WO2007140696A1 - Kit elisa pour détecter le rouge soudan et procédé correspondant - Google Patents

Kit elisa pour détecter le rouge soudan et procédé correspondant Download PDF

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Publication number
WO2007140696A1
WO2007140696A1 PCT/CN2007/001530 CN2007001530W WO2007140696A1 WO 2007140696 A1 WO2007140696 A1 WO 2007140696A1 CN 2007001530 W CN2007001530 W CN 2007001530W WO 2007140696 A1 WO2007140696 A1 WO 2007140696A1
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Prior art keywords
antibody
sudan red
solution
enzyme
liquid
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PCT/CN2007/001530
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English (en)
French (fr)
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WO2007140696A8 (fr
Inventor
Jianzhong Shen
Fangyang He
Caiwei Feng
Yuping Wan
Xiaoping Wu
Caimao Feng
Zhengmiao Zhao
Guikun Luo
Shanliang Wang
Shuangyang Ding
Haiyang Jiang
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Beijing Wanger Biotechnology Co., Ltd.
Beijing Wanger Kangtai Biotechnology Co., Ltd.
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Application filed by Beijing Wanger Biotechnology Co., Ltd., Beijing Wanger Kangtai Biotechnology Co., Ltd. filed Critical Beijing Wanger Biotechnology Co., Ltd.
Priority to AU2007257105A priority Critical patent/AU2007257105B8/en
Publication of WO2007140696A1 publication Critical patent/WO2007140696A1/zh
Publication of WO2007140696A8 publication Critical patent/WO2007140696A8/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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the invention relates to an enzyme-linked immunoassay kit for detecting Sudan red and a detection method thereof. Background information
  • Sudani is a synthetic red dye commonly used as an industrial dye. It is widely used in coloring such as solvents, oils, waxes, gasoline, and for lighting such as shoes and floors.
  • Sudan Red No. 1 dye contains “azobenzene”. When “azobenzene” is degraded, it produces “aniline”, a moderately toxic carcinogen. Excessive amounts of "aniline” are inhaled into the body, which may cause tissue hypoxia, poor breathing, damage to the central nervous system, cardiovascular system and other organs, and even infertility. Due to its serious carcinogenicity, sensitization and genotoxicity, countries such as China and the European Union have expressly banned the addition of pigments to foods. On February 18, 2005, The Food Standard Agency (FSA) warned consumers about foods containing reddane reddish and posted a list of products that may contain Sudan Red I on its website.
  • FSA Food Standard Agency
  • the object of the present invention is to provide a method which is simple in operation, low in cost, high in sensitivity, capable of on-site monitoring and suitable for screening a large number of samples, and is used for detecting Sudan red content in foods such as chili sauce, chili oil, chili powder and the like. .
  • the detection principle of the present invention is:
  • the Sudan red-conjugated antigen When the Sudan red-conjugated antigen is pre-coated on the microporous strip, add the sample solution or the standard solution, then add the Sudan red specific antibody solution, and the residual Sudan Red or Sudan Red standard and the ELISA plate in the sample.
  • the coated Sudan red-conjugated antigen competes with the Sudan red-specific antibody, and the enzyme-labeled anti-antibody is added for amplification.
  • the color is developed by the color developing solution, and the absorbance of the sample is negatively correlated with the content of Sudan red in the sample, which is compared with the standard curve.
  • the content of Sudan red in the sample can be obtained.
  • the ratio of the color of the series of Sudan red standard solution It is more crude to judge the concentration range of the Sudan red content in the sample.
  • the enzyme labeled Sudan red hapten solution is added, and the residual Sudan Red or Sudan Red standard and the enzyme labeled antigen are added in the sample.
  • the competition was coated with a Sudan red specific antibody on the plate, and the color was developed with a color developing solution.
  • the absorbance of the sample was negatively correlated with the content of Sudan red.
  • the content of Sudan red in the sample was obtained by comparison with the standard curve.
  • the concentration range of the Sudan red standard in the sample can be roughly judged by comparing the color of the series of Sudan red standard solution.
  • the Sudan red-conjugated antigen When the Sudan red-conjugated antigen is pre-coated on the microporous strip, add the sample solution or the standard 'solution, then add the enzyme-labeled Sudan red specific antibody solution, the Sudan red or Sudan red standard and the enzyme label in the sample.
  • the Sudan red antigen coated on the plate competes with the Sudan red specific antibody, and the color is developed by the color developing solution.
  • the absorbance of the sample is negatively correlated with the content of Sudan red in the sample, and the content of Sudan red in the sample can be obtained by comparison with the standard curve. .
  • the color concentration of the Sudan red standard solution in the sample can be roughly judged by comparing the color of the concentration of the Sudan red standard solution.
  • the anti-antibody When the anti-antibody is pre-coated on the microporous strip, add the sample solution or standard solution after adding the Sudan red antibody, and then add the enzyme-labeled Sudan red hapten solution, the Sudan Red or Sudan Red standard and enzyme in the sample.
  • the Sudan red hapten was labeled to compete with Sudan red specific antibody, and the color was developed with a color developing solution.
  • the absorbance value of the sample was negatively correlated with the content of Sudan red in the sample, and the content of Sudan red in the sample was obtained by comparison with the standard curve.
  • the color concentration of the Sudan red standard in the sample can be roughly judged by comparing the color of the series of Sudan red standard solution.
  • the present invention provides an enzyme-linked immunoassay kit for detecting Sudan red, which comprises:
  • an enzyme label (either an enzyme labeled antigen, an enzyme labeled antibody or an enzyme labeled anti-antibody),
  • the enzyme-linked immunoassay kit further comprises:
  • the enzyme-linked immunosorbent kit for detecting Sudan red comprises an enzyme-labeled plate coated with a coating, an optional Sudan red specific antibody concentrate (coated with an antigen on an enzyme label, and an enzyme label)
  • the coating may be a coupling antigen of Sudan red with a carrier protein, a Sudan red specific antibody or an anti-antibody
  • the enzyme label is an enzyme-labeled anti-antibody, a Sudan red hapten or a Sudan red specific antibody
  • the anti-antibody may be a goat anti-antibody Mouse anti-antibody or goat anti-rabbit anti-antibody.
  • the Sudan red hapten is obtained by acylation of Sudan red and succinic anhydride.
  • the labeling enzyme of the enzyme label is horseradish peroxidase or alkaline phosphatase, wherein horseradish peroxidase is preferred; enzyme-labeled goat anti-mouse anti-antibody or goat anti-rabbit anti-antibody is glutaraldehyde.
  • the method or the sodium periodate method is used to couple the labeling enzyme with the anti-antibody, and the horseradish peroxidase can be coupled with the anti-antibody by various methods in the prior art, such as the glutaraldehyde method.
  • the sodium periodate method and the like have been improved by the long-term labor creation by the sodium periodate method, which saves time and reduces the concentration ratio of horseradish peroxidase (HRP) to anti-antibody, thereby saving raw materials. .
  • the Sudan red specific antibody may be a Sudan red monoclonal antibody or a Sudan red polyclonal antibody; both of which are obtained by using a conjugate of a Sudan red hapten and a carrier protein by a mixed acid anhydride method as an immunogen;
  • the red polyclonal antibody may be a mouse, a horse source, a sheep source, a rabbit source or a guinea pig source antibody, and the Sudan red monoclonal antibody is preferably a Sudan red mouse monoclonal antibody, and the Sudan red polyclonal antibody is preferably a Sudan red rabbit.
  • Polyclonal antibody may be a Sudan red monoclonal antibody or a Sudan red polyclonal antibody; both of which are obtained by using a conjugate of a Sudan red hapten and a carrier protein by a mixed acid anhydride method as an immunogen;
  • the red polyclonal antibody may be a mouse, a horse source, a sheep source, a rabbit source or a guinea pig source antibody
  • the Sudan red monoclonal antibody is preferably a monoclonal antibody secreted by a monoclonal hybridoma cell line of Sudan Red B-1-1 CGMCC No. 1706.
  • the Sudan Hybrid monoclonal hybridoma cell line B-1-1 CGMCC No. 1706 was deposited on April 30, 2006 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee.
  • the above antibodies can be immunized with a conjugate of Sudan red hapten and carrier protein. It was prepared by the mixed acid anhydride method.
  • the carrier protein may be murine serum protein, thyroprotein
  • Common carrier protein such as (BCG), bovine serum albumin, rabbit serum albumin, human serum albumin, ovalbumin or hemocyanin; the Sudan red hapten and carrier protein conjugate can be passed through the Sudan red hapten It is obtained by coupling with a carrier protein by a mixed acid anhydride method.
  • the kit further includes a Sudan red standard solution, a substrate color developing solution, a stop solution, a concentrated washing solution, and a concentrated antibody dilution solution (coated with an antigen on the ELISA plate).
  • the enzyme label is an enzyme-labeled anti-antibody or an anti-antibody coated on the plate, the enzyme label is an enzyme-labeled antigen, and the concentrated solution is a phosphate buffer, for example, 2 ⁇ 0.04 mol/L phosphate buffer.
  • the concentrated washing liquid is preferably a phosphate buffer, such as a phosphate buffer containing Tween and sodium azide, further such as pH 7.4, 0.01 M phosphoric acid containing 0.8% to 1.2% Tween 20 and 0.5% sodium azide.
  • the salt buffer may also be other concentrated wash solutions commonly used in the art.
  • the concentrated complex solution is preferably a phosphate buffer, such as a 2x0.04 mol/L phosphate buffer, and may be other concentrated complex solutions commonly used in the art.
  • the substrate coloring liquid is composed of the coloring liquid liquid A and the coloring liquid liquid B, and the coloring liquid liquid A is preferably a hydrogen peroxide or a urea peroxide solution.
  • the liquid B liquid is preferably a solution of o-phenylenediamine or tetramethylbenzidine, and the stop solution is preferably a sulfuric acid solution having a concentration of, for example, 1 to 2 mol/L ; and when the labeling enzyme is preferably an alkaline phosphatase, the color developing solution A p-nitrophenyl phosphate buffer is preferred, and the stop solution is preferably a sodium hydroxide solution having a concentration of, for example, 1 to 2 mol/L, more particularly, for example, 2 mol/L.
  • the chromogenic and stop solutions can also be other chromogenic and stop solutions commonly used in the art.
  • the enzyme label is coated with an antigen
  • the enzyme label is an enzyme label anti-antibody or the anti-antibody is coated on the label plate
  • the enzyme label is an enzyme-labeled antigen
  • the diluted solution of the concentrated antibody is preferably a phosphate buffer.
  • a solution for example, a phosphate buffer containing bovine serum or methanol, further such as pH 8.6, 0.05 mol/L phosphate buffer containing 2% calf serum, 10% methanol, or other concentrated substances commonly used in the art. A dilution of the antibody.
  • the coating buffer used in the preparation of the microplate is preferably sodium citrate buffer, such as pH 6.6, 0.01-0.03 mol/L sodium citrate buffer; the blocking solution used is preferably phosphate buffer.
  • the coating buffer and blocking solution can also be other coating buffers and blocking solutions commonly used in the art.
  • the preparation process of the microplate in the invention may be: diluting the coating with a coating buffer, adding to each well, incubating for several hours at 37 ° C, and then overnight at about 4 ° C, pouring the coating solution Wash with washing solution, pat dry, then add blocking solution to each well, incubate, pour out the liquid in the well, dry it, and vacuum-seal it with aluminum film.
  • the coating is diluted with a coating buffer to 0.05-0.
  • ⁇ is added to each well, incubated at 37 ° C for 2 h, and then at 4 ° C overnight, the coating solution is decanted and washed with washing solution 2 Once, each time for 30 seconds, pat dry, then add 150-200 ⁇ 1 blocking solution to each well, incubate at 37 °C for l-2h, pour off the liquid in the well, dry it, and vacuum-seal it with aluminum film.
  • the synthesis process of the antigen in the present invention is, for example:
  • Sudan red hapten was synthesized by Sudan succinic anhydride method.
  • the Sudan red hapten and the carrier protein were coupled by a mixed acid anhydride method (isobutyl chloroformate) to obtain an immunogen.
  • Sudan red is a small molecule, only immunoreactive, non-immunogenic, unable to induce the body to produce an immune response, must be coupled with the macromolecular carrier protein to be immunogenic. Therefore, Sudan red and succinic anhydride were synthesized into Sudan red hapten, and a red carbon-containing spacer arm was attached to Sudan red, which highlighted the characteristic groups in the Sudan red molecular structure, and the prepared Sudan red antibody against Sudan red The specificity is very high.
  • Animal immunization program Balb/c mice were used as immunized animals, and the Sudan red hapten and carrier protein conjugate were used as immunogens to obtain a better polyclonal antibody, and the spleen was taken out for cell fusion.
  • New Zealand white rabbits were used as immunized animals, and New Zealand white rabbits were immunized with Sudan red and carrier protein conjugates as immunogens. After multiple immunizations, serum antibody titers were determined to obtain polyclonal antibodies.
  • the anti-antibody preparation process of the invention the goat anti-mouse anti-antibody is a sheep as an immunized animal, and the murine-derived antibody is used as an immunogen to immunize the disease-free pathogenic sheep to obtain a goat anti-mouse anti-antibody; the goat anti-rabbit anti-antibody is a sheep As an immunized animal, a rabbit-derived antibody is used as an immunogen to immunize a disease-free sheep, and a goat anti-rabbit anti-antibody is obtained.
  • the Sudan Red Standard solution in the kit of the present invention may be a standard solution of various concentrations within a certain concentration range, and the concentration thereof may preferably be, for example, between 0 and 5 ( ⁇ g/L.
  • concentration thereof may be contained 6 bottles of standard solutions at concentrations of 0 g/L, 0.1 g/L, 0.4 g/L, 1.6 g/L, 6.4 g/L and 25.6 g/L, 3 ml/bottle.
  • the present invention also provides a method for detecting a Sudan red content in a sample using the above-described enzyme-linked immunoassay kit, which comprises the steps of:
  • the sample in the above step (1) may be various products for animal or human consumption, particularly foods or raw materials for making foods. More particularly, it is a food such as chili sauce, chili oil, paprika or a raw material for making food.
  • the invention provides a treatment of a food sample (water base, oil base and powder) with Sudan red as a food additive: the sample is taken in a centrifuge tube, shaken by acetonitrile, the supernatant is removed, the nitrogen is blown dry, and the solution is reconstituted. , sampling analysis.
  • the kit for detecting in the present invention is: when the coating is originally a Sudan red-conjugated antigen, a standard solution or a sample solution is added to the microplate of the microplate, and then the antibody is added, and after washing, the sample is washed, and then the enzyme label is added.
  • the solution is further added with an enzyme-labeled Sudan red hapten. After the incubation, wash and dry, color and terminate, measure the absorbance value with the microplate reader; when the original anti-antibody is coated, add the Sudan red antibody to the microplate of the microplate, wash and pat dry, then add After the standard solution or the sample solution, the enzyme-labeled Sudan red hapten is added, and after washing, the sample is washed and dried, color-developed, terminated, and the absorbance value is measured by a microplate reader.
  • the detection result of the analysis process with the obtained standard solution of each concentration the average absorbance (B) divided by the standard solutions (0 standard) absorbance value (B 0) and multiplied by 100% , that is, the percent absorbance value.
  • the calculation formula is:
  • the semi-logarithmic value of the concentration (g/L) of the Sudan Red Standard solution is the X-axis, and the percent absorbance is the Y-axis, and a standard curve is drawn. In the same way, the percent absorbance of the sample solution is calculated, and the concentration of each sample is read from the standard curve to read the amount of Sudan red in the sample.
  • the analysis of the detection results in the present invention can also be carried out by using the regression equation method to calculate the concentration of the sample solution.
  • the analysis of the detection results in the present invention can also utilize computer professional software, which is more convenient for rapid analysis of a large number of samples, and the entire detection process can be completed in a short time, such as 1.5 hours, and the minimum detection limit of the sample is l ( ⁇ g/ L.
  • the enzyme-linked immunosorbent kit for detecting Sudan red in foods of the present invention mainly uses an indirect competitive ELISA method to qualitatively or quantitatively detect the products of animals or humans, especially the contents of Sudan red dyes in foods such as chili sauce, chili oil, and chili powder.
  • the sample preparation process is simple and can simultaneously detect a large number of samples.
  • the invention adopts a highly specific Sudan red monoclonal antibody or a polyclonal antibody, and the main reagent is provided in the form of a working fluid.
  • the test method is simple and convenient, and has the characteristics of high specificity, high sensitivity and high precision, and will be residual in Sudan red. The detection plays an important role.
  • Sudan red hapten was synthesized by Sudan succinic anhydride method. Specific steps: 5 g of Sudan Red is dissolved in 20 ml of DMF, 3 g of succinic anhydride and 50 ml of pyridine are added, and the reaction is refluxed at 80 ° C for 70 to 80 hours, and the solvent is removed by suction and washed with pure water 5 times (30 ml / time), and dried. That is.
  • the Sudan red hapten and ovalbumin were coupled by a mixed acid anhydride method to obtain an immunogen.
  • the Sudan red hapten and the thyroxin were coupled by a mixed acid anhydride method to obtain a coating antigen.
  • the immunogen is injected into Balb / c mice, immunization dose of 80 ⁇ ⁇ / only, to produce polyclonal antibodies.
  • spleen cells of the immunized Balb/c mice were fused with SP2/0 myeloma cells in a ratio of 5:1, and the cell supernatant was assayed by indirect competition ELISA to screen positive wells.
  • the positive wells were cloned by the limiting dilution method until a monoclonal antibody hybrid cell line B-1-1 CGMCC No. 1706, which stably secretes the monoclonal antibody, was obtained.
  • the Sudan hybrid monoclonal hybridoma cell line B-1-1 CGMCC No. 1706 was made into a lx10 6 /ml cell suspension with a cryopreservation solution and stored in liquid nitrogen for a long time. When resuscitation, remove the cryotube and immediately put it into a 37 °C water bath for rapid fusion. After centrifugation to remove the frozen solution, transfer it to a culture flask for cultivation.
  • the Balb / c mice were injected intraperitoneally sterile paraffin oil 0.4ml / mouse 7 days after intraperitoneal injection of Sudan monoclonal hybridoma cell line B-1-1 CGMCC No. 1706 5> 10 5 cells / animal, collected after 7 days ascites. Purification was carried out by the octanoic acid-saturated ammonium sulfate method, and the purified ascites was stored in an environment of -20 °C.
  • New Zealand white rabbits were used as immunized animals, and the Sudan red hapten and ovalbumin conjugate were used as immunogens.
  • the immunization dose was 1.5 mg/kg.
  • Emulsifier multiple injections of the neck and back subcutaneous, at the interval of 3 to 4 weeks, take the same dose of immunogen plus equal amount of Freund's incomplete adjuvant mixed emulsification, strengthen the immunization once, a total of 5 immunizations, the last time without adjuvant.
  • Blood was collected 10 days after the last immunization, serum antibody titer was measured, blood was collected from the heart, and purified polyclonal antibody was obtained by fractionation with ammonium sulfate.
  • Goats were used as immunized animals, and murine-derived antibodies were used as immunogens to immunize pathogen-free goats to obtain goat anti-mouse anti-antibodies.
  • Preparation of goat anti-rabbit anti-antibodies goats were used as immunized animals, and rabbit-derived antibodies were used as immunogens.
  • the pathogen goat is immunized to obtain a goat anti-rabbit anti-antibody.
  • the goat anti-mouse anti-antibody is coupled with horseradish peroxidase (HRP) by a modified sodium periodate method as follows:
  • Example 1 Each of the components prepared in Example 1 was used to construct an enzyme-linked immunosorbent kit for detecting Sudan red, which comprises the following components:
  • the concentrations are ( ⁇ g / L, 0.1 g / L, 0.4 g / L, 1.6 g / L, 6.4 g / L and 25.6 g / L;
  • the substrate color developing solution is composed of liquid A and liquid B, the substrate coloring liquid A liquid is 2% urea peroxide solution, and the substrate color developing liquid liquid B is 1% tetramethylbenzidine solution;
  • the stop solution is 2 mol/L hydrochloric acid
  • the concentrated washing solution is pH 7.4, 0.01 M phosphate buffer containing 0.8% to 1.2% Tween 20 and 0.5% sodium azide;
  • the antibody dilution is pH 8.6, 0.05 mol/L containing 2% calf serum, 10% methanol Phosphate buffer
  • the absorbance average ( ⁇ ) of each standard solution used was divided by the absorbance value (B Q ) of the first standard solution (0 standard) and multiplied by 100% to obtain a percent absorbance value.
  • the semi-logarithmic value of the Sudan red standard concentration (g/L) is the X-axis, and the percent absorbance is the Y-axis.
  • a standard curve is drawn. The same method is used to calculate the percent absorbance of the sample solution.
  • the residual amount of Sudan red in the sample can be read from the standard curve.
  • kits used in the experimental examples and the experimental examples described later were prepared from the formulation of Example 2.
  • a batch of enzyme-labeled plates were extracted from the microplates prepared in three different time periods, and 10 kits were taken from each batch. 20 microwells were extracted from each plate, and the absorbance value of the standard solution of 4.5 g/L was determined. , Calculate the coefficient of variation, the results are shown in Table 1.
  • the Sudan red standard was taken and added to the sample to be a concentration of 15 g/L sample.
  • the test results of the three samples are shown in Table 2, Table 3 and Table 4, respectively.
  • Cross-Reaction Rate Test Two drugs with similar structures and similar functions to Sudan Red were selected for cross-reactivity. The 50% inhibitory concentration was obtained by the standard curve of each drug. The cross-reactivity rate of the kit for other drugs was calculated by the following formula.
  • the kit was stored at 2-8 ° C. After 6 months of measurement, the maximum absorbance (zero standard), 50% inhibitory concentration, and actual measured value of Sudan Red were within the normal range. Considering that during the transportation and use, abnormal storage conditions will occur, and the kit will be stored under the condition of 37 ° C for 6 days to carry out accelerated aging test. The results show that the indicators of the kit are in full compliance with the requirements. Considering the freezing of the kit, the kit was frozen in a -20 °C refrigerator for 5 days. The results also showed that the kit parameters were completely normal. From the above results, the kit can be stored for at least 6 months at 2-8 °C.

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Description

检测苏丹红的酶联免疫试剂盒及检测方法 技术领域
本发明涉及一种检测苏丹红的酶联免疫试剂盒及检测方法。 背景资料
苏丹红（Sudani)是一种人工合成的红色染料， 常作为一种工业染 料， 被广泛用于如溶剂、油、蜡、汽油的增色以及鞋、地板等增光方面。 苏丹红一号染色剂含有"偶氮苯"， 当"偶氮苯 "被降解后， 就会产生"苯 胺"， 这是一种中等毒性的致癌物。 过量的"苯胺"被吸入人体， 可能会 造成组织缺氧， 呼吸不畅， 引起中枢神经系统、心血管系统和其他脏器 受损， 甚至导致不孕症。 因其严重的致癌性、 致敏性和遗传毒性， 我国 及欧盟等国已明文规定禁止其作为色素在食品中进行添加。 2005年 2月 18日， 英国食品标准局 (The Food Standard Agency, FSA)就含有添加苏 丹红色素的食品向消费者发出警告，并在其网站上公布了可能含有苏丹 红 I的产品清单。
目前检测苏丹红含量的化学方法有高效液相色谱法，由于复杂的仪 器设备和繁琐的过程， 不适合现场监控和大量样本筛查。 发明内容
本发明的目的是提供一种操作简便、 费用低廉、灵敏度高、 能够现 场监控且适合大量样本筛查的方法， 用于检测食品中， 例如辣椒酱、辣 椒油、 辣椒粉等中的苏丹红含量。
本发明的检测原理为：
当在微孔条上预包被苏丹红偶联抗原时，加入样本溶液或标准品溶 液后，再加入苏丹红特异性抗体溶液，样本中残留的苏丹红或苏丹红标 准品与酶标板上包被的苏丹红偶联抗原竞争苏丹红特异性抗体，加入酶 标记抗抗体进行放大作用，用显色液显色，样本吸光值与样本中苏丹红 的含量成负相关，与标准曲线比较即可得出样本中苏丹红的含量。 同时 也可根据酶标板上颜色的深浅，与系列浓度苏丹红标准品溶液颜色的比 较可粗略判断样品中苏丹红含量的浓度范围。
当在微孔条上预包被苏丹红特异性抗体时，加入样本溶液或标准品 溶液后，再加入酶标记苏丹红半抗原溶液，样本中残留的苏丹红或苏丹 红标准品与酶标记抗原竞争包被在酶标板上的苏丹红特异性抗体，用显 色液显色，样本吸光值与苏丹红的含量成负相关，与标准曲线比较即可 得出样本中苏丹红的含量。同时也可根据酶标板上的颜色深浅，与系列 浓度的苏丹红标准品溶液颜色的比较可粗略判断样品中苏丹红含量的 浓度范围。
当在微孔条上预包被苏丹红偶联抗原时，加入样本溶液或标准品 '溶 液后，再加入酶标记苏丹红特异性抗体溶液，样本中的苏丹红或苏丹红 标准品与酶标板上包被的苏丹红抗原竞争苏丹红特异性抗体，用显色液 显色，样本吸光值与样本中苏丹红的含量成负相关，与标准曲线比较即 可得出样本中苏丹红的含量。同时也可根据酶标板上的颜色深浅，与系 列浓度苏丹红标准品溶液颜色的比较可粗略判断样品中苏丹红含量的 浓度范围。
当在微孔条上预包被抗抗体时，加入苏丹红抗体孵育后，加入样本 溶液或标准品溶液，再加入酶标记苏丹红半抗原溶液，样本中的苏丹红 或苏丹红标准品与酶标记苏丹红半抗原竞争苏丹红特异性抗体，用显色 液显色，样本吸光度值与样本中苏丹红的含量成负相关，与标准曲线比 较即可得出样本中苏丹红的含量。 同时也可根据酶标板上的颜色深浅， 与系列浓度苏丹红标准品溶液颜色的比较可粗略判断样品中苏丹红含 量的浓度范围。
本发明提供了一种用于检测苏丹红的酶联免疫试剂盒， 它含有：
(1)包被有包被原的酶标板 （包被原为抗原、 抗体或抗抗体），
(2)酶标记物 （为酶标记抗原、 酶标记抗体或酶标记抗抗体），
(3)苏丹红标准品溶液，
(4)底物显色液，
(5)终止液，
(6)浓缩洗涤液，
(7)浓缩复溶液。 当酶标板上包被抗原且酶标记物为酶标记抗抗体或酶标板上包被 抗抗体且酶标记物为酶标记抗原时， 所述的酶联免疫试剂盒中还含有：
(8)苏丹红特异性抗体浓縮液， 和
(9)苏丹红特异性抗体浓缩液的稀释液。
本发明所提供的检测苏丹红的酶联免疫试剂盒， 包括包被有包被 原的酶标板、 任选的苏丹红特异性抗体浓缩液 （酶标板上包被抗原， 并且酶标记物为酶标记抗抗体时； 或酶标板上包被抗抗体， 并且酶标 记物为酶标记抗原时， 含有苏丹红特异性抗体浓缩液）和酶标记物工 作液； 所述包被原可为苏丹红与载体蛋白的偶联抗原、 苏丹红特异性 抗体或抗抗体； 所述酶标记物为酶标记的抗抗体、 苏丹红半抗原或苏 丹红特异性抗体； 所述抗抗体可为羊抗鼠抗抗体或羊抗兔抗抗体。
所述苏丹红半抗原是将苏丹红和琥珀酸酐通过酰化反应得到的。 所述酶标记物的标记酶为辣根过氧化物酶或碱性磷酸酯酶， 其中 优选辣根过氧化物酶； 酶标记的羊抗鼠抗抗体或羊抗兔抗抗体是采用 戊二醛法或过碘酸钠法将标记酶与抗抗体进行偶联得到的，辣根过氧 化物酶可采用现有技术中的多种方法将其与抗抗体进行偶联， 如戊二 醛法， 过碘酸钠法等， 本发明经过长期的劳动创造将过碘酸钠法进行 了改良， 使其省时、 降低辣根过氧化物酶 （HRP) 与抗抗体的浓度比 率， 节省了原材料。 .
所述苏丹红特异性抗体可为苏丹红单克隆抗体或苏丹红多克隆 抗体； 它们均是用苏丹红半抗原与载体蛋白采用混合酸酐法得到的偶 联物作为免疫原得到的； 所述苏丹红多克隆抗体可为鼠源、 马源、 羊 源、 兔源或豚鼠源抗体， 所述苏丹红单克隆抗体优选为苏丹红鼠单克 隆抗体， 所述苏丹红多克隆抗体优选为苏丹红兔多克隆抗体。
所述苏丹红单克隆抗体优选为苏丹红的单克隆杂交瘤细胞株 B-1-1 CGMCC No. 1706分泌的单克隆抗体。
所述苏丹红的单克隆杂交瘤细胞株 B-1-1 CGMCC No. 1706已于 2006年 4月 30日保藏于中国微生物菌种保藏管理委员会普通微生物 中心 （简称 CGMCC)。
以上抗体均可以用苏丹红半抗原与载体蛋白的偶联物作为免疫 原按混合酸酐法制备。 所述载体蛋白可为鼠血清蛋白、 甲状腺蛋白
( BCG)、 牛血清白蛋白、 兔血清蛋白、 人血清白蛋白、 卵清蛋白或 血蓝蛋白等常用载体蛋白；所述苏丹红半抗原与载体蛋白的偶联物可 通过将苏丹红半抗原和载体蛋白用混合酸酐法进行偶联得到。
为了更方便现场监控和大量样本筛査， 所述试剂盒还包括苏丹红 标准品溶液、底物显色液、终止液、浓縮洗涤液、浓缩抗体稀释液（酶 标板上包被抗原， 酶标记物为酶标记抗抗体时或酶标板上包被的抗抗 体， 酶标记物为酶标记抗原）， 浓缩复溶液为磷酸盐缓冲液， 例如 2χ 0.04mol/L的磷酸盐缓冲液。
所述浓缩洗涤液优选为磷酸盐缓冲液，例如含有吐温和叠氮钠的 磷酸盐缓冲液， 进一步的例如 pH 7.4， 含有 0.8 %〜1.2 %吐温 20和 0.5 %叠氮钠的 0.01M磷酸盐缓冲液， 还可以是本领域常用的其它浓 缩洗涤液。
所述浓缩复溶液优选为磷酸盐缓冲液， 例如 2x0.04mol/L的磷酸 盐缓冲液， 还可以是本领域常用的其它浓縮复溶液。
当标记酶为辣根过氧化物酶时，底物显色液由显色液 A液和显色 液 B液组成， 显色液 A液优选为过氧化氢或过氧化脲溶液， 所述显 色液 B液优选为邻苯二胺或四甲基联苯胺溶液，终止液优选为硫酸溶 液， 其浓度例如为 l〜2mol/L; 当标记酶优选为碱性磷酸酯酶时， 显 色液优选为对硝基苯磷酸酯缓冲液， 终止液优选为氢氧化钠溶液， 其 浓度例如为 l〜2mol/L， 更特别的例如为 2mol/L。 显色液和终止液也 可以是本领域常用的其它显色液和终止液。
当酶标板上包被抗原， 酶标记物为酶标记抗抗体时或酶标板上包 被抗抗体， 酶标记物为酶标记抗原时， 所述浓縮抗体的稀释液优选为 磷酸盐缓冲液， 例如含有牛血清、 甲醇的磷酸盐缓冲液， 进一步的例 如 pH8.6、 含有 2 %小牛血清、 10 %甲醇的 0.05mol/L磷酸盐缓冲液， 还可以是本领域常用的其它浓缩抗体的稀释液。
其中酶标板在制备过程中所用的包被缓冲液优选为柠檬酸钠缓 冲液， 例如 pH 6.6、 0.01〜0.03mol/L的柠檬酸钠缓冲液； 所用的封闭 液优选为磷酸盐缓冲液， 例如含有马血清、 叠氮化钠、 酪蛋白的磷酸 盐缓冲液， 进一步的例如含有 0.5 %马血清、 1%。叠氮化钠、 3 %酪蛋 白的磷酸盐缓冲液。包被缓冲液和封闭液也可以是本领域常用的其它 包被缓冲液和封闭液。
本发明中酶标板的制备过程可以为： 用包被缓冲液将包被原稀 释， 并加入各孔中， 37°C左右温育数小时， 再 4°C左右过夜， 倾去包 被液， 用洗涤液洗涤， 拍干， 然后在每孔中加入封闭液， 温育， 倾去 孔内液体拍干， 干燥后用铝膜真空密封保存。 例如， 用包被缓冲液将 包被原稀释为 0.05-0. l g/ml, 每孔加入 ΙΟΟμΙ, 37°C温育 2h， 再 4°C 过夜， 倾去包被液， 用洗涤液洗涤 2次， 每次 30秒， 拍干， 然后在 每孔中加入 150-200μ1封闭液， 37°C温育 l-2h， 倾去孔内液体拍干， 干燥后用铝膜真空密封保存。 本发明中抗原的合成过程例如为：
1.半抗原的合成
将苏丹红用琥珀酸酐法合成苏丹红半抗原。
2. 苏丹红抗体的制备
将苏丹红半抗原与载体蛋白采用混合酸酐法（氯甲酸异丁酯）进 行偶联得到免疫原。
苏丹红是小分子物质， 只有免疫反应性， 没有免疫原性， 不能诱 发机体产生免疫应答， 必须与大分子载体蛋白偶联后才具有免疫原 性。 因此将苏丹红和琥珀酸酐合成苏丹红半抗原， 给苏丹红接出了一 个含 4个碳的间隔臂， 这样突出了苏丹红分子结构中的特征基团， 使 制备的苏丹红抗体对苏丹红的特异性很高。
苏丹红鼠单克隆抗体的制备
动物免疫程序： 采用 Balb/c小鼠作为免疫动物， 以苏丹红半抗原 与载体蛋白偶联物为免疫原， 得到较好的多克隆抗体， 取出脾脏进行 细胞融合。
细胞融合与克隆化： 取免疫 BALB/c小鼠脾细胞与 SP2/0骨髓瘤 细胞融合， 筛选细胞株， 直到得到稳定分泌单克隆抗体的杂交瘤细胞 株。 苏丹红兔多克隆抗体的制备
采用新西兰大白兔作为免疫动物， 以苏丹红与载体蛋白偶联物为 免疫原对新西兰大白兔进行免疫， 多次免疫后测定血清抗体效价得到 多克隆抗体。
本发明抗抗体的制备过程： 羊抗鼠抗抗体是以羊作为免疫动物， 以鼠源抗体为免疫原对无病菌体羊进行免疫， 得到羊抗鼠抗抗体； 羊 抗兔抗抗体是以羊作为免疫动物， 以兔源抗体为免疫原对无病菌体羊 进行免疫， 得到羊抗兔抗抗体。
本发明所述试剂盒中苏丹红标准品溶液， 其可以是一定浓度范围 内的多种浓度的标准溶液，其浓度优选的例如可以在 0〜5(^g/L之间。 例如可以是含有 6瓶的标准品溶液， 其浓度分别为 0 g/L、 0.1 g/L、 0.4 g/L、 1.6 g/L、 6.4 g/L和 25.6 g/L， 3ml/瓶。
本发明还提供了一种应用上述酶联免疫试剂盒检测样品中苏丹 红含量的方法， 其包括以下步骤：
(1)样品前处理；
(2)用试剂盒进行检测；
(3)分析检测结果。
上述步骤 (1)中的样品可以是动物或人食用的各种产品，特别是食 品或制作食品的原料。 更特别地是辣椒酱、 辣椒油、 辣椒粉等食品或 制作食品的原料。
本发明提供以苏丹红为食品添加剂的食品样本（水基质、 油基质 及粉末状） 的处理： 称取样本于离心管中， 加入乙腈震荡离心， 移取 上清液， 氮气吹干， 复溶， 取样分析。
本发明中用试剂盒检测是： 当包被原为苏丹红偶联抗原时， 向酶 标板微孔中加入标准品溶液或样本溶液再加入抗体， 温育后洗涤拍 干， 再加入酶标抗抗体， 温育后洗涤拍干， 显色、 终止， 用酶标仪测 定吸光度值； 当包被原为苏丹红偶联抗原时， 向酶标板微孔中加入标 准品溶液或样本溶液再加入酶标记抗体， 温育后洗涤拍干， 显色、 终 止， 用酶标仪测定吸光度值； 当包被原为苏丹红特异性抗体时， 向酶 标板微孔中加入标准品溶液或样本溶液再加入酶标记苏丹红半抗原， 温育后洗涤拍干， 显色、 终止， 用酶标仪测定吸光度值； 当包被原为 抗抗体时， 向酶标板微孔中加入苏丹红抗体， 温育后洗涤拍干， 再加 入标准品溶液或样品溶液后加入酶标苏丹红半抗原， 温育后洗涤拍 干， 显色、 终止， 用酶标仪测定吸光度值。
本发明中检测结果分析过程为： 用所获得的每个浓度的标准品溶 液的吸光度平均值 （B ) 除以第一个标准溶液 （0 标准） 的吸光度值 (B0) 再乘以 100%， 即百分吸光度值。 计算公式为：
百分吸光度值 (％) = (B/B。） X 100%
以苏丹红标准品溶液的浓度（ g/L) 的半对数值为 X轴， 百分吸 光度值为 Y轴， 绘制标准曲线图。用同样的办法计算样品溶液的百分 吸光度值， 相对应每一个样品的浓度则可从标准曲线上读出样本中苏 丹红的含量。
本发明中检测结果的分析也可以采用回归方程法， 计算出样品溶 液浓度。
本发明中检测结果的分析还可以利用计算机专业软件， 此法更便 于大量样品的快速分析， 整个检测过程只需较短时间， 如 1.5小时即 可以完成， 样本最低检测限为 l(^g/L。
本发明中检测食品中苏丹红的酶联免疫试剂盒主要采用间接竞 争 ELISA方法定性或定量检测动物或人食用的产品， 特别是辣椒酱、 辣椒油、 辣椒粉等食品中苏丹红染料的含量， 样品前处理过程简单， 能同时检测大批样品。
本发明采用高特异性的苏丹红单克隆抗体或多克隆抗体， 主要试 剂以工作液形式提供，检验方法简便易行，具有特异性高、灵敏度高、 精确度高等特点， 将在苏丹红的残留检测中发挥重要作用。 附图说明
图 1 : 苏丹红的检测标准曲线图。 具体实施方式
下面结合具体的实施例来进一步阐述本发明。应理解，这些实施例 仅用于说明本发明， 而不是限制本发明的范围。
实施例 1 试剂盒组分的制备
1. 抗原的合成
a. 半抗原的合成
将苏丹红用琥珀酸酐法合成苏丹红半抗原。 具体步骤： 取苏丹红 5g溶于 20ml DMF中，加入琥珀酸酐 3g及 50ml吡啶， 80°C回流反应 70〜80小时， 抽干除去溶剂用纯水洗涤 5次（30ml/次）， 烘干， 即得。
b. 免疫原合成
将苏丹红半抗原和卵清蛋白， 采用混合酸酐法进行偶联得到免疫 原。
免疫原的制备过程： 取苏丹红半抗原 2g溶于 30ml， 50%的 Ν,Ν- 二甲基甲酰胺溶液中，再取 0.5ml 氯甲酸异丁酯溶于 5ml无水二噁烷 中，加到半抗原溶液中室温搅拌反应 4小时，取载体蛋白 32g溶于 70ml pH9.6碳酸盐缓冲液中，再将人血清白蛋白滴加到半抗原中， 4°C搅拌 过夜。 将反应完的人工抗原用透析袋装好放入 0.2M的磷酸盐缓冲液 中， 透析 7天， 每天换液 3〜4次。 最后将抗原冻干保存。
c 包被原苏丹红偶联抗原的制备
将苏丹红半抗原和甲状腺蛋白， 采用混合酸酐法进行偶联得到包 被抗原。
包被原的制备过程： 取苏丹红半抗原 2g溶于 30ml, 50 %的 Ν,Ν- 二甲基甲酰胺溶液中，再取 0.5ml 氯甲酸异丁酯溶于 5ml无水二噁烷 中，加到半抗原溶液中室温搅拌反应 4小时，取载体蛋白 32g溶于 70ml pH9.6碳酸盐缓冲液中，再将卵清蛋白滴加到半抗原中， 4°C搅拌过夜。 将反应完的人工抗原用透析袋装好放入 0.2M的磷酸盐缓冲液中透析 7天， 每天换液 3〜4次。 最后将抗原冻干保存。
2. 单克隆抗体的制备
a. 动物免疫
将免疫原注入到 Balb/c小鼠体内， 免疫剂量为 80μ§/只， 使其产 生多克隆抗体。
b. 细胞融合和克隆化 取免疫 Balb/c小鼠脾细胞， 按 5 ： 1比例与 SP2/0骨髓瘤细胞融 合， 采用间接竞争 ELISA测定细胞上清液， 筛选阳性孔。 利用有限 稀释法对阳性孔进行克隆化，直到得到稳定分泌单克隆抗体的杂交瘤 细胞株—苏丹红的单克隆杂交瘤细胞株 B-1-1 CGMCC No. 1706。
c. 细胞冻存和复苏
将苏丹红的单克隆杂交瘤细胞株 B-1-1 CGMCC No. 1706用冻存 液制成 l x lO6个 /ml的细胞悬液， 在液氮中长期保存。 复苏时取出冻 存管， 立即放入 37°C水浴中速融， 离心去除冻存液后， 移入培养瓶内 培养。
d. 单克隆抗体的制备与纯化
将 Balb/c小鼠腹腔注入灭菌石蜡油 0.4ml/只， 7天后腹腔注射苏 丹红的单克隆杂交瘤细胞株 B-1-1 CGMCC No. 1706 5> 105个 /只， 7 天后采集腹水。 用辛酸 -饱和硫酸铵法进行纯化， 纯化后的腹水放入 -20°C环境保存。
3. 多克隆抗体的制备
采用新西兰大白兔作为免疫动物， 以苏丹红半抗原与卵清蛋白偶 联物为免疫原， 免疫剂量为 1.5mg/kg， 首免时将免疫原与等量的弗氏 完全佐剂混合制成乳化剂， 颈背部皮下多点注射， 间隔 3〜4周取相 同剂量免疫原加等量弗氏不完全佐剂混合乳化， 加强免疫一次， 共免 疫 5次， 最后一次不加佐剂。 最后一次免疫 10天后采血， 测定血清 抗体效价， 心脏采血， 用硫酸铵分级沉淀得到纯化的多克隆抗体。
4. 羊抗鼠抗抗体的制备过程
以山羊作为免疫动物， 以鼠源抗体为免疫原对无病原体山羊进行 免疫， 得到羊抗鼠抗抗体； 羊抗兔抗抗体的制备： 以山羊作为免疫动 物， 以兔源抗体为免疫原对无病原体山羊进行免疫， 得到羊抗兔抗抗 体。
5. 酶标记羊抗鼠抗抗体的制备过程
将羊抗鼠抗抗体与辣根过氧化物酶 （HRP) 进行偶联， 采用的方 法是改良的过碘酸钠法， 方法如下：
a、 8mg辣根过氧化物酶溶解于 2mL蒸馏水中。 b、 加入现配制的 lOOmmol/L NaI04溶液 0.4mL， 室温搅拌反应 0分钟。
c、用 lmmol/L醋酸盐缓冲液于 4°C透析过夜，除去多余的 NaI04， 同时使自身偶联的酶还原。
d、加入磷酸盐缓冲液（pH8.6、 0.5mol/L) 40 μ 1和含有 IgG 16mg 的磷酸盐缓冲液 （pH8.6、 5mol/L) 2.0mL， 室温搅拌反应 4小时。
e、 加入现配制的 NaBH4水溶液 （lmol/L) O.lmL, 在 4°C反应 4 小时， 以还原 Schiff碱。
f、 纯化保存。
6、 酶标板的制备
用包被缓冲液将苏丹红偶联抗原、 抗体或抗抗体稀释成 0.05-0.^g/ml, 每孔加入 ΙΟΟμΙ, 37°C温育 2h或 4°C过夜， 倾去包被液， 用浓缩洗涤液稀释 20倍后洗涤 2次， 每次 30秒， 拍干， 然后在每孔中加 入 150μ1封闭液， 37°C温育 2h， 倾去孔内液体， 干燥后用铝膜真空密封 保存。 实施例 2 检测苏丹红的酶联免疫试剂盒的组建
用实施例 1制备的各组分组建检测苏丹红的酶联免疫试剂盒，使其 包含下述组分：
(1) 包被苏丹红偶联抗原的酶标板；
(2) 用辣根过氧化物酶标记的羊抗鼠抗抗体；
(3) 苏丹红单抗体浓缩液；
(4) 苏丹红标准品溶液 6瓶， 浓度分别为 (^g/L、 0.1 g/L、 0.4 g/L、 1.6 g/L、 6.4 g/L和 25.6 g/L;
(5) 底物显色液由 A液和 B液组成， 底物显色液 A液为 2%过氧 化脲溶液， 底物显色液 B液为 1%四甲基联苯胺溶液；
(6) 终止液为 2mol/L盐酸；
(7) 浓缩洗涤液为 pH 7.4、 含有 0.8%〜1.2%吐温 20和 0.5%叠氮 化钠的 0.01M磷酸盐缓冲液；
(8)抗体稀释液为 pH8.6、含有 2%小牛血清、 10%甲醇的 0.05mol/L 磷酸盐缓冲液；
( 9) 浓縮复溶液为 2x0.04mol/L的磷酸盐缓冲液。 实施例 3 样品中苏丹红含量的检测
1. 样品前处理
以苏丹红为食品添加剂食品的样本（水基质、油基质及粉末状）处 理步骤：
称取 2g样本于离心管中， 加入 10ml乙腈， 剧烈震荡 10min， 室 温 3000rpm以上离心 lOmin。移取 1ml上清液于 50°C环境下氮气吹干。 加入 4ml正己垸涡动 30s溶解干燥的残留物， 加入 lml 1M NaOH涡 动 15s， 室温 3000rpm离心 10min， 移取 lml上清液， 于 50°C环境下 氮气吹干， 加入 0.5ml DMF充分溶解干燥的残留物， 取 ΙΟΟμΙ加入 900μ1稀释好的复溶液充分混匀， 得到待测样品溶液。
2. 用试剂盒检测
本试验用实施例 2的试剂盒进行检测。 向苏丹红偶联抗原包被的 酶标板微孔中加入苏丹红标准品溶液或样本溶液 50μ1，再加入苏丹红 单克隆抗体工作液 50μ1， 用盖板膜封板， 37°C恒温箱中反应 30min， 倒出孔中液体， 每孔加入 250μ1洗涤液， 30秒后倒出孔中液体， 如此 重复操作共洗板 5次， 用吸水纸拍干。 加入辣根过氧化物酶标记的羊 抗鼠抗抗体工作液 100μ1， 37°C恒温箱中反应 30min， 倒出孔中液体， 重复洗涤步骤， 每孔加入底物显色液 A液过氧化脲， 底物显色液 B 液四甲基联苯胺（TMB )，轻轻振荡混匀， 37°C恒温箱避光显色 15min， 每孔加入 2mol/L终止液盐酸 50μ1， 轻轻振荡混匀， 用酶标仪， 测定 每孔吸光度值。
3. 检测结果分析
用得的每个浓度的标准品溶液的吸光度平均值（Β ) 除以第一个标 准品溶液（0标准）的吸光度值（BQ)再乘以 100 %，得到百分吸光度值。 以苏丹红标准品浓度（ g/L) 的半对数值为 X轴， 百分吸光度值为 Y轴， 绘制标准曲线图。用同样的办法计算样品溶液的百分吸光度值，相对应 每一个样品的浓度， 则可从标准曲线上读出样本中苏丹红的残留量。 实验例 1 标准品精密度试验
本实验例及后述实验例所用的试剂盒由实施例 2的配方制备的。 分 别从三个不同的时间段制备的酶标板中各抽出一批酶标板，每批各抽取 10个试剂盒， 每板各抽出 20个微孔， 测定 4.5 g/L标准溶液的吸光度值， 计算变异系数， 结果见表 1。
表 1 标准品精密度试验结果 (cv%)
Figure imgf000014_0001
通过上述试验结果可以得出，每批试剂盒各测定 10次标准品变异系 数在 3.8%〜11.7%之间， 符合精密度小于或等于 20%的规定。 实验例 2 样本精密度和准确度试验
a.样品精密度试验：
取苏丹红标准品，将其添加至样品中成为 15 g/L样品的浓度， 分别 取三个不同批次的试剂盒各三个， 每个浓度重复 5次， 分别计算变异系 数。 三种样品的试验结果分别见表 2、 表 3、 表 4。
表 2 水基质样品添加苏丹红的精密度试验结果
试剂盒 实测值 （ g/L) 变异系数 批号 cv%
8.4 14.2 9.5 10.4 14.6 24.6
0506008 10.7 11.6 8.9 9.5 16.7 27.0
11.7 9.6 10.4 9.6 13.7 15.8
13.4 9.4 11.7 8.4 15.4 24.5
0507003 14.8 8.2 9.7 10.8 13.7 24.1
8.6 9.4 11.6 13.5 15.4 24.1
14.8 12.9 11.7 9.7 8.2 22.7
0507009 8.4 11.7 14.6 13.8 12.5 19.7
15.4 12.6 9.4 13.6 10.8 19.0 表 3 油基质样品添加苏丹红的精密度试验结果
Figure imgf000015_0001
表 4 粉末状样品添加苏丹红的精密度试验结果
Figure imgf000015_0002
结果表明，水基质样品添加苏丹红的变异系数均低于 25 %，油基质 样品添加苏丹红的变异系数均低于 25 %，粉末状样品添加苏丹红的变异 系数均低于 25 %。实验使用的水基质样品为食用辣椒酱，油基质样品为 食用辣椒油， 粉末状样品为食用辣椒粉。 b.样本回收率试验
取三个浓度的苏丹红标准品溶液分别为 lO g/kg ( L)、 50μ§/]¾ ( L) 和 10(^g/kg (L)， 分别对不含苏丹红的样品进行添加回收试验， 每个浓 度做 4个平行， 具体方法如实施例 3中 "样品前处理"步骤， 分别计算准 确度， 结果见表 5。
从表中结果可见， 水基质样品添加苏丹红的添加回收率在 60.8%-102.3%之间， 油基质样品添加苏丹红的添加回收率在 63.5%-94.8%之间，粉末状样品添加苏丹红的添加回收率在 62.8%-87.6% 之间。
表 5 试剂盒的样本回收率测定
Figure imgf000016_0001
实验例 3
交叉反应率试验- 选择与苏丹红有类似结构和类似功能的 2种药物测定交叉反应率。 通过各种药物的标准曲线分别得到其 50%抑制浓度。用下式计算试剂盒 对其它药物的交叉反应率。
^ ^八 引起 50%抑制的苏丹红浓度
交叉反应率 (%) = ―—— χ 100 %
引起 50%抑制的类似物浓度 试剂盒的特异性
Figure imgf000017_0001
实验例 4
试剂盒保存条件为 2-8°C， 经过 6个月的测定， 试剂盒的最大吸 光度值 （零标准）、 50%抑制浓度、 苏丹红添加实际测定值均在正常 范围之内。 考虑到运输和使用过程中， 会有非正常保存条件出现， 将 试剂盒在 37°C保存的条件下放置 6天，进行加速老化实验， 结果表明 该试剂盒的各项指标完全符合要求。 考虑到试剂盒冷冻情况发生， 将 试剂盒放入 -20°C冰箱冷冻 5 天， 测定结果也表明试剂盒各项指标完 全正常。 从以上结果可得出试剂盒可以在 2-8 °C至少可以保存 6个月 以上。

Claims

权利要求书
1、 一种检测苏丹红的酶联免疫试剂盒， 其特征在于， 该试剂盒含 有：
(1)包被有包被原的酶标板，
(2)酶标记物，
(3)苏丹红标准品溶液，
(4)底物显色液，
(5)终止液，
(6)浓缩洗涤液， 和
(7)浓缩复溶液；
所述的包被原为抗原、抗体或抗抗体； 所述的酶标记物为酶标记抗 原、 酶标记抗体或酶标记抗抗体。
2、 如权利要求 1 所述的酶联免疫试剂盒， 其特征在于， 当酶标 板上包被抗原，并且酶标记物为酶标记抗抗体；或酶标板上包被抗抗体， 并且酶标记物为酶标记抗原时，该试剂盒还含有苏丹红特异性抗体浓缩 液和苏丹红特异性抗体浓缩液的稀释液。
3、如权利要求 1或 2 所述的试剂盒， 其特征在于： 所述酶标板中 包被的包被原为苏丹红半抗原与载体蛋白的偶联物、苏丹红特异性抗体 或抗抗体。
4、 如权利要求 3所述的试剂盒， 其特征在于： 所述的包被原中的 载体蛋白为鼠血清蛋白、 甲状腺蛋白、 牛血清白蛋白、 兔血清白蛋白、 人血清白蛋白、卵清蛋白或血蓝蛋白；所述的苏丹红特异性抗体为苏丹 红单克隆抗体或多克隆抗体；所述的抗抗体为羊抗鼠抗抗体或羊抗兔抗 抗体。
5、 如权利要求 4所述的试剂盒， 其特征在于： 所述的苏丹红单克 隆抗体为苏丹红的单克隆杂交瘤细胞株 B-1-1 CGMCC No. 1706分泌的 单克隆抗体。
6、 如权利要求 1〜5任意一项所述的试剂盒， 其特征在于： 制备酶 标板时，所用的包被缓冲液为柠檬酸缓冲液，所用的封闭液为含有马血 7、 如权利要求 1〜6任意一项所述的试剂盒， 其特征在于： 酶标记 物为酶标记苏丹红抗抗体、酶标记苏丹红半抗原或酶标记苏丹红特异性 抗体。
8、 如权利要求 7所述的试剂盒， 其特征在于： 其中抗抗体为羊抗 鼠抗抗体或羊抗兔抗抗体；酶标记物中的标记酶为辣根过氧化物酶或细 菌提取碱性磷酸酯酶。
9、 如权利要求 8所述的试剂盒， 其特征在于， 酶标记物中的标记 酶为辣根过氧化物酶时，底物显色液由显色液 A液和显色液 B液组成， 显色液 A液为过氧化氢或过氧化脲溶液， 显色液 B液为邻苯二胺或四 甲基联苯胺， 终止液为硫酸或盐酸溶液。
10、 如权利要求 8 所述的试剂盒， 其特征在于， 酶标记物中的标 记酶为细菌提取碱性磷酸酯酶时，此时的底物显色液为对硝基磷酸盐缓 冲液、 终止液为氢氧化钠溶液。
11、 如权利要求 1〜10任意一项所述的试剂盒， 其特征在于， 浓缩 洗涤液为含有吐温和叠氮化钠的磷酸盐缓冲液；浓缩复溶液为的磷酸盐 缓冲液。
12、 一种检测样品中苏丹红含量的方法， 包括以下步骤：
(1)样品前处理；
(2)用权利要求 1-11任意一项所述的试剂盒进行检测；
(3)分析检测结果。
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