CN108845131B - Colloidal gold immunochromatography detection card for detecting bisphenol B, and preparation method and application thereof - Google Patents
Colloidal gold immunochromatography detection card for detecting bisphenol B, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a colloidal gold immunochromatographic assay detection card for detecting bisphenol B, belonging to the technical field of additive detection. The detection card is provided with a sample pad, a colloidal gold combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped and stuck on the bottom plate; the colloidal gold bonding pad is adsorbed with a colloidal gold labeled anti-bisphenol B monoclonal antibody; and a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from the sample pad end to the water absorption pad end, the detection line is coated with a bisphenol B-carrier protein conjugate, and the quality control line is coated with a mouse-resistant second antibody. The detection card can be used for quickly and specifically qualitatively detecting bisphenol B contained in plastic products such as plastic feeding bottles and the like, has the advantage of strong specificity, is wide in application range, easy to popularize and use and capable of detecting in batches or in time by using a single sample due to the fact that the sample size is small, the detection speed is high, and the detection time is 5-10 min.
Description
Technical Field
The invention relates to the technical field of additive detection, in particular to a colloidal gold immunochromatography detection card for detecting bisphenol B, and a preparation method and application thereof.
Background
Bisphenol A, known under the chemical name 2, 2-bis (4-hydroxyphenyl) propane, abbreviated BPA, may be present in many plastic articles. However, bisphenol A is an estrogen analogue, can cause feminization of boys and sexual precocity of girls, and has an influence on the growth and development of infants. The use of bisphenol a as a chemical substance in food and beverage containers such as baby bottles has been banned in the united states. Bisphenol B, 2, 2-bis (4-hydroxyphenyl) butane, abbreviated as BPB, is very similar in chemical structure to bisphenol a. Animal experiments show that bisphenol B also has similar estrogenic effects to bisphenol A in biological properties.
GB 14942 + 1994 polycarbonate molded product sanitary standard for food containers and packaging materials stipulates: the free amount of phenols in the milk bottle dissolved out by refluxing distilled water for 6 hours is not more than 0.05 mg/kg.
At present, the detection of bisphenol B in the prior art comprises a titration method, a colorimetric method, an oscillography polarography, a liquid chromatography fluorescence method and a high performance liquid chromatography-tandem mass spectrometry method, and the methods generally have the defects of complex operation, high requirement on experimental instruments and equipment, need of special technical personnel for operation and the like.
Therefore, it is highly desirable to develop a method for rapidly detecting bisphenol B in situ, which is capable of conveniently and rapidly detecting bisphenol B.
Disclosure of Invention
Therefore, it is necessary to provide a colloidal gold immunochromatographic assay detection card for detecting bisphenol B, which has the characteristics of convenience, rapidness, intuition and low cost and is suitable for rapid field detection.
A colloidal gold immunochromatographic assay detection card for detecting bisphenol B is provided with a sample pad, a colloidal gold combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped and stuck on a bottom plate;
the colloidal gold bonding pad is adsorbed with a colloidal gold labeled anti-bisphenol B monoclonal antibody;
and a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from the sample pad end to the water absorption pad end, the detection line is coated with a bisphenol B-carrier protein conjugate, and the quality control line is coated with a mouse-resistant second antibody.
The detection card detects the bisphenol B by adopting a chromatography type antibody immune competition method, has the characteristics of convenience, rapidness, intuition and low cost, and is suitable for rapid field detection.
In one embodiment, the sample pad end and the absorbent pad end of the detection card are further provided with a sample end protection film and a handle end protection film respectively, the sample end protection film covers the sample pad, the colloidal gold bonding pad and the overlapping end of the nitrocellulose membrane and the colloidal gold bonding pad, and the handle end protection film covers the absorbent pad and the overlapping end of the nitrocellulose membrane and the absorbent pad. Through the practicality of above-mentioned protection film, interference and the pollution that receive in can avoiding the testing process ensure the reliable and stable of testing result.
In one embodiment, the base plate is a PVC strip; the sample pad and the colloidal gold combined pad are made of glass fiber cotton; the water absorption pad is made of water absorption filter paper; the protective film is an opaque adhesive film.
In one embodiment, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, or human serum albumin; the anti-mouse secondary antibody is a goat anti-mouse IgG antibody, a rabbit anti-mouse IgG antibody or a donkey anti-mouse IgG antibody.
In one embodiment, the bisphenol B-carrier protein conjugate is prepared by: dispersing bisphenol B, 3-bromopropylamine and anhydrous potassium carbonate into acetone, heating and refluxing for reaction to obtain bisphenol B hapten, adding the purified bisphenol B hapten, N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) into dimethyl sulfoxide (DMSO) for dissolving, stirring for reaction, weighing carrier protein, and dissolving in NaHCO solution3Obtaining a carrier protein solution, adding a solution containing bisphenol B hapten into the carrier protein solution for reaction, and dialyzing the obtained solution to obtain the bisphenol B hapten;
the anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) animal immunization: taking the bisphenol B-carrier protein conjugate, emulsifying the conjugate with Freund complete adjuvant completely, immunizing a female Balb/C mouse with the age of 6-8 weeks, breaking the tail after immunization, taking blood, measuring the titer, and selecting the mouse with the best result for fusion;
(B) cell fusion: fusing selected mouse spleen cells and mouse myeloma SP2/0 cells, screening the fused cells by HAT culture medium, determining supernatant by indirect ELISA method, screening out hybridoma cell strains which stably secrete monoclonal antibodies against bisphenol B;
(C) preparing an antibody: and (3) taking the hybridoma cell strain, performing in-vitro conventional culture and passage, injecting the hybridoma cell strain into the abdominal cavity of a Balb/C mouse, extracting ascites, purifying the ascites by using caprylic acid-ammonium sulfate precipitation, and obtaining the purified anti-bisphenol B-monoclonal antibody by using an affinity chromatography.
The colloidal gold labeled anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) preparing a colloidal gold solution: adding trisodium citrate into water, boiling, adding chloroauric acid, stirring and heating, stopping heating when the solution color becomes transparent mauve, adding water to original volume, cooling, and storing for use;
(B) preparation of colloidal gold marker: taking the prepared colloidal gold solution, and using K2CO3Adjusting the pH value of the colloidal gold solution; mixing the anti-bisphenol B monoclonal antibody solution with the colloidal gold solution, incubating at room temperature, centrifuging and then discarding the supernatant; and dissolving and diluting the obtained precipitate by using PBS buffer solution to obtain the anti-bisphenol B monoclonal antibody marked by the colloidal gold.
In one embodiment, the bisphenol B-carrier protein conjugate is prepared by: dispersing 0.24g of bisphenol B, 0.15g of 3-bromopropylamine and 0.70g of anhydrous potassium carbonate into 20mL of dry acetone, heating and refluxing for 8h to obtain a bisphenol B hapten, adding the purified bisphenol B hapten, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a beaker, adding 3mL of dimethyl sulfoxide to dissolve the mixture, magnetically stirring the mixture overnight, then weighing 0.12g of carrier protein, and dissolving the carrier protein in 5mL of 0.1mol/LNaHCO with the pH value of 7.03Adding a solution containing bisphenol B hapten into the carrier protein solution, slowly dripping while stirring, reacting overnight, and obtaining a solution with the temperature of 4 DEG CDialyzing for 3 days to obtain;
the anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) animal immunization: taking the bisphenol B-carrier protein conjugate, emulsifying the conjugate with equivalent volume of Freund complete adjuvant completely, immunizing female Balb/C mice of 6-8 weeks old, immunizing for 1 time at intervals of 2 weeks, cutting off the tail after three times of immunization, taking blood to measure titer, and selecting the mice with the best result for fusion;
(B) cell fusion: fusing selected mouse spleen cells and mouse myeloma SP2/0 cells, screening the fused cells by HAT culture medium, determining supernatant by indirect ELISA method, screening out hybridoma cell strains which stably secrete monoclonal antibodies against bisphenol B;
(C) preparing an antibody: taking hybridoma cell strains, performing in-vitro conventional culture and passage, injecting into the abdominal cavity of a Balb/C mouse, extracting ascites after 10 days, purifying the ascites by caprylic acid-ammonium sulfate precipitation, and obtaining a purified anti-bisphenol B-monoclonal antibody by an affinity chromatography;
the colloidal gold labeled anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) preparing a colloidal gold solution: adding 1mL of 1% trisodium citrate into 100mL of ultrapure water, boiling, quickly adding 1mL of 1% chloroauric acid, continuously stirring and heating, maintaining for 5min when the color of the solution is changed into transparent mauve, stopping heating, replenishing water to the original volume, cooling to room temperature, and storing at 4 ℃ for later use;
(B) preparation of colloidal gold marker: taking 40mL of prepared colloidal gold solution, and using 0.1mol/L K2CO3Adjusting the pH value of the colloidal gold solution to 8.2; mixing the equivalent anti-bisphenol B monoclonal antibody solution with the colloidal gold solution under magnetic stirring, incubating at room temperature for 15min, centrifuging at 10000r/min for 30min, and removing the supernatant; dissolving and diluting the obtained precipitate with PBS buffer solution containing 2% BSB and 0.1% sodium azide and having pH of 7.4 to obtain the colloidal gold labeled anti-bisphenol B monoclonal antibody, and storing at 4 ℃ for later use.
In one embodiment, the concentration of the colloidal gold labeled anti-bisphenol B monoclonal antibody is 40-80 μ g/mL, preferably 60 μ g/mL, and the dosage on the detection card is 4-6 μ L/cm, preferably 5 μ L/cm;
the concentration of the bisphenol B-carrier protein conjugate coated on the detection line is 0.1-0.3 mu g/mL, preferably 0.2mg/mL, and the dosage on the detection card is 0.5-1.5 mu L/cm, preferably 1.0 mu L/cm;
the concentration of the anti-mouse secondary antibody coated by the quality control line is 0.1-0.3, preferably 0.2mg/mL, and the dosage on the detection card is 0.5-1.5 muL/cm, preferably 1.0 muL/cm.
The invention also discloses a preparation method of the colloidal gold immunochromatographic assay detection card for detecting bisphenol B, which comprises the following steps:
(1) preparing a colloidal gold bonding pad: spraying the anti-bisphenol B monoclonal antibody-colloidal gold marker on carrier glass fiber cotton, and drying for later use;
(6) preparing a nitrocellulose membrane: spraying the anti-mouse secondary antibody and the bisphenol B-carrier protein conjugate on a nitrocellulose membrane in sequence, and drying for later use;
(7) assembling the detection card: and sequentially adhering a sample pad, a colloidal gold combined pad, a nitrocellulose membrane and a water absorption pad on the bottom plate, respectively adhering a sample end protective film and a handle end protective film on the sample pad end and the water absorption pad end of the detection card, assembling the detection card, and cutting the detection card into a preset width to obtain the detection card.
In one embodiment, in the step of preparing the colloidal gold conjugate pad, the prepared anti-bisphenol B monoclonal antibody-colloidal gold marker is uniformly sprayed on carrier glass fiber cotton at the concentration of 5 muL/cm, dried at 37 ℃ for 3h, sealed and stored at 4 ℃ for later use;
in the preparation step of the nitrocellulose membrane, the anti-mouse secondary antibody and the bisphenol B-carrier protein conjugate with proper concentration are sequentially sprayed on the nitrocellulose membrane at the concentration of 1 mu L/cm, dried for 8h at 37 ℃, sealed and stored for later use at 4 ℃.
The invention also discloses a use method of the colloidal gold immunochromatographic assay detection card for detecting bisphenol B, which comprises the following pretreatment steps: weighing a sample to be detected, adding water, heating in a water bath for reflux, and taking back a distillate obtained by reflux to obtain a solution to be detected.
The detection principle of the detection card of the invention is as follows:
the detection card of the invention applies the chromatographic antibody immune competition principle, and specifically detects the bisphenol B residue in the sample through the color reaction of the antigen (bisphenol B) and the gold-labeled antibody. If the sample solution contains bisphenol B residues, bisphenol B firstly reacts with the anti-bisphenol B monoclonal antibody on the colloidal gold particles, so that when the colloidal gold particles are diffused to the detection line along with the sample solution, the active sites of the antibody on the colloidal gold particles cannot be combined with the bisphenol B specific antigen (bisphenol B-carrier protein conjugate) on the detection line because the active sites are occupied by bisphenol B in the sample solution; when the content of bisphenol B in the sample exceeds the detection limit of the reagent plate, the detection line on the detection card shows lighter color than the quality control line and even colorless, and the detection card is judged to be positive. On the contrary, when the content of bisphenol B in the sample is below the detection limit of the detection card or no residue exists, the color development of the detection line on the reagent plate is close to or deep with the quality control line, and the detection line is judged to be negative.
Compared with the prior art, the invention has the following beneficial effects:
the colloidal gold immunochromatographic assay detection card for detecting bisphenol B adopts the principle of an immunological competition method, combines a colloidal gold labeling technology and an immunochromatographic assay design, and can quickly and specifically detect the bisphenol B contained in plastic products such as plastic feeding bottles in a semi-quantitative manner. Has the advantage of strong specificity, and can avoid the false positive false detection problem of other detection methods. And the detection card has the advantages of small sample amount, high detection speed, 5-10min detection time, wide application range, easy popularization and use and capability of batch or timely detection of a single sample.
Drawings
FIG. 1 is a schematic top view of a detection card in embodiment 1;
FIG. 2 is a schematic cross-sectional view of the detection card of embodiment 1;
in the figure: 1. a base plate; 2. a sample pad; 3. a colloidal gold bonding pad; 4. a nitrocellulose membrane; 5. detecting lines; 6. a quality control line; 7. a water absorbent pad; 8. a sample end protective film; 9. handle end protection film.
FIG. 3 is a schematic diagram of reading the result of the bisphenol B colloidal gold immunochromatographic assay card of the present invention;
in the figure: t is a detection line, and C is a quality control line; in FIG. 3-a, line T is darker or equally darker than line C, and is negative; in FIG. 3-b, the color of line T is lighter than that of line C or line T disappears, and the result is positive; line C disappears in FIG. 3-C, which is invalid.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The starting materials used in the following examples are all commercially available.
Example 1
A colloidal gold immunochromatographic assay detection card for detecting bisphenol B is shown in figure 1-2, and is provided with a sample pad 2, a colloidal gold combination pad 3, a nitrocellulose membrane 4 and a water absorption pad 7 which are sequentially lapped and stuck on a bottom plate 1, wherein the sample pad 2, the colloidal gold combination pad 3, the nitrocellulose membrane 4 and the water absorption pad 7 are overlapped by 1-2 mm in end-to-end manner; the colloidal gold combined pad 3 is adsorbed with a colloidal gold-labeled anti-bisphenol B monoclonal antibody; the nitrocellulose membrane 4 is sequentially provided with a detection line 5 and a quality control line 6 which are parallel to each other from the sample pad end to the water absorption pad end, the detection line 5 is coated with a bisphenol B-carrier protein conjugate, and the quality control line 6 is coated with a mouse-resistant secondary antibody.
In this embodiment, in order to avoid the interference in the detection process, preferably, the detection card sample pad end and the absorbent pad end are further provided with a sample end protection film 8 and a handle end protection film 9, respectively, the sample end protection film 8 covers the overlapping end of the sample pad 2, the gold colloid bonding pad 3, the nitrocellulose 4 film and the gold colloid bonding pad 3, and the handle end protection film 9 covers the overlapping end of the absorbent pad 7, the nitrocellulose 4 film and the absorbent pad 7, as shown in fig. 2.
In this embodiment, the bottom plate is a PVC strip; the sample pad 2 and the colloidal gold combined pad 3 are made of glass fiber cotton; the absorbent pad 7 is made of absorbent filter paper; the sample end protective film 8 and the handle end protective film 9 are opaque films.
When the detection card is used for detection, firstly, a solution to be detected of a sample to be detected is dripped into the sample pad 2 of the bisphenol B colloidal gold immunochromatography detection card, the solution to be detected and the anti-bisphenol B monoclonal antibody-colloidal gold marker contained in the colloidal gold conjugate pad 3 are diffused to the nitrocellulose membrane 4 together due to the siphon action principle, and the result is observed within 5-10 minutes.
The main reaction of the detection card is immunological antigen and antibody reaction, and the colloidal gold labeled antibody migrating on the nitrocellulose membrane reacts with the bisphenol B protein conjugate and the quality control line anti-mouse secondary antibody on the detection line respectively to form a brownish red strip. If the bisphenol B to be detected exists in the sample, the sample reacts with the antibody after being added, and does not react with the bisphenol B contained in the detection line, so that the color is not developed. As shown in fig. 3, the following 3 cases are the results of the main reaction:
1. when the detection line T and the quality control line C are displayed to show a brownish red blotting belt at the same time, and the color of the detection line T is darker than or as dark as the quality control line C, the detection result is negative, which indicates that the content of bisphenol B in the detected sample is not over-standard or bisphenol B is not in the detected sample, as shown in figure 3-a;
2. when the detection line T does not display color, only the quality control line 6 displays a brownish red blot, the detection result is positive, or the color of the detection line is lighter than that of the quality control line C, which indicates that the content of bisphenol B in the detected sample exceeds the standard, and is shown in a figure 3-B;
3. when the quality control line C does not develop color, the test strip is failed, as shown in FIG. 3-C.
Example 2
The preparation method of the colloidal gold immunochromatographic assay card for detecting bisphenol B in example 1 comprises the following steps:
(1) synthesis of bisphenol B-carrier protein conjugate: firstly, 0.24g of bisphenol B, 0.15g of 3-bromopropylamine and 0.70g of anhydrous potassium carbonate are dispersed into 20mL of dry acetone, heated and refluxed for reaction for 8 hours, and purified by a silica gel chromatographic column to obtain the bisphenol B hapten. The purified bisphenol B hapten, NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) were added to a beaker, dissolved by adding 3ml dmsd so (dimethyl sulfoxide), and stirred magnetically overnight. Then 0.12g of carrier protein (bovine serum albumin, commercially available) was weighed out and dissolved in 5mL of 0.1mol/LNaHCO3(pH 7.0) to obtain a carrier protein solution, adding a solution containing bisphenol B hapten into the carrier protein solution, slowly dripping while stirring, reacting overnight, dialyzing the obtained solution at 4 ℃ for 3 days to obtain a purified bisphenol B-carrier protein conjugate, subpackaging, and storing at-20 ℃.
(2) Preparation of anti-bisphenol B monoclonal antibody:
(a) animal immunization: taking the bisphenol B-carrier protein conjugate, emulsifying the conjugate with equivalent volume of Freund complete adjuvant completely, immunizing female Balb/C mice of 6-8 weeks old, immunizing for 1 time at intervals of 2 weeks, breaking tail after three times of immunization, taking blood to measure titer, and selecting the mice with the best result for fusion;
(b) cell fusion: fusing selected mouse spleen cells and mouse myeloma SP2/0 cells, screening the fused cells by HAT culture medium, determining supernatant by indirect ELISA method, screening hybridoma cell strains which stably secrete monoclonal antibodies against bisphenol B;
(c) preparing an antibody: and (3) taking the hybridoma cell strain, performing in-vitro conventional culture and passage, injecting the hybridoma cell strain into the abdominal cavity of the Balb/C mouse, and extracting ascites after 10 days. The ascites is purified by caprylic acid-ammonium sulfate precipitation, and the purified anti-bisphenol B-monoclonal antibody is obtained by affinity chromatography.
(3) Preparing a colloidal gold solution: adding 1mL of 1% trisodium citrate into 100mL of ultrapure water, boiling, rapidly adding 1mL of 1% chloroauric acid, stirring and heating, maintaining for 5min when the solution color is completely changed into transparent mauve, stopping heating, replenishing water to original volume, cooling to room temperature, and storing at 4 deg.C for use.
(4) Preparation of colloidal gold marker: taking 40mL of prepared colloidal gold solution, and using 0.1mol/L K2CO3Adjusting the pH value of the colloidal gold solution to 8.2; mixing the equivalent anti-bisphenol B monoclonal antibody solution with the colloidal gold solution under magnetic stirring, incubating at room temperature for 15min, centrifuging at 10000r/min for 30min, and removing the supernatant; the obtained precipitate was dissolved and diluted with PBS buffer (pH7.4) containing 2% BSA and 0.1% sodium azide to obtain a colloidal gold-labeled anti-bisphenol B monoclonal antibody, which was stored at 4 ℃ for further use.
(5) Preparing a colloidal gold bonding pad: the prepared 60mg/ml anti-bisphenol B monoclonal antibody-colloidal gold marker is uniformly sprayed on carrier glass fiber cotton at the concentration of 5 mu L/cm, dried for 3h at 37 ℃, sealed and stored for later use at 4 ℃.
(6) Preparing a nitrocellulose membrane: mixing the following components in parts by weight: 100 diluted anti-mouse secondary antibody (goat anti-mouse IgG, commercially available, concentration of 0.2mg/ml) bisphenol B-carrier protein conjugate (concentration of 0.2. mu.g/ml) was sequentially sprayed on a nitrocellulose membrane at a concentration of 1. mu.L/cm, dried at 37 ℃ for 8h, sealed, and stored at 4 ℃ for further use.
(7) Assembling the detection card: a sample pad, a colloidal gold combined pad, a nitrocellulose membrane and a water absorption pad are sequentially adhered on the PVC base plate, a sample end protective film and a handle end protective film are respectively adhered at the sample pad end and the water absorption pad end of the detection card, the detection card is assembled, the detection card is cut into a preset width, and the detection card is sealed and stored.
Example 3
Bisphenol B minimum detection limit test.
First, experiment method
Accurately weighing 0.500g of bisphenol B standard product, dissolving in ultrapure water and fixing the volume to 100mL, marking as B1 solution, taking 1mL of B1 solution, adding ultrapure water in a volumetric flask and fixing the volume to 1000mL to form B2 solution, taking 1mL of B2 solution, adding ultrapure water in the volumetric flask and fixing the volume to 1000mL to form B3 solution, wherein the concentration of the B3 solution is 5ng/mL (5 ppb).
Taking 1mL of B3 solution in 5 test tubes, respectively marking by 1-5, respectively adding 19mL, 9mL, 4mL, 1mL and 0mL of ultrapure water into the test tubes to prepare bisphenol B standard solution with the concentrations respectively as follows: 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5 ng/mL; another empty tube was taken and 5mL of ultrapure water, labeled test tube No. 6, was added.
And taking the liquid in the No. 1-6 test tube as a sample, dropwise adding the sample to the colloidal gold immunochromatographic detection card for detecting bisphenol B in example 1, and observing the result 5min after adding the sample.
And II, obtaining a result.
The color development results of the detection line T and the quality control line C are shown in Table 1.
TABLE 1 bisphenol B minimum detection limit test
As can be seen from the above results, the detection limit of the colloidal gold immunochromatographic detection card for semi-quantitatively detecting bisphenol B of example 1 was 0.5 ng/mL.
Example 4
The sample is detected by using the colloidal gold immunochromatographic detection card for detecting bisphenol B in example 1, and the operation method is as follows: weighing about 1g of sample to be detected (food packaging product and baby bottle) respectively, accurately measuring to 0.01g, adding 100mL of distilled water, heating in water bath, boiling slightly, refluxing for 6 hr, taking the distillate as the solution to be detected, adding dropwise onto the detection card, and observing the result 5min after sample addition.
And simultaneously, the sample to be detected is subjected to parallel detection by a liquid chromatography tandem mass spectrometry, and the detection method is referred to the literature (ultra high performance liquid chromatography-tandem mass spectrometry is used for rapidly determining 7 bisphenol compounds [ J ] in paper food packaging materials and containers, J.China health inspection, 2017, 27(13): 1858-.
The results are shown in the following table:
TABLE 2 results of sample testing
From the above results, it can be seen that the detection card of example 1 can detect bisphenol B in food packaging products, and has good consistency compared with the detection method by liquid chromatography tandem mass spectrometry.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A colloidal gold immunochromatographic assay detection card for detecting bisphenol B is characterized in that a sample pad, a colloidal gold bonding pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped and stuck on a bottom plate are arranged on the detection card;
the colloidal gold bonding pad is adsorbed with a colloidal gold labeled anti-bisphenol B monoclonal antibody;
a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from the sample pad end to the water absorption pad end, the detection line is coated with a bisphenol B-carrier protein conjugate, and the quality control line is coated with a mouse-resistant second antibody:
the bisphenol B-carrier protein conjugate is prepared byThe preparation method comprises the following steps: dispersing 0.24g of bisphenol B, 0.15g of 3-bromopropylamine and 0.70g of anhydrous potassium carbonate into 20mL of dry acetone, heating and refluxing for 8h to obtain a bisphenol B hapten, adding the purified bisphenol B hapten, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a beaker, adding 3mL of dimethyl sulfoxide to dissolve the mixture, magnetically stirring the mixture overnight, then weighing 0.12g of carrier protein, and dissolving the carrier protein in 0.1mol/LNaHCO dissolved in 5mLpH7.03Adding a solution containing bisphenol B hapten into the carrier protein solution, slowly dripping while stirring, reacting overnight, and dialyzing the obtained solution at 4 ℃ for 3 days to obtain a carrier protein solution;
the anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) animal immunization: taking the bisphenol B-carrier protein conjugate, emulsifying the conjugate with Freund complete adjuvant completely, immunizing a female Balb/C mouse with the age of 6-8 weeks, breaking the tail after immunization, taking blood, measuring the titer, and selecting the mouse with the best result for fusion;
(B) cell fusion: fusing selected mouse spleen cells and mouse myeloma SP2/0 cells, screening the fused cells by HAT culture medium, determining supernatant by indirect ELISA method, screening out hybridoma cell strains which stably secrete monoclonal antibodies against bisphenol B;
(C) preparing an antibody: taking hybridoma cell strains, performing in-vitro conventional culture and passage, injecting the hybridoma cell strains into the abdominal cavity of a Balb/C mouse, extracting ascites, purifying the ascites by caprylic acid-ammonium sulfate precipitation, and obtaining a purified anti-bisphenol B-monoclonal antibody by an affinity chromatography;
the colloidal gold labeled anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) preparing a colloidal gold solution: adding trisodium citrate into water, boiling, adding chloroauric acid, stirring and heating, stopping heating when the solution color becomes transparent mauve, adding water to original volume, cooling, and storing for use;
(B) preparation of colloidal gold marker: taking 40mL of prepared colloidal gold solution, and using 0.1mol/L K2CO3Adjusting the pH value of the colloidal gold solution to 8.2; get etcWeighing the anti-bisphenol B monoclonal antibody solution, mixing with the colloidal gold solution under magnetic stirring, incubating at room temperature for 15min, centrifuging at 10000r/min for 30min, and removing the supernatant; dissolving and diluting the obtained precipitate with PBS (phosphate buffer solution) containing 2% BSA and 0.1% sodium azide and having pH of 7.4 to obtain a colloidal gold-labeled anti-bisphenol B monoclonal antibody;
the concentration of the colloidal gold labeled anti-bisphenol B monoclonal antibody is 40-80 mu g/ml, and the dosage on the detection card is 4-6 mu L/cm;
the concentration of the bisphenol B-carrier protein conjugate coated by the detection line is 0.1-0.3 mu g/ml, and the dosage of the bisphenol B-carrier protein conjugate on the detection card is 0.5-1.5 mu L/cm;
the concentration of the anti-mouse secondary antibody coated by the quality control line is 0.1-0.3mg/ml, and the dosage on the detection card is 0.5-1.5 muL/cm.
2. The immunochromatographic detection card for detecting bisphenol B according to claim 1, wherein the detection card sample pad end and the water absorption pad end are further provided with a sample end protective film and a handle end protective film, respectively, the sample end protective film covers the sample pad, the colloidal gold conjugate pad, and the overlapping end of the nitrocellulose membrane and the colloidal gold conjugate pad, and the handle end protective film covers the water absorption pad, and the overlapping end of the nitrocellulose membrane and the water absorption pad.
3. The colloidal gold immunochromatographic detection card for detecting bisphenol B according to claim 2, wherein the base plate is a PVC strip;
the sample pad and the colloidal gold combined pad are made of glass fiber cotton;
the water absorption pad is made of water absorption filter paper;
the protective film is an opaque adhesive film.
4. The colloidal gold immunochromatographic assay card for detecting bisphenol-B according to claim 1, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin or human serum albumin; the anti-mouse secondary antibody is a goat anti-mouse IgG antibody, a rabbit anti-mouse IgG antibody or a donkey anti-mouse IgG antibody.
5. The colloidal gold immunochromatographic detection card for detecting bisphenol B according to claim 4, wherein said anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) animal immunization: taking the bisphenol B-carrier protein conjugate, emulsifying the conjugate with equivalent volume of Freund complete adjuvant completely, immunizing female Balb/C mice of 6-8 weeks old, immunizing for 1 time at intervals of 2 weeks, cutting off the tail after three times of immunization, taking blood to measure titer, and selecting the mice with the best result for fusion;
(B) cell fusion: fusing selected mouse spleen cells and mouse myeloma SP2/0 cells, screening the fused cells by HAT culture medium, determining supernatant by indirect ELISA method, screening hybridoma cell strains which stably secrete monoclonal antibodies against bisphenol B;
(C) preparing an antibody: taking hybridoma cell strains, performing in-vitro conventional culture and passage, injecting into the abdominal cavity of a Balb/C mouse, extracting ascites after 10 days, purifying the ascites by caprylic acid-ammonium sulfate precipitation, and obtaining a purified anti-bisphenol B-monoclonal antibody by an affinity chromatography;
the colloidal gold labeled anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) preparing a colloidal gold solution: adding 1mL of 1% trisodium citrate into 100mL of ultrapure water, boiling, quickly adding 1mL of 1% chloroauric acid, continuously stirring and heating, maintaining for 5min when the color of the solution is changed into transparent mauve, stopping heating, replenishing water to the original volume, cooling to room temperature, and storing at 4 ℃ for later use;
(B) preparation of colloidal gold marker: taking 40mL of prepared colloidal gold solution, and using 0.1mol/L K2CO3Adjusting the pH value of the colloidal gold solution to 8.2; mixing the equivalent anti-bisphenol B monoclonal antibody solution with the colloidal gold solution under magnetic stirring, incubating at room temperature for 15min, centrifuging at 10000r/min for 30min, and removing the supernatant; dissolving and diluting the obtained precipitate with PBS buffer solution containing 2% BSA and 0.1% sodium azide and having pH of 7.4 to obtain the colloidal gold labeled anti-bisphenol B monoclonal antibody, and storing at 4 ℃ for later use.
6. The method for preparing a colloidal gold immunochromatographic test card for detecting bisphenol B according to any of claims 1 to 5, comprising the steps of:
(1) preparing a colloidal gold bonding pad: spraying the anti-bisphenol B monoclonal antibody-colloidal gold marker on carrier glass fiber cotton, and drying for later use;
(2) preparing a nitrocellulose membrane: spraying the anti-mouse secondary antibody and the bisphenol B-carrier protein conjugate on a nitrocellulose membrane in sequence, and drying for later use;
(3) assembling the detection card: sequentially adhering a sample pad, a colloidal gold combined pad, a nitrocellulose membrane and a water absorption pad on the bottom plate, respectively adhering a sample end protective film and a handle end protective film on the sample pad end and the water absorption pad end of the detection card, assembling the detection card, and cutting the detection card into a preset width to obtain the detection card;
the bisphenol B-carrier protein conjugate is prepared by the following method: dispersing 0.24g of bisphenol B, 0.15g of 3-bromopropylamine and 0.70g of anhydrous potassium carbonate into 20mL of dry acetone, heating and refluxing for 8h to obtain a bisphenol B hapten, adding the purified bisphenol B hapten, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a beaker, adding 3mL of dimethyl sulfoxide to dissolve the mixture, magnetically stirring the mixture overnight, then weighing 0.12g of carrier protein, and dissolving the carrier protein in 0.1mol/LNaHCO dissolved in 5mLpH7.03Adding a solution containing bisphenol B hapten into the carrier protein solution, slowly dripping while stirring, reacting overnight, and dialyzing the obtained solution at 4 ℃ for 3 days to obtain a carrier protein solution;
the anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) animal immunization: taking the bisphenol B-carrier protein conjugate, emulsifying the conjugate with Freund complete adjuvant completely, immunizing a female Balb/C mouse with the age of 6-8 weeks, breaking the tail after immunization, taking blood, measuring the titer, and selecting the mouse with the best result for fusion;
(B) cell fusion: fusing selected mouse spleen cells and mouse myeloma SP2/0 cells, screening the fused cells by HAT culture medium, determining supernatant by indirect ELISA method, screening out hybridoma cell strains which stably secrete monoclonal antibodies against bisphenol B;
(C) preparing an antibody: taking hybridoma cell strains, performing in-vitro conventional culture and passage, injecting the hybridoma cell strains into the abdominal cavity of a Balb/C mouse, extracting ascites, purifying the ascites by caprylic acid-ammonium sulfate precipitation, and obtaining a purified anti-bisphenol B-monoclonal antibody by an affinity chromatography;
the colloidal gold labeled anti-bisphenol B monoclonal antibody is prepared by the following method:
(A) preparing a colloidal gold solution: adding trisodium citrate into water, boiling, adding chloroauric acid, stirring and heating, stopping heating when the solution color becomes transparent mauve, adding water to original volume, cooling, and storing for use;
(B) preparation of colloidal gold marker: taking 40mL of prepared colloidal gold solution, and using 0.1mol/L K2CO3Adjusting the pH value of the colloidal gold solution to 8.2; mixing the equivalent anti-bisphenol B monoclonal antibody solution with the colloidal gold solution under magnetic stirring, incubating at room temperature for 15min, centrifuging at 10000r/min for 30min, and removing the supernatant; the obtained precipitate was dissolved and diluted with 2% BSA, 0.1% sodium azide in PBS buffer, pH7.4, to obtain a colloidal gold-labeled anti-bisphenol B monoclonal antibody.
7. The method for preparing a colloidal gold immunochromatographic detection card for detecting bisphenol B according to claim 1,
in the step of preparing the colloidal gold conjugate pad, the prepared anti-bisphenol B monoclonal antibody-colloidal gold marker is uniformly sprayed on carrier glass fiber cotton at the concentration of 5 mu L/cm, dried for 3h at 37 ℃, sealed and stored for later use at 4 ℃;
in the preparation step of the nitrocellulose membrane, the anti-mouse secondary antibody and the bisphenol B-carrier protein conjugate with preset concentrations are sequentially sprayed on the nitrocellulose membrane at the concentration of 1 mu L/cm, dried for 8h at 37 ℃, sealed and stored for later use at 4 ℃.
8. The use of the colloidal gold immunochromatographic test card for detecting bisphenol B according to any of claims 1 to 5, characterized by comprising the following pretreatment steps: weighing a sample to be detected, adding water, heating in a water bath for reflux, and taking back a distillate obtained by reflux to obtain a solution to be detected.
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