CN112129956B - Colloidal gold labeling solution for colloidal gold immunochromatography, and preparation method and application thereof - Google Patents
Colloidal gold labeling solution for colloidal gold immunochromatography, and preparation method and application thereof Download PDFInfo
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- CN112129956B CN112129956B CN202010907899.0A CN202010907899A CN112129956B CN 112129956 B CN112129956 B CN 112129956B CN 202010907899 A CN202010907899 A CN 202010907899A CN 112129956 B CN112129956 B CN 112129956B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
Abstract
The invention relates to a colloidal gold labeling solution for colloidal gold immunochromatography, a preparation method and application thereof, wherein the colloidal gold labeling solution contains a colloidal gold compound, and the colloidal gold compound is obtained by crosslinking colloidal gold of which the outer side is connected with a coating protein and an anti-25-hydroxy vitamin D antibody. And spraying the marking solution on a combination pad and assembling the combination pad, the nitrocellulose membrane, the water absorption pad and the sample pad to obtain the 25-hydroxyvitamin D colloidal gold immunochromatographic test strip which can be used for detecting the content of 25-hydroxyvitamin D. According to the invention, the indirect marking of the colloidal gold and the antibody is realized through the coating protein, the coating protein not only obviously improves the stability of the colloidal gold solution, but also enables the connection to be tighter due to covalent crosslinking of the coating protein and the antibody, and compared with the direct marking of the colloidal gold, the influence on the conformation and the immunocompetence of the antibody is smaller, so that the sensitivity and the repeatability of the detection test strip are obviously improved, and the detection performance of the test strip is effectively improved.
Description
Technical Field
The invention belongs to the technical field of in vitro diagnostic reagents for immunological detection, and particularly relates to a colloidal gold labeling solution for colloidal gold immunochromatography, and a preparation method and application thereof.
Background
Vitamin D is a steroid derivative, belongs to fat-soluble vitamins, and is a cyclopentane polyhydrophenanthrene compound. Vitamin D is mainly synthesized by the skin of a human body after being irradiated by ultraviolet rays, and a small part of vitamin D is taken in food or supplements. Vitamin D not only affects calcium and phosphorus metabolism, but also has a wide range of physiological effects, such as anti-proliferative, anti-differentiative, apoptosis-regulating, immune response-mediating, secretion-regulating of various endocrine gland hormones, and hematopoietic regulation of hematopoietic tissues, is an essential substance for maintaining human health, cell growth and development, and is closely related to various diseases. The active form of vitamin D in humans is 1, 25 dihydroxyvitamin D because it has two hydroxyl groups at positions 1 and 25, where 25 hydroxylation is performed in the liver and 1 hydroxylation is performed in the kidney, where the level of 25 hydroxyvitamin D in serum reflects the level of vitamin D storage in humans and correlates with clinical symptoms of vitamin D deficiency, and thus can be used as an indicator of vitamin D deficiency in the body.
Due to the tight binding of 25 hydroxyvitamin D in the blood circulation to the binding protein, 25 hydroxyvitamin D must be thoroughly separated from the binding protein in advance for accurate quantification. The dissociation method is mainly a protein denaturation method, i.e. a high-concentration denaturation dissociation agent is added into a sample solution, and the denaturation dissociation agent comprises perfluorohexanoate, perfluorooctanoate, guanidine hydrochloride, urea or sodium dodecyl sulfate and the like. However, when the content of 25-hydroxyvitamin D is detected by using the colloidal gold immunochromatography, the use of a dissociation agent can lead to a complex matrix of a sample solution to be detected, the stability of a colloidal gold compound can be significantly affected by the ionic surfactant and the denaturant, the anti-25-hydroxyvitamin D antibody connected with the colloidal gold based on physical adsorption and the colloidal gold fall off or the activity of the anti-25-hydroxyvitamin D antibody and the colloidal gold is weakened, the immunoreaction color development strength based on agglutination color development of the colloidal gold is weakened, the natural conformation of the antibody can be affected by the direct adsorption of the colloidal gold to the antibody, the reaction sites of the antibody can be affected by the steric hindrance of colloidal gold particles, the immunoreaction activity and the specificity of the antibody can be affected, and the detection sensitivity and the repeatability of the colloidal gold immunochromatography test paper can be significantly affected.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the colloidal gold labeling solution for 25-hydroxy vitamin D colloidal gold immunochromatography, and the preparation method and the application thereof, which effectively improve the stability of the solution after the colloidal gold labeling and the activity of an antibody, thereby effectively weakening the influence of a sample matrix to be detected on a colloidal gold compound and the influence of the activity of the antibody, and obviously improving the sensitivity and the repeatability of a detection test strip.
In order to achieve the purpose, the invention adopts the technical scheme that:
the colloidal gold labeling solution for 25-hydroxyvitamin D colloidal gold immunochromatography comprises a colloidal gold compound, wherein the colloidal gold compound is obtained by crosslinking colloidal gold of which the outer side is connected with coating protein and an anti-25-hydroxyvitamin D antibody.
The principle of the scheme is as follows: the colloidal metal is in the nanometer microsphere, the nanometer microsphere has extremely strong adsorbability due to the huge specific surface area, the extremely strong adsorbability can obviously influence the conformation and the immunocompetence of the adsorbed protein, and the colloidal gold solution is extremely easy to be influenced by ionic strength, pH and the like to generate obvious aggregation, discoloration and precipitation phenomena. According to the invention, the wrapping protein is added firstly to be connected with the colloidal gold, and after the colloidal gold is sealed by the wrapping protein in advance, the colloidal gold solution becomes more stable and cannot be influenced by the ionic strength, pH and the like of the solution. And then the envelope protein outside the colloidal gold is crosslinked with the anti-25-hydroxyvitamin D antibody, so that the indirect labeling of the colloidal gold and the antibody to be labeled is realized, and the envelope protein and the antibody are covalently crosslinked, so that the connection is tighter, and the influence on the antibody conformation and the immune activity is smaller.
Further, the coating protein is one or more of bovine serum albumin, casein, chicken ovalbumin and fish glue protein.
Further, the envelope protein outside the colloidal gold and the anti-25-hydroxyvitamin D antibody are subjected to covalent crosslinking through EDC.
The invention also provides a colloidal gold immunochromatographic test strip for 25-hydroxy vitamin D, and the colloidal gold labeling solution is attached to the test strip.
The invention also provides a preparation method of the colloidal gold immunochromatographic test strip for 25-hydroxy vitamin D, which comprises the steps of preparing the colloidal gold labeling solution and attaching the colloidal gold labeling solution to the test strip.
Further, the method specifically comprises the following steps: s1, taking a colloidal gold solution with the mass concentration of 4%, and adjusting the pH of the solution to 7.5-8.5;
s2, adding the solution obtained in the step S1 with a volume ratio of 1 mL: adding 10-30 mu L of a wrapping protein solution with the mass concentration of 10%, reacting for 20-40 min, centrifuging, and re-dissolving the precipitate with a buffer solution;
s3, adding the solution obtained in the step S2 into a mixed solution according to the volume-to-mass ratio of 1 mL: adding an anti-25-hydroxyvitamin D antibody in 10-30 mu g, and uniformly mixing;
s4, adding the solution obtained in the step S3 with the volume ratio of 1 mL: adding 10mg/mL of EDC solution into 10-30 mu L of the solution, uniformly mixing, reacting at room temperature for 2-6 hours, centrifuging, and re-dissolving the precipitate with a working solution to obtain a colloidal gold compound solution;
s5, spraying the colloidal gold compound solution on a bonding pad, and assembling the bonding pad, the nitrocellulose membrane, the water absorption pad and the sample pad to prepare the 25-hydroxy vitamin D colloidal gold immunochromatographic test strip.
Further, in step S4, the working fluid includes: 10-100 mM buffer solution, 1-10% protein protective agent and 0.05-0.5% preservative, wherein the percentages are mass percentages.
Further, the buffer solution is one or more of PBS buffer solution, borate buffer solution, phosphate buffer solution, ammonia-ammonium chloride buffer solution and HEPES buffer solution.
Further, the protein protective agent is one or more of sucrose, trehalose, glucose, bovine serum albumin, casein and glycine.
The invention also provides the application of the 25-hydroxy vitamin D colloidal gold immunochromatographic test strip in detecting the content of 25-hydroxy vitamin D.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the coating protein is added to seal the colloidal gold before the colloidal gold is used for marking the antibody, and then the coating protein on the outer side of the colloidal gold is covalently cross-linked with the anti-25-hydroxyvitamin D antibody, so that the indirect marking of the colloidal gold and the antibody to be marked is realized, the stability of the colloidal gold solution is obviously improved, the phenomenon of colloidal gold aggregation and precipitation caused by the ionic strength, pH and the like of the solution is avoided, the covalent cross-linking of the coating protein and the antibody ensures that the connection is tighter, the influence on the conformation and the immunocompetence of the antibody is smaller, the specificity and the immunocompetence of the antibody are obviously provided, the sensitivity and the repeatability of the detection test strip are further obviously improved, and the detection performance of the test strip is effectively improved.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the specific embodiment, the experimental materials used in the comparative example and the example are the same, and include: colloidal gold solution: the mass concentration is 4%; ② 0.1M potassium carbonate solution; ③ Bovine Serum Albumin (BSA) solution: the mass concentration is 10 percent; borate buffer: 0.02M, pH 8.0, wherein the PEG20000 with the mass concentration of 0.05 percent; anti-25 hydroxy vitamin D antibody; sixth, EDC solution: 10 mg/mL; the working solution: 10-100 mM phosphate buffer (pH 8.0), 10% trehalose, 5% BSA, 0.1% sodium azide, wherein the percentages are mass percentages.
Example 1
The embodiment provides a preparation method of a 25-hydroxy vitamin D colloidal gold immunochromatographic test strip, which specifically comprises the following steps:
s1, taking 10mL of colloidal gold solution, and mixing the solution according to the volume ratio of 1 mL: mu.L of 0.1M potassium carbonate solution was added to 15. mu.L, the pH was adjusted to 7.5, and the mixture was vortexed and mixed. The pH value of the colloidal gold solution is adjusted, so that the subsequent connection of the coating protein and the colloidal gold is facilitated.
S2, adding the solution obtained in the step S1 with a volume ratio of 1 mL: mu.L of 200. mu.L of 10% BSA solution was added, vortexed quickly for 1 minute, then mixed slowly for 30 minutes, and then centrifuged at 9000rpm for 20 minutes, the supernatant was removed, and the precipitate was reconstituted to 10mL with borate buffer and sonicated for 1 minute. Wherein, BSA is used as a wrapping protein to be connected with the colloidal gold and is used for maintaining the stability of the colloidal gold.
S3, adding 200 mu g of anti-25-hydroxyvitamin D antibody into the solution obtained in the step S2, quickly adding the anti-25-hydroxyvitamin D antibody, and uniformly mixing the anti-25-hydroxyvitamin D antibody in a vortex manner;
s4, taking the prepared EDC solution (ready for use), quickly taking 200 mu L of the prepared EDC solution, adding the solution into the solution obtained in the step S3, uniformly mixing the solution in a vortex manner, then placing the mixture in a turntable to slowly rotate and uniformly mix the mixture, reacting the mixture at room temperature for 2 to 6 hours, centrifuging the mixture at 9000rpm for 20 minutes after the reaction is finished, removing supernatant, and redissolving the precipitate with working solution to 2mL to obtain the colloidal gold compound solution. EDC promotes the condensation reaction of amino and carboxyl between the envelope protein and the antibody, thereby realizing the indirect labeling of the antibody by colloidal gold.
S5, spraying the colloidal gold compound solution obtained in the step S4 on the treated bonding pad by an XYZ three-dimensional gold spraying machine according to the volume of 3 mu l/cm to prepare a gold pad; and (3) assembling the gold pad with a nitrocellulose membrane, a water absorption pad and a sample pad which are obtained by a conventional preparation method to obtain the 25-hydroxy vitamin D colloidal gold immunochromatographic test strip.
Example 2
The present embodiment is different from embodiment 1 in that: the volume of the EDC solution added in step S4 was 100 μ L.
Example 3
The present embodiment is different from embodiment 1 in that: the volume of the EDC solution added in step S4 was 300 μ L.
Comparative example 1
The test strip prepared according to the comparative example by a conventional method comprises the following specific steps:
s1, taking 10mL of colloidal gold solution, adding 150 mu L of potassium carbonate solution, adjusting the pH value to 7.5, and uniformly mixing by vortex.
S2, adding anti-25-hydroxy vitamin D antibody 200ug, quickly mixing by vortex for 1 minute, and slowly mixing for reaction for 30 minutes. Namely, the colloidal gold is directly connected with the antibody through the adsorption effect of the colloidal gold.
S3, 1mL of the volume ratio: mu.L of 200. mu.L of 10% BSA solution was added, vortexed quickly for 1 minute, and then mixed slowly for 30 minutes. The effect of adding BSA in this step is to block the excess sites on the colloidal gold.
S4, centrifuging at 9000rpm for 20 minutes, removing supernatant, and redissolving the precipitate to 2mL by using working solution to obtain the colloidal gold compound solution.
S5, spraying the colloidal gold compound solution obtained in the step S4 on the treated bonding pad by an XYZ three-dimensional gold spraying machine according to the volume of 3 mu l/cm to prepare a gold pad; and (3) assembling the gold pad with a nitrocellulose membrane, a water absorption pad and a sample pad which are obtained by a conventional preparation method to obtain the 25-hydroxy vitamin D colloidal gold immunochromatographic test strip.
Namely, the main difference between comparative example 1 and example 1 is that: in the process of labeling the antibody with the colloidal gold, in the embodiment 1, the BSA is firstly added to wrap the colloidal gold, and then the BSA is connected with the antibody to be labeled through EDC, so that the indirect labeling of the colloidal gold and the antibody is realized, in the comparative example 1, the antibody is firstly added into the colloidal gold solution, so that the colloidal gold is directly labeled with the antibody, and then the BSA is added to seal redundant sites on the colloidal gold, so that the colloidal gold is prevented from adsorbing other substances.
Evaluation protocol
Taking 500 mu L of a 25-hydroxyvitamin D positive serum sample, adding 500 mu L of dissociation liquid, uniformly mixing by vortex, and then incubating for 10 minutes at 37 ℃, wherein the components of the dissociation liquid are as follows: 0.3% perfluorooctanoic acid sodium salt, 0.1% SDS, 0.2% Triton X-100, 0.5% isopropanol, 0.1% mercaptoethanol, 0.05M PBS buffer (pH7.4) to obtain an antigen sample to be tested.
And (3) testing the sensitivity: the test strips of example 1 and comparative example 1 were used to detect dissociated negative, weakly positive, medium positive and strongly positive antigen samples, and the degree of color development of the test strip was observed, with the results shown in table 1.
And (3) repeatability test: the test strips of example 1 and comparative example 1 were used to test the same sample for 10 times, and the results of observing the color development degree of the test strips are shown in Table 2,
in tables 1 and 2: "-" indicates negative, and "-/+" indicates that the T line is absent or not; "+" indicates a weak sun, more pronounced than "-/+"; "+" indicates positive, more obvious in color than "+"; "+ + + +" indicates strong yang, more pronounced than "+ +".
TABLE 1 sensitivity test
TABLE 2 repeatability tests
According to the determination results in table 1, the test strips of examples 1-3 are more accurate in determination when detecting the samples of weak positive and medium positive, while the color development of comparative example 1 is poor, and the detection effect on the samples of weak positive, medium positive and strong positive is not accurate, i.e., the sensitivity of examples 1-3 is higher than that of comparative example 1. According to the determination results in table 2, when the same sample is repeatedly determined, the repeatability of the example 1-3 is better, and the results are all positive or strong positive, wherein the repeatability of the example 1 is optimal, and the results of 10 times of detection are all strong positive, while the comparative example 1 shows a plurality of different results, including three results of weak positive, positive and strong positive, namely, compared with the comparative example 1, the repeatability of the detection test strip prepared by the method of the invention is better.
By combining the results, the detection test strip prepared by the method avoids the adverse effect of colloidal gold on the antibody through indirect labeling, improves the specificity and immunogenicity of the antibody, has higher sensitivity and better stability, is not easily influenced by a sample matrix in the detection process, and obviously improves the repeatability.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (7)
1. The colloidal gold labeling solution for 25-hydroxyvitamin D colloidal gold immunochromatography is characterized by comprising a colloidal gold compound, wherein the colloidal gold compound is obtained by crosslinking colloidal gold of which the outer side is connected with a coating protein and an anti-25-hydroxyvitamin D antibody;
the coating protein is any one or more of bovine serum albumin, casein, chicken ovalbumin and fish glue protein;
covalent crosslinking of the encapsulating protein and the anti-25-hydroxyvitamin D antibody is realized through EDC;
the preparation process of the colloidal gold compound comprises the following steps:
s1, taking a colloidal gold solution with the mass concentration of 4%, and adjusting the pH of the solution to 7.5-8.5;
s2, adding a 10% mass concentration encapsulating protein solution into the solution obtained in the step S1 according to the volume ratio of 1mL to 10-30 mu L, reacting for 20-40 min, centrifuging, and re-dissolving the precipitate with a buffer solution;
s3, adding an anti-25-hydroxyvitamin D antibody into the solution obtained in the step S2 according to the volume-to-mass ratio of 1mL to 10-30 μ g, and uniformly mixing;
s4, adding 10mg/mL EDC solution into the solution obtained in the step S3 according to the volume ratio of 1 mL: 10-30 μ L, uniformly mixing, reacting at room temperature for 2-6 hours, centrifuging, and re-dissolving the precipitate with a working solution to obtain a colloidal gold compound solution.
2. A colloidal gold immunochromatographic test strip for 25-hydroxyvitamin D, which is characterized in that the colloidal gold labeled solution of claim 1 is attached to the test strip.
3. A method for preparing a 25-hydroxyvitamin D colloidal gold immunochromatographic test strip, comprising preparing the colloidal gold-labeled solution of claim 1, and attaching the colloidal gold-labeled solution to the test strip.
4. The method for preparing a 25 hydroxy vitamin D colloidal gold immunochromatographic test strip according to claim 3, which is characterized in that the method specifically comprises:
s1, taking a colloidal gold solution with the mass concentration of 4%, and adjusting the pH of the solution to 7.5-8.5;
s2, adding the solution obtained in the step S1 with a volume ratio of 1 mL: adding 10-30 mu L of a wrapping protein solution with the mass concentration of 10%, reacting for 20-40 min, centrifuging, and re-dissolving the precipitate with a buffer solution;
s3, adding the solution obtained in the step S2 into a mixed solution with a volume-to-mass ratio of 1 mL: adding an anti-25-hydroxyvitamin D antibody in 10-30 mu g, and uniformly mixing;
s4, adding the solution obtained in the step S3 with the volume ratio of 1 mL: adding 10mg/mL of EDC solution into 10-30 mu L of the solution, uniformly mixing, reacting at room temperature for 2-6 hours, centrifuging, and re-dissolving the precipitate with a working solution to obtain a colloidal gold compound solution;
s5, spraying the colloidal gold compound solution on a bonding pad, and assembling the bonding pad, the nitrocellulose membrane, the water absorption pad and the sample pad to prepare the 25-hydroxy vitamin D colloidal gold immunochromatographic test strip.
5. The method for preparing a 25 hydroxy vitamin D colloidal gold immunochromatographic test strip according to claim 4, wherein the working solution in step S4 comprises: 10-100 mM buffer solution, 1-10% protein protective agent and 0.05-0.5% preservative, wherein the percentages are mass percentages.
6. The method for preparing a 25 hydroxy vitamin D colloidal gold immunochromatographic strip according to claim 4 or 5, wherein the buffer is one or more of PBS buffer, borate buffer, phosphate buffer, ammonia-ammonium chloride buffer, HEPES buffer.
7. The method for preparing a 25 hydroxy vitamin D colloidal gold immunochromatographic test strip according to claim 5, wherein the protein protective agent is one or more of sucrose, trehalose, glucose, bovine serum albumin, casein and glycine.
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