CN109633159B - Method for detecting melamine content and special colloidal gold test paper thereof - Google Patents
Method for detecting melamine content and special colloidal gold test paper thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting melamine content and a special colloidal gold test paper thereof. The colloidal gold test paperThe quenching antibody combination pad is coated with colloidal gold marked by a melamine antibody, the quality control line position on the reaction membrane is coated with the antibody of the melamine antibody, the detection line position is coated with the antigen of the melamine antibody, and the back plate is coated with a fluorescent substance; the melamine antibody is an antibody prepared by taking a conjugate of a compound shown in a formula (I) and a carrier protein as an immunogen. Experiments prove that the colloidal gold test paper provided by the invention can be used for rapidly detecting the melamine content in a milk sample, is simple to operate, short in time consumption, high in accuracy and high in sensitivity, and the minimum detection limit can reach 5 mug/L. The colloidal gold test paper provided by the invention has important application value in rapidly detecting the melamine content.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a method for detecting melamine content and a special colloidal gold test paper thereof.
Background
Melamine is a triazine organic compound containing nitrogen heterocyclic ring, an important nitrogen heterocyclic ring organic chemical raw material, is called triamine for short, and is commonly called melamine or protamine, and the molecular formula isC3N6H6And the molecular weight is 126.12. Because the molecular formula of melamine contains about 66% of nitrogen and the average nitrogen content of protein is about 16%, melamine is often used as a food additive by illegal people due to the defects of a protein content testing method in the dairy industry so as to improve the protein content index in food detection. Although melamine is less toxic, its long-term intake is not negligible. The international chemical safety program, volume three and the international chemical safety card, compiled by the international chemical safety program, 1994 and by the european union committee, indicate that prolonged or repeated ingestion of large quantities of melamine may have an effect on the kidneys and bladder, leading to the production of stones.
At present, the method for detecting the melamine residue in the dairy product mainly adopts an instrumental analysis method and an immunoassay method. Instrumental analysis methods include High Performance Liquid Chromatography (HPLC), gas mass spectrometry (GC-MS) and liquid mass spectrometry (LC-MS/MS). The HPLC method has the advantages of accurate quantification and the like, but the pretreatment is relatively complex during detection, and the method has the defects of low sensitivity and the like in practical application. The GC-MS method can be used for confirmation and detection and has high sensitivity, but the pretreatment process is complex and needs derivatization treatment. The LC-MS/MS method has the advantages of high sensitivity, good selectivity, accurate qualitative and quantitative determination, strong anti-interference capability and the like, but the method also has the defects of complex pretreatment process, long time consumption, high cost and the like. The immunoassay method comprises an enzyme-linked immunosorbent assay and a colloidal gold immunochromatography, has the advantages of rapidness, convenience, simple operation, lower cost and high efficiency of screening samples, but antibodies and enzymes used by the enzyme-linked immunosorbent assay belong to bioactive substances, need to be stored at low temperature and have poor stability; the colloidal gold immunochromatography method is low in detection sensitivity and mainly used for rapid screening and analysis, and both the enzyme-linked immunosorbent assay and the colloidal gold immunochromatography method have nonspecific reaction, so that the problem that false positive is easily generated in the detection result is solved. Therefore, a simple, convenient, rapid and accurate quantitative detection method is urgently needed to be made to improve the current situation, fill up the defects of the original rapid detection method, and improve and standardize the technical level and the product quality of the whole rapid detection industry.
Disclosure of Invention
The invention aims to quickly detect the melamine content in a solution to be detected.
The invention firstly protects a colloidal gold test paper for detecting melamine content, wherein a quenching antibody combination pad is coated with colloidal gold marked by a melamine antibody, a quality control line position on a reaction membrane is coated with the antibody of the melamine antibody, a detection line position is coated with the antigen of the melamine antibody, and a fluorescent substance is sprayed on a backboard or under the reaction membrane;
the melamine antibody is an antibody prepared by taking a conjugate of a compound shown in a formula (I) and a carrier protein as an immunogen;
in the above colloidal gold test paper, the melamine antibody may be a1) or a 2):
a1) the compound shown in the formula (I) and the conjugate of the carrier protein are used as immunogen to immunize animals and then are subjected to somatic cell hybridization to obtain a melamine monoclonal antibody;
a2) the melamine polyclonal antibody obtained by using the conjugate of the compound shown in the formula (I) and carrier protein as immunogen to strengthen immunity of animals.
The animal may be any one of a mouse, rabbit, goat, sheep or horse.
In the colloidal gold test paper, the melamine monoclonal antibody consists of a heavy chain and a light chain. The amino acid sequence of the variable region of the heavy chain can be shown as a sequence 1 in a sequence table. The amino acid sequence of the variable region of the light chain can be shown as a sequence 2 in a sequence table.
In the colloidal gold test paper, the antibody of the melamine antibody can be goat anti-mouse IgG antibody. The goat anti-mouse IgG antibody may be a product of Jackson ImmunoResearch, USA.
In the above colloidal gold test paper, the antigen of the melamine antibody may be a conjugate of a compound represented by formula (I) and a carrier protein.
Any of the carrier proteins may be bovine serum albumin or ovalbumin.
In the above method, the compound represented by the formula (I) is linked to the carrier protein via an amide bond. The amido bond is formed by carboxyl of the compound shown in the formula (I) and amino on carrier protein through active ester.
The conjugate can be melamine-bovine serum albumin conjugate (compound shown in formula (II)) or melamine-ovalbumin conjugate (compound shown in formula (III)).
In the above colloidal gold test paper, the fluorescent substance may be an organic fluorescent dye (e.g., tetraethylrhodamine).
In the above colloidal gold test paper, instead of spraying fluorescent substance on the back plate or under the reaction membrane, ProClin 300 (as preservative) may be coated.
The specific steps of "spraying fluorescent substance on the back plate" can be as follows:
(1) dissolving tetraethyl rhodamine with Tris-HCl buffer solution with the concentration of 0.05-0.2M (such as 0.2M) to obtain a tetraethyl rhodamine solution with the concentration of 0.05-0.2% (such as 0.01%) (M/v).
(2) After the step (1) is finished, adding ProClin 300 (serving as a preservative) into the tetraethyl rhodamine solution to obtain a working solution; the concentration of ProClin 300 in the working solution is 0.01-0.03% (e.g. 0.02%) (v/v).
(3) And (3) uniformly spraying the working solution obtained in the step (2) on a PVC plate to obtain the back plate sprayed with the fluorescent substance. In order to prevent the fluorescent material from falling off in the storage and use processes, after the working solution is uniformly sprayed on the PVC plate, a plastic packaging film with good light transmission can be covered above the PVC plate.
In the above colloidal gold test paper, the reaction membrane may be a nitrocellulose membrane.
In the above colloidal gold test paper, the quenching antibody binding pad may be made of a glass cellulose membrane.
In the above colloidal gold test paper, the concentration of the antibody coating the melamine antibody at the position of the control line may be 0.1-0.3mg/mL (e.g., 0.2 mg/mL). In the embodiment of the invention, the goat anti-mouse secondary antibody is diluted by PBS buffer solution with the concentration of 0.1-1mM and the pH value of 7.4 to obtain the quality control membrane-scribing liquid with the concentration of 0.2 mg/mL. The amount of scratching can be 0.5-0.6. mu.L/cm.
In the above colloidal gold test paper, the concentration of the antigen coating the melamine antibody at the position of the detection line may be 0.1-0.2mg/mL (e.g., 0.15 mg/mL). In the embodiment of the invention, specifically, the melamine-bovine serum albumin conjugate is diluted by PBS buffer solution with pH7.4 and 150mM to obtain the detection scribing solution with the concentration of 0.15 mg/mL. The amount of scratching can be 0.5-0.6. mu.L/cm.
In the above colloidal gold test paper, the distance between the quality control line and the detection line may be 2-4mm (e.g., 2-3mm, 3-4mm, 2mm, 3mm, or 4 mm).
The invention also provides a method for detecting the melamine content in the solution to be detected by adopting any one of the above colloidal gold test paper, which comprises the following steps:
(1) immersing the sample pad of the colloidal gold test paper into a solution to be tested, standing for more than 10min, and then inserting the colloidal gold test paper into a fluorescence immunoassay quantitative analyzer to obtain the ratio of F1/F2;
F1/F2 ratio is the fluorescence intensity of the blank space between the quality control line and the detection line (F1)/the fluorescence intensity of the detection line (F2);
(2) immersing the sample pad of any one of the colloidal gold test paper in a melamine standard solution, standing for more than 10min, and then inserting the colloidal gold test paper into a fluorescence immunoassay quantitative analyzer to obtain a corresponding F1/F2 ratio;
(3) and (3) drawing a standard curve according to the concentration of melamine in the melamine standard solution and the corresponding F1/F2 ratio, and then substituting the F1/F2 ratio obtained in the step (1) into the standard curve to obtain the melamine content in the solution to be detected.
In the above method, the solution to be tested may be a milk sample.
Experiments prove that the colloidal gold test paper provided by the invention can be used for rapidly detecting the melamine content in a milk sample, is simple to operate, short in time consumption, high in accuracy and high in sensitivity, and the minimum detection limit can reach 5 mug/L. The colloidal gold test paper provided by the invention has important application value in rapidly detecting the melamine content.
Drawings
FIG. 1 is a schematic structural diagram of a colloidal gold test paper for detecting melamine.
Figure 2 is a standard curve for melamine.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
F1/F2 ratio (F1) of fluorescence intensity of blank space between quality control line and detection line/fluorescence intensity of detection line (F2)
Example 1 preparation of colloidal gold test paper for detecting Melamine
First, preparation of antigen
1. Preparation of Compounds of formula (I) (i.e., Melamine haptens)
The specific steps for preparing the compound of formula (I) (i.e. the melamine hapten) are as follows:
(1) 100mg of 2-chloro-4, 6-diaminotriazine was dissolved in 10mL of ethanol to give solution 1.
(2) 130mg of sodium 4-aminobenzoate was sufficiently dissolved in 10mL of a 0.1mol/L aqueous sodium hydrogencarbonate solution to obtain solution 2.
(3) After the steps (1) and (2) are completed, slowly adding the solution 1 into the solution 2, uniformly mixing, and then stirring and reacting for 24 hours at 70 ℃.
(4) And (4) taking the reaction system which finishes the step (3), adding 0.1mol/L hydrochloric acid aqueous solution to adjust the pH value to 6.0, filtering, and collecting precipitate.
(5) And (4) adding a small amount of water into the precipitate collected in the step (4) for washing, and then carrying out vacuum drying to obtain powder, namely the melamine hapten.
2. Preparation of Compound of formula (II) (i.e., Melamine-bovine serum Albumin conjugate)
The specific steps for preparing the compound shown in the formula (II) (namely, the melamine-bovine serum albumin conjugate) are as follows:
(1) taking 40mg of melamine hapten, adding 5mL of N, N-Dimethylformamide (DMF) and fully dissolving; then adding 38 mu L of tri-n-butylamine under the ice bath condition, and stirring for reaction for 10 min; finally, 22 mu L of isobutyl chloroformate is added to react for 1h at room temperature, and the activated melamine hapten solution is obtained.
(2) Taking 100mg of Bovine Serum Albumin (BSA), adding 10mL of 0.1mol/L sodium carbonate aqueous solution, and fully dissolving; dropwise adding the melamine hapten solution obtained in the step (1), and stirring at room temperature overnight; finally, the mixture was dialyzed at 4 ℃ for 72 hours (during which the dialyzate was changed 6 times) using 0.01MPBS buffer solution of pH7.4 as a dialyzate to obtain a dialyzed product.
(3) And (3) taking the dialysis product obtained in the step (2), and passing through a filter membrane of 0.2 mu m under the aseptic condition to obtain a filtrate.
The filtrate is the melamine-bovine serum albumin conjugate.
The melamine-bovine serum albumin conjugate is used as a melamine immune antigen.
3. Preparation of Compound of formula (III) (i.e., Melamine-ovalbumin conjugate)
The specific steps for preparing the compound represented by the formula (III) (i.e. the melamine-ovalbumin conjugate) are as follows:
(1) taking 40mg of melamine hapten, adding 5mL of N, N-Dimethylformamide (DMF) and fully dissolving; then adding 38 mu L of tri-n-butylamine under the ice bath condition, and stirring for reaction for 10 min; finally, 22 mu L of isobutyl chloroformate is added to react for 1h at room temperature, and the activated melamine hapten solution is obtained.
(2) Adding 10mL of 0.1mol/L sodium carbonate aqueous solution into 100mg of Ovalbumin (OVA), and fully dissolving; dropwise adding the melamine hapten solution obtained in the step (1), and stirring at room temperature overnight; finally, the mixture was dialyzed at 4 ℃ for 72 hours (during which the dialyzate was changed 6 times) using 0.01MPBS buffer solution of pH7.4 as a dialyzate to obtain a dialyzed product.
(3) And (3) taking the dialysis product obtained in the step (2), and passing through a filter membrane of 0.2 mu m under the aseptic condition to obtain a filtrate.
The filtrate is the melamine-ovalbumin conjugate.
A melamine-ovalbumin conjugate as the melamine coating antigen.
Preparation of melamine monoclonal antibody
The Balb/c mice are 8-week-old healthy Balb/c female mice (product of Beijing Wittingle laboratory animal technology Co., Ltd.).
The melamine-bovine serum albumin conjugate was diluted with 0.1MPBS buffer, pH7.4, to give solution A.
1. Basic immunity
(1) 1mL of the solution A and 1mL of a complete adjuvant (product of Sigma Co., USA) were mixed, and stirred at 4 ℃ until sufficiently emulsified (the mixture was not dispersed in water, i.e., sufficiently emulsified), to obtain an emulsified complete antigen.
(2) Taking the emulsified complete antigen, injecting Balb/c mice into the neck and back part of the patient at multiple subcutaneous points, wherein the injection dose is 50-100 mu g per mouse.
2. Boosting immunity
(1) 1mL of the solution A and 1mL of an incomplete adjuvant (product of sigma company, USA) are mixed, and stirred at 4 ℃ until the mixture is fully emulsified (the mixture is not dispersed in water, namely, the mixture is fully emulsified), so that emulsified incomplete antigen is obtained.
(2) Taking emulsified incomplete antigen, and immunizing Balb/c mice after completing the steps for one or two weeks for 4 times, wherein each immunization is separated by two weeks. The steps of each immunization are as follows: the emulsified incomplete antigen was injected subcutaneously into Balb/c mice at multiple points on the back of the neck. The injection dose was 50-100. mu.g/mouse (calculated as BSA).
(3) Taking emulsified incomplete antigen, and injecting the Balb/c mice two weeks after the completion of the step (2) into the abdominal cavity. The injection dose was 50-100. mu.g/mouse (calculated as BSA).
3. Obtaining and cloning of hybridoma cells
(1) On day 4 when step 2 was completed, Balb/c mice were enucleated and bled to death, and spleen cells from Balb/c mice were collected.
(2) After completion of step (1), spleen cells of Balb/c mice were subjected to cell fusion with SP2/0 myeloma cells (ratio (4-8): 1) to give several fused cells.
(3) And (3) measuring the supernatant of the fusion cells obtained in the step (2) by adopting indirect competitive ELISA (melamine-ovalbumin conjugate as coating antigen), and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody. The hybridoma cells were subjected to scale-up culture and then cryopreserved.
4. Preparation and purification of melamine monoclonal antibody
The melamine monoclonal antibody is prepared by adopting a method of inducing an animal endocrine monoclonal antibody. The method comprises the following specific steps:
(1) 0.5mL of sterile paraffin oil was injected into the abdominal cavity of each Balb/c mouse.
(2) And (3) injecting the hybridoma cells obtained in the step (3) into the abdominal cavity of the Balb/c mouse 7-14 days after the step (1) is completed. The injection dose of hybridoma cells was 5X 105-106One/only.
(3) After 7-10 days from the completion of step (1), ascites was collected.
(4) And (4) taking the ascites collected in the step (3), and purifying the ascites by using an octanoic acid-saturated ammonium sulfate method to obtain the melamine monoclonal antibody.
Through detection, the amino acid sequence of the variable region of the heavy chain of the melamine monoclonal antibody is shown as the sequence 1 in the sequence table, and the amino acid sequence of the variable region of the light chain of the melamine monoclonal antibody is shown as the sequence 2 in the sequence table.
Preparation of colloidal gold test paper for detecting melamine
1. Preparation of quenched antibody conjugate pad
(1) Adding 2-4mL of 0.5-5% trisodium citrate solution into boiling 100mL of 1% chloroauric acid aqueous solution to obtain colloidal gold solution; the diameter of the colloidal gold particles in the colloidal gold solution is about 20-50 nm.
(2) Mu.g of the melamine monoclonal antibody was diluted to 5mL with 150mM PBS buffer solution (pH 7.4) to obtain a monoclonal antibody dilution.
(3) And (3) adding 10-25g of colloidal gold solution into the monoclonal antibody diluent obtained in the step (2), uniformly mixing, and standing overnight at 4 ℃ to obtain a gold-labeled antibody suspension.
(4) And (3) spreading and soaking the glass cellulose membrane in the gold-labeled antibody suspension, taking out the glass cellulose membrane, baking the glass cellulose membrane at 37 ℃ overnight, and cutting the glass cellulose membrane into strips with the width of 18mm and the length of 300mm to obtain the quenched antibody binding pads.
2. Preparation of nitrocellulose membrane coating quality control line and detection line
(1) The melamine-bovine serum albumin conjugate was diluted with PBS buffer (pH 7.4, 150 mM) to obtain a detection membrane-scribing solution with a concentration of 0.15 mg/mL.
(2) Goat anti-mouse secondary antibody (product of Jackson ImmunoResearch, USA) was diluted with 0.1-1mM PBS buffer at pH7.4 to obtain a quality control membrane-cutting solution at a concentration of 0.2 mg/mL.
(3) And respectively and uniformly scratching the quality control scratching solution and the detection scratching solution on the nitrocellulose membrane (the scratching amount is 0.5-0.6 mu L/cm) to form a quality control line (C) and a detection line (T) which are separated from each other, and naturally drying at room temperature to obtain the nitrocellulose membrane coated with the quality control line and the detection line. And the nitrocellulose membrane is coated with the quality control line and the detection line, and the distance between the quality control line and the detection line is 2-4 mm.
3. Preparation of fluorescent backing sheet
(1) And dissolving tetraethyl rhodamine by using Tris-HCl buffer solution with the concentration of 0.1M to obtain tetraethyl rhodamine solution with the concentration of 0.01% (M/v).
(2) After the step (1) is finished, adding ProClin 300 (serving as a preservative) into the tetraethyl rhodamine solution to obtain a working solution; the concentration of ProClin 300 in the working solution was 0.02% (v/v).
(3) And (3) uniformly spraying the working solution obtained in the step (2) on a PVC plate, and covering a plastic packaging film with good light transmission (so as to prevent the fluorescent substance from falling off in the storage and use processes).
4. Assembly of colloidal gold test paper for detecting melamine
The colloidal gold test paper for detecting melamine consists of a water absorption pad (namely a water absorption material such as filter paper), a nitrocellulose membrane coating a quality control line and a detection line, a quenching antibody combination pad, a sample pad (made of glass fiber cotton) and a fluorescent back plate.
The assembly steps of the colloidal gold test paper for detecting melamine are as follows: adhering a water absorption pad to one end (the overlapping length is 0.2cm) of a nitrocellulose membrane coating a quality control line and a detection line by using double-sided adhesive, adhering one end (the overlapping length is 0.2cm) of a quenching antibody combination pad to the other end of the nitrocellulose membrane coating the quality control line and the detection line by using double-sided adhesive, adhering a sample pad (the overlapping length is 0.2cm) to the other end of the quenching antibody combination pad by using double-sided adhesive, and finally adhering the sample pad and the quenching antibody combination pad to a fluorescent backboard (the nitrocellulose membrane coating the quality control line and the detection line is positioned in the center of the fluorescent backboard) by using double-sided adhesive and cutting the sample pad into the colloidal gold test paper for detecting melamine, wherein the width of the colloidal gold test paper is 4-5mm (specifically 4.72 mm). The colloidal gold test paper is sealed by a plastic package bag and then is stored in a drying box for later use.
The overlapping area in the colloidal gold test paper ensures the continuity of the flow of the sample liquid, so that the reaction can fully occur, and the experimental error is effectively reduced. The fluorescence area of the fluorescence backboard releases fluorescence signals under the action of corresponding exciting light, in the colloidal gold immunochromatography reaction, the detection line and the quality control line are combined with the aggregated colloidal gold particles to quench the fluorescence signals at the position, and the quenching degree of the fluorescence signals is positively correlated with the color development intensity of the detection line and the quality control line, so that the purpose of quantitative detection of melamine is realized.
The structure of the colloidal gold test paper for detecting melamine is schematically shown in figure 1. According to the requirement, a sample adding hole can be arranged on the sample pad.
Example 2 method for detecting melamine content in solution to be detected by colloidal gold test paper
1. Drawing a melamine standard curve
(1) Dissolving and diluting a melamine standard product (a product of Shanghai' an spectral laboratory science and technology Co., Ltd.) with water to obtain a melamine standard product diluent; the concentration of the melamine standard dilution is 0. mu.g/L, 5. mu.g/L, 10. mu.g/L, 25. mu.g/L, 50. mu.g/L or 100. mu.g/L.
(2) And (3) taking the colloidal gold test paper for detecting melamine, adding 120 mu L of melamine standard substance diluent on the sample pad, standing for 10min, and inserting the colloidal gold test paper into a fluorescence immunoassay quantitative analyzer to obtain a corresponding F1/F2 ratio.
(3) And drawing a melamine standard curve by taking the concentration of the melamine standard product diluent as an abscissa and the corresponding F1/F2 ratio as an ordinate.
The standard curve for melamine is shown in figure 2.
The standard curve equation of melamine is as follows: y ═ 0.04+ (3.12+0.04)/(1+ (x/7.85)1.24)。
2. Detecting the content of melamine in a solution to be detected
(1) Taking the colloidal gold test paper for detecting melamine, adding 120 mu L of solution to be detected (such as liquid milk sample and milk powder solution) on the sample pad, standing for 10min, and inserting the colloidal gold test paper into a fluorescence immunoassay quantitative analyzer to obtain the ratio of F1/F2 of the solution to be detected.
(2) And substituting the F1/F2 ratio of the solution to be detected into a melamine standard curve equation to obtain the melamine content in the solution to be detected.
The solution to be tested can be a milk-like or milk powder solution.
Example 3 minimum detection Limit of colloidal gold test paper for Melamine detection
1. Drawing a melamine standard curve
Same as step 1 in example 2.
2. The melamine content in 20 fresh milk samples was measured separately.
The steps for detecting the melamine content in each raw milk sample are as follows: taking colloidal gold test paper for detecting melamine, adding 120 mu L of raw milk sample on the sample pad, standing for 10min, and inserting the colloidal gold test paper into a fluorescence immunoassay quantitative analyzer to obtain the ratio of F1/F2 of the raw milk sample; and substituting the F1/F2 ratio of the raw and fresh milk sample into a melamine standard curve equation to obtain the melamine content in the raw and fresh milk sample.
The results are shown in Table 1.
TABLE 1
| Fresh milk sample number | Melamine content (μ g/L) | Fresh milk sample number | Melamine content (μ g/L) |
| 1 | 1.10 | 11 | 1.56 |
| 2 | 2.30 | 12 | 1.71 |
| 3 | 2.92 | 13 | 2.96 |
| 4 | 1.57 | 14 | 1.35 |
| 5 | 3.25 | 15 | 1.18 |
| 6 | 4.34 | 16 | 1.34 |
| 7 | 1.87 | 17 | 1.28 |
| 8 | 1.40 | 18 | 1.09 |
| 9 | 2.03 | 19 | 2.49 |
| 10 | 2.82 | 20 | 2.55 |
3. After completion of step 2, the average and standard deviation of the melamine content in 20 samples of raw milk were calculated.
The results show that the average melamine content in 20 raw milk samples was 2.06. mu.g/L, and the standard deviation was 0.88. mu.g/L.
4. And (4) after the step 3 is completed, further obtaining the minimum detection limit of the colloidal gold test paper.
The minimum detection limit is the mean +3 × standard deviation.
The results show that the minimum detection limit of the colloidal gold test paper is 4.70 mug/L. In order to ensure the stability of the product, the detection limit of the colloidal gold test paper for detecting melamine in the fresh milk is about 5 mug/L.
Example 4 accuracy test of colloidal gold test paper for detecting Melamine
First, accuracy experiment
1. Drawing a melamine standard curve
The same procedure as in step two 1 of example 2.
2. Detecting the content of melamine in a solution to be detected
The solution to be tested is fresh milk containing 5 mug/L melamine or fresh milk containing 10 mug/L melamine.
The experiment was repeated 3 times to take the average (i.e. between batches). For each replicate, 5 replicates of each test solution were averaged (i.e., within the batch). The steps for each repetition are as follows:
(1) and (3) taking the colloidal gold test paper for detecting the melamine, adding 120 mu L of the solution to be detected to the sample pad, standing for 10min, and inserting the colloidal gold test paper into a fluorescence immunoassay quantitative analyzer to obtain the F1/F2 ratio of the solution to be detected.
(2) And substituting the F1/F2 ratio of the solution to be detected into a melamine standard curve equation to obtain the melamine content (average value +/-standard deviation) in the solution to be detected.
(3) The recovery (%) and the coefficient of variation CV (%) were counted.
Recovery rate (measured average value/added concentration) × 100%.
Coefficient of variation CV ═ (measured value standard deviation/measured average) × 100%.
The results are shown in Table 2. The results show that the recovery rate is 88.70-109.20%, and the coefficient of variation CV is lower than 10%.
TABLE 2
Second, accuracy experiment second
The solution to be tested was 50 parts of fresh breast blindness sample. 50 parts of fresh milk blind sample are numbered as 1-50 in sequence.
1. The content of melamine in 50 parts of fresh milk blind sample is detected by adopting colloidal gold test paper for detecting melamine
(1) Drawing a melamine standard curve
The same procedure as in step two 1 of example 2.
(2) Detecting the melamine content in 50 parts of fresh milk blind sample
(1) And (3) taking colloidal gold test paper for detecting melamine, respectively adding 120 mu L of fresh milk blind sample on the sample pad, standing for 10min, and inserting the colloidal gold test paper into a fluorescence immunoassay quantitative analyzer to obtain the ratio of F1/F2 of the solution to be detected.
(2) And substituting the F1/F2 ratio of the solution to be detected into a melamine standard curve equation to obtain the melamine content in the solution to be detected.
The results of the colloidal gold test paper are shown in Table 3, column 2.
2. The operation is carried out according to the liquid chromatography-mass spectrometry/mass spectrometry in GBT 22388-2008 < method for detecting melamine in raw milk and dairy products >, and the content of melamine in 50 parts of raw and fresh milk blind samples is detected by adopting the gas chromatography-mass spectrometry/mass spectrometry.
The results of the gas chromatography-mass spectrometry/mass spectrometry are shown in column 3 of table 3.
The result shows that the results of the melamine content detection by the colloidal gold test paper for detecting melamine provided by the invention are basically consistent with the results of the gas chromatography-mass spectrometry/mass spectrometry melamine content. The colloidal gold test paper provided by the invention has higher accuracy in detecting the melamine content.
TABLE 3
Note: "ND" means that no melamine was detected.
<110> Peking Weideweikang biotechnological company Beijing market feed monitoring institute
<120> method for detecting melamine content and special colloidal gold test paper thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223>
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Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
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Ser Ser Ala Ser Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu
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Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro
35 40 45
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
50 55 60
Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met
65 70 75 80
Tyr Tyr Cys Ala Arg His Phe Tyr Tyr Tyr Ser Tyr Ala Met Asp Tyr
85 90 95
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
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<210> 2
<211> 117
<212> PRT
<213> Artificial sequence
<220>
<223>
<400> 2
Met Ala Trp Ile Ser Leu Ile Leu Ser Leu Leu Ala Leu Ser Ser Gly
1 5 10 15
Gly Ala Gly Ala Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr
20 25 30
Ser Pro Gly Glu Thr Val Gly Glu Thr Cys Arg Ser Ser Thr Gly Ala
35 40 45
Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His
50 55 60
Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val
65 70 75 80
Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
85 90 95
Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu
100 105 110
Trp Tyr Ser Asn His
115
Claims (8)
1. The colloidal gold test paper for detecting the melamine content is characterized in that: the quenching antibody combination pad is coated with colloidal gold marked by melamine antibody, the quality control line position on the reaction membrane is coated with the antibody of the melamine antibody, the detection line position is coated with the antigen of the melamine antibody, and fluorescent substance is sprayed on the back plate or under the reaction membrane;
the melamine antibody is a melamine monoclonal antibody;
the melamine monoclonal antibody consists of a heavy chain and a light chain; the amino acid sequence of the variable region of the heavy chain is shown as a sequence 1 in a sequence table; the amino acid sequence of the variable region of the light chain is shown as a sequence 2 in a sequence table.
2. The colloidal gold test paper according to claim 1, wherein: the antibody of the melamine antibody is a goat anti-mouse IgG antibody.
3. The colloidal gold test paper according to claim 1, wherein: the antigen of the melamine antibody is a conjugate of a compound shown as a formula (I) and a carrier protein;
the carrier protein is bovine serum albumin or egg white albumin.
4. The colloidal gold test paper according to claim 1, wherein: the fluorescent substance is tetraethyl rhodamine.
5. The colloidal gold test paper according to claim 1, wherein: the reaction membrane is a nitrocellulose membrane.
6. The colloidal gold test paper according to claim 1, wherein: the material of the quenching antibody combining pad is glass cellulose membrane.
7. A method for detecting the melamine content in a solution to be detected by using the colloidal gold test paper of any one of claims 1 to 6, comprising the following steps:
(1) immersing the sample pad of the colloidal gold test paper of any one of claims 1 to 6 in a solution to be tested, standing for more than 10min, and then inserting the colloidal gold test paper into a quantitative fluorescence immunoassay analyzer to obtain a ratio of F1/F2;
F1/F2 ratio is the fluorescence intensity of the blank space between the quality control line and the detection line (F1)/the fluorescence intensity of the detection line (F2);
(2) immersing the sample pad of the colloidal gold test paper of any one of claims 1 to 6 in a melamine standard solution, standing for more than 10min, and then inserting the colloidal gold test paper into a fluorescence immunoassay quantitative analyzer to obtain a corresponding F1/F2 ratio;
(3) and (3) drawing a standard curve according to the concentration of melamine in the melamine standard solution and the corresponding F1/F2 ratio, and then substituting the F1/F2 ratio obtained in the step (1) into the standard curve to obtain the melamine content in the solution to be detected.
8. The method of claim 7, wherein: the solution to be tested is a milk sample.
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| CN102401829B (en) * | 2010-09-17 | 2015-04-29 | 上海鑫谱生物科技有限公司 | Immune response analysis method based on principle of fluorescent quenching |
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