WO2014005527A1 - 一种提高竞争免疫分析灵敏性的方法 - Google Patents

一种提高竞争免疫分析灵敏性的方法 Download PDF

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Publication number
WO2014005527A1
WO2014005527A1 PCT/CN2013/078772 CN2013078772W WO2014005527A1 WO 2014005527 A1 WO2014005527 A1 WO 2014005527A1 CN 2013078772 W CN2013078772 W CN 2013078772W WO 2014005527 A1 WO2014005527 A1 WO 2014005527A1
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reaction
sensitivity
improving
molecule
reacted
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PCT/CN2013/078772
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English (en)
French (fr)
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朱海
李细清
李金峰
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深圳市易瑞生物技术有限公司
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Priority to US14/412,681 priority Critical patent/US20150148249A1/en
Publication of WO2014005527A1 publication Critical patent/WO2014005527A1/zh

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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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to the field of biomedical testing, and more particularly to a method for improving the sensitivity of competitive immunoassays. Background technique
  • the immunological detection method is a series of experimental methods for the determination of antigens, antibodies, immune cells and their secreted cytokines by immunological theory.
  • the antigen is specifically bound by the complementarity of the surface antigenic determinant and the hypervariable region of the antibody molecule in spatial configuration.
  • the same antigen molecule can have a plurality of different antigenic determinants. If two different antigen molecules have one or more identical antigenic determinants, a cross reaction can occur when reacting with the antibody.
  • the antigen-antibody binding is mainly combined by non-covalent means such as hydrogen bonding, electrostatic attraction, van der Waals force and hydrophobic bond.
  • the complex formed after the combination can be dissociated under certain conditions.
  • the dissociated antigens and antibodies retain their original properties.
  • the degree of dissociation of antigen-antibody complexes depends on the degree of complementation of the three-dimensional configuration of the specific antibody hypervariable region and the corresponding antigenic determinant. The higher the degree of complementarity, the smaller the molecular spacing and the greater the force. The stronger the combination of the two, the easier it is to dissociate; on the contrary, the dissociation is prone to occur.
  • the small molecule antigen or the semi-anti-cause antigenic epitope is small, and can not be measured by the sandwich method, and only the competition method detection mode can be used.
  • the existing methods for detecting small molecule antigens mainly include enzymes. Methods such as immunosorbent assay, immunochromatographic assay, and instrumental analysis. Enzyme-linked immunosorbent assays are widely used because of their specificity, sensitivity, and low cost of detection. Although the immunochromatographic test is simple and easy to use, its accuracy is not high, which limits its application. Instrumental analysis is accurate and reliable, but it requires limited use of expensive equipment and professional technicians, and is often used to confirm testing.
  • a method for improving the sensitivity of competitive immunoassay wherein the steps of improving the sensitivity of the competitive immune analysis are as follows:
  • Pre-reaction pre-reacting the substance to be detected with the receptor/antibody
  • Re-reaction reacting the pre-reacted reaction product with a second molecule on the solid phase that participates in a competitive reaction;
  • the pre-reacted reaction product is divided into one or more portions during the re-reaction, and reacted with the second molecule, respectively.
  • the method for improving the sensitivity of competitive immunoassay wherein the pre-reacted reaction product flows in a flowing manner through the surface of the solid phase to react with the second molecule.
  • the method for improving the sensitivity of competitive immunoassay wherein, during the re-reaction, the reaction time of each pre-reacted reaction product and the second molecule is within 20 min.
  • reaction time of each pre-reacted reaction product and the second molecule is within 5 minutes during the re-reaction.
  • the method for improving the sensitivity of the competitive immunoassay wherein the pre-reaction process has a reaction temperature of 0 ° C to 65 ° C and a reaction time of 0 to 3 hours.
  • reaction time of the pre-reaction process is 30 min.
  • the method for improving the sensitivity of competitive immunoassay wherein the second molecule refers to a conjugate of a small molecule involved in a competitive reaction with a carrier protein.
  • the present invention proceeds from a reaction mode, and provides a method for improving the sensitivity of competitive immunoassay, mainly by first reacting a small molecule substance to be detected with an antibody/receptor first, and then on a solid phase.
  • the second molecule involved in the competitive response reacts in a short period of time, which greatly increases the sensitivity of the immunoassay method.
  • the method of the invention is simple in operation, low in cost, does not require expensive equipment and professional technicians, and has high sensitivity and is suitable for various immunoassay methods. detailed description
  • the present invention provides a method for improving the sensitivity of competitive immunoassay. In order to make the objects, technical solutions and effects of the present invention clearer and more clear, the present invention will be further described in detail below. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • the present invention starting from the reaction mode, provides a method for improving the sensitivity of competitive immunoassay, mainly by first reacting the small molecule substance to be detected with the antibody/receptor first, and then participating with the solid phase. Another molecule that competes for the reaction reacts in a short period of time, which greatly increases the sensitivity of the immunoassay.
  • the method steps for improving the sensitivity of the competitive immunoassay are as follows: Pre-reaction: pre-reacting the substance to be detected with the receptor/antibody;
  • Re-reaction The pre-reacted reaction product is reacted with a second molecule on a solid phase that participates in a competitive reaction.
  • the pre-reaction is to fully react the substance to be detected with the receptor/antibody first, that is, when the substance to be tested is an antigen, it fully reacts with the antibody, and when the substance to be tested is a complement, React fully with its receptor.
  • the pre-reaction is specifically a reaction between 0 ° C and 65 ° C for 0-3 hours, and the reaction time is preferably 30 min.
  • the re-reaction is specifically to divide the pre-reacted reaction product into one or more portions and react with the second molecule participating in the competitive reaction.
  • the pre-reacted The reaction product is divided into an average of 2 to 6 parts, and then each reaction product is separately reacted with the second molecule on the solid phase.
  • the pre-reacted reaction product preferably flows in a flowing manner over the surface of the solid phase coated with the second molecule involved in the competition, and it has been experimentally proved that the pre-reaction product flows through the solid phase surface.
  • Reaction with the second molecule can increase the sensitivity of competitive immunoassays.
  • the second molecule involved in the competitive reaction refers to a conjugate formed by a small molecule involved in the competitive reaction with the carrier protein.
  • the small molecule involved in the competitive reaction refers to complement corresponding to the receptor or an antigen corresponding to the receptor.
  • the reaction time of each of the pre-reacted reaction product and the second molecule is controlled within 20 min, preferably within 5 min.
  • the main improvement of the present invention is that the pre-reaction product is divided into one or more portions in the re-reaction process, and then reacted with the second molecule participating in the competitive reaction in a short time, and after a large number of experiments, it is obvious that Improve the sensitivity of the competitive immune response.
  • the reacted liquid should be removed, and then another pre-reaction product is taken to react with the second molecule. In this way, the influence of the remaining reaction liquid on the next reaction can be reduced, and the sensitivity of the analysis and detection can be improved.
  • the method of increasing the sensitivity of the competitive immunoassay prior to performing the re-reaction step further comprises the steps of:
  • the second molecule is transferred to a solid phase.
  • the microplates involved in the competitive reaction are coated with an ELISA plate.
  • the solid phase may be blocked with a blocking solution, thereby effectively preventing the antibody from binding to the non-specific antigen and causing false positives.
  • the blocking solution may be BSA, skim milk powder or the like.
  • the blocking step can be incubated for 30 min at 37 °C with blocking solution.
  • the method of increasing the sensitivity of the competitive immunoassay after performing the re-reaction step further comprises the steps of:
  • Color development adding a substrate color developing solution to the reaction product of the re-reaction;
  • Termination of reaction 2 can be terminated by adding an acid solution
  • the OD value of the product can be determined by a microplate reader.
  • the method for improving the sensitivity of competitive immunoassay is applicable to enzyme-linked immunoassay, time-resolved fluorescence immunoassay, radioimmunoassay, chemiluminescence immunoassay, magnetochemiluminescence immunoassay, protein chip analysis, liquid phase chip analysis, and Other immunological analysis methods.
  • the method of the invention is simple in operation, low in cost, does not require expensive equipment and professional technicians, and has high sensitivity and is suitable for various immunoassay methods.
  • Example 1 Whether the existing pre-reaction mode can improve sensitivity (using ELISA for detection of ciprofloxacin)
  • the plate was coated with ciprofloxacin BSA conjugate, 1 ⁇ g/mL, 100 ⁇ L per well, and blocked overnight at 4 °C. The next day was blocked with 3% skim milk powder.
  • the standard of ciprofloxacin Oppb, 1 ppb, 3 ppb, 9 ppb, 27 ppb, 81 ppb was prepared with washing solution (PBST).
  • Group 1 Pre-incubation of 60 ⁇ L of standard and 60 ⁇ L of ciprofloxacin antibody at 37 °C for 20 min; then 100 ⁇ L of ELISA plate coated with ciprofloxacin conjugate In the 37 ° C reaction for 30 min; wash the plate, add enzyme-labeled secondary antibody, 37 ° C reaction for 30 min, wash the plate; add The toner 100 L, reacted at 37 ° C for 15 min; finally, 50 stop solution was added, and OD450 was measured by a microplate reader.
  • Group 2 50 ⁇ L of standard and 50 ⁇ L of antibody were sequentially added to the plate containing ciprofloxacin conjugate, and reacted at 37 ° C for 30 min to wash the plate; add enzyme-labeled secondary antibody, 37 ° C The reaction was carried out for 30 min, and the plate was washed; 100 L of the lip developer was reacted at 37 ° C for 15 min; finally, 50 stop solution was added, and the OD450 was measured by a microplate reader.
  • Example 2 Effect of different re-reaction time (example of ciprofloxacin by ELISA) Enzyme plate coated with ciprofloxacin BSA conjugate, 1 ⁇ g/mL, 100 ⁇ L per well, 4 °C Closed overnight. The next day was blocked with 3% skim milk powder. Prepare ciprofloxacin standard products with washing solution 0ppb, lppb, 5ppb, 10ppb.
  • the experiment was divided into 5 groups. Each group took 100 ⁇ L of standard and 100 ⁇ L of ciprofloxacin antibody, pre-reacted at 37 °C for 20 min in the well plate, and then added to the ciprofloxacin plate:
  • Group 1, group 2, group 3, group 4 took 120 ⁇ L of pre-reaction product into ciprofloxacin plate and then reacted for 30 min, 20 min, 10 min, 5 min respectively; wash the plate; add enzyme-labeled secondary antibody, 37° C reaction for 30 min, wash the plate; force port developer 100 L, 37 ° C reaction for 15 min; finally add 50 L stop solution, determine OD450.
  • Group 5 Take 20 ⁇ L of antibody and standard pre-reaction solution into the ciprofloxacin plate, react for 1 min, pour off the liquid in the well, pat dry on absorbent paper, and add 20 ⁇ ⁇ to react for 1 min. Repeat a total of 6 times; wash the plate; add the enzyme-labeled secondary antibody, react at 37 ° C for 30 min, wash the plate; Lip filter 100 L, 37 ° C reaction for 15 min; finally add 50 stop solution, determination
  • test results are shown in the table below. It can be seen from the table that the antibody and the standard can react with the solid phase antigen on the microplate after a short reaction in advance, which can significantly improve the sensitivity of the reaction, and the IC50 can be reduced from lOppb to lppb.
  • Example 3 Effect of different pre-reaction time (example of ciprofloxacin by ELISA) Enzyme plate coated with ciprofloxacin BSA conjugate, 1 ⁇ g/mL, 100 ⁇ L per well, 4 °C Closed overnight. The next day was blocked with 3% skim milk powder. Prepare ciprofloxacin standard with washing solution 0ppb, lppb, 5 pb, 10ppb.
  • the experiment was divided into 4 groups. Each group took 100 ⁇ L of standard and 100 ⁇ L of ciprofloxacin antibody, and pre-incubated for 5 min, 10 min, 15 min, 20 min in a microplate at 37 °C, then took 20 ⁇ L of antibody and Mix the standard solution into the ciprofloxacin plate, react for 1 min, pour Remove the liquid from the well, pat dry on absorbent paper, then add 20 mixed solution for 1 min, repeat 6 times; wash the plate; add enzyme-labeled secondary antibody, react at 37 °C for 30 min, wash the plate; add developer 100 L, The reaction was carried out at 37 ° C for 15 min; finally, 50 stop solution was added to determine the OD450.
  • ractopamine microplate Add ractopamine coating to the microplate, 100 ⁇ ⁇ 7 well, react at 37 ° C for 2 h; discard the supernatant; add blocking solution to the microplate, ⁇ ⁇ pupil, 37 ° C Block lh; wash 3 times with washing solution; spare.
  • the ractopamine standard was prepared at a concentration of 0 ppb, 0.05 ppb, 0.15 ppb, 0.45 ppb, 1.35 ppb, and 4.05 ppb using a washing solution.
  • Group 1 60 ⁇ L of each standard and ractopamine antibody were added to a new well plate and pre-reacted for 20 min. Take 20 ⁇ L of the mixture of antibody and standard into the ractopamine microplate, react for 1 min, pour Remove the liquid from the well, pat dry on the absorbent paper, add 20 IA L mixture for 1 min, repeat 5 times, and add the pre-reaction products in each well. ⁇ ; Add enzyme standard secondary antibody, ⁇ ⁇ pupil, reaction at 37 ° C for 45 min; wash the plate; add color reagent 100 L, 37 ° C reaction for 15 min; finally add 50 stop solution, determine OD450.
  • Group 2 60 ⁇ L of the standard and ractopamine antibody were separately added to a new microplate, pre-reacted for 20 min; Take: 100 ⁇ L of the mixture of antibody and standard into the ractopamine microplate for 30 min. Pour the liquid in the well, pat dry on absorbent paper; wash; add enzyme-labeled secondary antibody, 100 ⁇ ⁇ 7 well, react at 37 ° C for 45 min, wash the plate; add developer 100 L, 37 ° C reaction for 15 min; Add 50 ⁇ L of stop solution and measure OD450.
  • the experimental results are shown in the table below. As can be seen from the table, Group 1 can significantly improve the sensitivity of the detection method.
  • chloramphenicol microplate Add chloramphenicol-BSA conjugate to 100 ⁇ 7 well to the plate, overnight at 4 ° C, wash 3 times, each time 3 min, pat dry; follow 130 ⁇ 7 wells Add 5% skim milk powder, block at 37 ° C for 1 h, pat dry; wash 3 times with washing solution, pat dry, spare.
  • the chloramphenicol standard solution was prepared by washing and stripping: Oppb, 0.01 ppb, 0.03 ppb, 0.09 ppb, 0.27 ppb, 0.81 ppb.
  • Group 1 The product was transferred to the plate according to the amount of 20 ⁇ 7 well, and reacted at 37 ° C for 1 min to remove the liquid in the well. This operation was repeated 5 times, and the pre-reaction product added in each well was ⁇ ;
  • Group 2 The reaction product 100 was sub-transformed into the chloramphenicol microplate, and the reaction was reversed at 37 ° C. Should be 20min;
  • the sensitivity of the method for detecting chloramphenicol by the method of the present invention is higher than that of the conventional method.

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Abstract

一种提高竞争免疫分析灵敏性的方法，是将待检测的小分子物质首先与抗体/受体发生充分反应，然后再与位于固相上的参与竞争反应的第二分子在短时间内反应。该方法具有操作简单、灵敏性高和成本低的特点。

Description

一种提高竟争免疫分析灵敏性的方法 技术领域
本发明涉及生物医学检测领域，尤其涉及一种提高竟争免疫分析 灵敏性的方法。 背景技术
免疫学检测方法是应用免疫学理论设计的一系列测定抗原、 抗 体、免疫细胞及其分泌的细胞因子的实验方法。抗原借助表面的抗原 决定簇与抗体分子超变区在空间构型上的互补，发生特异性结合。 同 一抗原分子可具有多种不同的抗原决定簇，若两种不同的抗原分子具 有一个或多个相同的抗原决定簇， 则与抗体反应时可出现交叉反应。
抗原抗体结合除以空间构型互补外， 主要以氢键、 静电引力、 范 德华力和疏水键等分子表面的非共价方式结合，结合后形成的复合物 在一定条件下可发生解离， 回复抗原抗体的游离状态。解离后的抗原 和抗体仍保持原有的性质。抗原抗体复合物解离度在 ^[艮大程度上取决 于特异性抗体超变区与相应抗原决定簇三维空间构型的互补程度，互 补程度越高， 分子间距越小， 作用力越大， 两者结合越牢固， 不易解 离； 反之， 则容易发生解离。
而小分子抗原或半抗原因抗原表位少， 不能用夹心法进行测定 , 只能采用竟争法检测模式。而现有的检测小分子抗原的方法主要有酶 联免疫吸附试验、 免疫层析试验、 仪器分析等方法。 酶联免疫吸附试 验因其特异、 灵敏、 检测成本低而得到广泛应用。 免疫层析试验虽然 简便易用， 但因其准确性不高， 同样限制了它的应用。 仪器分析法准 确、 可靠， 但因需要用到昂贵的设备和专业的技术人员， 限制了它的 应用， 多用于确认检测。
因此， 现有技术还有待于改进和发展。 发明内容
鉴于上述现有技术的不足，本发明的目的在于提供一种提高竟争 免疫分析灵敏性的方法， 旨在解决现有免疫分析方法灵敏性偏低、 不 适于高灵敏检测的问题。
本发明的技术方案如下：
一种提高竟争免疫分析灵敏性的方法， 其中， 所述提高竟争免疫 分析灵敏性的方法步骤如下：
预反应： 将待检测物质与受体 /抗体进行预反应；
再反应：将预反应的反应产物与位于固相上的参与竟争反应的第 二分子进行反应；
所述再反应过程中将预反应的反应产物分成一份或多份，分别与 所述第二分子进行反应。
所述的提高竟争免疫分析灵敏性的方法， 其中， 所述再反应过程 中，所述预反应的反应产物是以流动的方式流过所述固相表面与所述 第二分子反应。 所述的提高竟争免疫分析灵敏性的方法， 其中， 所述再反应过程 中， 每一份预反应的反应产物与所述第二分子的反应时间在 20min 以内。
所述的提高竟争免疫分析灵敏性的方法， 其中， 所述再反应过程 中， 每一份预反应的反应产物与所述第二分子的反应时间在 5min以 内。
所述的提高竟争免疫分析灵敏性的方法， 其中，每一份所述预反 应的反应产物与所述第二分子进行反应后， 将反应后的液体去除。
所述的提高竟争免疫分析灵敏性的方法， 其中， 所述预反应过程 的反应温度为 0 °C -65 °C , 反应时间为 0-3小时。
所述的提高竟争免疫分析灵敏性的方法， 其中， 所述预反应过程 的反应时间为 30min。
所述的提高竟争免疫分析灵敏性的方法， 其中， 所述第二分子是 指参与竟争反应的小分子与载体蛋白形成的偶联物。
有益效果： 本发明从反应方式上着手， 提供一种提高竟争免疫分 析灵敏性的方法， 主要是将待检测的小分子物质首先与抗体 /受体发 生充分反应，然后再与位于固相上的参与竟争反应的第二分子在短时 间内反应， 这样能大大提高了免疫分析方法的灵敏度。 本发明方法操 作简单， 成本低， 不需要用到昂贵的设备和专业的技术人员， 而且灵 敏性高， 适用于多种免疫分析方法。 具体实施方式 本发明提供一种提高竟争免疫分析灵敏性的方法，为使本发明的 目的、 技术方案及效果更加清楚、 明确， 以下对本发明进一步详细说 明。 应当理解， 此处所描述的具体实施例仅仅用以解释本发明， 并不 用于限定本发明。
灵敏性偏低、 不适于高灵敏检测是免疫学检测普遍存在的问题， 因而人们开发出了多种免疫学检测信号放大方法， 如： 酶标记、 稀土 荧光标记、放射物标记等方式提高检测信号。 而本发明从反应方式上 着手， 提供一种提高竟争免疫分析灵敏性的方法， 主要是将待检测的 小分子物质首先与抗体 /受体发生充分反应， 然后再与位于固相上的 参与竟争反应的另一种分子在短时间内反应 ,这样能大大提高了免疫 分析方法的灵敏度。
具体的， 所述提高竟争免疫分析灵敏性的方法步骤如下： 预反应： 将待检测物质与受体 /抗体进行预反应；
再反应：将预反应的反应产物与位于固相上的参与竟争反应的第 二分子进行反应。
其中， 所述预反应， 是将待检测物质与受体 /抗体先进行充分的 反应， 即当待测物质为抗原时， 则与其抗体进行充分反应， 当所述待 测物质为补体时， 则与其受体进行充分反应。
所述预反应具体是在 0°C-65°C之间反应 0-3小时，反应时间优选 为 30min„
所述再反应， 具体是将预反应的反应产物分成 1份或多份， 分别 与参与竟争反应的第二分子进行反应。优选地， 可以将所述预反应的 反应产物平均分成 2〜6份，然后将每一份反应产物分别与位于固相上 的所述第二分子进行反应。所述预反应的反应产物优选为以流动的方 式流过表面包被有参与竟争的第二分子的固相表面， 经过实验证明， 所述预反应产物流动的方式流过所述固相表面与所述第二分子进行 反应， 能提高竟争免疫分析的灵敏性。 其中， 所述参与竟争反应的第 二分子是指参与竟争反应的小分子与载体蛋白形成的偶联物。所述参 与竟争反应的小分子具是指与所述受体相对应的补体或与所述受体 相对应的抗原。
所述再反应中，将每一份所述预反应的反应产物与所述第二分子 的反应时间控制在 20min以内， 优选 5min以内。 本发明的主要改进 在于， 所述再反应过程中将预反应产物分一份或多份， 然后与参与竟 争反应的第二分子在短时间内进行反应， 经过大量的实验证明， 这样 能明显提高竟争免疫反应的灵敏性。而每一份所述预反应的反应产物 与第二分子进行反应后， 都应将反应后的液体去除， 然后再取另一份 预反应产物与所述第二分子进行反应。 这样， 能减少剩余的反应液体 对下一次反应的影响， 提高分析检测的灵敏性。
在进行所述再反应步骤以前 ,所述提高竟争免疫分析灵敏性的方 法还包括以下步骤：
将所述第二分子转移到固相上。
如， 用所述参与竟争反应的小分子包被酶标板。 进一步的， 当所 述参与竟争反应的小分子包被酶标板后，可以用封闭液对所述固相进 行封闭 ,这样能有效地避免抗体和非特异性的抗原结合而造成假阳性 结果， 使检测的效果更加准确。 所述封闭液可以是 BSA、 脱脂奶粉 等。 所述封闭的步骤可以为加封闭液 37 °C孵育 30min。
在进行所述再反应步骤以后，所述提高竟争免疫分析灵敏性的方 法还包括以下步骤：
显色： 在所述再反应的反应产物中加入底物显色液；
终止反应二 可加入疏酸溶液终止反应；
检测： 可通过酶标仪测定所述产物的 OD值。
所述提高竟争免疫分析灵敏性的方法， 适用于酶联免疫分析、 时 间分辨荧光免疫分析、 放射免疫分析、 化学发光免疫分析、 磁化学发 光免疫分析、 蛋白质芯片分析、 液相芯片分析， 以及其它免疫学分析 方法。 本发明方法操作简单， 成本低， 不需要用到昂贵的设备和专业 的技术人员， 而且灵敏性高， 适用于多种免疫分析方法。
实施例 1 现有预反应方式是否能够提高灵敏性（以 ELISA检测 环丙沙星为例）
用环丙沙星 BSA偶联物包被酶标板， 1 μ g/mL, 每孔 100 μ L, 4 °C封闭过夜。 第二天用 3%的脱脂奶粉封闭。 用洗涤液（PBST )配制 环丙沙星的标准品 Oppb、 1 ppb、 3ppb、 9ppb、 27ppb、 81 ppb。
实马 分为两组：
组 1 : 把 60 μ L标准品和 60 μ L稀译好的环丙沙星抗体 37°C预 孵育 20min; 然后取 100 μ L加到包被有环丙沙星偶联物的酶标板中， 37°C反应 30min; 洗板， 加酶标二抗， 37°C反应 30min, 洗板； 加显 色剂 100 L, 37°C反应 15min; 最后加 50 终止液， 用酶标仪测 定 OD450。
组 2: 往包被有环丙沙星偶联物的酶标板中依次加入 50 μ L标准 品、 50 μ L抗体， 37°C反应 30min,洗板；加酶标二抗， 37°C反应 30min, 洗板； 力口显色剂 100 L, 37°C反应 15min; 最后加 50 终止液， 用酶标仪测定 OD450。
检测结果如下表所示。可以看出组 1与组 2相比并不能提高检测 的灵敏性。
Figure imgf000008_0001
实施例 2 不同再反应时间的影响（以 ELISA检测环丙沙星为例） 用环丙沙星 BSA偶联物包被酶标板， 1 μ g/mL, 每孔 100 μ L, 4 °C封闭过夜。 第二天用 3%的脱脂奶粉封闭。 用洗涤液配制环丙沙星 标准品 0ppb、 lppb、 5ppb、 10ppb。
实验分为 5组， 每组取 100 μ L标准品与 100 μ L环丙沙星抗体， 37 °C于 孔板中预反应 20min, 然后加到环丙沙星酶标板中：
组 1、 组 2、 组 3、 组 4取 120 μ L预反应产物到环丙沙星酶标板 中后分别再反应 30min、 20min、 10min、 5min; 洗板； 加酶标二抗， 37°C反应 30min, 洗板； 力口显色剂 100 L, 37°C反应 15min; 最后加 50 L终止液， 测定 OD450。
组 5取 20 μ L抗体和标准品的预反应液到环丙沙星酶标板中 ,反 应 lmin, 倒去孔中液体， 在吸水纸上拍干， 再加入 20 μ Ι^ 合液反 应 lmin,共重复 6次； 洗板； 加酶标二抗， 37°C反应 30min, 洗板； 力口显色剂 100 L, 37°C反应 15min; 最后加 50 终止液， 测定
OD450。
检测结果如下表所示。从表中可以看出， 抗体和标准品预先预反 应后短时间内与酶标板上固相抗原竟争反应，能明显提高反应的灵敏 度， IC50可以从 lOppb降为 lppb„
Figure imgf000009_0001
实施例 3 不同预反应时间的影响（以 ELISA检测环丙沙星为例） 用环丙沙星 BSA偶联物包被酶标板， 1 μ g/mL, 每孔 100 μ L, 4 °C封闭过夜。 第二天用 3%的脱脂奶粉封闭。 用洗涤液配制环丙沙星 标准品 0ppb、 lppb、 5 pb, 10ppb。
实验分为 4组， 每组取 100 μ L标准品与 100 μ L环丙沙星抗体， 37°C于微孔板中分别预孵育 5min、 10min、 15min、 20min, 然后取 20 μ L抗体和标准品的混合液到环丙沙星酶标板中， 反应 1 min , 倒 去孔中液体， 在吸水纸上拍干， 再加入 20 混合液反应 lmin,共 重复 6次； 洗板； 加酶标二抗， 37°C反应 30min, 洗板； 加显色剂 100 L, 37°C反应 15min; 最后加 50 终止液， 测定 OD450。
实验结果如下表所示。 从表中可以看出预孵育时间超过 5min对 结果影响不大， 考虑到加样时间的差别， 可以提高到 20min。
Figure imgf000010_0001
实施例 4 ELISA检测莱克多巴胺
制备莱克多巴胺微孔板： 往微板中加入莱克多巴胺包被原， 100 μ Ι7孔， 37°C反应 2h; 弃上清； 往微孔板中加入封闭液， ΙΟΟ μ ϋ孔， 37°C封闭 lh; 用洗涤液洗涤 3次； 备用。 用洗涤液配制浓度分别为 0ppb、 0.05ppb、 0.15ppb、 0.45ppb、 1.35ppb、 4.05 ppb的莱克多巴胺 标准品。
分两组：
组 1 : 将标准品和莱克多巴胺抗体各 60 μ L分别加入一新的 孔 板中 , 预反应 20min; 取 20 μ L抗体和标准品的混合液到莱克多巴胺 微孔板中， 反应 lmin, 倒去孔中液体， 在吸水纸上拍干， 再加入 20 IA L混合液反应 lmin,共重复 5次， 每孔中所加入的预反应产物共 ΙΟΟμΙ^; 加入酶标二抗， ΙΟΟ μ ϋ孔， 37°C反应 45min; 洗板； 加显色 剂 100 L, 37°C反应 15min; 最后加 50 终止液， 测定 OD450。 组 2: 将标准品和莱克多巴胺抗体各 60 μ L分别加入一新的微孔 板中， 预反应 20min; 取: 100 μ L抗体和标准品的混合液到莱克多巴 胺微孔板中， 反应 30min, 倒去孔中液体， 在吸水纸上拍干； 洗涤； 加酶标二抗， 100 μ Ι7孔， 37°C反应 45min, 洗板； 加显色剂 100 L, 37°C反应 15min; 最后加 50 μ L终止液， 测定 OD450。 实验结果如下表所示。从表中可以看出， 组 1可显著提高检测方 法的灵敏性。
Figure imgf000011_0001
实施例 5 在化学发光免疫检测中的应用 （以检测氯霉素为例）
1)氯霉素微孔板的制备： 按照 100μΙ7孔加入氯霉素 -BSA偶联物 到酶标板中， 4°C过夜，洗涤 3次，每次间隔 3 min,拍干；按照 130μΙ7 孔加入 5%脱脂奶粉， 37°C封闭 l h,拍干； 用洗涤液洗涤 3次，拍干， 备用。 用洗)、条液配制氯霉素标准溶液： Oppb, 0.01 ppb, 0.03ppb, 0.09ppb, 0.27ppb、 0.81 ppb。
2)标准品与氯霉素单抗等体积混合， 37°C孵育 20 min; 然后将产 物转至氯霉素微孔板中：
组 1 : 按照 20μΙ7孔的量将产物转至酶标板中， 37°C反应 lmin, 去除孔中液体， 重复本操作 5 次， 每孔中所加入的预反应产物共 ΙΟΟμΙ^; 组 2: 将反应产物 100 —次性转入氯霉素微孔板中， 37°C反 应 20min;
3 ) 洗涤 3次； 加入 HRP标记二抗， ΙΟΟμϋ孔， 37 °C , 45 min; 洗涤 5次； 加入发光底物后用化学发光测定仪测定发光强度。
下表为测试所得数据。
Figure imgf000012_0001
由以上数据可知，采用本发明方法检测氯霉素的灵敏度比常规方 法检测的灵敏性更高。
应当理解的是， 本发明的应用不限于上述的举例， 对本领域普通 技术人员来说， 可以根据上述说明加以改进或变换， 所有这些改进和 变换都应属于本发明所附权利要求的保护范围。

Claims

权 利 要 求 书
1、 一种提高竟争免疫分析灵敏性的方法， 其特征在于， 所述提 高竟争免疫分析灵敏性的方法步骤如下：
预反应： 将待检测物质与受体 /抗体进行预反应；
再反应：将预反应的反应产物与位于固相上的参与竟争反应的第 二分子进行反应；
所述再反应过程中将预反应的反应产物分成一份或多份，分别与 所述第二分子进行反应。
2、 根据权利要求 1所述的提高竟争免疫分析灵敏性的方法， 其 特征在于， 所述再反应过程中， 所述预反应的反应产物是以流动的方 式流过所述固相表面与所述第二分子反应。
3、 根据权利要求 2所述的提高竟争免疫分析灵敏性的方法， 其 特征在于， 所述再反应过程中， 每一份预反应的反应产物与所述第二 分子的反应时间在 20min以内。
4、 根据权利要求 2所述的提高竟争免疫分析灵敏性的方法， 其 特征在于， 所述再反应过程中， 每一份预反应的反应产物与所述第二 分子的反应时间在 5min以内。
5、 根据权利要求 1所述的提高竟争免疫分析灵敏性的方法， 其 特征在于， 每一份所述预反应的反应产物与所述第二分子进行反应 后， 将反应后的液体去除。
6、 根据权利要求 1所述的提高竟争免疫分析灵敏性的方法， 其 特征在于， 所述预反应过程的反应温度为 0 °C -65 °C , 反应时间为 0-3 小时。
7、 根据权利要求 6所述的提高竟争免疫分析灵敏性的方法， 其 特征在于， 所述预反应过程的反应时间为 30min。
8、 根据权利要求 1所述的提高竟争免疫分析灵敏性的方法， 其 特征在于，所述第二分子是指参与竟争反应的小分子与载体蛋白形成 的偶联物。
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