CN114217072A - Colloidal gold immunochromatographic test strip for detecting freshness of fish and preparation and application thereof - Google Patents
Colloidal gold immunochromatographic test strip for detecting freshness of fish and preparation and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of food, and particularly relates to a colloidal gold immunochromatographic test strip for detecting freshness of fish meat, and a preparation method and application thereof. The colloidal gold immunochromatographic test strip comprises a bottom plate, a detection pad, a gold label pad, a sample pad and a water absorption pad; a T line and a C line are arranged on the detection pad, the T line is sprayed with parvalbumin, and the C line is sprayed with goat anti-mouse polyclonal antibody; and the gold label pad is sprayed with a colloidal gold-labeled parvalbumin monoclonal antibody. The method uses the parvalbumin to reflect the freshness of the fish meat objectively and accurately; the colloidal gold immunochromatographic test paper has small detection sample amount and small damage to a detection sample; the sensitivity is high, and the operation is simple and convenient; the detection time is short, and the detection result is visual; the target analyte is definite, and the protein can be specifically combined with the antibody, so that specific detection can be realized; the test strip has good stability, and the experimental result can be stored for a long time; the detection process has no participation of harmful substances, and is a green and environment-friendly detection means.
Description
Technical Field
The invention belongs to the technical field of food, and particularly relates to a colloidal gold immunochromatographic test strip for detecting freshness of fish meat, and a preparation method and application thereof.
Background
The fish is rich in nutrition, high in water content, high in protease activity in muscle tissues and easy to decay, so that the evaluation and classification of the freshness of the fish meat are very necessary.
At present, a plurality of methods for evaluating the freshness of fish meat mainly comprise sensory evaluation, microorganisms and physical and chemical changes. For example, patent CN112285237A discloses a squid freshness evaluation method based on biogenic amine content, which adopts biogenic amine to determine the freshness of squids; CN111523542A adopts the total number of bacterial colonies as the basis for judging the freshness of the fish meat; CN103424374A establishes a detection model by using the TVB-N (volatile basic nitrogen) content in the hairtail body, and evaluates the freshness grade of the hairtail according to the TVB-N content. However, the deterioration of the fish meat is a comprehensive changing process, and the single index is taken as the basis for dividing the freshness grade, so that the one-sidedness is realized, and the more objective and comprehensive result can be brought by reflecting the freshness of the fish meat from multiple aspects by using different indexes.
Protein is one of the basic components of fish meat and is also a main factor of quality change, and the degradation or denaturation degree of the protein is closely related to the fish meat quality. With the development of proteomics technology, research on screening freshness indicator protein in the storage process of aquatic products has been carried out quite a lot, however, the research results of freshness indicator protein are difficult to popularize and apply due to the fact that the proteomics technology is directly applied to evaluate freshness technology, which has strong specialization, high instrument dependence, time-consuming property and high cost. The colloidal gold immunochromatography technology combines antigen-antibody specific reaction with colloidal gold, a visible marker, and realizes detection by using a membrane as a medium through chromatography. The colloidal gold immunochromatographic test paper for the freshness indicator protein can be prepared, and the change of the protein in different storage time can be detected. The detection method has the advantages of less sampling amount, simple and convenient operation, no need of professional operation and complex precision equipment, applicability to field detection and large sample amount detection, and important practical significance and practical value in detecting the freshness of aquatic products by combining the freshness indicator protein with the colloidal gold immunochromatography technology.
Disclosure of Invention
The invention aims to provide a colloidal gold immunochromatographic test strip for detecting fish freshness and a preparation method thereof.
According to the technical scheme of the invention, the colloidal gold immunochromatographic test strip for detecting the freshness of fish meat comprises a bottom plate, a detection pad, a gold label pad, a sample pad and a water absorption pad; the detection pad is arranged in the middle of the bottom plate, a T line and a C line are arranged on the detection pad, the T line is sprayed with parvalbumin, and the C line is sprayed with goat anti-mouse polyclonal antibody; the sample pad is arranged on one side, close to the T line of the detection pad, of the bottom plate, the gold-labeled pad is arranged between the sample pad and the detection pad in an overlapped mode, and the colloidal gold-labeled parvalbumin monoclonal antibody is sprayed on the gold-labeled pad; the water absorption pad is arranged on one side, close to the line C, of the detection pad on the bottom plate and partially overlapped on the detection pad.
The second aspect of the invention provides a preparation method of a colloidal gold immunochromatographic test strip for detecting freshness of fish, which comprises the following steps,
s1: extracting purified fish parvalbumin;
s2: spraying the parvalbumin and the goat anti-mouse polyclonal antibody on a detection pad base material to form a T line and a C line respectively, and drying to obtain a detection pad;
s3: assembling the sample pad, the water absorption pad and the detection pad on a bottom plate, and cutting to obtain a test strip with the width of 3-4 mm;
s4: labeling a parvalbumin monoclonal antibody (primary antibody) with colloidal gold to obtain a gold-labeled antibody, and spraying the gold-labeled antibody on an activated gold-labeled pad to obtain a gold-labeled pad;
and assembling the gold label pad into the test strip to obtain the colloidal gold immunochromatographic test strip.
Specifically, the material of the detection pad base material is an NC membrane (nitrocellulose membrane), a polyvinylidene fluoride membrane, a polyester membrane or a cellulose acetate membrane, and preferably is an NC membrane; the bottom plate is a PVC (polyvinyl chloride) lining plate.
Further, the specific steps of step S1 are as follows,
s1.1: fully crushing fish, adding a buffer solution I, homogenizing, and centrifuging to obtain a supernatant A;
s1.2: heating the supernatant A and then centrifuging to obtain a supernatant B;
s1.3: salting out the supernatant B, and separating to obtain a precipitate;
s1.4: dissolving the precipitate in buffer solution II, and dialyzing overnight at 4 ℃;
s1.5: and (3) filtering the dialyzed liquid by using a microporous filter membrane, loading the liquid to an ion exchange column, carrying out gradient elution on eluent at the flow rate of 1mL/min, collecting all components, determining the ultraviolet absorption values of the components at 220nm and 280nm, carrying out SDS-PAGE detection, determining the components where the fish parvalbumin is located, and completing the extraction and purification of the fish parvalbumin.
Further, the buffer I and the buffer II are boric acid buffer, phosphate buffer or Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride) buffer, preferably 0.02mol/L Tris-HCl (pH7.5) buffer.
Furthermore, the volume ratio of the fish meat to the buffer solution I is 1: 2-4.
Further, in the step S1.3, ammonium sulfate solution with mass percent concentration of 40-100% is adopted for salting out.
Further, in step S1.5, the eluates are mixed by a gradient mixer at a volume ratio of 0.02mol/L Tris-HCl (containing 0.5mol/L NaCl) and 0.02mol/L Tris-HCl 1: 1, respectively.
Further, the formula for calculating the concentration of the eluent by the gradient mixer is as follows:
C＝(C2-C1)(1-V)A2/A1wherein A is1、A2Is the cross-sectional area of two containers, C1、C2Is the sodium chloride concentration of the solution in the vessel and V is the ratio of the outflow volume to the total volume.
Further, the gradient elution volume was 600-.
Specifically, the operation of step S1 may be as follows:
1) fully mincing fish, adding 0.02mol/L Tris-HCl (pH7.5) buffer solution, homogenizing for 1-2min, standing at 4 deg.C for 30min, freezing at 10000g, centrifuging for 20min, and collecting supernatant;
2) putting the supernatant obtained in the step 1) into a water bath kettle at 85-90 ℃ for stirring and water bathing for 15-30min, and centrifuging at 10000g for 20min to obtain the supernatant;
3) salting out the supernatant in the step 2) by 40-100% ammonium sulfate, standing for 2h at 4 ℃, and centrifuging by 10000 g;
4) the pellet was redissolved in buffer and dialyzed overnight at 4 ℃ during which time the buffer was changed 4-5 times (0.02mol/L Tris-HCl (pH 7.5));
5) filtering dialyzed liquid through a 0.22 mu m microporous filter membrane, loading the dialyzed liquid on a DEAE-Sepharose ion exchange column of which the sample loading buffer solution (0.02mol/L Tris-HCl, pH7.5) is balanced overnight, carrying out gradient elution on eluent at the flow rate of 1mL/min, collecting all components, determining ultraviolet absorption values of the components at 220nm and 280nm, carrying out SDS-PAGE detection, determining the components of the grass carp parvalbumin, collecting, storing and freeze-drying the components.
Further, DEAE-Sepharose ion exchange column eluate was received at 4 mL/tube.
Further, in the step S2, the parvalbumin is dissolved by PB (phosphate buffer, 0.02MpH7.4) to a concentration of 0.5-1.5mg/mL and sprayed as a T line, wherein the spraying amount is 1 muL/cm; the goat anti-mouse polyclonal antibody is diluted by PB to the concentration of 1.0-2.0mg/mL and sprayed as a C line, and the spraying amount is 1 muL/cm.
Further, in the step S2, the drying temperature is 37 ℃ and the time is 1.5-4 h.
Further, in step S3, the sample pad is obtained by soaking the sample pad without activation in the sample treatment solution and then drying; the sample treatment solution is a buffer solution containing surface active substances, saccharides and macromolecules.
Specifically, the soaking time is 30-60min, and the drying temperature is 37 ℃; the sample processing solution is 0.01M Tris-HCl (pH8.0) buffer solution containing 0.4-0.6% (m/v) BSA, 3-5% (v/v) Trixton, and 0.1-0.3% (m/v) NaCl.
Further, in order to stabilize the protein and improve the re-dissolving performance of the colloidal gold, in step S4, the activated gold label pad is soaked in the gold label pad treatment solution from the non-activated gold label pad and dried; the gold-labeled pad treatment solution is a buffer solution containing surface active substances, saccharides and macromolecules.
Specifically, the soaking time is 30-60min, and the drying temperature is 37 ℃; the gold pad treatment solution is 0.02MPB (pH7.4), which contains 4-6% (m/v) sucrose, 0.5-1.5% (m/v) trehalose, 0.2-0.3% (m/v) PEG20000, and 0.2-0.4% (v/v) Tween-20.
Specifically, the non-activated sample pad and the non-activated gold label pad are independently selected from NC membrane (nitrocellulose membrane), polyvinylidene fluoride membrane, polyester membrane or cellulose acetate membrane, preferably NC membrane.
Further, in step S4, the gold-labeled antibody is prepared as follows:
SS 1: adding a trisodium citrate solution into the boiled chloroauric acid solution, and after the reaction is finished, adjusting the pH to 5.0-9.0 (preferably, the pH is 7.0) to obtain a colloidal gold solution;
SS 2: adding the parvalbumin monoclonal antibody into the colloidal gold solution, uniformly mixing and standing; adding BSA solution, mixing uniformly and standing; adding PEG20000 solution, mixing, and standing; obtaining a mixed solution;
SS 3: separating the mixed solution to obtain a precipitate, and carrying out heavy suspension to obtain the gold-labeled antibody.
In step SS2, adding parvalbumin monoclonal antibody into the colloidal gold solution to label the protein on the colloidal gold particles; BSA was added to block the sites where colloidal gold was not bound by the protein; the added PEG20000 is macromolecule, which can provide a skeleton function, and better stabilize the system and the gold-labeled antibody.
Further, the mass ratio of the chloroauric acid to the trisodium citrate in the step SS1 is 1: 1.5-3.
Specifically, the operation of step SS1 is as follows: adding 1mL of 1% chloroauric acid solution into 100mL of ultrapure water, heating to boil under magnetic stirring, rapidly adding 1% trisodium citrate solution with a certain volume, continuing to heat for 10-20min after the color change is stable, naturally cooling to room temperature, and supplementing with ultrapure water, heating and evaporating water. The volume ratio of the 1 percent chloroauric acid solution to the 1 percent trisodium citrate solution is 1: 1.5-3.
The operations of steps SS2 and SS3 are as follows:
adjusting the pH value of the colloidal gold solution to 5.0-9.0 (preferably 7.0) by using 0.2M potassium carbonate solution;
adding 12 μ L of 1mg/mL parvalbumin monoclonal antibody into 500 μ L colloidal gold solution, vortex mixing for 1min, and standing at room temperature for 20 min;
adding 10% BSA solution until the final concentration of BSA is 1%, vortex mixing for 1min, and standing at room temperature for 30 min;
adding 10% PEG20000 solution to PEG20000 final concentration of 1%, mixing with vortex for 1min, and standing at room temperature for 30 min;
centrifuging at 4 deg.C under 10000g for 30min, removing supernatant, adding resuspension solution to resuspend the enriched gold-labeled antibody.
Further, the composition of the resuspension solution was 0.02M PB (pH7.4), 1% (M/v) BSA, 1% (M/v) PEG20000, 1% (v/v) Tween-20, 1% (M/v) sucrose.
Further, in the step S4, the labeled amount of the colloidal gold-labeled parvalbumin monoclonal antibody is 18 to 30 μ g/mL, and the sprayed amount of the gold-labeled antibody is 6 to 8 μ L.
The third aspect of the invention provides the application of the colloidal gold immunochromatographic test strip for detecting the freshness of fish, which comprises the following steps of diluting fish exudation juice or extracted sarcoplasmic protein, dripping the diluted fish exudation juice or extracted sarcoplasmic protein on a sample pad, carrying out lateral chromatography for 10-15min at room temperature, measuring a T/C value after color development is stable, and judging the freshness of the fish according to the T/C value.
Further, the freshness detection device is used for detecting the freshness of the refrigerated or ice fish; specifically, the refrigeration temperature is 4 ℃, and the ice storage temperature is 0 ℃.
Further, the extraction method of the sarcoplasmic proteins comprises the steps of fully stirring fish, adding the buffer solution III, homogenizing for 1-2min, standing at 4 ℃, freezing and centrifuging, and taking supernatant.
Specifically, the extraction method of sarcoplasmic proteins comprises the steps of fully stirring fish meat, adding 0.02mol/L Tris-HCl (pH7.5) buffer solution, homogenizing for 1-2min, standing at 4 ℃ for 30min, freezing and centrifuging at 10000g for 20min, and taking supernatant;
the exuded juice obtaining method comprises the following steps: wrapping fish meat with 200 mesh filter cloth, suspending in a centrifuge tube, centrifuging at room temperature for 15min at 3000g, and collecting liquid at the bottom of the centrifuge tube.
Further, the buffer III is preferably 0.02mol/L Tris-HCl (pH7.5) buffer.
Further, taking grass carp as an example, the method for judging the freshness of the fish meat according to the T/C value is as follows:
taking sarcoplasmic protein as a test solution, and obtaining the first-class freshness T/C value of the ice-stored grass carp: 0.66-0.67, secondary freshness T/C value: 0.58-0.65, and three-level freshness T/C value: 0.52-0.57;
the first-grade freshness T/C value of the refrigerated grass carp is as follows: 0.64-0.67, secondary freshness T/C value: 0.56-0.63, and T/C value of three-level freshness: 0.49-0.55;
and (3) taking the exuded juice as a test solution, and obtaining the first-class freshness T/C value of the iced grass carp: 0.65-0.66, secondary freshness T/C value: 0.55-0.64, and three-level freshness T/C value: 0.51-0.54;
the first-grade freshness T/C value of the refrigerated grass carp is as follows: 0.62-0.66, secondary freshness T/C value: 0.56-0.61, and three-level freshness T/C value: 0.51-0.55.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the fish freshness degree is comprehensively reflected in a mode of combining physicochemical, sensory and protein, each index is subjected to cluster analysis, and the freshness degree grade is divided according to the similarity among data. The grade division result of the clustering analysis among the sensory indexes is consistent with the grade division result of comprehensive physicochemical, sensory and protein indexes (figure 1 and figure 2), so that the freshness of the fish meat is objectively and accurately reflected by the parvalbumin; the colloidal gold immunochromatographic test paper has small detection sample amount and small damage to a detection sample; the sensitivity is high, and the operation is simple and convenient; the detection time is short, and the detection result is visual; the target analyte is definite, and the protein can be specifically combined with the antibody, so that specific detection can be realized; the test strip has good stability, and the experimental result can be stored for a long time; the detection process has no participation of harmful substances, and is a green and environment-friendly detection means.
Drawings
FIG. 1 is an electrophoretogram of sarcoplasmic proteins of cryopreserved grass carp fillets and an immunoblot analysis of parvalbumin.
FIG. 2 is an HCA analysis chart of the freshness index of the refrigerated grass carp fillet.
FIG. 3 is a schematic view of the assembly of the colloidal gold immunochromatographic test strip.
FIG. 4 is a pH-optimized graph of colloidal gold labeled primary antibody.
FIG. 5 is a diagram showing the optimization of the amount of the colloidal gold-labeled antibody.
FIG. 6 is a graph showing the optimization of the amount of gold-labeled antibody sprayed.
FIG. 7 is an T, C-line film density optimization chart.
FIG. 8 is an optimized pore size diagram of an NC membrane tube.
Fig. 9(a) is elution volume 200: 200mL protein elution profile; (b) is an electrophoretogram.
Fig. 10(a) is elution volume 300: 300mL protein elution profile; (b) is an electrophoretogram.
Description of reference numerals: 1-bottom plate, 2-detection pad, 3-gold label pad, 4-sample pad, 5-water absorption pad, 6-T line and 7-C line.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1 extraction of purified grass carp parvalbumin
(1) Fully mincing grass carp meat, adding 0.02mol/L Tris-HCl (pH7.5) buffer solution, homogenizing for 1-2min, standing at 4 deg.C for 30min, freezing at 10000g, centrifuging for 20min, and collecting supernatant;
(2) putting the supernatant obtained in the step (1) into a water bath kettle at 85-90 ℃ for stirring and water bathing for 15-30min, and centrifuging at 10000g for 20min to obtain the supernatant;
(3) salting out the supernatant in the step (2) by 40-100% ammonium sulfate, standing for 2h at 4 ℃, and centrifuging by 10000 g;
(4) the pellet was redissolved in buffer and dialyzed overnight at 4 ℃ during which time the buffer was changed 4-5 times (0.02mol/L Tris-HCl (pH 7.5));
(5) filtering dialyzed liquid through a 0.22 mu m microporous membrane, loading the dialyzed liquid on a DEAE-Sepharose ion exchange column which is balanced by loading buffer solution (0.02mol/LTris-HCl, pH7.5) overnight, carrying out gradient elution on eluent at the flow rate of 1ml/min, collecting all components, determining ultraviolet absorption values of the components at 220nm and 280nm, carrying out SDS-PAGE detection, determining the components of the grass carp meat parvalbumin, collecting, storing and freeze-drying the components.
Wherein the eluates are mixed by a gradient mixer at a volume ratio of 0.02mol/L Tris-HCl (containing 0.5mol/L NaCl) and 0.02mol/L Tris-HCl 1: 1, respectively.
Example 2 preparation of gold-labeled antibody
(1) Adding 1mL of 1% chloroauric acid solution into 100mL of ultrapure water, heating to boiling under magnetic stirring, and mixing the chloroauric acid solution and the trisodium citrate solution according to the volume ratio of 1: 1.5-3, quickly adding a trisodium citrate solution with a certain volume of 1%, continuing to heat for 10-20min after the color changes stably, naturally cooling to room temperature, supplementing with ultrapure water, heating and evaporating water to obtain a colloidal gold solution;
(2) adjusting the pH value of the colloidal gold solution by using 0.2M potassium carbonate solution;
(3) adding 12 μ L of 1mg/mL parvalbumin monoclonal antibody into 500 μ L of colloidal gold solution with pH7.0, vortex mixing for 1min, and standing at room temperature for 20 min;
(4) adding 10% BSA solution until the final concentration of BSA is 1%, vortex mixing for 1min, and standing at room temperature for 30 min;
(5) adding 10% PEG20000 solution to PEG20000 final concentration of 1%, mixing with vortex for 1min, and standing at room temperature for 30 min;
(6) centrifuging at 4 deg.C under 10000g for 30min, removing supernatant, adding resuspension solution to resuspend the enriched gold-labeled antibody.
Example 3 preparation of colloidal gold immunochromatographic test strip for detecting freshness of grass carp
Dissolving the parvalbumin obtained in example 1 by PB (0.02M pH7.4), spraying T line 6 on an NC membrane, diluting goat anti-mouse polyclonal antibody by PB, spraying C line 7 on the NC membrane, and drying at 37 ℃ to obtain a detection pad 2;
attaching a detection pad 2 to a base plate 1(PVC base plate); the water absorption pad 5 is stuck above the detection pad, and the overlapping length of the water absorption pad 5 and the detection pad 2 is 1-2 mm; the activated sample pad 4 is stuck above the detection pad, and the overlapping length of the sample pad 4 and the detection pad 2 is 0.5-2 mm; cutting the assembled test strip into strips with the width of 3-4mm by a strip cutting machine; the gold-labeled pad 3 labeled with the gold-labeled antibody obtained in example 2 was assembled between the sample pad and the detection pad to obtain a colloidal gold immunochromatography, which was then placed in a card case.
Example 4
The test paper strip obtained in the embodiment 3 is used for detecting the freshness of the refrigerated and iced grass carps, and whether the division intervals of the test paper strip are consistent with the division intervals of the freshness of the refrigerated and iced grass carps according to comprehensive indexes such as sense, physicochemical indexes and the like is judged. Wherein, sensory description analysis evaluation criteria are shown in Table 1,
TABLE 1
The refrigerated grass carp fillet sarcoplasmic protein electrophoresis image and the parvalbumin immunoblot analysis are shown in figure 1 (a: refrigerated grass carp fillet sarcoplasmic protein electrophoresis image, b: refrigerated grass carp fillet parvalbumin immunoblot image, c: refrigerated grass carp fillet parvalbumin immunoblot grey-scale analysis). The HCA analysis of the freshness index of the refrigerated grass carp fillet is shown in FIG. 2 (a: HCA analysis of sensory, physicochemical and protein index; b: HCA analysis of sensory, physicochemical and physicochemical).
The parvalbumin is a preferable scheme for detecting the freshness of the grass carp meat. The division of the freshness of the refrigerated and iced grass carps is consistent with the division of the freshness of the refrigerated and iced grass carps by comprehensive indexes such as sense, physicochemical indexes and the like (figure 2).
Example 5
The pH in example 2 and step (2) was adjusted to optimize the pH for colloidal gold-labeled primary antibody optimum labeling, and the results are shown in FIG. 4 and Table 2,
TABLE 2 wavelength and absorption value of the peak of the UV-Vis scanning spectrum after marking with different pH values
Indicating that the pH of the colloidal gold-labeled primary antibody is 7.
Example 6
The results of UV-visible spectrum scanning by adjusting the amount of the colloidal gold-labeled parvalbumin monoclonal antibody obtained in example 2 are shown in FIG. 5 (preferably, 20. mu.g/mL, 25. mu.g/mL, 35. mu.g/mL, 15. mu.g/mL, 30. mu.g/mL, 40. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL, and 0 in the order from the top to the bottom in the peak), and Table 3.
TABLE 3 UV-VISIBLE SCANNING Peak wavelength and Absorbance for optimal protein labeling
The optimal condition is that the labeling quantity of the parvalbumin monoclonal antibody is 20 mug/mL.
Example 7
The labeling amount of the parvalbumin monoclonal antibody labeled with colloidal gold was 20. mu.g/mL, and the freshness test was carried out by adjusting the spraying amount of the gold-labeled antibody on the gold-labeled pad in example 3, and the results are shown in FIG. 6 (0-0 ppb, 1-1ppb, 2-10ppb, 3-100ppb, 4-1000ppb, and 5-10000ppb in each test).
The result shows that the TC line is uniformly and clearly developed when 7 mu L of the paint is sprayed, and the sensitivity is better.
Example 8
The freshness was measured by adjusting the concentration of dissolved parvalbumin and diluted goat anti-mouse polyclonal antibody (secondary antibody) in example 3, and the results are shown in fig. 7.
The result shows that when the concentration of the parvalbumin sprayed on the T line is 1.5mg/mL, and the concentration of the secondary antibody of the C line is 1.0mg/mL, the TC line is uniform and clear in color development and good in sensitivity.
Example 9
The pore size of the NC film in example 3 was adjusted to detect freshness, and the results are shown in fig. 8.
The results show that the pore size of the NC membrane of Pall90 is large, the chromatographic speed is high, the C-line color development is normal, the T-line color development intensity is generally weak, and the color development effects and sensitivity differences of Pall120, CN140 and Pall170 are not large. Wherein the membranes of different specifications correspond to flow rates: pall90:90s/4cm, Pall120:120s/4cm, CN140:140s/4cm and Pall170:170s/4 cm. Wherein the English preceding the number represents the brand (Pall represents Pall, CN is Saedolis brand), and the numeral following the English represents the chromatography speed of the NC membrane, i.e. the time for which water is separated by 4cm in the NC membrane.
Example 10
The volume of the eluate in example 1 was adjusted and conditions for parvalbumin extraction were optimized, and the results are shown in FIGS. 9 and 10.
The results show that parvalbumin and heteroproteins cannot be separated when the elution volume is 200mL:200 mL; when the elution volume is 300mL to 300mL, the separation effect of parvalbumin is good.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (10)
1. A colloidal gold immunochromatographic test strip for detecting freshness of fish is characterized by comprising a bottom plate, a detection pad, a gold label pad, a sample pad and a water absorption pad; the detection pad is arranged in the middle of the bottom plate, a T line and a C line are arranged on the detection pad, the T line is sprayed with parvalbumin, and the C line is sprayed with goat anti-mouse polyclonal antibody; the sample pad is arranged on one side, close to the T line of the detection pad, of the bottom plate, the gold-labeled pad is arranged between the sample pad and the detection pad in an overlapped mode, and the colloidal gold-labeled parvalbumin monoclonal antibody is sprayed on the gold-labeled pad; the water absorption pad is arranged on one side, close to the line C, of the detection pad on the bottom plate and partially overlapped on the detection pad.
2. A preparation method of a colloidal gold immunochromatographic test strip for detecting freshness of fish meat is characterized by comprising the following steps,
s1: extracting purified fish parvalbumin;
s2: spraying the parvalbumin and the goat anti-mouse polyclonal antibody on a detection pad base material to form a T line and a C line respectively, and drying to obtain a detection pad;
s3: assembling the sample pad, the water absorption pad and the detection pad on a bottom plate, and cutting to obtain a test strip with the width of 3-4 mm;
s4: labeling the parvalbumin monoclonal antibody with colloidal gold to obtain a gold-labeled antibody, and spraying the gold-labeled antibody on an activated gold-labeled pad to obtain a gold-labeled pad;
and assembling the gold label pad into the test strip to obtain the colloidal gold immunochromatographic test strip.
3. The method according to claim 2, wherein the step S1 is specifically performed as follows,
s1.1: fully crushing fish, adding a buffer solution I, homogenizing, and centrifuging to obtain a supernatant A;
s1.2: heating the supernatant A and then centrifuging to obtain a supernatant B;
s1.3: salting out the supernatant B, and separating to obtain a precipitate;
s1.4: dissolving the precipitate in buffer solution II, and dialyzing overnight at 4 ℃;
s1.5: and (3) filtering the dialyzed liquid by using a microporous filter membrane, loading the liquid to an ion exchange column, carrying out gradient elution by using eluent at the flow rate of 1mL/min, collecting all components, determining the ultraviolet absorption values of the components at 220nm and 280nm, carrying out SDS-PAGE detection, determining the components where the fish parvalbumin is located, and completing the extraction and purification of the fish parvalbumin.
4. The method according to claim 2, wherein in step S2, the parvalbumin is dissolved with PB to a spray T-line concentration of 0.5 to 1.5mg/mL at a spray amount of 1 μ L/cm; and (3) diluting the goat anti-mouse polyclonal antibody to the concentration of 1.0-2.0mg/mL by using PB, and spraying C lines with the spraying amount of 1 mu L/cm.
5. The method of claim 2, wherein in the step S3, the sample pad is obtained by soaking an unactivated sample pad in the sample treatment solution and then drying the sample pad; the sample treatment solution is a buffer solution containing surface active substances, saccharides and macromolecules.
6. The method according to claim 2, wherein in step S4, the activated gold label pad is soaked in the gold label pad treatment solution from an unactivated gold label pad and dried; the gold-labeled pad treatment solution is a buffer solution containing surface active substances, saccharides and macromolecules.
7. The method of claim 2, wherein in step S4, the gold-labeled antibody is prepared as follows:
SS 1: adding a reducing agent into the chloroauric acid solution, heating for reaction, and adjusting the pH value to 5.0-9.0 to obtain a colloidal gold solution;
SS 2: adding the parvalbumin monoclonal antibody into the colloidal gold solution, uniformly mixing and standing; adding BSA solution, mixing uniformly and standing; adding PEG20000 solution, mixing, and standing; obtaining a mixed solution;
SS 3: separating the mixed solution to obtain a precipitate, and carrying out heavy suspension to obtain the gold-labeled antibody.
8. The method according to claim 2 or 7, wherein in step S4, the labeled amount of the colloidal gold-labeled parvalbumin monoclonal antibody is 18 to 30 μ g/mL, and the sprayed amount of the gold-labeled antibody is 6 to 8 μ L.
9. The application of the colloidal gold immunochromatographic test strip of claim 1 for detecting freshness of fish meat, comprising the steps of diluting the juice of fish meat exudation or the extracted sarcoplasmic protein, dripping the diluted solution on a sample pad, performing lateral chromatography at room temperature for 10-15min, measuring a T/C value after color development is stabilized, and judging the freshness of fish meat according to the T/C value.
10. The use of claim 9, wherein the sarcoplasmic proteins are extracted by thoroughly mincing fish meat, adding buffer III, homogenizing for 1-2min, standing at 4 ℃, freezing and centrifuging to obtain supernatant.
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