproheptadine hydrochloride reached 0.096 ng/mL.
进一步,本实用新型提供的基于量子点荧光微球的免疫层析试纸条中,所述检测线和质控线之间的间隔为3-8 mm。Further, in the immunochromatographic test strip based on quantum dot fluorescent microspheres provided by the present invention, the interval between the detection line and the quality control line is 3-8 mm.
其次,本实用新型还提供了上述基于量子点荧光微球的免疫层析试纸条在检测盐酸赛庚啶中的应用。其具体检测方法如下:Secondly, the present invention also provides the application of the above-mentioned immunochromatographic test strip based on quantum dot fluorescent microspheres in the detection of cyproheptadine hydrochloride. The specific detection method is as follows:
(1)将抗盐酸赛庚啶单克隆抗体-量子点荧光探针分别与盐酸赛庚啶标准品混合后孵育1min,然后滴于样品垫,15min后,若在紫外灯照射下试纸条的质控线显色,即以荧光读数仪记录试纸条检测线(T线)、质控线(C线)荧光强度,并通过计算,建立抑制率-盐酸赛庚啶标品浓度线性关系,绘制标准曲线:y=95.81-99.22/(1+(x/0.9)0.83);若在紫外灯照射下试纸条的质控线(C线)不显色,则试纸条判定为无效;所述盐酸赛庚啶标准品的浓度为0-50 ng/mL(图5),优选盐酸赛庚啶标准品的浓度依次为0,0.78,1.56,3.125,6.25,12.5,25,50 ng/mL(图4)。(1) The anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent probe was mixed with the cyproheptadine hydrochloride standard and incubated for 1 min, and then dropped on the sample pad. Color development of the quality control line, that is, record the fluorescence intensity of the test strip detection line (T line) and the quality control line (C line) with a fluorescence reader, and establish a linear relationship between the inhibition rate and the concentration of the cyproheptadine hydrochloride standard through calculation. Draw the standard curve: y=95.81-99.22/(1+(x/0.9) 0.83 ); if the quality control line (C line) of the test strip does not develop color under UV light irradiation, the test strip is judged to be invalid; The concentration of the standard cyproheptadine hydrochloride is 0-50 ng/mL (Fig. 5), preferably the concentration of the standard cyproheptadine hydrochloride is 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 ng/mL. mL (Figure 4).
(2)取待测样品至于离心管中,3000 g离心10 min;吸取上清,与稀释液按体积比1:4混合后,获得样品稀释液,重复步骤(1)进行检测:若在紫外灯的照射下试纸条的质控线显色,则根据检测线显色程度高低可判断盐酸赛庚啶含量的高低,并用荧光读数仪记录试纸条检测线、质控线荧光强度,根据荧光数值计算抑制率,代入步骤(1)曲线方程中,即获得样品中盐酸赛庚啶含量;若在紫外定的照射下质控线不显色,则试纸条判定为无效。进一步,上述检测方法中,所述抗盐酸赛庚啶单克隆抗体-量子点荧光微球探针是通过如下方法获得:(2) Put the sample to be tested in a centrifuge tube, centrifuge at 3000 g for 10 min; aspirate the supernatant and mix it with the diluent in a volume ratio of 1:4 to obtain a sample diluent, and repeat step (1) for detection: Under the illumination of the lamp, the quality control line of the test strip develops color, and the content of cyproheptadine hydrochloride can be judged according to the degree of color development of the test line, and the fluorescence intensity of the test line and the quality control line of the test strip is recorded by a fluorescence reader. Calculate the inhibition rate by fluorescence numerical value, and substitute it into the curve equation of step (1) to obtain the content of cyproheptadine hydrochloride in the sample; if the quality control line does not develop color under UV irradiation, the test strip is judged to be invalid. Further, in the above detection method, the anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent microsphere probe is obtained by the following method:
将0.12 mg量子点荧光微球溶解于pH 6.0,2.7 mL 0.01 mol/L磷酸盐缓冲液中,加入1μg EDC进行活化,然后加入150 μg盐酸赛庚啶单抗,混匀后,磁力搅拌反应30 min,13500 rpm离心15 min,去掉上清,离心沉淀用600 μL复溶液复溶,并4℃保存。Dissolve 0.12 mg of quantum dot fluorescent microspheres in pH 6.0, 2.7 mL of 0.01 mol/L phosphate buffer, add 1 μg of EDC for activation, and then add 150 μg of cyproheptadine hydrochloride. After mixing, magnetically stir for 30 minutes. min, centrifuge at 13,500 rpm for 15 min, remove the supernatant, reconstitute the centrifuged pellet with 600 μL of reconstituted solution, and store at 4°C.
复溶液配方:以质量百分比计算,5% 蔗糖,2% 果糖,1% PEG 20000,1% BSA,0.4%吐温-20,以去离子水补足余量。Compound solution formula: Calculated by mass percentage, 5% sucrose, 2% fructose, 1%
本步骤抗盐酸赛庚啶单克隆抗体-量子点荧光探针的方法也可以参见文献:“RenM , Xu H , Huang X , et al. Immunochromatographic assay for ultrasensitivedetection of aflatoxin B1 in maize by highly luminescent quantum dot beads.[J]. Acs Applied Materials & Interfaces, 2014, 6(16):14215-14222.”。The method of anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent probe in this step can also be found in the literature: "RenM , Xu H , Huang X , et al. Immunochromatographic assay for ultrasensitive detection of aflatoxin B 1 in maize by highly luminescent quantum dot beads.[J]. Acs Applied Materials & Interfaces, 2014, 6(16):14215-14222.”.
本实用新型中,量子点荧光微球优选通过掺杂CdSe/ZnS量子点所得,直径为247nm ±13 nm,该量子点荧光微球为现有技术,可市场购买或自行制备,制备方法参见文献:“Ren M , Xu H , Huang X , et al. Immunochromatographic assay forultrasensitive detection of aflatoxin B1 in maize by highly luminescentquantum dot beads.[J]. Acs Applied Materials & Interfaces, 2014, 6(16):14215-14222.”。In the present invention, the quantum dot fluorescent microspheres are preferably obtained by doping CdSe/ZnS quantum dots, with a diameter of 247 nm ± 13 nm. The quantum dot fluorescent microspheres are the prior art and can be purchased in the market or prepared by yourself. For the preparation method, please refer to the literature. : "Ren M , Xu H , Huang X , et al. Immunochromatographic assay for ultrasensitive detection of aflatoxin B 1 in maize by highly luminescentquantum dot beads. [J]. Acs Applied Materials & Interfaces, 2014, 6(16):14215-14222 .".
与现有检测方法相比,本实用新型提供的试纸条及其检测有益效果:Compared with the existing detection method, the test strip provided by the utility model and the detection beneficial effects thereof:
(1) 本实用新型提供的一种基于量子点荧光微球的盐酸赛庚啶免疫层析试纸条,较传统分析技术, 可实现对盐酸赛庚啶的更快速、简便、灵敏检测,利用便携式的荧光读卡仪即可进行现场实时定量检测。该试纸条可用于检测多种样品来源中的盐酸赛庚啶,如猪尿、动物饲料,猪肉组织等。(1) A kind of cyproheptadine hydrochloride immunochromatographic test strip based on quantum dot fluorescent microspheres provided by the present utility model, compared with traditional analysis technology, can realize more rapid, convenient and sensitive detection of cyproheptadine hydrochloride, using A portable fluorescence card reader can perform on-site real-time quantitative detection. The test strip can be used to detect cyproheptadine hydrochloride in various sample sources, such as pig urine, animal feed, pork tissue, etc.
(2)本实用新型提供的一种基于量子点荧光微球的盐酸赛庚啶荧光免疫层析试纸条,由于引入了量子点荧光探针,大大提高了该方法的检测灵敏度,其最低检测限好于同抗体的ELISA方法。(2) The cyproheptadine hydrochloride fluorescent immunochromatography test strip based on quantum dot fluorescent microspheres provided by the present utility model greatly improves the detection sensitivity of the method due to the introduction of quantum dot fluorescent probes. Limited to ELISA methods that are better than the same antibody.
(3) 本实用新型基于量子点荧光微球的盐酸赛庚啶免疫层析试纸条的检测方法可实现对样品中盐酸赛庚啶的定量检测。(3) The detection method of the cyproheptadine hydrochloride immunochromatographic test strip based on the quantum dot fluorescent microspheres of the present invention can realize the quantitative detection of cyproheptadine hydrochloride in the sample.
附图说明Description of drawings
图1为采用间接竞争ELISA方法检测单抗对盐酸赛庚啶的标准抑制曲线。Fig. 1 is the standard inhibition curve of monoclonal antibody against cyproheptadine hydrochloride detected by indirect competitive ELISA method.
图2为本实用新型基于量子点荧光微球的盐酸赛庚啶免疫层析试纸条原理示意图Fig. 2 is the principle schematic diagram of cyproheptadine hydrochloride immunochromatographic test strip based on quantum dot fluorescent microspheres of the present utility model
图3为本实用新型快速检测盐酸赛庚啶结果判读示意图。3 is a schematic diagram of the interpretation of the results of rapid detection of cyproheptadine hydrochloride according to the present invention.
图4为检测不同浓度的盐酸赛庚啶灵敏度实验图。Figure 4 is a graph of the sensitivity experiment for detecting cyproheptadine hydrochloride at different concentrations.
图5为本实用新型的免疫层析试纸条对盐酸赛庚啶的检测标准曲线。Fig. 5 is the detection standard curve of cyproheptadine hydrochloride by the immunochromatographic test strip of the present invention.
具体实施方式Detailed ways
实施例1盐酸赛庚啶单克隆抗体的制备Example 1 Preparation of Cyproheptadine Hydrochloride Monoclonal Antibody
1、免疫原(CYP-BSA)、竞争抗原 (CYP-OVA)的合成1. Synthesis of immunogen (CYP-BSA) and competing antigen (CYP-OVA)
1.1选取0.05 mmol的盐酸赛庚啶半抗原溶于1 mL二甲基甲酰胺(DMF),再依次添加N-羟基琥珀酰亚胺(NHS)(0.06 mmol)和N,N-二环己基碳二亚胺 (DCC) (0.06 mmol),在常温避光环境下进行搅拌并放置过夜。1.1 Dissolve 0.05 mmol of cyproheptadine hydrochloride hapten in 1 mL of dimethylformamide (DMF), and then add N-hydroxysuccinimide (NHS) (0.06 mmol) and N,N-dicyclohexyl carbon in sequence Diimine (DCC) (0.06 mmol) was stirred at room temperature and protected from light and left overnight.
1.2第2天,将放置过夜的混合物,10000 g,10 min离心处理,选取上清液备用。1.2 On the second day, the mixture left overnight was centrifuged at 10,000 g for 10 min, and the supernatant was selected for use.
1.3将BSA(0.15×10-3 mmol)与OVA(0.15×10-3 mmol)分别溶于5 mL PBS中,制得蛋白溶液;将步骤1.2获取的溶液缓慢滴加到蛋白溶液中,置于常温环境下搅拌4小时;1.3 Dissolve BSA (0.15×10 -3 mmol) and OVA (0.15×10 -3 mmol) in 5 mL of PBS respectively to prepare a protein solution; slowly add the solution obtained in step 1.2 dropwise to the protein solution and place it in the Stir at room temperature for 4 hours;
1.4反应完成之后,将生成物置于4oC的PBS中透析三天,每天更换透析液3-4次,透析后获得的产物即为免疫原(CYP-BSA)、竞争抗原 (CYP-OVA),置于-20oC环境下保存。1.4 After the reaction was completed, the resultant was dialyzed in PBS at 4 oC for three days, and the dialysate was replaced 3-4 times a day. The products obtained after dialysis were the immunogen (CYP-BSA) and the competing antigen (CYP-OVA). , and stored at -20 o C.
本步骤中,盐酸赛庚啶半抗原可商业购买或自行制备,制备方法为本领域常规技术,如见文献“杨茜茹. 新型“瘦肉精”盐酸赛庚啶免疫检测技术的研究[D].”所公开的制备方法。In this step, the cyproheptadine hydrochloride hapten can be purchased commercially or prepared by itself, and the preparation method is a conventional technique in the field, such as the literature "Yang Qianru. Research on the new "clenbuterol" cyproheptadine hydrochloride immunodetection technology [D]. "The disclosed preparation method.
2、盐酸赛庚啶人工抗原的鉴定2. Identification of Cyproheptadine Hydrochloride Artificial Antigen
将合成的盐酸赛庚啶免疫抗原和竞争抗原和载体蛋白分别进行紫外200-400 nm扫描,并通过比较三者在同一波长下的吸光值计算其结合比。The synthetic cyproheptadine hydrochloride immunization antigen, competition antigen and carrier protein were scanned by ultraviolet 200-400 nm respectively, and their binding ratios were calculated by comparing the absorbance values of the three at the same wavelength.
免疫原和竞争抗原的紫外光谱图与载体蛋白相比最大吸收波长发生位移,则说明半抗原已经与载体蛋白成功偶联制得盐酸赛庚啶人工抗原。经计算,所获人工抗原盐酸赛庚啶半抗原分子与BSA分子的结合比为20:1,盐酸赛庚啶半抗原分子与OVA分子的结合比为18:1,证明有较好的偶联效果。Compared with the carrier protein, the UV spectra of the immunogen and the competing antigen shifted the maximum absorption wavelength, indicating that the hapten had been successfully coupled with the carrier protein to obtain the cyproheptadine hydrochloride artificial antigen. After calculation, the binding ratio of the obtained artificial antigen cyproheptadine hydrochloride hapten molecule and BSA molecule is 20:1, and the binding ratio of cyproheptadine hydrochloride hapten molecule and OVA molecule is 18:1, which proves that there is a good coupling. Effect.
3、盐酸赛庚啶单抗的制备3. Preparation of Cyproheptadine hydrochloride
选用6-8周龄Balb/c雌鼠,通过四次免疫,每次免疫原(步骤1.4获得的CYP-BSA)用量为100 μg/只,每次免疫间隔时间为3周。初次免疫分别将免疫抗原与弗氏完全佐剂等体积混匀,腹腔注射。融合前3天每只腹腔注入150 μg免疫原进行加强免疫,免疫后第6天断尾取血,采用ELISA法测定效价。取效价最高的免疫小鼠脾细胞与小鼠骨髓瘤细胞SP2/0细胞混合,以融合剂50% PEG进行细胞融合,待细胞长到培养孔面积的1/4时,采用分步筛选法筛选杂交瘤细胞。采用间接ELISA方法进行细胞上清中抗体性质鉴定,若经盐酸赛庚啶阻断后的OD450 nm值下降到对照孔的50%以下,则为阳性,经2-3次检测都为阳性的孔,立即用有限稀释法进行亚克隆。将2-3次亚克隆建株后的杂交瘤细胞扩大培养,收集上清液用间接ELISA测定效价,冻存;取8-10周龄Balb/c小鼠腹腔注射液体石蜡0.5 mL/只,7-10日后腹腔注射杂交瘤细胞1-2×106/只,7-10日后抽取小鼠腹水,离心取上清,测定效价后冻存备用。Select 6-8-week-old Balb/c female mice and immunize them four times. The dose of each immunogen (CYP-BSA obtained in step 1.4) is 100 μg/mouse, and the interval between each immunization is 3 weeks. For the primary immunization, the immunizing antigen and Freund's complete adjuvant were mixed in equal volumes and injected intraperitoneally. Three days before fusion, 150 μg of immunogen was injected into each animal for booster immunization. On the 6th day after immunization, blood was collected from the tail, and the titer was determined by ELISA. The immunized mouse spleen cells with the highest titer were mixed with mouse myeloma cells SP2/0 cells, and the cells were fused with 50% PEG as a fusion agent. When the cells grew to 1/4 of the culture well area, the step-by-step screening method Screening of hybridoma cells. Indirect ELISA method was used to identify the properties of antibodies in the cell supernatant. If the OD 450 nm value after blocking by cyproheptadine hydrochloride dropped to less than 50% of the control well, it was positive, and it was positive after 2-3 tests. Wells were immediately subcloned by limiting dilution. The hybridoma cells after 2-3 times of subcloning were expanded and cultured, and the supernatant was collected to determine the titer by indirect ELISA, and then cryopreserved; 8-10-week-old Balb/c mice were injected intraperitoneally with 0.5 mL of liquid paraffin per mouse 7-10 days later, intraperitoneal injection of 1-2 × 10 6 hybridoma cells per mouse was performed, and ascites was extracted from mice 7-10 days later, and the supernatant was collected by centrifugation.
步骤2、3的制备方法也可以参见文献“杨茜茹. 新型“瘦肉精”盐酸赛庚啶免疫检测技术的研究[D]”及“闫丽婷. 通用有机磷、喜树碱单克隆抗体的制备及免疫检测技术研究[D].”所公开的方法。The preparation methods of
4、腹水的纯化4. Purification of ascites
4.1将5 mL腹水与等体积的PBS混合,向其中加入5 mL饱和硫酸铵溶液,边搅拌边逐滴加入,再在室温下继续搅30 min,将其置于4℃冰箱冷却过夜,使免疫球蛋白充分沉淀。4.1
4.2将充分沉淀后的混合液离心,4oC,10000 g/min,20 min,将沉淀用5 mL PBS溶解,再向其中边搅拌边滴加3 mL饱和硫酸铵溶液,并继续搅拌30 min,置4oC冰箱过夜。4.2 Centrifuge the fully precipitated mixture, 4 o C, 10000 g/min, 20 min, dissolve the precipitate with 5 mL of PBS, add 3 mL of saturated ammonium sulfate solution dropwise to it while stirring, and continue to stir for 30 min , refrigerate at 4 o C overnight.
4.3重复步骤4.2两次,用5 mL PBS溶解末次离心后的沉淀,将其装入透析袋,用PBS透析3天,每天换液3-4次,充分除盐。4.3 Repeat step 4.2 twice, dissolve the precipitate after the last centrifugation with 5 mL PBS, put it into a dialysis bag, dialyze it with PBS for 3 days, change the medium 3-4 times a day, and fully remove the salt.
4.4将透析产物离心,4 oC,5000 g/min,50 min,上清液即为粗提得到的盐酸赛庚啶单抗,将其分装保存于-20oC,以备后续使用。4.4 Centrifuge the dialysis product, 4 o C, 5000 g/min, 50 min. The supernatant is the crudely extracted cyproheptadine hydrochloride, which is stored in aliquots at -20 o C for subsequent use.
采用间接竞争ELISA方法鉴定单抗的检测灵敏度,检测方法参见文献“闫丽婷. 通用有机磷、喜树碱单克隆抗体的制备及免疫检测技术研究[D].”所公开的方法。检测结果如图1所示,同时绘制标准曲线公式为:y=90.6-90.23/(1+(x/6.52)0.95),其中,x表示盐酸赛庚啶标准品的浓度,y表示盐酸赛庚啶标准品对单抗的抑制率。如图1,盐酸赛庚啶标准品对单抗抑制率为50%时所对应的盐酸赛庚啶的浓度 (IC50)为8.05 ng/mL,最低检测线(IC10)为0.70 ng/mL,检测范围(IC20-IC80)在1.69-54.46 ng/mL。The detection sensitivity of monoclonal antibody was identified by indirect competitive ELISA. For the detection method, please refer to the method disclosed in the literature "Yan Liting. Preparation of universal organophosphorus and camptothecin monoclonal antibodies and research on immunodetection technology [D].". The detection results are shown in Figure 1, and the formula for drawing a standard curve at the same time is: y=90.6-90.23/(1+(x/6.52) 0.95 ), where x represents the concentration of the standard cyproheptadine hydrochloride, and y represents the cyproheptadine hydrochloride Inhibition rate of monoclonal antibody against pyridine standard. As shown in Figure 1, the concentration of cyproheptadine hydrochloride standard (IC 50 ) is 8.05 ng/mL when the inhibition rate of monoclonal antibody is 50%, and the lowest detection line (IC 10 ) is 0.70 ng/mL , the detection range (IC 20 -IC 80 ) is 1.69-54.46 ng/mL.
实施例2 抗盐酸赛庚啶单克隆抗体-量子点荧光探针的制备Example 2 Preparation of anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent probe
将0.12 mg量子点荧光微球溶解于pH 6.0,2.7 mL 0.01 mol/L磷酸盐缓冲液中,加入1μg EDC进行活化,然后加入150 μg盐酸赛庚啶单抗,混匀后,磁力搅拌反应30 min,13500 rpm离心15 min,去掉上清,离心沉淀用600 μL复溶液复溶,并4℃保存。Dissolve 0.12 mg of quantum dot fluorescent microspheres in pH 6.0, 2.7 mL of 0.01 mol/L phosphate buffer, add 1 μg of EDC for activation, and then add 150 μg of cyproheptadine hydrochloride. After mixing, magnetically stir for 30 minutes. min, centrifuge at 13,500 rpm for 15 min, remove the supernatant, reconstitute the centrifuged pellet with 600 μL of reconstituted solution, and store at 4°C.
复溶液配方:以质量百分比计算,5% 蔗糖,2% 果糖,1% PEG 20000,1% BSA,0.4%吐温-20,以去离子水补足余量。Compound solution formula: Calculated by mass percentage, 5% sucrose, 2% fructose, 1
上述量子点荧光微球的制备方法也可参见文献“Duan H , Chen X , Xu W , etal. Quantum-DoT submicrobead-based immunochromatographic assay forquantitative and sensitive detection of zearalenone[J]. Talanta, 2015, 132:126-131.”。所公开的方法制备;上述抗盐酸赛庚啶单克隆抗体-量子点荧光探针的方法也可以参见文献“Ren M , Xu H , Huang X , et al. Immunochromatographic assay forultrasensitive detection of aflatoxin B1in maize by highly luminescentquantum dot beads.[J]. Acs Applied Materials & Interfaces, 2014, 6(16):14215-14222.”The preparation method of the above quantum dot fluorescent microspheres can also be found in the literature "Duan H , Chen X , Xu W , etal. Quantum-DoT submicrobead-based immunochromatographic assay for quantitative and sensitive detection of zearalenone [J]. Talanta, 2015, 132:126 -131.". The disclosed method is prepared; the above-mentioned anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent probe method can also refer to the document "Ren M, Xu H, Huang X, et al. Immunochromatographic assay for ultrasensitive detection of aflatoxin B 1 in maize by highly luminescentquantum dot beads.[J]. Acs Applied Materials & Interfaces, 2014, 6(16):14215-14222.”
实施例3 试纸条的制备Example 3 Preparation of test strips
如图2A所示,一种基于量子点荧光微球的免疫层析试纸条,包括PVC底板,样品垫1、硝酸纤维素膜4和吸水垫5,样品垫1搭接于硝酸纤维素膜4的一端上表面(相邻垫交叠2mm),吸水垫5接于硝酸纤维素膜4的另一端上表面(相邻垫交叠2 mm),样品垫1端部与吸水垫5端部之间外露的硝酸纤维素膜4上包被有与底板端面平行的检测线2(T线)和质控线3(C线)。As shown in Figure 2A, an immunochromatographic test strip based on quantum dot fluorescent microspheres includes a PVC bottom plate, a
如图2B所示,利用切条仪将组装好的试纸条切成4 mm宽,60mm长的小条,置于白色的长条状扁平检测卡中,所述检测壳6的表面设有加样孔7和检查窗8,待检测样本和荧光探针适于从所述加样孔7注入到试纸条上;通过所述检查窗8,便于操作人员观察实验结果。As shown in Figure 2B, the assembled test strip is cut into 4 mm wide and 60 mm long strips using a strip cutter, and placed in a white long strip-shaped flat detection card. The surface of the
所述检测线表面固定有1 mg/mL盐酸赛庚啶竞争抗原(实施例1步骤1.4制备的CYP-OVA);The surface of the detection line is immobilized with 1 mg/mL cyproheptadine hydrochloride competition antigen (CYP-OVA prepared in step 1.4 of Example 1);
所述质控线表面固定有浓度为0.3 mg/mL羊抗鼠IgG;The surface of the quality control line is immobilized with goat anti-mouse IgG at a concentration of 0.3 mg/mL;
本实施例中,样品垫组装前要经缓冲液浸泡1小时,常温晾干后再组装;浸泡步骤可以减少样本差异,提高实验灵敏度,避免复杂辨识剂、追迹缓冲液的麻烦,使检测一步完成。In this embodiment, the sample pad should be soaked in buffer solution for 1 hour before assembly, and then assembled after drying at room temperature; the soaking step can reduce sample differences, improve experimental sensitivity, avoid the trouble of complex identifiers and tracking buffers, and make detection in one step. Finish.
缓冲液成分为:以质量百分比计,2% BSA,2.5% 蔗糖, 0.02% 叠氮化钠NaN3,以PBS补足余量,pH7.4。The components of the buffer solution are: 2% BSA, 2.5% sucrose, 0.02% sodium azide NaN3 in mass percentage, the balance is made up with PBS, pH 7.4.
上述羊抗鼠IgG可商业购买,或参考文献“Liu B , Gong H , Wang Y , et al. Agold immunochromatographic assay for simultaneous detection of parathion andtriazophos in agricultural products[J]. ANALYTICAL METHODS, 2018, 10.”。The above-mentioned goat anti-mouse IgG can be purchased commercially, or refer to the literature "Liu B, Gong H, Wang Y, et al. Agold immunochromatographic assay for simultaneous detection of parathion and triazophos in agricultural products [J]. ANALYTICAL METHODS, 2018, 10.".
实施例4量子点荧光微球试纸条检测应用Example 4 Quantum dot fluorescent microsphere test strip detection application
(1) 结果判断(1) Result judgment
将实施例2制备的量子点荧光探针取1μL后分别与含5%甲醇PBS缓冲液及溶有不同浓度盐酸赛庚啶标品的缓冲液混合,室温孵育1min后,加入试纸条加样孔,反应 15 min 后用试纸条读取仪进行检测。以不添加盐酸赛庚啶标品的实验组作为阴性对照组。将试纸条进入读取仪(干式荧光免疫分析仪,型号:FIC-S2011-L44,厂家:苏州和迈精密仪器有限公司)后,经过光学元件激发,试纸条 T、C 线处量子点荧光微球发射出荧光信号,仪器将荧光信号转换成数字信号显示在读取仪显示器上。15 min后,若C线显色,T线色泽强度较阴性对照组的T线相当(如图3B),则为阴性结果;若T、C线均显色,且T线色泽强度较阴性对照组的T线小(如图3C),或者C线显色,T线不显色(如图3D),则为阳性结果;若C线不显色,则试纸条判定为无效,(如图3 E、F),其中图3A为阴性对照。如图4所示,从左到右,标品浓度分别为0,0.78,1.56,3.125,6.25,12.5,25,50 ng/mL, T线亮度逐渐变暗直到肉眼不可见,符合实验预期结果,可初步实现对赛庚啶残留的定性检测。Take 1 μL of the quantum dot fluorescent probe prepared in Example 2 and mix it with PBS buffer containing 5% methanol and buffer containing cyproheptadine hydrochloride with different concentrations respectively. After incubation at room temperature for 1 min, add test strips for sample loading. After 15 min of reaction, it was detected with a test strip reader. The experimental group without cyproheptadine hydrochloride standard was used as the negative control group. After entering the test strip into the reader (dry fluorescence immunoassay analyzer, model: FIC-S2011-L44, manufacturer: Suzhou Hemai Precision Instrument Co., Ltd.), after excitation by optical components, the quantum dots at the T and C lines of the test strip are The fluorescent microspheres emit fluorescent signals, and the instrument converts the fluorescent signals into digital signals and displays them on the display of the reader. After 15 minutes, if the C line develops color and the color intensity of the T line is comparable to that of the T line in the negative control group (as shown in Figure 3B), the result is a negative result; if both the T and C lines develop color, and the color intensity of the T line is higher than that of the negative control If the T line in the group is small (as shown in Figure 3C), or if the C line develops color and the T line does not develop color (as shown in Figure 3D), it is a positive result; if the C line does not develop color, the test strip is judged to be invalid, (such as Figure 3 E, F), wherein Figure 3A is a negative control. As shown in Figure 4, from left to right, the concentration of the standard is 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 ng/mL, and the T-line brightness gradually dims until it is invisible to the naked eye, which is in line with the expected results of the experiment , the qualitative detection of cyproheptadine residues can be initially realized.
(2) 标准曲线的确定(2) Determination of standard curve
用含体积比为5%甲醇的PBS缓冲液2倍梯度稀释盐酸赛庚啶标品,浓度分别为:0.048828125, 0.09765625, 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25,12.5, 25, 50每个浓度测定五个平行重复,15 min后用荧光读数仪记录试纸条T、C线荧光强度,设定浓度为0标准液的FIT/FIC比值为B0,其它加标浓度的FIT/FIC比值为B,以(B0-B)/B0×100(%)为纵坐标,盐酸赛庚啶浓度为横坐标做图,绘制试纸条竞争抑制曲线,得到的标准曲线如图5所示。抑制曲线公式:y=95.81-99.22/(1+(x/0.9)0.83),根据公式算得:IC50为1.09 ng/mL,最低检测线为0. 096 ng/mL,检测范围在0.22-6.68 ng/mL。Cyproheptadine hydrochloride standard was diluted 2-fold in PBS buffer containing 5% methanol by volume, the concentrations were: 0.048828125, 0.09765625, 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 each The concentration was measured in five parallel repetitions. After 15 minutes, the fluorescence intensity of the T and C lines of the test strip was recorded with a fluorescence reader. The FIT/FIC ratio of the standard solution with a concentration of 0 was set to be B 0 , and the FIT of other standard concentrations was set as B 0 . The ratio of /FI C is B, take (B 0 -B)/B 0 ×100 (%) as the ordinate and the concentration of cyproheptadine hydrochloride as the abscissa to draw the graph, and draw the test strip competition inhibition curve, and the obtained standard curve is as follows shown in Figure 5. Inhibition curve formula: y=95.81-99.22/(1+(x/0.9) 0.83 ), calculated according to the formula: IC 50 is 1.09 ng/mL, the lowest detection line is 0.096 ng/mL, and the detection range is 0.22-6.68 ng/mL.
(3)样品处理(3) Sample processing
取猪尿样本2 mL于聚苯乙烯离心管中,3000 g离心10 min;吸取上清,与稀释液按1:4体积混合;取适量混合液(样品稀释液)用于检测。Take 2 mL of pig urine sample into a polystyrene centrifuge tube and centrifuge at 3000 g for 10 min; aspirate the supernatant and mix it with the diluent at a volume of 1:4; take an appropriate amount of the mixture (sample diluent) for detection.
所述稀释液为含体积比为5%甲醇的PBS缓冲液。The diluent was a PBS buffer containing 5% methanol by volume.
(4) 样品的检测(4) Detection of samples
向不含盐酸赛庚啶的尿液样品中添加盐酸赛庚啶标准品,使标准品在样品中的终浓度分别为1、5、20 mg/L;将添加后的样品分别按照上述方法进行检测,每个实验重复5次,分别计算变异系数及添加回收率。结果分别见表1。Add cyproheptadine hydrochloride standard to the urine sample without cyproheptadine hydrochloride, so that the final concentration of the standard in the sample is 1, 5, and 20 mg/L; Detection, each experiment was repeated 5 times, and the coefficient of variation and recovery rate of addition were calculated respectively. The results are shown in Table 1, respectively.
表1加标回收实验结果Table 1 The experimental results of standard addition and recovery
结果表明,猪尿样品的平均添加回收率在94-104%,变异系数在1.5-7%。这一检测结果说明该方法可准确检测猪尿中的盐酸赛庚啶含量。The results showed that the average additive recovery of pig urine samples was 94-104%, and the coefficient of variation was 1.5-7%. This test result shows that the method can accurately detect the content of cyproheptadine hydrochloride in pig urine.
(4) 交叉反应率试验(4) Cross-reaction rate test
选择与赛庚啶结构或功能类似的药物如弗雷他定、富马酸酮替芬、盐酸可乐定进行交叉反应试验,通过各种药物的标准曲线分别得到其IC50。用IC50盐酸赛庚啶/IC50类似物计算试纸条对其它赛庚啶类似物的交叉反应率。与其他药物的交叉反应率越小,说明量子点荧光微球免疫层析试纸条对盐酸赛庚啶的检测特异性越好。结果量子点荧光微球免疫层析试纸条对弗雷他定、酮替芬、盐酸可乐定交叉反应率< 1%,表明该方法具有较好的特异性,本实用新型可用于检测猪尿中的盐酸赛庚啶残留量。Drugs similar in structure or function to cyproheptadine, such as fretadine, ketotifen fumarate, and clonidine hydrochloride, were selected for cross-reaction test, and their IC 50 was obtained through the standard curve of each drug. The IC50 Cyproheptadine HCl/ IC50 Analogue was used to calculate the cross-reactivity of the test strips to other Cyproheptadine analogs. The smaller the cross-reaction rate with other drugs, the better the detection specificity of the quantum dot fluorescent microsphere immunochromatographic test strip for cyproheptadine hydrochloride. As a result, the quantum dot fluorescent microsphere immunochromatographic test strip has a cross-reaction rate<1% to fretadine, ketotifen, and clonidine hydrochloride, indicating that the method has good specificity, and the utility model can be used to detect pig urine residual cyproheptadine hydrochloride.
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