[0050] Example 1:
[0051] 1. Antigen design and synthesis
[0052] The bifunctional chelating agent benzyl ethylene diamine tetraacetic acid isothiocyanate (ITCBE) is used to couple heavy metal cadmium ions with keyhole hemocyanin (KLH). The specific method is:
[0053] Weigh 1.4 mg ITCBE and dissolve it in 1 mL DMSO, slowly add 710 μL of 1000 mg/L cadmium solution standard dropwise to the ITCBE solution, adjust the pH to 7.4 with triethylamine, and stir at 25° C. with a magnetic stirrer for 24 hours. 10mg KLH was dissolved in 20mmol/L Hepes buffer solution, and the chelated reaction solution was slowly added dropwise to the KLH solution, and the pH was adjusted to 9.6 with triethylamine. After stirring for 24h with a magnetic stirrer at 25°C, dialyzed with 5mmol/L HEPES buffer solution for three days.
[0054] 2. Preparation of coating antigen
[0055] The bifunctional chelating agent benzyl ethylene diamine tetraacetic acid isothiocyanate (ITCBE) is used to couple heavy metal cadmium ions with bovine serum albumin (BSA). The specific method is:
[0056] Weigh 1.33 mg of ITCBE and dissolve it in 1 mL of DMSO, slowly add 716 μL of 1000 mg/L cadmium solution standard to the ITCBE solution, adjust the pH to 7.4 with triethylamine, and stir at 25°C with a magnetic stirrer for 24 hours. 10mg BSA was dissolved in 20mmol/L Hepes buffer solution, and the chelated reaction solution was slowly added dropwise to the BSA solution, and the pH was adjusted to 9.6 with triethylamine. After stirring for 24 hours with a magnetic stirrer at 25° C., it was dialyzed with 5 mmol/L Hepes buffer solution for three days.
[0057] 3. Immunization and specific antibody preparation
[0058] Immunization: Two New Zealand white rabbits, 3 months old and weighing about 1.5 kg, were selected as immunized animals. They were kept in a standard laboratory animal room and observed for 4 consecutive days. After confirming that their physical condition was normal, they were immunized. Immunization is performed jointly by subcutaneous and intramuscular injection.
[0059] Initial immunization: Weigh the dose of 1 mg, dissolve it in 1 mL of 0.9% NaCl, and mix with 1 mL of Freund's complete adjuvant for emulsification. The emulsion is used for immunization.
[0060] Boost immunization: boost immunization on 15th, 30th and 45th days after the initial immunization, weigh 0.5mg, dissolve in 1mL 0.9% NaCl, then mix with 1mL of Freund’s incomplete adjuvant to emulsify, and emulsify for immunization , A total of 4 immunizations. Starting from the third immunization, 8-10 days after each immunization, blood was collected from the animals for serum titer determination. After the last immunization, whole blood was collected from the animal by the femoral artery blood sampling method. After centrifugation, all serum was collected, and the proteinA-SepharoseCL-4B immunoaffinity chromatography column was used for antibody purification. The antibody obtained was added with 1:1 (v/v) glycerol Store at -20°C for later use.
[0061] 4. Establishment of immunoassay methods
[0062] The item to be tested is: Chinese medicine Angelica dahurica. Using the rapid immunoassay method, the steps to detect its heavy metal cadmium ion content are as follows:
[0063] The Angelica dahurica was pulverized into a powder using a Chinese medicine pulverizer, and 2 g was soaked in 10 mL of mixed acid of perchloric acid overnight, and then nitrated at 160-180°C in a digestion tank until the solution was clear. Use TRIS to adjust the pH to neutral, pass through a 0.22μm filter membrane and dilute to 50 mL with double distilled water. The solution after constant volume was diluted 1.6 times and used for ELISA test.
[0064] Coating: Dissolve the prepared Cd-ITCBE-BSA coating in a buffer of pH 9.6Na2CO3-NaHCO3, and dilute the Cd-ITCBE-BSA coating to 1.0μg/100μL as a coating solution, 96-well micro Add 100 μL to each well of the well plate, place overnight at room temperature or incubate at 37°C for 2 to 3 hours, and wash three times with PBST (phosphate buffer saline 0.05% (V/V), Tween20 washing solution);
[0065] Blocking: add 200μL 0.5% skimmed milk powder/PBS blocking solution to each well, block for 1 hour, and wash three times with lotion;
[0066] Adding samples: dissolve the test sample in PBS after chelating EDTA, and dissolve the heavy metal cadmium ion standard (Cd-EDTA) in PBS. Add 50μL of the above test sample or standard sample to each well when adding samples;
[0067] Competitive reaction: Dissolve the heavy metal cadmium ion antibody in PBS buffer, add 50μL of antibody solution to each well after adding the test sample or standard sample, incubate for 60min, wash with PBST four times;
[0068] Secondary antibody: Add 100μL goat anti-rabbit enzyme-labeled secondary antibody to each microwell, react at 37°C for 30 minutes, and wash five times with PBST;
[0069] Add substrate: Tetramethylbenzidine (TMB) is used as the color substrate. Add 100μL of tetramethylbenzidine-hydrogen peroxide solution (5mg tetramethylbenzidine dissolved in 1ml substrate buffer) to each well, and react for 0.5 hours at room temperature;
[0070] Stop: Add 50μL of sulfuric acid stop solution to each microwell to stop the reaction, and read the absorbance value with a microplate reader.
[0071] Sensitivity and specificity analysis:
[0072] (1) Sensitivity
[0073] For the indirect competition ELISA method, the sensitivity refers to the concentration of the standard substance when the inhibition rate is 50%, and the IC of the indirect competition ELISA method for heavy metal cadmium ion has been researched and established. 50 = 11.33μg/L, so its sensitivity is 11.33μg/L. The detection limit refers to the concentration of the standard substance when the inhibition rate is 15%. The IC of the indirect competition ELISA detection method for heavy metal cadmium ion has been established 15 =0.76μg/L, so the detection limit is 0.76μg/L.
[0074] (2) Specificity
[0075] The antibody prepared by the present invention and the established immunoassay method have a cross-reaction to Pb, Mg, Ni, Ag, Cu, Cr, Fe less than 1.2%, and a cross-reaction to Hg 10.9%. The analysis may be due to the three-dimensional analysis of heavy metals Cd and Hg The structure is similar. This indicates that the antibody prepared by the present invention is highly specific to the heavy metal cadmium ion of the target detection object.
