技术领域Technical Field
本技术方案涉及化学发光检测领域，更具体地，本技术方案涉及一种人类免疫缺陷病毒抗体检测试剂盒及其应用。The present technical solution relates to the field of chemiluminescence detection, and more specifically, to a human immunodeficiency virus antibody detection kit and its application.
背景技术Background Art
免疫分析经过半个多世纪的发展，已经发展出了很多种类。根据测定过程中是否要将待测物质与反应体系分离可以分为非均相(Heterogenous)免疫分析和均相(Homogeneous)免疫分析。非均相免疫分析，是指引入探针进行标记的操作过程中，各种相关试剂混合反应后需要进行分离，将待测物与反应体系分离后再进行检测，是现在免疫分析中的主流方法。如广为人们熟知的酶联免疫吸附法(ELISA法)和磁微粒化学发光法等。均相免疫分析则是指在测定过程中将待测物与反应体系中的相关试剂混合反应后直接测定，而没有多余的分离或清洗的步骤。截止目前，多种灵敏的检测方法被应用于均相免疫分析，比如光学检测方法、电化学检测方法等。After more than half a century of development, immunoassays have developed into many types. Depending on whether the analyte needs to be separated from the reaction system during the determination process, it can be divided into heterogeneous immunoassays and homogeneous immunoassays. Heterogeneous immunoassays refer to the process of introducing probes for labeling, in which various related reagents need to be separated after mixed reaction, and the analyte is separated from the reaction system before detection. It is the mainstream method in immunoassays now. Such as the well-known enzyme-linked immunosorbent assay (ELISA) and magnetic particle chemiluminescence method. Homogeneous immunoassays refer to the process of directly measuring the analyte after mixing the analyte with the related reagents in the reaction system during the determination process, without any extra separation or cleaning steps. Up to now, a variety of sensitive detection methods have been applied to homogeneous immunoassays, such as optical detection methods, electrochemical detection methods, etc.
获得性免疫缺陷综合征(AIDS)是一种由HIV病毒即人类免疫缺陷病毒(简称HIV)侵入人体后破坏人体免疫功能，使人体发生多种不可治愈的感染和肿瘤，最后导致被感染者死亡的一种严重传染病。HIV感染后主要表现为免疫系统受到严重损伤，T4淋巴细胞遭破坏，机体抵抗力下降，诱发严重感染和一些少见的癌瘤，病人容易患各种罕见的疾病，最终因长期消耗，全身衰竭而死亡。因此，HIV检测时预防和治疗获得性免疫缺陷综合征最重要的环节。Acquired immunodeficiency syndrome (AIDS) is a serious infectious disease caused by the human immunodeficiency virus (HIV) that invades the human body and destroys the human immune function, causing a variety of incurable infections and tumors, and ultimately leading to the death of the infected person. HIV infection is mainly manifested by severe damage to the immune system, destruction of T4 lymphocytes, decreased body resistance, inducing severe infections and some rare cancers, and patients are prone to various rare diseases, and eventually die due to long-term consumption and systemic failure. Therefore, HIV testing is the most important part of preventing and treating acquired immunodeficiency syndrome.
目前，HIV的检测方法情况如下：Currently, HIV testing methods are as follows:
A.酶联免疫吸附试验(ELISA)A. Enzyme-linked immunosorbent assay (ELISA)
目前应用的ELISA法有8种之多。它们的特异性和敏感性超过99％。There are currently 8 types of ELISA methods in use, with specificity and sensitivity exceeding 99%.
B.颗粒凝集法(PA)B. Particle Agglutination Assay (PA)
PA为快速、简便的一种筛选方法。如属阳性，应经WB证实。PA不需任何特殊仪器，其结果用肉眼可判别。全过程仅需5分钟。缺点有假阳性，且价格昂贵。PA is a fast and simple screening method. If positive, it should be confirmed by WB. PA does not require any special equipment and the results can be judged by the naked eye. The whole process takes only 5 minutes. The disadvantages are false positives and high cost.
C.快速试剂C. Rapid Reagents
a)人类免疫缺陷病毒(HIV)1+2型抗体诊断试剂(胶体硒法)a) Human immunodeficiency virus (HIV) type 1+2 antibody diagnostic reagent (colloidal selenium method)
仅用于无偿献血员现场初筛及临床紧急情况的使用，本品检测阳性者，需进行进一步筛查确认。It is only used for on-site initial screening of voluntary blood donors and in clinical emergencies. Those who test positive for this product need further screening and confirmation.
b)InstantCHEKTM-HIVl+2金标快速诊断试剂b) InstantCHEKTM - HIV1+2 Gold Label Rapid Diagnostic Reagent
InstantCHEKTM-HIV1+2是一种快速、简单、灵敏的检验方法，用以检测艾滋病病毒(HIV-1和HIV-2)的抗体。该方法适用于初筛检测，凡由该试剂测定为阳性者，需用另一种方法检测如ELISA或用蛋白印记法确定。InstantCHEKTM -HIV1+2 is a rapid, simple and sensitive test method for detecting antibodies to HIV (HIV-1 and HIV-2). This method is suitable for primary screening. Those who are positive by this test need to be confirmed by another method such as ELISA or protein blotting.
D.HIV-抗体确认实验D. HIV-antibody confirmation test
免疫印迹试验(WB)、条带免疫试验(LIATEKHIVⅢ)、放射免疫沉淀试验(RIPA)及免疫荧光试验(IFA)。国内常用的确认试验方法是WB。Western blot test (WB), strip immunoassay (LIATEKHIVⅢ), radioimmunoprecipitation assay (RIPA) and immunofluorescence assay (IFA). The commonly used confirmation test method in China is WB.
a)免疫印迹实验(westernblot，WB)a) Western blot (WB)
广泛用于许多传染病诊断的实验方法，就HIV的病原学诊断而言，它是首选的用以确认HIV抗体的确认实验方法，WB的检测结果常常被作为鉴别其他检验方法优劣的“金标准”。It is an experimental method widely used in the diagnosis of many infectious diseases. As far as the etiological diagnosis of HIV is concerned, it is the preferred confirmatory experimental method for confirming HIV antibodies. The test results of WB are often used as the "gold standard" to identify the advantages and disadvantages of other testing methods.
WB的敏感性一般不低于初筛实验，但它的特异性很高，这主要是基于HIV不同抗原组分的分离以及浓缩和纯化，能够检测针对不同抗原成分的抗体，因而能够用WB方法鉴别初筛实验的准确性。从WB确认试验结果看出，初筛试验尽管选择质量较好的试剂，如第三代ELISA，仍会有假阳性出现，必须通过确认试验才能得出准确结果。The sensitivity of WB is generally not lower than that of the initial screening test, but its specificity is very high. This is mainly based on the separation, concentration and purification of different HIV antigen components, and the ability to detect antibodies against different antigen components. Therefore, the accuracy of the initial screening test can be identified by the WB method. From the results of the WB confirmation test, it can be seen that even if the initial screening test uses reagents of better quality, such as the third-generation ELISA, false positives will still occur, and the confirmation test must be carried out to obtain accurate results.
b)免疫荧光实验(IFA)b) Immunofluorescence assay (IFA)
IFA法经济、简便、快速，曾被FDA推荐用于WB不确定样品的诊断。但需要昂贵的荧光显微镜，需要受过良好训练的技术人员、观察和解释结果易受主观因素的影响，结果不宜长期保存，IFA不宜在一般的实验室开展和应用。IFA is economical, simple, and rapid, and was recommended by the FDA for the diagnosis of WB uncertain samples. However, it requires expensive fluorescence microscopes, well-trained technicians, and the observation and interpretation of results are easily affected by subjective factors. The results are not suitable for long-term storage, and IFA is not suitable for development and application in general laboratories.
以上这些方法都存在一些难以克服的困难，化学发光法具有高灵敏度、高特异性、宽线性、快速、影响因素少、结果准确等优点，是近年来运用最为广泛的一种疾病检测方法。目前逐渐运用于HIV的检测，有助于实现感染的早发现、早诊断、早治疗，减少假阳性和漏检率。All of the above methods have some difficulties that are difficult to overcome. Chemiluminescence has the advantages of high sensitivity, high specificity, wide linearity, rapidity, few influencing factors, and accurate results. It is the most widely used disease detection method in recent years. It is gradually being used in HIV detection, which helps to achieve early detection, early diagnosis, and early treatment of infection, and reduce false positives and missed detection rates.
光激化学发光的基础原理是一种均相免疫反应。它是基于两种微粒表面包被的抗原或抗体，在液相中形成免疫复合物而将两种微粒拉近。在激光的激发下，发生微粒之间的离子氧的转移，进而产生高能级的红光，通过单光子计数器和数学拟合将光子数换算为靶分子浓度。而当样本不含靶分子时，两种微粒间无法形成免疫复合物，两种微粒的间距超出离子氧传播范围，离子氧在液相中迅速淬灭，检测时则无高能级红光产生。利用光激化学发光法检测HIV，目前存在以下问题难以解决：(1)制备工艺复杂，尤其是微球制备及修饰工艺过于复杂；(2)假阳性率高。The basic principle of photochemiluminescence is a homogeneous immune reaction. It is based on the antigen or antibody coated on the surface of two microparticles, which forms an immune complex in the liquid phase and brings the two microparticles closer. Under the excitation of the laser, the transfer of ionized oxygen between the microparticles occurs, thereby generating high-energy red light. The number of photons is converted into the concentration of the target molecule through a single photon counter and mathematical fitting. When the sample does not contain the target molecule, the two microparticles cannot form an immune complex, the distance between the two microparticles exceeds the propagation range of ionized oxygen, and the ionized oxygen is rapidly quenched in the liquid phase, and no high-energy red light is generated during detection. The use of photochemiluminescence to detect HIV currently has the following problems that are difficult to solve: (1) The preparation process is complicated, especially the preparation and modification process of the microspheres is too complicated; (2) The false positive rate is high.
因此，亟需开发一种能够大批量生产、成本低廉、质量合格、性能稳定，既能满足灵敏度要求、又能满足线性范围要求的HIV检测试剂盒。Therefore, there is an urgent need to develop an HIV detection kit that can be mass-produced, has low cost, qualified quality, stable performance, and can meet both sensitivity requirements and linear range requirements.
发明内容Summary of the invention
本发明所要解决的技术问题是针对现有技术的不足，提供一种人类免疫缺陷病毒抗体检测试剂盒。当将该试剂盒应用于均相化学发光分析检测时，本申请的发明人意外发现其既有超高的灵敏度，又具有很宽的检测量程。The technical problem to be solved by the present invention is to provide a human immunodeficiency virus antibody detection kit in view of the deficiencies of the prior art. When the kit is applied to homogeneous chemiluminescence analysis and detection, the inventor of the present application unexpectedly discovered that it has both ultra-high sensitivity and a wide detection range.
基于此，本发明一方面提供一种人类免疫缺陷病毒抗体检测试剂盒，其包括试剂R1，所述试剂R1包含第一缓冲溶液以及悬浮于其中的能够与活性氧作用产生化学发光信号的受体颗粒，所述受体颗粒包括载体，所述载体的内部填充有发光组合物，所述载体的表面键合有HIV抗原；所述受体颗粒在受体试剂中的粒径分布变异系数C.V值不低于5％且不高于20％；每毫克质量的所述受体颗粒中的糖含量不高于25μg。Based on this, on one hand, the present invention provides a human immunodeficiency virus antibody detection kit, which includes a reagent R1, wherein the reagent R1 contains a first buffer solution and receptor particles suspended therein that can react with active oxygen to produce chemiluminescent signals, the receptor particles include a carrier, the interior of the carrier is filled with a luminescent composition, and the surface of the carrier is bonded with an HIV antigen; the coefficient of variation C.V value of the particle size distribution of the receptor particles in the receptor reagent is not less than 5% and not more than 20%; the sugar content in the receptor particles per milligram mass is not more than 25μg.
所述受体颗粒在所述试剂R1中的粒径分布变异系数C.V值不高于15％。The coefficient of variation C.V of the particle size distribution of the receptor particles in the reagent R1 is no higher than 15%.
所述受体颗粒在所述试剂R1中的粒径分布变异系数C.V值不低于8％。The coefficient of variation C.V of the particle size distribution of the receptor particles in the reagent R1 is not less than 8%.
每毫克质量的所述受体颗粒中的糖含量不高于20μg。The sugar content in the receptor particles is no more than 20 μg per mg mass.
所述第一缓冲溶液中多糖的含量为0.01～1wt％，优选为0.05～0.5wt％。The content of polysaccharide in the first buffer solution is 0.01-1 wt %, preferably 0.05-0.5 wt %.
所述载体的表面没有包被糖分子而直接键合HIV抗原。The surface of the carrier is not coated with sugar molecules but directly bonded to HIV antigens.
所述载体的表面上带有键合官能团，其用于将HIV抗原直接化学键合在所述载体的表面上，所述键合官能团选自胺基、酰胺基、羟基、醛基、羧基、环氧基、马来酰亚胺基和巯基中的至少一种；优选选自醛基、羧基、环氧基和马来酰亚胺基。The carrier has a bonding functional group on its surface, which is used to directly chemically bond the HIV antigen to the surface of the carrier. The bonding functional group is selected from at least one of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, an epoxy group, a maleimide group and a thiol group; preferably selected from an aldehyde group, a carboxyl group, an epoxy group and a maleimide group.
所述多糖选自含有三个或更多个未修饰或修饰的单糖单元的碳水化合物；优选选自葡聚糖、淀粉、糖原、菊粉、果聚糖、甘露聚糖、琼脂糖、半乳聚糖、羧基葡聚糖和氨基葡聚糖；更优选选自葡聚糖、淀粉、糖原和聚核糖，最优选为葡聚糖或葡聚糖衍生物。所述糖含量可以利用蒽酮法检测。The polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose, most preferably dextran or dextran derivatives. The sugar content can be detected by anthrone method.
所述试剂盒还包括试剂R2，试剂R2包含生物素标记的HIV抗原。The kit further comprises a reagent R2, which contains a biotin-labeled HIV antigen.
所述试剂盒还包括Anti-HIV样品稀释液。The kit also includes an Anti-HIV sample diluent.
所述试剂盒还包括Anti-HIV阴性对照。The kit also includes an Anti-HIV negative control.
所述试剂盒还包括Anti-HIV-1阳性对照，其含有小牛血清和HIV-1阳性血清。The kit also includes an Anti-HIV-1 positive control, which contains calf serum and HIV-1 positive serum.
所述试剂盒还包括Anti-HIV-2阳性对照，其含有小牛血清和HIV-2多克隆抗体。The kit also includes an Anti-HIV-2 positive control, which contains calf serum and HIV-2 polyclonal antibodies.
所述试剂盒还包括Anti-HIV弱阳性对照，其含有小牛血清和HIV-1阳性血清。The kit also includes an Anti-HIV weak positive control, which contains calf serum and HIV-1 positive serum.
本发明的有益效果为：本发明所述试剂盒中R1试剂的受体颗粒粒径分布变异系数C.V值不低于5％且不高于20％，每毫克质量的所述受体颗粒中的糖含量不高于25μg；进而上述R1试剂既能满足大批量生产的商业需要，同时也有超高的灵敏度，又有很宽的检测量程。同时，本发明对受体颗粒中的糖含量进行控制，降低了糖对检测信号的影响，使检测精密度提高，也降低了生产成本。The beneficial effects of the present invention are as follows: the coefficient of variation C.V of the receptor particle size distribution of the R1 reagent in the kit of the present invention is not less than 5% and not more than 20%, and the sugar content in the receptor particles per milligram is not more than 25 μg; and the above R1 reagent can meet the commercial needs of mass production, and also has ultra-high sensitivity and a wide detection range. At the same time, the present invention controls the sugar content in the receptor particles, reduces the influence of sugar on the detection signal, improves the detection precision, and reduces the production cost.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为实施例2制备的受体颗粒的Gaussian分布图。FIG. 1 is a Gaussian distribution diagram of the receptor particles prepared in Example 2.
图2是一种糖含量测定标准曲线图。FIG. 2 is a standard curve diagram for determining sugar content.
具体实施方式DETAILED DESCRIPTION
为使本发明容易理解，下面将详细说明本发明。但在详细描述本发明前，应当理解本发明不限于描述的具体实施方式。还应当理解，本文中使用的术语仅为了描述具体实施方式，而并不表示限制性的。本发明的实施并不局限于下面的实施例，对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be restrictive. The implementation of the present invention is not limited to the following embodiments, and any formal modifications and/or changes made to the present invention will fall within the scope of protection of the present invention.
在提供了数值范围的情况下，应当理解所述范围的上限和下限和所述规定范围中的任何其他规定或居间数值之间的每个居间数值均涵盖在本发明内。这些较小范围的上限和下限可以独立包括在较小的范围中，并且也涵盖在本发明内，服从规定范围中任何明确排除的限度。在规定的范围包含一个或两个限度的情况下，排除那些包括的限度之任一或两者的范围也包含在本发明中。Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicitly excluded limits in the specified ranges. Where a specified range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the present invention.
除非另有定义，本文使用的所有术语与本发明所属领域的普通技术人员的通常理解具有相同的意义。虽然与本文中描述的方法和材料类似或等同的任何方法和材料也可在本发明的实施或测试中使用，但是现在描述了优选的方法和材料。Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials are now described.
Ⅰ.术语I. Terminology
本发明所述用语“活性氧”是指机体内或者自然环境中由氧组成，含氧并且性质活泼的物质的总称，主要为一种激发态的氧分子，包括氧的一电子还原产物超氧阴离子(O2·-)、二电子还原产物过氧化氢(H2O2)、三电子还原产物羟基自由基(·OH)以及一氧化氮和单线态氧(1O2)等。The term "active oxygen" as used in the present invention refers to a general term for substances composed of oxygen, containing oxygen and active in the body or in the natural environment, mainly an excited oxygen molecule, including the one-electron reduction product of oxygen, superoxide anion (O2 ·-), the two-electron reduction product of oxygen, hydrogen peroxide (H2 O2 ), the three-electron reduction product of oxygen, hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen (1O2 ) and the like.
本发明所述用语“受体颗粒”是指含有能够与活性氧反应可以产生可检测信号的化合物的颗粒。供体颗粒被能量或者活性化合物诱导激活并释放高能态的活性氧，该高能态的活性氧被近距离的受体颗粒俘获，从而传递能量以激活所述受体颗粒。在本发明的一些具体实施方式中，所述受体颗粒包含发光组合物和载体，所述发光组合物填充于载体中和/或包被于载体表面。本发明所述“载体”选自带、片、棒、管、孔、微滴定板、珠、粒子和微球，其可以是本领域技术人员所公知的微球或微粒，其可以是任何尺寸的，其可以是有机的或是无机的，其可以是可膨胀或不可膨胀的，其可以是多孔的或非多孔的，其具有任何密度，但优选具有和水接近的密度，优选能漂浮于水中，且由透明、部分透明或不透明的材料构成。所述载体可以有或没有电荷，当带有电荷时，优选是负电荷。所述载体可以是乳胶颗粒或是含有有机或无机聚合物的其他颗粒、脂双层如脂质体、磷脂囊泡、小油滴、硅颗粒、金属溶胶、细胞和微晶染料。The term "acceptor particles" used in the present invention refers to particles containing compounds that can react with active oxygen to produce detectable signals. Donor particles are activated by energy or active compounds and release high-energy active oxygen, which is captured by acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some specific embodiments of the present invention, the acceptor particles include a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and/or coated on the surface of the carrier. The "carrier" used in the present invention is selected from strips, sheets, rods, tubes, holes, microtiter plates, beads, particles and microspheres, which can be microspheres or microparticles known to those skilled in the art, which can be of any size, which can be organic or inorganic, which can be expandable or non-expandable, which can be porous or non-porous, and which has any density, but preferably has a density close to that of water, preferably can float in water, and is composed of transparent, partially transparent or opaque materials. The carrier may have or have no charge, and when it has a charge, it is preferably negatively charged. The carrier may be a latex particle or other particle containing an organic or inorganic polymer, a lipid bilayer such as a liposome, a phospholipid vesicle, an oil droplet, a silica particle, a metal sol, a cell, and a microcrystalline dye.
本发明中，所述“发光组合物”即一种被称作为标记物的化合物，可进行化学反应以便引起发光，比如通过被转化为在电子激发态下形成的另一种化合物。激发态可以是单线态或是三重激发态。激发态可弛豫到基态直接发光，或者是通过将激发能量传递到发射能量受体，从而自身恢复到基态。在此过程中，能量受体颗粒将被跃迁为激发态而发光。In the present invention, the "luminescent composition" is a compound called a marker that can undergo a chemical reaction to cause luminescence, such as by being converted into another compound formed in an electronic excited state. The excited state can be a singlet state or a triplet excited state. The excited state can relax to the ground state to emit light directly, or by transferring the excitation energy to the emission energy acceptor, thereby restoring itself to the ground state. In this process, the energy acceptor particle will be transitioned to the excited state and emit light.
本发明所述“粒径分布变异系数C.V值”是指在纳米粒度仪的检测结果中，粒径在Gaussian分布中的变异系数。变异系数的计算公式为：C.V值＝(标准偏差SD/平均值Mean)×100％。标准偏差(Standard Deviation，SD)也被称为标准差，它描述各数据偏离平均数的距离(离均差)的平均数，它是离差平方和平均后的方根，用σ表示。标准差是方差的算术平方根。标准偏差能反映一个数据集的离散程度，标准偏差越小，这些值偏离平均值就越少，反之亦然。标准偏差σ为正态分布曲线上的拐点(0.607倍峰高处)至峰高与时间轴的垂线间的距离，即正态分布曲线上两拐点间距离的一半。半高峰宽(Wh/2)是指峰高一半处的峰宽，Wh/2＝2.355σ。通过正态分布曲线两侧的拐点作切线，在基线上的截距称为峰宽或称基线宽度，W＝4σ或W＝1.699Wh/2。The "coefficient of variation of particle size distribution C.V value" described in the present invention refers to the coefficient of variation of particle size in Gaussian distribution in the detection results of nanoparticle size analyzer. The calculation formula of coefficient of variation is: C.V value = (standard deviation SD/mean value Mean) × 100%. Standard deviation (SD) is also called standard deviation, which describes the average of the distance (deviation from the mean) of each data from the mean. It is the square root of the average sum of squares of deviations, represented by σ. Standard deviation is the arithmetic square root of variance. Standard deviation can reflect the degree of dispersion of a data set. The smaller the standard deviation, the less these values deviate from the mean, and vice versa. Standard deviation σ is the distance between the inflection point (0.607 times the peak height) on the normal distribution curve and the vertical line between the peak height and the time axis, that is, half the distance between the two inflection points on the normal distribution curve. Half peak width (Wh/2) refers to the peak width at half the peak height, Wh/2 = 2.355σ. Tangent lines are drawn through the inflection points on both sides of the normal distribution curve, and the intercept on the baseline is called the peak width or baseline width, W = 4σ or W = 1.699Wh/2.
本发明所述用语“待测样品”是指待测的含有或疑似含有待测目标分子的一种混合物。可以被用在本发明的待测样品包括体液，如血液(可以是在收集的血液样品中通常看到的抗凝血)、血浆、血清、尿、精液、唾液、细胞培养物、组织提取物等。其他类型的待测样品包括溶剂、海水、工业水样、食品样品、环境样本诸如土或水、植物材料、真核细胞、细菌、质粒、病毒、真菌、及来自于原核的细胞。待测样品可以在使用前根据需要利用稀释液进行稀释。例如，为了避免HOOK效应，可以在上机检测前使用稀释液对待测样品进行稀释后再在检测仪器上进行检测。The term "test sample" of the present invention refers to a mixture containing or suspected to contain the target molecule to be tested. The test sample that can be used in the present invention includes body fluids, such as blood (can be anticoagulated blood commonly seen in the collected blood sample), plasma, serum, urine, semen, saliva, cell culture, tissue extracts, etc. Other types of test samples include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The test sample can be diluted with diluent as needed before use. For example, in order to avoid the HOOK effect, the test sample can be diluted with a diluent before being tested on the machine and then tested on the detection instrument.
本发明所述用语“待测目标分子”是指检测时待检测样本中的物质。与待测目标分子具有特异性结合亲合力的一种或多种物质会被用于检测该目标分子。待测目标分子可以是蛋白、肽、抗体或可以使其与抗体结合的半抗原。待测目标分子可以是与互补核酸或寡聚核苷酸结合的核酸或寡聚核苷酸。待测目标分子可以是可形成特异性结合配对成员的任何其他物质。其他典型的待测目标分子的例子包括：药物，诸如类固醇、激素、蛋白、糖蛋白、粘蛋白、核蛋白、磷蛋白、滥用的药物、维生素、抗细菌药、抗真菌药、抗病毒药、嘌呤、抗肿瘤试剂、安非他命、杂氮化合物、核酸和前列腺素，以及任何这些药物的代谢物；杀虫剂及其代谢物；以及受体。分析物也包括细胞、病毒、细菌和真菌。The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity to the target molecule to be detected will be used to detect the target molecule. The target molecule to be detected may be a protein, a peptide, an antibody, or a hapten that can bind to an antibody. The target molecule to be detected may be a nucleic acid or an oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected may be any other substance that can form a specific binding pair member. Other typical examples of target molecules to be detected include: drugs, such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial drugs, antifungal drugs, antiviral drugs, purines, antitumor agents, amphetamines, nitrogen compounds, nucleic acids and prostaglandins, and metabolites of any of these drugs; pesticides and their metabolites; and receptors. Analytes also include cells, viruses, bacteria and fungi.
本发明所述用语“抗体”以最广含义使用，包括任何同种型的抗体，保留对抗原的特异性结合的抗体片段，包括但不限于Fab、Fv、scFv、和Fd片段、嵌合抗体、人源化抗体、单链抗体、双特异性抗体、和包含抗体的抗原结合部分和非抗体蛋白的融合蛋白。在任何需要的情况下，抗体可以进一步与其它部分，诸如特异性结合配对成员中的一员，例如生物素或亲和素(生物素-亲和素特异性结合配对成员中的一员)等缀合。The term "antibody" as used herein is used in the broadest sense, including antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any desired case, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, for example, biotin or avidin (a member of a biotin-avidin specific binding pair member), etc.
本发明所述用语“抗原”是指能够刺激机体产生免疫应答，并能与免疫应答产物抗体和致敏淋巴细胞在体内外结合，发生免疫效应的物质。The term "antigen" as used in the present invention refers to a substance that can stimulate the body to produce an immune response and can combine with the immune response products antibodies and sensitized lymphocytes in vivo or in vitro to produce an immune effect.
本发明所述用语“结合”指由于例如共价、静电、疏水、离子和/或氢键等相互作用，包括但不限于如盐桥和水桥等相互作用引起的两个分子间的直接联合。As used herein, the term "binding" refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonds, including but not limited to interactions such as salt bridges and water bridges.
本发明所述用语“特异性结合”，是指两种物质之间的相互辨别和选择性结合反应，从立体结构角度上说就是相应的反应物之间构象的对应性。在本发明公开的技术思想下，特异性结合反应的检测方法包括但不限于：双抗体夹心法、竞争法、中和竞争法、间接法或捕获法。The term "specific binding" used in the present invention refers to the mutual recognition and selective binding reaction between two substances, which is the correspondence of the conformations between the corresponding reactants from the perspective of the three-dimensional structure. Under the technical concept disclosed in the present invention, the detection method of the specific binding reaction includes but is not limited to: double antibody sandwich method, competition method, neutralization competition method, indirect method or capture method.
Ⅱ.具体实施方案II. Specific implementation plan
下面将结合实施例更详细地说明本发明。The present invention will be described in more detail below with reference to the embodiments.
现有的常识为：微球的粒径尺寸越均一，利用该微球进行的均相化学发光检测的性能就越好。因此目前针对均相化学发光中采用的微球的研究趋于获得更均一粒径的微球。本申请的发明人通过研究后发现，采用粒径尺寸均一的微球进行均相化学发光检测时，检测结果的灵敏度和检测量程难以同时保障。但是通过采用粒径尺寸均一性合适的微球(如微球粒径分布的变异系数＞5％)，反而既能保障光激化学发光检测的灵敏度，又能拓宽检测量程。The existing common sense is that the more uniform the particle size of the microspheres, the better the performance of homogeneous chemiluminescence detection using the microspheres. Therefore, current research on microspheres used in homogeneous chemiluminescence tends to obtain microspheres with more uniform particle sizes. After research, the inventors of this application found that when using microspheres with uniform particle sizes for homogeneous chemiluminescence detection, it is difficult to guarantee both the sensitivity and detection range of the detection results. However, by using microspheres with appropriate particle size uniformity (such as the coefficient of variation of the microsphere particle size distribution>5%), it is possible to both guarantee the sensitivity of photoinduced chemiluminescence detection and broaden the detection range.
本发明的发明人通过控制受体试剂，即R1试剂中受体颗粒的粒径分布和糖含量，进而控制每个受体颗粒表面报告分子(如，抗体/抗原)的量(小粒径微球比表面积大，单位质量微球表面报告分子的量多，大粒径微球比表面积小，单位质量微球表面报告分子的量少)。受体颗粒在受体试剂中的粒径分布的变异系数越大，不均一程度越高，相当于体系中存在各种尺寸不一的受体颗粒，从而既有较高的灵敏度，又有很宽的检测量程。The inventors of the present invention control the particle size distribution and sugar content of the receptor particles in the receptor reagent, i.e., the R1 reagent, and thereby control the amount of reporter molecules (e.g., antibodies/antigens) on the surface of each receptor particle (small-diameter microspheres have a large specific surface area, and a large amount of reporter molecules on the surface per unit mass of microspheres; large-diameter microspheres have a small specific surface area, and a small amount of reporter molecules on the surface per unit mass of microspheres). The larger the coefficient of variation of the particle size distribution of the receptor particles in the receptor reagent, the higher the degree of heterogeneity, which is equivalent to the presence of receptor particles of various sizes in the system, thereby having both high sensitivity and a wide detection range.
本发明一方面提供一种人类免疫缺陷病毒抗体检测试剂盒，其包括试剂R1，所述试剂R1包含第一缓冲溶液以及悬浮于其中的能够与活性氧作用产生化学发光信号的受体颗粒，所述受体颗粒的表面结合有HIV抗原，其特征在于：所述受体颗粒在受体试剂中的粒径分布变异系数C.V值不低于5％且不高于20％；每毫克质量的所述受体颗粒中的糖含量不高于25μg。On one hand, the present invention provides a human immunodeficiency virus antibody detection kit, which includes a reagent R1, wherein the reagent R1 contains a first buffer solution and receptor particles suspended therein that can react with active oxygen to generate chemiluminescent signals, wherein HIV antigens are bound to the surface of the receptor particles, and is characterized in that: the coefficient of variation C.V value of the particle size distribution of the receptor particles in the receptor reagent is not less than 5% and not more than 20%; and the sugar content in each milligram of the receptor particles is not more than 25 μg.
所述受体颗粒包括载体，所述载体的内部填充有发光组合物，所述载体的表面键合有HIV抗原。The receptor particle comprises a carrier, the interior of the carrier is filled with a luminescent composition, and the surface of the carrier is bonded with HIV antigens.
在一些具体实施例中，所述载体表面包被有多糖分子，所述HIV抗原通过与多糖分子的化学键合而间接结合到受体颗粒的表面。In some specific embodiments, the surface of the carrier is coated with polysaccharide molecules, and the HIV antigen is indirectly bound to the surface of the receptor particle by chemical bonding with the polysaccharide molecules.
上述受体颗粒，其在所述试剂R1中的粒径分布变异系数C.V值可以为5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％。The coefficient of variation C.V of the particle size distribution of the above-mentioned receptor particles in the reagent R1 can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.
优选的所述受体颗粒在所述试剂R1中的粒径分布变异系数C.V值不高于15％。Preferably, the coefficient of variation C.V of the particle size distribution of the receptor particles in the reagent R1 is no higher than 15%.
优选的所述受体颗粒在所述试剂R1中的粒径分布变异系数C.V值不低于8％。Preferably, the coefficient of variation C.V of the particle size distribution of the receptor particles in the reagent R1 is not less than 8%.
优选的每毫克质量的所述受体颗粒中的糖含量不高于20μg；更优选不高于10μg。Preferably, the sugar content per milligram of the receptor particles is no more than 20 μg; more preferably no more than 10 μg.
优选的所述第一缓冲溶液中多糖的含量为0.01～1wt％，优选为0.05～0.5wt％。Preferably, the content of polysaccharide in the first buffer solution is 0.01-1 wt%, preferably 0.05-0.5 wt%.
在优选实施例中，所述载体的表面没有包被糖分子而直接键合HIV抗原。In a preferred embodiment, the surface of the carrier is not coated with sugar molecules but directly bonded to HIV antigens.
所述载体的表面上带有键合官能团，其用于将HIV抗原直接化学键合在所述载体的表面上，所述键合官能团选自胺基、酰胺基、羟基、醛基、羧基、环氧基、马来酰亚胺基和巯基中的至少一种；优选选自醛基、羧基、环氧基和马来酰亚胺基。The carrier has a bonding functional group on its surface, which is used to directly chemically bond the HIV antigen to the surface of the carrier. The bonding functional group is selected from at least one of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, an epoxy group, a maleimide group and a thiol group; preferably selected from an aldehyde group, a carboxyl group, an epoxy group and a maleimide group.
上述实施例中，所述多糖选自含有三个或更多个未修饰或修饰的单糖单元的碳水化合物；优选选自葡聚糖、淀粉、糖原、菊粉、果聚糖、甘露聚糖、琼脂糖、半乳聚糖、羧基葡聚糖和氨基葡聚糖；更优选选自葡聚糖、淀粉、糖原和聚核糖，最优选为葡聚糖或葡聚糖衍生物。In the above embodiments, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose, and most preferably dextran or a dextran derivative.
上述实施例中，所述糖含量利用蒽酮法检测。In the above examples, the sugar content was detected using the anthrone method.
所述试剂盒还包括试剂R2，试剂R2包含生物素标记的HIV抗原。The kit further comprises a reagent R2, which contains a biotin-labeled HIV antigen.
所述试剂盒还包括Anti-HIV样品稀释液。The kit also includes an Anti-HIV sample diluent.
所述试剂盒还包括Anti-HIV阴性对照。The kit also includes an Anti-HIV negative control.
所述试剂盒还包括Anti-HIV-1阳性对照，其含有小牛血清和HIV-1阳性血清。The kit also includes an Anti-HIV-1 positive control, which contains calf serum and HIV-1 positive serum.
所述试剂盒还包括Anti-HIV-2阳性对照，其含有小牛血清和HIV-2多克隆抗体。The kit also includes an Anti-HIV-2 positive control, which contains calf serum and HIV-2 polyclonal antibodies.
所述试剂盒还包括Anti-HIV弱阳性对照，其含有小牛血清和HIV-1阳性血清。The kit also includes an Anti-HIV weak positive control, which contains calf serum and HIV-1 positive serum.
所述试剂盒中含有多个试剂条，每个试剂条上开设有若干盛装试剂的试剂孔槽，其中至少一个所述试剂孔槽用于盛装所述试剂R1。The reagent kit contains a plurality of reagent strips, each of which is provided with a plurality of reagent wells for containing reagents, wherein at least one of the reagent wells is used for containing the reagent R1.
本发明还提供一种上述受体试剂或上述试剂盒在化学发光分析仪上的应用。The present invention also provides a use of the receptor reagent or the kit on a chemiluminescence analyzer.
本发明还提供一种上述受体试剂或上述试剂盒在POCT检测中的应用。POCT是指现场快速检验或在病人旁边进行的临床检测。The present invention also provides an application of the above receptor reagent or the above kit in POCT detection. POCT refers to a rapid test on site or a clinical test performed next to the patient.
III.实施例III. Embodiment
实施例1:受体颗粒a的制备Example 1: Preparation of receptor particles a
1.1载体的合成及表征1.1 Synthesis and characterization of carrier
1)准备100mL的三口烧瓶，加入40mmol苯乙烯、3mmol甲基丙烯酸、10mL水，搅拌10min后通N230min；1) Prepare a 100 mL three-necked flask, add 40 mmol styrene, 3 mmol methacrylic acid, and 10 mL water, stir for 10 min, and then pass N2 for 30 min;
2)称取0.11g过硫酸铵和0.2g氯化钠，溶于40mL水中配制成水溶液。将该水溶液加入到步骤1)的反应体系中，继续通N230min；2) Weigh 0.11 g of ammonium persulfate and 0.2 g of sodium chloride, dissolve in 40 mL of water to prepare an aqueous solution. Add the aqueous solution to the reaction system in step 1), and continue to pass N2 for 30 min;
3)将反应体系升温至70℃，反应15h；3) The reaction system was heated to 70°C and reacted for 15 hours;
4)将反应完成后的乳液冷却至室温，用合适的滤布过滤。得到的乳液用去离子水多次离心沉降清洗，直至离心初的上清液的电导率接近去离子水，然后用水稀释，以乳液形式保存；4) After the reaction is completed, the emulsion is cooled to room temperature and filtered with a suitable filter cloth. The obtained emulsion is washed by centrifugation with deionized water for multiple times until the conductivity of the supernatant at the beginning of the centrifugation is close to that of deionized water, and then diluted with water and stored in the form of an emulsion;
1.2.发光组合物的填埋过程1.2. Filling process of luminescent composition
1)准备25mL的圆底烧瓶，加入0.1g二甲基噻吩衍生物和0.1g铕(Ⅲ)配合物(MTTA-EU3+)，10mL 95％乙醇，磁力搅拌，水浴升温至70℃，获得配合物溶液；1) Prepare a 25 mL round-bottom flask, add 0.1 g of dimethylthiophene derivative and 0.1 g of europium (III) complex (MTTA-EU3+ ), 10 mL of 95% ethanol, stir magnetically, and heat in a water bath to 70°C to obtain a complex solution;
2)准备100mL的三口烧瓶，加入10mL 95％乙醇、10mL水和10mL浓度为10％、步骤1.1中获得的羧基聚苯乙烯乳胶微球，磁力搅拌，水浴升温至70℃；2) Prepare a 100 mL three-necked flask, add 10 mL 95% ethanol, 10 mL water and 10 mL 10% carboxyl polystyrene latex microspheres obtained in step 1.1, stir magnetically, and heat the water bath to 70°C;
3)将步骤1)中的配合物溶液缓慢滴加至步骤2)中的三口烧瓶中，70℃反应2h后停止搅拌，自然冷却；3) slowly adding the complex solution in step 1) dropwise to the three-necked flask in step 2), reacting at 70° C. for 2 h, then stopping stirring and cooling naturally;
4)将上述乳液离心1h，30000G，离心后弃去上清液，得到填埋有发光组合物的羧基聚苯乙烯微球。用20mM HEPES缓冲液定容，使其终浓度为20mg/mL。4) The emulsion was centrifuged for 1 hour at 30000G, and the supernatant was discarded to obtain carboxyl polystyrene microspheres filled with the luminescent composition. The volume was fixed with 20mM HEPES buffer to a final concentration of 20mg/mL.
1.3发光微球偶联HIV抗原的包被1.3 Coating of luminescent microspheres coupled with HIV antigen
1)将HIV抗原透析至pH值为5.0的50mM MES缓冲液，测得浓度为1mg/mL。1) HIV antigen was dialyzed into 50 mM MES buffer at pH 5.0 and the concentration was measured to be 1 mg/mL.
2)在2mL离心管中加入0.5mL羧基发光微球和0.5mL透析后的HIV抗原，混匀后加入100μL 10mg/mL EDAC溶液(50mM MES缓冲液)，2-8℃反应4h。2) Add 0.5 mL of carboxyl luminescent microspheres and 0.5 mL of dialyzed HIV antigen to a 2 mL centrifuge tube, mix well, add 100 μL of 10 mg/mL EDAC solution (50 mM MES buffer), and react at 2-8° C. for 4 h.
3)反应完毕后加入0.5mL 100mg/mL BSA溶液(50mM MES缓冲液)，2-8℃反应2h。3) After the reaction is complete, add 0.5 mL of 100 mg/mL BSA solution (50 mM MES buffer) and react at 2-8°C for 2 h.
4)反应完毕后离心30min，20000G，离心后弃去上清液，用50mM MES缓冲液重新悬浮。重复离心清洗四次，并稀释至终浓度为100μg/mL，获得偶联HIV抗原的受体颗粒溶液，得到受体试剂A。4) After the reaction is completed, centrifuge for 30 minutes at 20000G, discard the supernatant, and resuspend in 50mM MES buffer. Repeat the centrifugation and washing four times, and dilute to a final concentration of 100μg/mL to obtain a receptor particle solution coupled to HIV antigen, and obtain receptor reagent A.
实施例2:受体颗粒b的制备Example 2: Preparation of receptor particles b
2.1聚苯乙烯乳胶微球的制备2.1 Preparation of polystyrene latex microspheres
1)准备100mL的三口烧瓶，加入40mmol苯乙烯、5mmol丙烯醛、10mL水，搅拌10min后通N230min；1) Prepare a 100 mL three-necked flask, add 40 mmol styrene, 5 mmol acrolein, and 10 mL water, stir for 10 min, and then pass N2 for 30 min;
2)称取0.11g过硫酸铵和0.2g氯化钠，溶于40mL水中配制成水溶液。将该水溶液加入到步骤1)的反应体系中，继续通N230min；2) Weigh 0.11 g of ammonium persulfate and 0.2 g of sodium chloride, dissolve in 40 mL of water to prepare an aqueous solution. Add the aqueous solution to the reaction system in step 1), and continue to pass N2 for 30 min;
3)将反应体系升温至70℃，反应15h；3) The reaction system was heated to 70°C and reacted for 15 hours;
4)将反应完成后的乳液冷却至室温，用合适的滤布过滤。得到的乳液用去离子水过次离心沉降清洗，直至离心初的上清液的电导率接近去离子水，然后用水稀释，以乳液形式保存；4) After the reaction is completed, the emulsion is cooled to room temperature and filtered with a suitable filter cloth. The obtained emulsion is washed with deionized water by centrifugal sedimentation until the conductivity of the supernatant at the beginning of the centrifugation is close to that of deionized water, and then diluted with water and stored in the form of an emulsion;
2.2发光组合物的填埋过程2.2 Filling process of luminescent composition
1)准备25mL的圆底烧瓶，加入0.1g二甲基噻吩衍生物和0.1g铕(Ⅲ)配合物(MTTA-EU3+)，10mL 95％乙醇，磁力搅拌，水浴升温至70℃，获得配合物溶液；1) Prepare a 25 mL round-bottom flask, add 0.1 g of dimethylthiophene derivative and 0.1 g of europium (III) complex (MTTA-EU3+ ), 10 mL of 95% ethanol, stir magnetically, and heat in a water bath to 70°C to obtain a complex solution;
2)准备100mL的三口烧瓶，加入10mL 95％乙醇、10mL水和10mL浓度为10％、步骤1.1中获得的醛基聚苯乙烯乳胶微球，磁力搅拌，水浴升温至70℃；2) Prepare a 100 mL three-necked flask, add 10 mL 95% ethanol, 10 mL water and 10 mL 10% aldehyde polystyrene latex microspheres obtained in step 1.1, stir magnetically, and heat the water bath to 70°C;
3)将步骤1)中的配合物溶液缓慢滴加至步骤2)中的三口烧瓶中，70℃反应2h后停止搅拌，自然冷却；3) slowly adding the complex solution in step 1) dropwise to the three-necked flask in step 2), reacting at 70° C. for 2 h, then stopping stirring and cooling naturally;
4)将上述乳液离心1h，30000G，离心后弃去上清液，得到填埋有发光组合物的醛基聚苯乙烯微球。4) The emulsion was centrifuged for 1 h at 30,000 G, and the supernatant was discarded to obtain aldehyde-based polystyrene microspheres filled with the luminescent composition.
2.3受体颗粒的表面包被多糖2.3 Surface coating of receptor particles
1)取50mg氨基葡聚糖固体于20mL圆底烧瓶中，加入5mL 50mM/pH＝10碳酸盐缓冲液，30℃避光搅拌溶解；1) Take 50 mg of aminodextran solid in a 20 mL round-bottom flask, add 5 mL of 50 mM/pH=10 carbonate buffer, stir and dissolve at 30°C in the dark;
2)取100mg已制备好的填埋有发光组合物的醛基聚苯乙烯微球，加入到氨基葡聚糖溶液中搅拌2h；2) taking 100 mg of the prepared aldehyde-based polystyrene microspheres filled with the luminescent composition, adding them to the aminodextran solution and stirring for 2 h;
3)将10mg硼氢化钠溶于0.5mL 50mM/pH＝10碳酸盐缓冲液后滴加到上述反应液中，30℃避光反应过夜；3) Dissolve 10 mg of sodium borohydride in 0.5 mL of 50 mM/pH = 10 carbonate buffer and add dropwise to the above reaction solution. React at 30°C in the dark overnight;
4)将反应后的混合液30000G离心后弃去上清液，加入50mM/pH＝10碳酸盐缓冲液超声分散。重复离心清洗三次后用50mM/pH＝10碳酸盐缓冲液定容，使其终浓度为20mg/mL；4) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50mM/pH=10 carbonate buffer was added for ultrasonic dispersion. After repeated centrifugation and washing three times, the volume was fixed with 50mM/pH=10 carbonate buffer to a final concentration of 20mg/mL;
5)取100mg醛基葡聚糖固体于20mL圆底烧瓶中，加入5mL 50mM/pH＝10碳酸盐缓冲液，30℃避光搅拌溶解；5) Take 100 mg of aldehyde dextran solid in a 20 mL round-bottom flask, add 5 mL of 50 mM/pH = 10 carbonate buffer, stir and dissolve at 30 ° C in the dark;
6)将上述微球加入到醛基葡聚糖溶液中搅拌2h；6) Add the above microspheres into the aldehyde dextran solution and stir for 2 hours;
7)将15mg硼氢化钠溶于0.5mL 50mM/pH＝10碳酸盐缓冲液后滴加到上述反应液中，30℃避光反应过夜；7) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM/pH = 10 carbonate buffer and add dropwise to the above reaction solution. React at 30° C. in the dark overnight;
8)将反应后的混合液30000G离心后弃去上清液，加入50mM/pH＝10碳酸盐缓冲液超声分散。重复离心清洗三次后用50mM/pH＝10碳酸盐缓冲液定容，使其终浓度为20mg/mL。8) The reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50mM/pH=10 carbonate buffer was added for ultrasonic dispersion. The centrifugation was repeated three times, and the volume was fixed with 50mM/pH=10 carbonate buffer to a final concentration of 20mg/mL.
9)由纳米粒度仪测得此时微球粒径的Gaussian分布平均粒径为241.6nm，变异系数(C.V)＝12.90％，Gaussian分布曲线如图1所示。9) The Gaussian distribution average particle size of the microspheres measured by the nanoparticle size analyzer is 241.6 nm, the coefficient of variation (CV) is 12.90%, and the Gaussian distribution curve is shown in FIG1 .
2.4HIV抗原与受体颗粒的偶联过程2.4 Coupling process of HIV antigen and receptor particles
1)将HIV抗原Ⅰ透析至pH＝9.0的50mM CB缓冲液，测得浓度为1mg/mL。1) HIV antigen I was dialyzed into 50 mM CB buffer at pH = 9.0 and the concentration was measured to be 1 mg/mL.
2)在2mL离心管中加入0.5mL步骤2.3中获得的受体颗粒以及0.5mL的HIV抗原Ⅰ，混匀后加入100μL 10mg/mL NaBH4溶液(50mM CB缓冲液)，2-8℃反应4h。2) Add 0.5 mL of the receptor particles obtained in step 2.3 and 0.5 mL of HIV antigen I to a 2 mL centrifuge tube, mix well, add 100 μL of 10 mg/mL NaBH4 solution (50 mM CB buffer), and react at 2-8°C for 4 h.
3)反应完毕后加入0.5mL 100mg/mL BSA溶液(50mM CB缓冲液)，2-8℃反应2h。3) After the reaction is complete, add 0.5 mL of 100 mg/mL BSA solution (50 mM CB buffer) and react at 2-8°C for 2 h.
4)反应完毕后将离心45min，30000G，离心后弃去上清液，用50mM MES缓冲液重新悬浮。重复离心清洗四次，并稀释至终浓度为100μg/mL，获得偶联HBsAg抗原Ⅰ的受体颗粒溶液，得到受体试剂B。4) After the reaction is completed, centrifuge for 45 minutes at 30000G, discard the supernatant, and resuspend with 50mM MES buffer. Repeat the centrifugation and washing four times, and dilute to a final concentration of 100μg/mL to obtain a receptor particle solution coupled to HBsAg antigen I, and obtain receptor reagent B.
实施例3:检测微球的糖含量Example 3: Detection of sugar content of microspheres
1)微球样品预处理：1) Microsphere sample pretreatment:
分别取实施例1中含有1mg受体微球a的受体试剂A和实施例2中含有1mg受体微球b的受体试剂B，20000G离心40min，倒去上层清液后用纯化水超声分散，重复离心分散三次后用纯化水定容至1mg/mL。Receptor reagent A containing 1 mg of receptor microsphere a in Example 1 and receptor reagent B containing 1 mg of receptor microsphere b in Example 2 were taken separately, centrifuged at 20000G for 40 min, the supernatant was discarded and ultrasonically dispersed with purified water, and the centrifugal dispersion was repeated three times and then fixed to 1 mg/mL with purified water.
2)葡萄糖标准溶液的配制：2) Preparation of glucose standard solution:
用纯化水将1mg/mL葡萄糖储备液配制成0mg/mL、0.025mg/mL、0.05mg/mL、0.075mg/mL、0.10mg/mL、0.15mg/mL的标准溶液。Use purified water to prepare 1 mg/mL glucose stock solution into 0 mg/mL, 0.025 mg/mL, 0.05 mg/mL, 0.075 mg/mL, 0.10 mg/mL, and 0.15 mg/mL standard solutions.
3)蒽酮溶液的配制：用80％硫酸溶液配制成2mg/mL(此溶液室温下24h内稳定，现用现配)。3) Preparation of anthrone solution: Prepare 2 mg/mL with 80% sulfuric acid solution (this solution is stable at room temperature for 24 hours and should be prepared immediately before use).
4)向离心管分别加入0.1mL各浓度的葡萄糖标准溶液及待测样品，每管各加1mL蒽酮试液。4) Add 0.1 mL of glucose standard solution of each concentration and the sample to be tested to the centrifuge tubes respectively, and add 1 mL of anthrone test solution to each tube.
5)85℃孵育30min。5) Incubate at 85°C for 30 min.
6)将样品反应管15000G离心40min，移液器枪头从管底吸取澄清液体进行吸光度的测定，避免将上部悬浮物吸出。6) Centrifuge the sample reaction tube at 15000G for 40 minutes, and use a pipette tip to aspirate the clear liquid from the bottom of the tube to measure the absorbance, avoiding aspirating the suspended matter on the top.
7)恢复至室温，测量620nm的吸光度(测量最好在2h内进行)。7) Return to room temperature and measure the absorbance at 620 nm (measurement should be performed preferably within 2 hours).
8)标准溶液的糖含量浓度与吸光度关系如表1所示，以标准溶液糖含量浓度为X值，吸光度为Y值，进行一次直线回归，获得如图2所示糖含量测定标准曲线，并以此为基础测定待测样品的糖含量浓度。8) The relationship between the sugar content concentration and absorbance of the standard solution is shown in Table 1. With the sugar content concentration of the standard solution as the X value and the absorbance as the Y value, a linear regression is performed to obtain the sugar content determination standard curve shown in Figure 2, and the sugar content concentration of the sample to be tested is determined based on this.
表1Table 1
检测结果：Test results:
实施例1中受体微球a的糖含量不高于25μg/mg微球；The sugar content of the acceptor microsphere a in Example 1 is not higher than 25 μg/mg microsphere;
实施例2中受体微球b的糖含量为60.5μg/mg微球。The sugar content of the acceptor microsphere b in Example 2 is 60.5 μg/mg microsphere.
实施例4:HIV检测试剂盒的制备及其性能验证Example 4: Preparation of HIV Detection Kit and Its Performance Verification
本试剂盒由试剂R1(由实施例1制备所得)、试剂R2(生物素标记HIV抗原)、另外包括含有供体颗粒的感光试剂R3、Anti-HIV阴性对照、Anti-HIV-1阳性对照、Anti-HIV-2阳性对照、Anti-HIV弱阳性对照、Anti-HIV样品稀释液组成。各组分分别制备，试剂R1和试剂R2组合成Anti-HIV试剂盒，阴、阳性对照、弱阳性对照及样品稀释液分别独立分装，再组装成盒。其制备工艺概括如下：This kit consists of reagent R1 (prepared in Example 1), reagent R2 (biotin-labeled HIV antigen), and also includes a photosensitive reagent R3 containing donor particles, an Anti-HIV negative control, an Anti-HIV-1 positive control, an Anti-HIV-2 positive control, an Anti-HIV weak positive control, and an Anti-HIV sample diluent. Each component is prepared separately, and reagent R1 and reagent R2 are combined into an Anti-HIV kit, and the negative and positive controls, weak positive controls, and sample diluents are separately packaged and then assembled into a box. The preparation process is summarized as follows:
⑴试剂R1(受体颗粒包被HIV抗原)：将实施例1中制备得到的受体颗粒，加入第一缓冲溶液配制而成，浓度为50μg/mL。(1) Reagent R1 (receptor particles coated with HIV antigen): The receptor particles prepared in Example 1 were added into the first buffer solution to prepare a reagent with a concentration of 50 μg/mL.
⑵试剂R2(生物素标记HIV抗原)：将处理好的HIV抗原与生物素按照一定的浓度和比例混匀反应形成连接物，经透析后，加入一定量的缓冲溶液配制而成。⑵ Reagent R2 (biotin-labeled HIV antigen): The treated HIV antigen and biotin are mixed at a certain concentration and ratio to form a linker, which is then dialyzed and then added with a certain amount of buffer solution to prepare the linker.
⑶Anti-HIV阴性对照：Anti-HIV定性参考品稀释液。⑶Anti-HIV negative control: Anti-HIV qualitative reference dilution solution.
⑷Anti-HIV-1阳性对照：使用Anti-HIV定性参考品稀释液稀释HIV-1阳性血清配制而成。⑷Anti-HIV-1 positive control: prepared by diluting HIV-1 positive serum with Anti-HIV qualitative reference diluent.
⑸Anti-HIV-2阳性对照：使用Anti-HIV定性参考品稀释液稀释HIV-2多克隆抗体配制而成。⑸Anti-HIV-2 positive control: prepared by diluting HIV-2 polyclonal antibody with Anti-HIV qualitative reference diluent.
⑹Anti-HIV弱阳性对照：使用Anti-HIV定性参考品稀释液稀释HIV-1阳性血清配制而成。⑹Anti-HIV weak positive control: prepared by diluting HIV-1 positive serum with Anti-HIV qualitative reference diluent.
本试剂盒可对人类免疫缺陷病毒HIV(1+2型)抗体进行定性检测，检测时请使用配套的Anti-HIV弱阳性对照、Anti-HIV阴性对照、Anti-HIV-1阳性对照、Anti-HIV-2阳性对照，Anti-HIV弱阳性对照加2孔、Anti-HIV阴性对照、Anti-HIV-1阳性对照、Anti-HIV-2阳性对照各加1孔进行实验。试剂使用前需平衡至环境温度。This kit can be used for qualitative detection of human immunodeficiency virus HIV (type 1+2) antibodies. When testing, please use the matching Anti-HIV weak positive control, Anti-HIV negative control, Anti-HIV-1 positive control, Anti-HIV-2 positive control. Add 2 wells of Anti-HIV weak positive control, 1 well of Anti-HIV negative control, Anti-HIV-1 positive control, and Anti-HIV-2 positive control for the experiment. The reagents must be balanced to ambient temperature before use.
步骤1：将待测样品用Anti-HIV样品稀释液进行11倍稀释，并充分混匀(如将10μL样品加入100μL样品稀释液中)；Step 1: Dilute the sample to be tested 11 times with Anti-HIV sample diluent and mix thoroughly (e.g., add 10 μL of sample to 100 μL of sample diluent);
步骤2：在反应孔中分别加入25μL稀释样品及Anti-HIV阴性对照、Anti-HIV-1阳性对照、Anti-HIV-2阳性对照、Anti-HIV弱阳性对照；Step 2: Add 25 μL of diluted sample and Anti-HIV negative control, Anti-HIV-1 positive control, Anti-HIV-2 positive control, and Anti-HIV weak positive control to the reaction wells;
步骤3：在反应孔中依次加入25μL试剂R1、25μL试剂R2和25μL感光试剂R3；Step 3: Add 25 μL of reagent R1, 25 μL of reagent R2, and 25 μL of photosensitive reagent R3 to the reaction wells in sequence;
步骤4：放入博阳生物科技(上海)有限公司生产的LiCA500全自动光激化学发光检测仪，由仪器自动操作，具体步骤如下：Step 4: Place the sample into the LiCA500 fully automatic photochemiluminescence detector produced by Boyang Biotechnology (Shanghai) Co., Ltd. and let the instrument operate automatically. The specific steps are as follows:
A.振动A. Vibration
B.37℃温育15minB. Incubate at 37℃ for 15min
C.自动加入感光试剂175μLC. Automatically add 175μL of photosensitizer
D.37℃温育10minD. Incubate at 37℃ for 10 min
E.激光照射微孔并计算每孔发出光子量E. Laser irradiation of microwells and calculation of the number of photons emitted from each well
F.由软件计算S/CO值(待测样本光信号值与Anti-HIV弱阳性对照光信号的比值)，并判定阴阳性。F. The software calculates the S/CO value (the ratio of the light signal value of the sample to be tested to the light signal of the Anti-HIV weak positive control) and determines the positive or negative.
【试剂盒有效性判定方法】【Method for determining the effectiveness of the test kit】
每次试验时均需加Anti-HIV弱阳性对照、Anti-HIV阴性对照、Anti-HIV-1阳性对照、Anti-HIV-2阳性对照Anti-HIV weak positive control, Anti-HIV negative control, Anti-HIV-1 positive control, Anti-HIV-2 positive control should be added in each test
Anti-HIV阴性对照的S/CO值应<0.6，Anti-HIV-1阳性对照的S/CO值应≥3，Anti-HIV-2阳性对照的S/CO值应≥3，如结果异常，则本次试验结果不可信，需重复。The S/CO value of the Anti-HIV negative control should be <0.6, the S/CO value of the Anti-HIV-1 positive control should be ≥3, and the S/CO value of the Anti-HIV-2 positive control should be ≥3. If the result is abnormal, the result of this test is unreliable and needs to be repeated.
【试剂盒检测结果判定方法】【Test kit test result determination method】
S：待测样本光信号值；S: optical signal value of the sample to be tested;
CO：Anti-HIV弱阳性对照光信号值(CUT OFF参考值)；CO: Anti-HIV weak positive control light signal value (CUT OFF reference value);
软件自动计算S/CO值，当S/CO<1时待测样品被判定为阴性，当S/CO≥1时待测样品被判定为阳性。The software automatically calculates the S/CO value. When S/CO < 1, the sample is judged as negative, and when S/CO ≥ 1, the sample is judged as positive.
经检测，本实施例的试剂盒具有以下良好性能：After testing, the kit of this embodiment has the following good properties:
(1)阴性参考品符合率：用国家参考品进行检定，20份HIV抗体阴性参考品符合率不低于18份；(1) Negative reference sample compliance rate: When using national reference samples for testing, the compliance rate of 20 HIV antibody negative reference samples should be no less than 18;
(2)阳性参考品符合率：用国家参考品进行检定，18份HIV-1型抗体阳性参考品不得出现假阴性，且RLUP12≥RLUP11；2份HIV-2型抗体阳性样品不得出现假阴性；(2) Compliance rate of positive reference materials: When testing with national reference materials, 18 HIV-1 antibody positive reference materials must not have false negatives, and RLUP12 ≥ RLUP11; 2 HIV-2 antibody positive samples must not have false negatives;
(3)灵敏度：用国家参考品进行检定，6份灵敏度参考品血清中，1份基质血清为阴性，5份稀释血清中至少3份出现阳性；(3) Sensitivity: When tested with national reference materials, one matrix serum out of six sensitivity reference serum samples was negative, and at least three out of five diluted serum samples were positive;
(4)精密性：用国家参考品进行检定，C.V≤15％(n＝10)；(4) Precision: Verified with national reference materials, C.V ≤ 15% (n = 10);
(5)临床灵敏度、临床特异性：对500份临床判定为阳性的血清样本的检测中，499例样本均被检出阳性，临床灵敏度达到99.8％，在对600份临床判定为阴性的血清样本的检测中，600例样本均被检测为阴性，临床特异性达到100％。(5) Clinical sensitivity and clinical specificity: Among the 500 serum samples that were clinically judged to be positive, 499 samples were detected as positive, and the clinical sensitivity reached 99.8%. Among the 600 serum samples that were clinically judged to be negative, all 600 samples were detected as negative, and the clinical specificity reached 100%.
实施例5：受体颗粒的C.V值对检测结果的影响Example 5: Effect of the C.V value of the receptor particles on the test results
5.1制备不同C.V值的受体颗粒5.1 Preparation of receptor particles with different C.V values
按照实施例1中所述方法，获得粒径分布的变异系数不同的偶联HIV抗原的受体颗粒溶液，具体为：According to the method described in Example 1, a receptor particle solution coupled to HIV antigen with different coefficients of variation of particle size distribution was obtained, specifically:
受体颗粒1：Gaussian分布平均粒径为211.8nm，粒径分布变异系数C.V值＝3.5％；Nicomp分布为单峰。Acceptor particles 1: Gaussian distribution average particle size is 211.8 nm, particle size distribution coefficient of variation C.V value = 3.5%; Nicomp distribution is unimodal.
受体颗粒2：Gaussian分布平均粒径为214.4nm，粒径分布变异系数C.V值＝5.0％；Nicomp分布为单峰。Acceptor particles 2: Gaussian distribution average particle size is 214.4 nm, particle size distribution coefficient of variation C.V value = 5.0%; Nicomp distribution is unimodal.
受体颗粒3：Gaussian分布平均粒径为212.1nm，粒径分布变异系数C.V值＝6.9％；Nicomp分布为单峰。Acceptor particles 3: Gaussian distribution average particle size is 212.1 nm, particle size distribution coefficient of variation C.V value = 6.9%; Nicomp distribution is unimodal.
受体颗粒4：Gaussian分布平均粒径为213.9nm，粒径分布变异系数C.V值＝8.3％；Nicomp分布为单峰。Acceptor particles 4: Gaussian distribution average particle size is 213.9 nm, particle size distribution coefficient of variation C.V value = 8.3%; Nicomp distribution is unimodal.
受体颗粒5：Gaussian分布平均粒径为212.4nm，粒径分布变异系数C.V值＝14.8％；Nicomp分布为单峰。Acceptor particles 5: Gaussian distribution average particle size is 212.4 nm, particle size distribution coefficient of variation C.V value = 14.8%; Nicomp distribution is unimodal.
受体颗粒6：Gaussian分布平均粒径为211.2nm，粒径分布变异系数C.V值＝20.0％；Nicomp分布为双峰。Acceptor particles 6: Gaussian distribution average particle size is 211.2 nm, particle size distribution coefficient of variation C.V value = 20.0%; Nicomp distribution is bimodal.
受体颗粒7：Gaussian分布平均粒径为210.8nm，粒径分布变异系数C.V值＝30.3％；Nicomp分布为双峰。Acceptor particles 7: Gaussian distribution average particle size is 210.8 nm, particle size distribution coefficient of variation C.V value = 30.3%; Nicomp distribution is bimodal.
5.2试剂盒检出限和HOOK样本的测定5.2 Kit detection limit and HOOK sample determination
定义最低检出限为0.5NCU康彻思坦质控品检出能力判定，当某一实验组样本测试信号刚好大于CO信号，即RLU(Cx)>RLU(C0)，则测试结果为阳性。定义HOOK样本为待测物浓度高于试剂中抗原抗体浓度的样本，测试结果存在假性偏低的风险，检测结果为S/CO＝RLU(Cx)/RLU(C0)。The minimum detection limit is defined as 0.5 NCU. The detection ability of the Conchestan quality control product is determined. When the test signal of a certain experimental group sample is just greater than the CO signal, that is, RLU (Cx)> RLU (C0), the test result is positive. The HOOK sample is defined as a sample with a concentration of the analyte higher than the concentration of the antigen and antibody in the reagent. The test result has the risk of false low, and the test result is S/CO＝RLU (Cx)/RLU (C0).
实验步骤如下：The experimental steps are as follows:
1、将0.5NCU康彻思坦质控品样品作为检出限样本通过S/CO值大小以及阴阳性判断检出能力差异；1. Use 0.5 NCU Conchestan quality control sample as the detection limit sample to judge the difference in detection ability by the S/CO value and positive and negative properties;
2、将HOOK样本以2倍梯度稀释成系列样本后待测；2. Dilute the HOOK sample by 2-fold gradient to form a series of samples for testing;
3、将不同粒径C.V的受体颗粒偶联HIV抗原后配制成20μg/mL的浓度，与稀释后的康彻思坦质控品和HOOK样本同步测试；3. The receptor particles of C.V with different particle sizes were coupled with HIV antigens and prepared into a concentration of 20μg/mL, and tested simultaneously with the diluted Conchestan quality control product and HOOK sample;
4、比较不同粒径C.V的受体颗粒对检出限和HOOK样本的影响。4. Compare the effects of C.V receptor particles of different sizes on the detection limit and HOOK samples.
检测结果如下表2所示：The test results are shown in Table 2 below:
表2Table 2
从表2可知，当所述受体颗粒粒径分布的变异系数(C.V值)大于等于5％且不高于20％时，包含该受体颗粒的试剂盒既有对低浓度样本检出能力，又有较好的抗HOOK能力。当所述受体颗粒粒径分布的变异系数大于等于8％且不高于15％时，包含该受体颗粒的试剂盒既有对低浓度样本检出能力，抗HOOK能力更好。As can be seen from Table 2, when the coefficient of variation (C.V. value) of the receptor particle size distribution is greater than or equal to 5% and not higher than 20%, the kit containing the receptor particles has both the ability to detect low-concentration samples and good anti-HOOK ability. When the coefficient of variation of the receptor particle size distribution is greater than or equal to 8% and not higher than 15%, the kit containing the receptor particles has both the ability to detect low-concentration samples and better anti-HOOK ability.
实施例6:糖含量对检测结果的影响Example 6: Effect of sugar content on test results
实验步骤：Experimental steps:
1、挑选10例肿瘤患者干扰样本，经过确证试剂验证为HIV无反应性的样本；以及20例经确证试剂验证的HIV阳性样本；1. Select 10 tumor patient interference samples, which were verified as HIV-unreactive by confirmation reagents; and 20 HIV-positive samples verified by confirmation reagents;
2、将不同糖含量的受体颗粒偶联HIV抗原后配制成20μg/mL的浓度与30例样本反应后测试信号值；2. The receptor particles with different sugar contents were coupled to HIV antigens and prepared into a concentration of 20 μg/mL, and then reacted with 30 samples to test the signal value;
3、计算每个样本的S/CO值后，当S/CO大于等于1时为检测结果为阳性，反之为阴性；3. After calculating the S/CO value of each sample, when S/CO is greater than or equal to 1, the test result is positive, otherwise it is negative;
4、当肿瘤干扰样本中出现阳性结果则为假阳结果，若HIV阳性样本出现阴性结果则为漏检。4. When a positive result appears in a tumor interference sample, it is a false positive result. If a negative result appears in an HIV-positive sample, it is a missed detection.
检测结果如下表3所示：The test results are shown in Table 3 below:
表3Table 3
从上表结果分析可见，当受体颗粒的糖含量不高于25μg的时候，灵敏度高，低值样本检出能力高，假阳样本受干扰较小。From the analysis of the results in the above table, it can be seen that when the sugar content of the receptor particles is not higher than 25μg, the sensitivity is high, the detection ability of low-value samples is high, and false positive samples are less interfered.
实施例7：正常人与疑似感染HIV病毒患者的样本中HIV抗体水平的检测Example 7: Detection of HIV antibody levels in samples from normal subjects and patients suspected of being infected with HIV
一、HIV试剂的临床验证(采用实施例1中制备得到的受体试剂A)：1. Clinical validation of HIV reagents (using the receptor reagent A prepared in Example 1):
1、随机选择100例正常体检样本，50例肿瘤患者样本，50例疑似感染HIV病毒患者的样本；1. Randomly select 100 normal physical examination samples, 50 samples from tumor patients, and 50 samples from patients suspected of being infected with HIV;
2、将受体试剂A配制成20μg/mL的浓度与200例样本反应后测试信号值；2. Prepare receptor reagent A at a concentration of 20 μg/mL and react with 200 samples to test the signal value;
3、计算每个样本的S/CO值后，当S/CO大于等于1时为检测结果为阳性，反之为阴性；3. After calculating the S/CO value of each sample, when S/CO is greater than or equal to 1, the test result is positive, otherwise it is negative;
检测结果如下表4所示：The test results are shown in Table 4 below:
表4Table 4
结果分析：150例阴性样本符合率为100％，不存在假阳性；50例阳性样本符合率为100％，不存在漏检。Result analysis: The conformity rate of 150 negative samples was 100%, and there was no false positive; the conformity rate of 50 positive samples was 100%, and there was no missed detection.
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