AU2007257105B8 - Elisa kit for detecting Sudan red and method thereof - Google Patents

Abstract

An ELISA kit for detecting Sudan red includes: a plate coated with a coating molecule, enzyme labeled molecule, Sudan red standard solution, substrate and color developing solution, stop solution, concentrated washing solution and concentrated diluting solution. The kit can also include: specific antibody concentrated solution to Sudan red and diluting solution for concentrated antibody. A detecting method for Sudan red with said ELISA kit, which includes the steps: firstly, pretreating samples; then detecting the samples with the kit; lastly, analyzing the results.

Description

ELISA KIT FOR DETECTING SUDAN I AND IMMUNOASSAY METHOD USING THE SAME TECHNICAL FIELD 5 The present invention relates to an ELISA kit for detecting Sudan I and the immunoassay method using the kit. BACKGROUNDART Sudan Red (Sudan I) is an artificially synthesized red dye, which is broadly 10 employed in enhancing the color of organic solvents (oil, wax, petrol, etc.) and the appearance of shoes and floor. Sudan I comprises azobenzene, whose degradation product is aniline, a carcinogen with medium toxicity. Superfluous aniline in the body would cause hypoxia, dyspnoea, and damage central nerve, cardiovascular system and other viscera, even result in barrenness. Due to its severe carcinogenicity, sensitization 15 and genotoxicity, Sudan I was prohibited to be used in food production as dyes in EU and China. The Food Standard Agency (FSA, UK) warned the consumer about Sudan Red-contaminated food on February 18, 2005, and published a list of products that might comprise Sudan I in the website. Currently, the common approaches to determine Sudan I are based on high 20 performance liquid chromatography technology, which is very cumbersome and complex, and not suitable for on-site inspection and large-scale sample screening. SUMMARY OF THIS INVENTION In the present invention, an immunoassay is used for detecting Sudan I in food 25 samples, particularly in hot pepper sauce, chili oil and chili powder, etc., which is convenient, economical, sensitive and suitable for on-site inspection and large-scale sample screening. The invention is based on indirect competitive enzyme linked immunosorbent assay (ELISA) principle, which is specified as follows: 30 (1) Sudan I-coupled coating antigen is immobilized on the microtiter wells. After adding sample solution (containing Sudan I) or Sudan I standard solution, and anti-Sudan I antibody solution, the Sudan I antigen coated on the wells will compete for anti-Sudan I antibody with the Sudan I from the sample or the standard solution. After adding enzyme-labeled secondary antibody and the substrate and color-developing 5 reagent, the absorbency signal is amplified and measured on an ELISA reader. The absorbency is inversely proportional to the Sudan I concentration in the sample, thus, the Sudan I content in the sample can be calculated by comparison with the standard curve. In addition, by comparing the color of samples with the color of the Sudan I standard series, a round estimate of the Sudan I concentration of the sample would be 10 obtained. (2) Anti-Sudan I antibody is immobilized on the microtiter wells. After adding sample solution (containing Sudan 1) or Sudan I standard solution, and enzyme-labeled Sudan I antigen solution, the added enzyme-labeled antigen will compete for anti-Sudan I antibody with the Sudan I from sample or standard solution. After adding 15 the substrate and color-developing reagent, the absorbency signal is measured on an ELISA reader. The absorbency is inversely proportional to the Sudan I concentration in the sample, thus, the Sudan I content in the sample can be calculated by comparison with the standard curve. In addition, by comparing the color of samples with the color of the Sudan I standard series, a round estimate of the Sudan I concentration of the 20 sample would be obtained. (3) Sudan I-coupled coating antigen is immobilized on the microtiter wells. After adding sample solution (containing Sudan I) or Sudan I standard solution, and enzyme-labeled anti-Sudan I antibody solution, the Sudan I antigen coated on the wells will compete for enzyme-labeled anti-Sudan I antibody with the Sudan I from sample or 25 standard solution. After adding the substrate and color-developing reagent, the absorbency signal is measured on an ELISA reader. The absorbency is inversely proportional to the Sudan I concentration in the sample, thus, the Sudan I content in the sample can be calculated by comparison with the standard curve. In addition, by comparing the color of samples with the color of the Sudan I standard series, a round 30 estimate of the Sudan I concentration of the sample would be obtained. 2 (4) Secondary antibody is immobilized on the microtiter wells. After adding anti-Sudan I antibody solution and incubation, sample solution (containing Sudan I) or Sudan I standard solution is added. After that, enzyme-labeled Sudan I antigen is added, the enzyme-labeled Sudan I antigen will compete for anti-Sudan I antibody with the 5 Sudan I from sample or standard solution. After adding the substrate and color-developing reagent, the absorbency signal is measured on an ELISA reader. The absorbency is inversely proportional to the Sudan I concentration in the sample, thus, the Sudan I content in the sample can be calculated by comparison with the standard curve. In addition, by comparing the color of samples with the color of the Sudan I 10 standard series, a round estimate of the Sudan I concentration of the sample would be obtained. An ELISA kit for detecting Sudan I containing: (1) Microtiter wells coated with coating antigen (the coating antigen is Sudan I antigen, anti-Sudan I antibody or secondary antibody); 15 (2) Enzyme conjugate (enzyme-labeled antigen, enzyme-labeled antibody or enzyme-labeled secondary antibody); (3) Sudan I standard solution series; (4) Substrate and color-developing solution; (5) Stop solution; 20 (6) Concentrated washing solution; (7) Concentrated redissolving solution; when Sudan I antigen is coated on the microtiter wells and the enzyme conjugate is enzyme-labeled secondary antibody, or secondary antibody is coated on the wells and the enzyme conjugate is enzyme-labeled Sudan I antigen, the said ELISA kit also 25 contains: (8) Concentrated anti-Sudan I antibody solution, and (9) Diluent solution for the concentrated anti-Sudan I antibody solution. The ELISA kit provided in the present invention is composed of a microtiter plates coated with coating antigen, optional concentrated anti-Sudan I antibody solution (only 30 when Sudan I antigen is coated on the microtiter wells and the enzyme conjugate is 3 enzyme-labeled secondary antibody, or secondary antibody is coated on the wells and the enzyme conjugate is enzyme-labeled Sudan I antigen) and enzyme conjugate solution. In the kit described above, the said coating antigen may be coupled antigen of 5 Sudan I and carrier protein, anti-Sudan I antibody or secondary antibody. In the kit described above, the said enzyme conjugate is enzyme-labeled secondary antibody, enzyme-labeled Sudan I antigen or enzyme-labeled anti-Sudan I antibody solution. In the kit described above, the said secondary antibody is goat-anti-mouse or 10 goat-anti-rabbit secondary antibody. In the kit described above, the said Sudan I hapten can be obtained from an acylation reaction of Sudan I and succinic anhydride. In the kit described above, the labeling enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (ALP), and preferably is HRP . 15 The enzyme-labeled secondary antibody (goat-anti-mouse or goat-anti-rabbit antibody) is obtained by conjugating the said labeling enzyme to the secondary antibody via glutaraldehyde or sodium periodate cross-linking. HRP can be conjugated with the secondary antibody via various methods in the art, such as glutaraldehyde, sodium periodate cross-linking, etc. The sodium periodate 20 cross-linking method used herein is optimized in terms of reaction time, concentration ratio of HRP to the secondary antibody, raw and processed materials, etc. The said anti-Sudan I antibody is monoclonal or polyclonal antibody. The immunogen to raise either of above antibodies is a Sudan I hapten-carrier protein conjugate created by mixed anhydride method. 25 The said anti-Sudan I polyclonal antibody is from mouse, equine, goat, rabbit or guinea pig; the said anti-Sudan I monoclonal antibody is preferable mouse monoclonal antibody, and the said anti-Sudan I polyclonal antibody is preferable rabbit polyclonal antibody. The said anti-Sudan I monoclonal antibody is preferably secreted by hybridoma 30 cell line B-1-1 CGMCC No. 1706. 4 The said hybridoma cell line that secretes the said preferable anti-Sudan I monoclonal antibody has been conserved at China General Microbiological Culture Collection Center (CGMCC) with a deposit number of B-I-I CGMCC No. 1706. The above said antibodies are raised with the said Sudan I hapten-carrier protein 5 immunogen, wherein the carrier protein may be an generally used carrier protein, such as mouse serum albumin, thyroglobulin, bovine serum albumin, rabbit serum albumin, human serum albumin, ovalbumin or hemocyanin from keyhole limpets, etc. The said Sudan I immunogen is a conjugate of Sudan I hapten and the carrier protein said above, which is obtained by mixed anhydride method. 10 The kit comprises Sudan I standard solution series, substrate and color-developing solution, stop solution, concentrated washing solution, and diluent solution for the concentrated anti-Sudan I antibody solution (only when Sudan I antigen is coated on the microtiter wells and the enzyme conjugate is enzyme-labeled secondary antibody, or secondary antibody is coated on the wells and the enzyme conjugate is 15 enzyme-labeled Sudan I antigen). The concentrated redissolving solution is phosphate buffer (PBS), e.g., 2x0.O4mol/L PBS. The said concentrated washing solution is preferably phosphate buffer, e.g., PBS containing Tween and sodium azide, more particularly, pH 7.4, 0.Olmol/L PBS containing 0.8-1.2% Tween-20 and 0.5% sodium azide, the said washing solution can 20 also be the common washing solution or buffer in the art. The said concentrated redissolving solution is preferably phosphate buffer, e.g., 2x0.O4mol/L PBS, the said concentrated redissolving solution can also be the common redissolving solution or buffer in the art. When the said labeling enzyme is horseradish peroxidase (HRP), the substrate and 25 color-developing solution comprises solutions A and B, wherein solution A is preferably hydrogen peroxide or carbamide peroxide solution, while solution B is preferably 0-phenylenediamine or tetramethylbenzidine (TMB) solution, the corresponding stop solution is preferably sulfuric acid with a concentration of -2 mol/L. When the said labeling enzyme is alkaline phosphatase, the substrate and 30 color-developing solution is preferably p-nitrophenyl phosphate buffer, and the 5 corresponding stop solution is preferably NaOH solution with a concentration of 1-2 mol/L, particularly 2 mol/L. The said substrate solution and stop solution can also be other common ones in the art. When Sudan I antigen is coated on the microtiter wells and the enzyme conjugate 5 is enzyme-labeled secondary antibody, or secondary antibody is coated on the wells and the enzyme conjugate is enzyme-labeled antigen, the corresponding diluent solution for concentrated antibody solution is preferably phosphate buffer solution, e.g., PBS containing bovine serum and methanol, more particularly, pH 8.6, 0.05mol/L PBS containing 2% calf serum and 10% methanol. The said diluent 10 solution can also be the common diluent solution in the art. The coating buffer hereinafter used in coating the microtiter wells is preferably sodium citrate buffer, e.g., pH 6.6 0.01-0.03mol/L sodium citrate buffer; the blocking buffer hereinafter is preferably PBS, e.g., PBS containing equine serum, sodium azide and casein, more particularly, PBS containing 0.5% equine serum, l%o sodium azide 15 and 3% casein. The coating buffer and blocking buffer can also be the common ones in the art. One way to produce the coated microtiter wells is as follows: (1) Dilute the coating antigen with coating buffer, and add the dilute coating antigen into the wells, incubate at about 37 for several hours, and incubate overnight at about 20 4 ; (2) Pour the coating buffer out, wash with washing buffer, absorb the residue liquid with absorbent paper; (3) Add blocking buffer into each well. After incubation, pour the liquid out, dry the wells and seal with aluminum membrane. 25 More particularly, (1) Dilute the coating antigen with coating buffer to 0.05-0.Ipg/ml, add the dilute coating antigen into the wells at 100tL per well, incubate at 37 for 2 hours, and incubate overnight at 4 ; (2) Pour the coating buffer out, wash with washing buffer twice, 30s each time, pour 30 the liquid out and absorb the residue liquid with absorbent paper; 6 (3) Add blocking buffer 150-200l into each well. After incubating at 37 for 1-2h, pour the liquid out, absorb the residue liquid with absorbent paper, and seal with aluminum membrane in vacuum for future use. In this invention, the antibodies are produced as follows: 5 1. Synthesis of Hapten An artificial hapten was obtained from Sudan I via succinic anhydride method. 2. Production of anti-Sudan I Antibody The Sudan I hapten said hereinbefore is coupled to carrier proteins via a mixed anhydride (isobutyl chloroformate) method to produce complete immunogens. 10 As a small molecule, Sudan I is immunoreactive, but it is not immunogenic, which can not trigger an immune response in the body. By coupling to macromolecular carrier proteins, it then becomes immunogenic. In the present invention, a 4-carbolic branch is combined to the Sudan I molecule by reacting with succinic anhydride, which exposes the characteristic structure of Sudan I molecule, and guarantees the 15 specificity of the anti-Sudan I antibody produced hereinafter. 2.1 Production of anti-Sudan I monoclonal antibody Immunization of animals: Immunize BALB/c mice with the immunogen (conjugate of Sudan I hapten and carrier proteins) for several times to obtain better poluclonal antibody, take the spleen and perform a cell fusion. 20 Cell fusion and cloning: take the spleen cells from the immunized BALB/c mice and fused with SP2/0 myeloma cells. Screen the fused cells till hybridomas secreting anti-Sudan I antibody stably are found. 2.2 Production of anti-Sudan I polyclonal antibody New Zealand white rabbit is immunized with the immunogens said hereinbefore, 25 bleeding was performed and the serum titer was tested after several immunizations to obtain polyclonal antibody. The secondary antibody is produced as follows: The goat-anti-mouse secondary antibody is produced by immunizing the mouse-sourced anti-Sudan I antibody to germfree goat. 30 The goat-anti-rabbit secondary antibody is produced by immunizing the 7 rabbit-sourced anti-Sudan I antibody to germfree goat. The Sudan I standard solution series in this invention is a solution series in certain concentration scales, the preferable scale is 0-50ig/L. e.g. the solution series is composed compose of 6 solutions with concentrations of 0, 0.1, 0.4, 1.6, 6.4 and 5 25.6pig/L, respectively, and the volume is 3ml. A method to detect Sudan I in a sample with the ELISA kit said hereinbefore is also provided in this invention, which includes the following steps: (1) Sample preparation; (2) Sample detection using the kit; 10 (3) Result interpretation. The samples mentioned in step (1) are foods for animals or human beings, in particular, food or food ingredients, more particularly, hot pepper sauce, chili oil, chili powder and other related foods or food ingredients, etc. In the present invention, sample preparation method for Sudan I-contaminated 15 food samples is provided. Samples in water or oil matrix and powder sample can be prepared and detected by the following steps: Weighing samples into centrifuge tubes, adding acetonitrile and shaking for a while, centrifuging, then taking the supernatant and drying with a nitrogen evaporator, dissolving the dry leftover and taking part of which for the assay. 20 The assay utilizing the kit may be as follows: (1) Sudan I-coupled coating antigen is immobilized on the microtiter wells: add the Sudan I standard solution or Sudan I-contained samples into the wells, and then add anti-Sudan I antibody solution, incubate and wash the wells, and remove the residual liquid. Add enzyme-labeled secondary antibody, incubate and wash the wells, 25 remove the residual liquid, add substrate and color-developing solution, stop the reaction after incubation, and measure the absorbencies; (2) Sudan I-coupled coating antigen is immobilized on the microtiter wells: add the Sudan I standard solution or Sudan I-contained samples into the wells, and then add enzyme-labeled anti-Sudan I antibody solution, incubate and wash the wells, and 30 remove the residual liquid. Add substrate and color-developing solution, stop the 8 reaction after incubation, and measure the absorbencies; (3) anti-Sudan I antibody is immobilized on the microtiter wells: add the Sudan I standard solution or Sudan I-contained samples into the wells, and then add enzyme-labeled Sudan I antigen solution, incubate and wash the wells, and remove 5 the residual liquid. Add substrate and color-developing solution, stop the reaction after incubation, and measure the absorbencies; (4) Secondary antibody is immobilized on the microtiter wells: add the anti-Sudan I antibody solution, incubate and wash the wells, and remove the residual liquid. Add Sudan I standard solution or Sudan I-contained samples into the wells, and then add 10 enzyme-labeled Sudan I antigen solution, incubate and wash the wells, remove the residual liquid. Add substrate and color-developing solution, stop the reaction after incubation, and measure the absorbencies. The result interpretation after assay is as follows: Calculate the percentage absorbance: divide absorbency of each standard (B) by 15 the absorbency of the Opg/L standard solution (Bo), and then multiply by 100%. The formula is shown as: Percentage Absorbency (%) = (B/Bo) x100 Plot the standard curve: take semi-log of the concentrations (pg/L) of Sudan I standards as X-axis, and the percentage absorbance as Y-axis. 20 Get the sample result: calculate the percentage absorbencies of the sample solution as described above, and read the corresponding concentration from the standard curve. A regression model can also be used to interpret the result and to calculate the concentration of the sample. 25 Special computer software is also developed for result interpretation, which is convenient and rapid for large-scale sample screening. The time cost for this method is short, e.g., only 1.5 hours, and the detection limit of samples is 10pg/L. The ELISA kit in the present invention is based on an indirect competitive ELISA 30 method to detect Sudan I-contaminated food samples for animals or human beings, in 9 particular, foods or food ingredients, e.g. hot pepper sauce, chili oil and chili powder. The sample preparation is simple and ready for screening of large quantities of samples simultaneously. In this invention, specific anti-Sudan I monoclonal or polyclonal antibody is used, 5 and major reagents in the kit are provided as solutions. The method using the kit to detect Sudan I is simple, convenient, specific, sensitive and precise, which would play very important roles in the detection of Sudan I-contaminated samples. DESCRIPTION OF THE DRAWINGS 10 FIG. I Standard curve for detection of Sudan I DETAILED DESCRIPTION OF THE INVENTION A number of examples are provided here to describe the present invention. It should be understood that the examples are only for describing, rather than limiting the scope 15 of the present invention. EXAMPLE 1: PREPARATION OF THE KIT COMPONENTS 1. SYNTHESIS OF ANTIGENS a. Synthesis of Hapten 20 The Sudan I hapten is synthesized from Sudan I by a succinic anhydride method. Briefly, take 5g Sudan I and dissolve in 20ml N,N-dimethyl formamide (DMF), the add 3g succinic anhydride and 50ml pyridine, reflux at 80 for 70-80 hours, drain the solvent, and wash with purified water for five times (30 ml each time), and dry the resultant to obtain the hapten. 25 b. Synthesis of the immunogen Sudan I hapten is coupled to ovalbumin (OVA) via a mixed anhydride method. Briefly, 2g Sudan I is dissolved in 30ml 50% DMF (Solution 1). Take 0.5ml isobutyl chloroformate and dissolve in 5ml anhydrous dioxane (Solution 2). Add solution 2 into solution 1, react for 4 hours under stirring (Solution 3). Then, take 32g carrier 30 protein and dissolve in 70ml pH 9.6 carbonate buffer solution, drip the carrier protein 10 solution into solution 3, and react overnight at 4 under stirring. Gather the resultants and dialyze in 0.2mol/L PBS for 7 days, and change the dialysis fluid 3-4 times/day. Finally, gather the obtained immunogen and lyophilize for future use. c. Synthesis of Sudan I-coupled coating antigen 5 The Sudan I hapten is coupled to thyroglobulin (TPR) via a mixed anhydrides method. Briefly, 2g Sudan I hapten is dissolved in 30ml 50% DMF (Solution 1). Take 0.5ml isobutyl chloroformate and dissolve in 5ml anhydrous dioxane (Solution 2). Add solution 2 into solution 1, react for 4 hours under stirring (Solution 3). Then take 10 32g carrier protein and dissolve in 70ml pH 9.6 carbonate buffer solution, drip the carrier protein solution into solution 3, react overnight at 4 under stirring. Gather the resultants and dialyze in 0.2mol/L PBS for 7 days, and change the dialysis fluid 3-4 times/day. Finally, gather the obtained immunogen and lyophilize for future use. 2. PRODUCTION OF ANTI-SUDAN I MONOCLONAL ANTIBODY 15 a. Immunization of animals Immunize several BALB/c mice with the immunogen at the quantity of 80gg/mouse to trigger immunoresponse of the mice. b. Cell fusion and cloning Take the spleen cells from immunized mice, and perform cell fusion with the 20 SP2/0 myeloma cells in the quantity ratio of 5:1, test the cell culture supernatant with indirect competitive ELISA to choose positive wells. Clone the positive wells with limited dilution method. Finally, a cell line with a deposit number of B-1-1 CGMCC No. 1706, which can secrete anti-Sudan I monoclonal antibody stably, was obtained. c. Cell cryopreservation and recovery 25 Produce a cell suspension with cell line B-1-1 CGMCC No. 1706 at the concentration of I x 106 cells /ml in cryopreservation solution, then store in liquid nitrogen. Take out the cryopreservation tube when recovering, then put into 37 water bath immediately to thaw it speedily. Centrifuge to remove the cryopreservation solution, 30 then transfer the cells to culture bottles, and start culturing. .11 d. Production and purification of monoclonal antibody Intraperitoneally inject sterile paraffin oil into several BALB/c mice at 0.4ml/mouse, and inject hybridoma cell line B-1-1 CGMCC No. 1706 into these mice 7 days later with quantity of 5x10 5 cells/mouse. Gather the ascites 7 days after cell 5 line injection. Purify the ascites via the octanoic acid-saturated ammonium sulfate method. Store the purified ascites at -20 . 3. Production of polyclonal antibody As an immunogen, the Sudan I hapten-OVA conjugate was injected into New Zealand white rabbit with a quantity of 1.5mg/kg body weight. In the first injection, 10 immunogen was mixed with equivalent Freund's Complete Adjuvant (FCA) to obtain an emulsified agent, which was then injected at multiple sites in the neck and the back of the rabbit. In subsequent injections (3-4 weeks after the former injection), the immunogen was mixed and emulsified with Freund's Incomplete Adjuvant (FIA) and injected. In the 5th injection, the animals were immunized with the immunogen 15 without any adjuvant. Bleeding was performed 10 days after the last immunization. The serum titer was tested and then the rabbits were sacrificed to gather the serum. The antibody was purified via ammonium sulfate fractional precipitation. 4. Production of secondary antibody The mouse-sourced antibody was injected into germfree goat to obtain 20 goat-anti-mouse secondary antibody; the rabbit-sourced antibody was injected into germfree goat to obtain goat-anti-rabbit secondary antibody. 5. Production of enzyme-labeled goat-anti-mouse secondary antibody The goat-anti-mouse secondary antibody is conjugated with HRP via modified sodium periodate method, which is described below: 25 a. 8g HRP is dissolved in 2ml distilled water. b. add fresh 100mmol/L NaIO 4 solution 0.4mL into the HRP solution, react at room temperature for 20min under stirring. c. Dialyze the reacted solution in Immol/L acetate buffer solution at 4 overnight to remove excess NalO 4 , meanwhile the conjugated HRP would be deoxidized. 30 d. add 40ptL PBS (pH 8.6, 0.5mol/L) and 2.OmL PBS (pH 8.6, 0.5mol/L) 12 containing 16mg mouse anti-Sudan I IgG, and react at room temperature for 4 hours under stirring. e. add freshly prepared NaBH 4 solution (lmol/L) 0.lmL, and react at 4 for 4 hours to deoxidize Schiff's base. 5 f. Purify the resultant and store at certain conditions. 6. Preparation of the microtiter plates Dissolve the Sudan I coating antigen or anti-Sudan I antibody or secondary antibody with coating buffer to 0.05-0.1 g/ml, add 100 l obtained solution into each well, and then incubate at 37 for 2h or at 4 overnight. 10 Pour the coating buffer out, and wash with 20 times-diluted washing solution twice, 30s each time, then remove the liquid and eliminate the residual liquid with absorbent paper. Add 150ptl blocking buffer into each well, incubate at 37 for 2h, and then dispose the liquid and seal with aluminum membrane after the plate is dry. 15 EXAMPLE 2: COMPOSITION OF THE ELISA KIT FOR THE DECTION OF SUDAN I Components said hereinabove (in EXAMPLE 1) are used to establish the said ELISA kit comprising: 20 (1) Microtiter plate coated with Sudan I-coupled coating antigen; (2) HRP-labeled goat-anti-mouse secondary antibody; (3) Concentrated anti-Sudan I monoclonal antibody solution; (4) Sudan I standard solution series, with concentrations of 0, 0.1, 0.4, 1.6, 6.4 and 25.6pg/L, respectively; 25 (5) Substrate and color-developing solutions are composed of Substrate A and Substrate B, wherein Substrate A is 2% carbamide peroxide solution and Substrate B is 1% TMB solution; (6) Stop solution is 2mol/L hydrochloric acid; (7) Concentrated washing solution is 0.01mol/L PBS (pH7.4, containing 0.8-1.2% 30 tween-20 and 0.5% sodium azide); 13 (8) Antibody diluent solution is 0.05mol/L PBS (pH8.6, containing 2% calf serum, 10% methanol); (9) Concentrated redissolving solution is 2x0.04mol/L PBS. 5 EXAMPLE 3: DETECTION OF SUDAN I IN SAMPLES 1. Sample preparation Sudan I-contaminated food samples (in water or oil matrix and powder) are treated as the following steps: Weighing 2g of the samples into a centrifuge tube, adding 10ml acetonitrile, 10 shaking fiercely for 10min, and then centrifuging for 10min at least at 3000rpm; transferring Iml of the supernatant and drying the supernatant at 50 with nitrogen evaporator; adding 4ml n-hexane to dissolve the dry leftover by shaking with an eddy shaker for 30s, then adding Iml Imol/L NaOH and shaking for 15s; after that, centrifuging at room temperature for 10min at 3000rpm; taking Iml of the supernatant, 15 drying the supernatant at 50 with nitrogen evaporator; dissolving the dry leftover with 0.5ml DMF, taking 100pl of the solution and dilute with 900pl diluted redissolving solution, and mixing completely to obtain the sample solution for detection. 2. Assay with the ELISA kit 20 The ELISA kit said hereinbefore in Example 2 is used here to proceed the assay: Add Sudan I standard solution or Sudan I-contaminated sample solution 50p1l into the Sudan I-coupled antigen coated microtiter wells, and then add anti-Sudan I monoclonal antibody solution 50pl, seal with a covering membrane, incubate in an incubator at 37 for 30min, after that, pour the liquid out from the wells, add 250pl 25 diluted washing solution to each well, pour the liquid out after 30s, repeat washing for 5 times, absorb the residual liquid with absorbent paper. Add HRP-labeled goat-anti-mouse secondary antibody solution 100p l to each well, and incubate in an incubator at 37 for 30min. Take out, remove the liquid and repeat the wash step. After that, add substrate solution A (carbamide peroxide) and B (TMB 30 solution) into each well, mix gently by hand, then incubate at 37 in dark for 15min. 14 Add 50pl stop solution (hydrochloric acid) to stop the reaction, and measure the absorbencies of each well with an ELISA reader. 3. Result Interpretation Divide the average absorbency of each standard (B) by the absorbency of 0ig/L 5 standard solution (Bo), and then multiply by 100% to get the Percentage Absorbance. Plot the standard curve; take semi-log of the concentrations (ptg/L) of Sudan I standards as X-axis, and the percentage absorbency as Y-axis. Get the sample result, calculate the percentage absorbencies of the sample solution as described above and read the corresponding concentration from the 10 standard curve. Experiment 1. PRECISION EXPERIMENT OF THE STANDARD SOLUTIONS The kits used herein and hereinafter were produced according to the method described hereinbefore in Example 2. 15 Randomly choose a number of microtiter plates from 3 different lots, 10 kits/lot. Take 20 microtiter wells from each plate; test the absorbance of the 4.5ptg/L standard solution, and calculate the coefficient of variation (C.V.). The results are listed in Table 1. Table 1. Precision Result of the 4.5pg/L Standard Solution (CV%) 1 2 3 4 5 6 7 8 9 10 Lot 01 10.4 5.7 7.4 9.3 6.5 11.4 10.7 5.2 4.8 6.5 CV% Lot 03 8.7 7.3 6.0 8.9 11.2 8.1 9.5 10.3 11.0 8.7 Lot 06 6.4 4.8 3.8 5.9 7.4 10.6 8.6 8.8 11.7 8.5 20 The results indicated that, CV% of the chosen kits was in the range of 3.8%-11.7%, which complied with the rules of precision being equal to or less than 20%. Experiment 2 PRECISION AND ACCURACY OF SAMPLES a. Precision test 25 Standard Sudan I was blended with the 3 kinds of samples at the concentration of 15 g/L, respectively. Take kits from 3 different lots (3 kits per lot), run 5 repeats for 15 each concentration, and calculate the CV% respectively. The results are listed in Tables 2, 3 and 4. Table 2 Precision of water matrix samples blended with Sudan I Lot No. Detected value (p.g/L) 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 Table 3 Precision of oil matrix samples blended with Sudan I Lot No. Detected value (pg/L) CV% 11.7 10.4 8.9 10.2 15.7 18.7 0506008 16.2 14.7 9.5 13.7 15.2 17.5 14.7 10.6 12.5 9.7 10.4 17.9 9.7 10.2 13.5 8.5 11.6 13.7 0507003 8.7 10.0 12.6 11.4 10.7 14.2 11.6 13.7 14.6 15.2 10.9 23 12.7 9.5 10.2 16.7 13.8 11.6 0507009 15.7 11.6 13.4 14.2 12.6 15.8 13.7 16.4 15.4 12.9 10.8 18.7 5 Table 4 Precision of powder samples blended with Sudan I 1.6 10.7 12.8 8.7 9.4 19.2 Lot No. CV% 10.7 16.4 13.8 14.2 10.6 18.9 16 13.7 11.6 12.8 10.4 9.5 14.7 10.7 8.4 12.6 9.8 8.6 17.1 11.6 12.4 10.8 9.5 11.7 9.9 0507009 10.7 9.3 12.2 9.5 8.0 15.9 12.4 11.6 13.8 10.6 8.7 16.8 The results indicated that CV% for each kind of samples tested with kits from different lots was less than 25%. The water matrix sample used in the test is edible hot pepper sauce, the oil matrix sample is edible chili oil, and the powder sample is edible chili powder. 5 b. Recovery Test Sudan I was blended into different samples at the concentration of 10, 50 and 100pg/kg (L), and 4 repeats were performed for each concentration. Carry out the assay as desctribed above (sample preparation of Example 3), and calculate the accuracy. The results are listed in Table 5. 10 The results indicated that for water matrix samples, the recovery rate was 60.8-102.3%; for oil matrix samples, it was 63.5-94.8%; for powder samples, it was 62.8-87.6%. 15 Table 5 Recovery test of samples Water matrix sample blended Oil matrix sample blended Samples with Sudan I with Sudan I Sudan I concentration 10 50 100 10 50 100 (4g/L) 1 63.7 83.4 83.4 68.5 78.4 94.8 2 71.6 74.6 102.3 73.6 92.0 82.7 Recovery rate% 3 69.5 86.5 87.4 64.7 84.7 79.4 4 60.8 82.4 93.5 63.5 72.6 83.5 Average % 66.4 81.7 91.7 67.6 81.9 85.1 17 Sample Power sample blended with Sudan I Sudan I concentration 10 50 100 (pig/L)______ ___ 1 67.4 72.4 84.7 2 73.5 76.4 72.4 Recovery rate % 3 74.9 81.4 92.6 4 62.8 87.6 87.4 Average % 69.7 79.5 84.3 Experiment 3 Cross reaction experiment Chemicals with similar structure or function as Sudan I were selected for the experiment. Calculate the 50% inhibition concentration of each chemical, and calculate 5 the percentage cross reaction according to the following equation. 50% inhibition concentration of Sudan I Percentage Cross reaction(%) X100% 50% inhibition concentration of tested chemical 10 Table 6 Specificity of the kit Chemicals Percentage Cross Reaction (%) Sudan I 100 Para Red 120 Sudan II <1 Sudan III <1 Sudan IV <1 EXPERIMENT 4 18 The ELISA kit hereinabove was stored at 2-8 . After 6 months, the maximum absorbance of the kit (the O g/L (kg) standard), 50% inhibition concentration and the results of the recovery test were within the normal range. Considering the abnormal conditions during transportation and practical use, the kit was stored at 37 for 6 days 5 as a speedy deterioration experiment, and the result indicated that the kit completely met the requirements for all parameters. The kits were frozen at -20 for 5 days, and the test results showed that the parameters said hereinbefore were all normal. The experiments above demonstrated the kits had a shelf life at 2-8 for at least 6 months. 19

Claims (12)

1. An anti-Sudan I monoclonal antibody, which is secreted by hybridoma cell line B-1-1 CGMCC No. 1706.

2. An ELISA kit, comprising: (1) A microtiter plate coated with Sudan I antigen, the anti-Sudan I monoclonal antibody of Claim 1 or the secondary antibody from the anti-Sudan I monoclonal antibody of Claim 1, (2) Enzyme conjugate, (3) Sudan I standard solution series, (4) Substrate and color-developing solution, (5) Stop solution, (6) Concentrated washing solution, and (7) Concentrated redissolving solution, when the microtiter plate is coated with Sudan I antigen, said enzyme conjugate is enzyme-labeled anti-Sudan I monoclonal antibody of Claim 1 or the enzyme labeled secondary antibody thereof; when the microtiter plate is coated with the anti Sudan I monoclonal antibody of Claim 1 or the secondary antibody thereof, said enzyme conjugate is enzyme-labeled Sudan I antigen.

3. The ELISA kit of Claim 2, further comprising concentrated anti-Sudan I antibody solution and the diluent thereof when the microtiter plate is coated with Sudan I antigen and the enzyme conjugate is enzyme-labeled secondary antibody, or when the microtiter plate is coated with the secondary antibody and the enzyme conjugate is enzyme-labeled Sudan I antigen.

4. The ELISA kit of Claim 2 or 3, wherein the Sudan I antigen is Sudan I hapten, which is conjugated with a carrier protein.

5. The ELISA kit of Claim 4, wherein the carrier protein is mouse serum albumin, thyroglobulin, bovine serum albumin, rabbit serum albumin, human serum albumin, ovalbumin or hemocyanin from keyhole limpets. 20

6. The ELISA kit of any one of Claims 2-5, wherein the microtiter plate is produced with citric acid buffer as the coating buffer, and with the solution containing equine serum, sodium azide and casein as the blocking buffer.

7. The ELISA kit of any one of Claims 2-6, wherein the enzyme-labeled Sudan I antigen is enzyme-labeled Sudan I hapten.

8. The ELISA kit of any one of Claims 2-6, wherein the secondary antibody is goat-anti-mouse or goat-anti-rabbit secondary antibody, and the enzyme used in the enzyme conjugate is horseradish peroxidase or bacterial alkaline phosphatase.

9. The ELISA kit of Claim 8, wherein when the enzyme is horseradish peroxidase, the substrate and color-developing solution is composed of substrate A and substrate B, and substrate A is hydrogen peroxide or carbamide peroxide solution, while substrate B is 0-phenylenediamine or tetramethylbenzidine, and the stop solution is sulfuric acid or hydrochloric acid solution.

10. The ELISA kit of Claim 8, wherein when the enzyme is bacterial alkaline phosphatase, the substrate and color-developing solution is p-nitrophenylphosphate buffer, and the stop solution is sodium hydroxide solution.

11. The ELISA kit of any one of Claims 2-10, wherein the concentrated washing solution is PBS containing Tween and sodium azide, and the concentrated redissolving solution is PBS.

12. A method for detecting Sudan I in a sample, comprising the following steps: (1) Preparing the samples; (2) Proceeding with the assay using the kit of any one of Claims 2-11; and (3) Analyzing the result of the assay. 21