CN112904000A - Colloidal gold labeling solution composition and application thereof - Google Patents
Colloidal gold labeling solution composition and application thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a colloidal gold-labeled solution composition and application thereof, wherein a buffer solution with the pH value of 8.0-9.5 is used as a solvent, and the colloidal gold-labeled solution composition contains colloidal gold particles labeled with antibodies capable of immunoreacting with protein to be detected, hydroxypropyl methylcellulose and a nonionic surfactant, wherein the mass volume percentage concentration of the hydroxypropyl methylcellulose in the colloidal gold-labeled solution composition is 0.03-0.05%. The colloidal gold labeling solution composition can obviously enhance the sensitivity of the colloidal gold test strip, for example, the labeled coronavirus N protein antibody reacts with an N protein quality control substance in a sample, so that higher sensitivity of coronavirus detection is realized.
Description
Technical Field
The invention belongs to the technical field of pathogenic organism detection reagents, and particularly relates to a colloidal gold labeling solution composition and application thereof.
Background
The immunoassay is based on the immunological principle of antigen-antibody specific binding, and the commonly used methods mainly comprise immunochromatography assay, ELISA assay or chemiluminescence assay. The colloidal gold test strip belongs to immunochromatography detection, and the detection method has the advantages of convenience, rapidness, low price, wide application range, high yield and the like, but also has the defect of low sensitivity. Sometimes, false detection can occur, for example, coronavirus detection, and the currently marketed gold detection product generally has low sensitivity and risk of false detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a colloidal gold labeling solution composition.
Another object of the present invention is to provide the use of the above-mentioned colloidal gold-labeled solution composition.
The technical scheme of the invention is as follows:
a colloidal gold-labeled solution composition takes a buffer solution with the pH value of 8.0-9.5 as a solvent, and contains colloidal gold particles labeled with an antibody capable of immunoreacting with a protein to be detected, hydroxypropyl methylcellulose and a nonionic surfactant, wherein the mass volume percentage concentration of the hydroxypropyl methylcellulose in the colloidal gold-labeled solution composition is 0.03-0.05%.
In a preferred embodiment of the invention, the non-ionic surfactant is Tween 20, Tween 40, Pluronic F-127, Triton X-405 or Triton X-100.
In a preferred embodiment of the present invention, the mass volume percentage concentration of the nonionic surfactant in the colloidal gold-labeled solution composition is 0.8 to 1.2%.
In a preferred embodiment of the invention, inert proteins are also included.
Further preferably, the inert protein is BSA.
In a preferred embodiment of the present invention, the buffer is Tris-HCl buffer, phosphate buffer or borate buffer.
Further preferably, the concentration of the buffer is 10 to 50 mM.
In a preferred embodiment of the present invention, the colloidal gold particles have a particle size of 30 to 80 nm.
The other technical scheme of the invention is as follows:
a colloidal gold labeled conjugate pad is prepared by dripping the above colloidal gold labeled solution composition onto a base film, and drying.
In a preferred embodiment of the present invention, the base membrane is a glass fiber membrane or a nitrocellulose membrane.
The invention has the beneficial effects that: the colloidal gold labeling solution composition can obviously enhance the sensitivity of the colloidal gold test strip, for example, the labeled coronavirus N protein antibody reacts with an N protein quality control substance in a sample, so that higher sensitivity of coronavirus detection is realized.
Detailed Description
The technical solution of the present invention is further illustrated and described by the following detailed description.
Example 1
The labeling process in this embodiment is divided into four groups:
the first group is the traditional standard gold labeling process: diluting an antibody protein which can perform antigen-antibody reaction with coronavirus N protein to 10 mu g/mL, marking the antibody protein on colloidal gold particles with the particle size of 30-80nm, adding 1 wt% BSA after marking, centrifuging at 9000r/min for 5min, removing supernatant, dissolving the colloidal gold precipitate with 150mM NaCl, 25mM Tris-HCl and pH 9.0 buffer solution, dropwise adding the redissolved colloidal gold solution to a glass cellulose membrane, and drying to obtain a marker binding pad;
the second group is a process of adding viscous agent to mark colloidal gold: diluting an antibody protein which can perform antigen-antibody reaction with coronavirus N protein to 10 mu g/mL, marking the diluted antibody protein on colloidal gold particles with the particle size of 30-80nm, adding 1 wt% BSA after marking, centrifuging at 9000r/min for 5min, removing supernatant, dissolving the colloidal gold precipitate with 150mM NaCl, 25mM Tris-HCl and pH 9.0 buffer solution, adding 0.04 (w/v)% HPMC for dissolving and mixing uniformly, dropwise adding the obtained colloidal gold solution to a glass cellulose membrane, and drying to obtain a marker binding pad;
the third group is a process of adding nonionic surfactant to mark colloidal gold: diluting an antibody protein which can perform antigen-antibody reaction with coronavirus N protein to 10 mu g/mL, marking the antibody protein on colloidal gold particles with the particle size of 30-80nm, adding 1 wt% BSA after marking, centrifuging at 9000r/min for 5min, removing supernatant, dissolving colloidal gold precipitate with 150mM NaCl, 25mM Tris-HCl and pH 9.0 buffer solution, adding 1% tween 20 for dissolving and mixing uniformly, dropwise adding the obtained colloidal gold solution to a glass cellulose membrane, and drying to obtain a marker binding pad;
the fourth group is the process of marking colloidal gold of the invention: diluting an antibody protein which can have antigen-antibody reaction with coronavirus N protein to 10 mu g/mL, then labeling the diluted antibody protein on colloidal gold particles with the particle size of 30-80nm, adding 1 wt% BSA after labeling, centrifuging for 5min at 9000r/min, removing supernatant, dissolving the colloidal gold precipitate with 150mM NaCl, 25mM Tris-HCl and pH 9.0 buffer solution, adding 0.04 (w/v)% HPMC and 1 (w/v)% tween 20, dissolving and uniformly mixing to obtain the colloidal gold labeling solution composition, dripping the colloidal gold labeling solution composition on a glass cellulose membrane, and drying to obtain the label binding pad.
To investigate the effect of the addition of HPMC and tween 20 to the colloidal gold-labeled solution composition of the present invention on sensitivity, comparison was made between the addition and non-addition. And (3) drying and then assembling to form an immunochromatography device, dripping the coronavirus N protein quality control product subjected to gradient dilution onto the marker binding pads prepared from the first group to the fourth group during detection, and recording the gold card reading value of the colloid, wherein the larger the numerical value is, the stronger the color development is, and the higher the sensitivity is. Specific results are shown in table 1, and the colloidal gold-labeled solution composition of the present invention significantly improves the detection sensitivity.
Table 1, test results of example 1
| Quality control product | 1μg/mL | 100ng/mL | 10ng/mL | 1ng/mL | 0.1ng/mL | 0.01ng/mL |
| First group | L9 | L8 | L7 | L5-L6 | L2 | L0 |
| Second group | L9 | L8 | L7-L8 | L6-L7 | L3-L4 | L0 |
| Third group | L9 | L8 | L7-L8 | L6 | L3 | L0 |
| Fourth group | L9 | L8-L9 | L8 | L7 | L4 | L0-L1 |
Example 2
The labeling process in this embodiment is divided into four groups:
the first group is the traditional standard gold labeling process: diluting an antibody protein which can perform antigen-antibody reaction with coronavirus S protein to 10 mu g/mL, marking the antibody protein on colloidal gold particles with the particle size of 30-80nm, adding 1% BSA9000r/min after marking, centrifuging for 5min, removing supernatant, dissolving colloidal gold precipitate with 150mM NaCl, 25mM Tris-HCl and pH 9.0 buffer solution, dropwise adding the redissolved colloidal gold solution to a glass cellulose membrane, and drying to obtain a marker binding pad;
the second group is a process of adding viscous agent to mark colloidal gold: diluting an antibody protein which can perform antigen-antibody reaction with coronavirus S protein to 10 mu g/mL, marking the diluted antibody protein on colloidal gold particles with the particle size of 30-80nm, adding 1% BSA9000r/min after marking, centrifuging for 5min, removing supernatant, dissolving colloidal gold precipitate with 150mM NaCl, 25mM Tris-HCl and pH 9.0 buffer solution, adding 0.04 (w/v)% HPMC for dissolving and uniformly mixing, dropwise adding the obtained colloidal gold solution to a glass cellulose membrane, and drying to obtain a marker conjugate pad;
the third group is a process of adding nonionic surfactant to mark colloidal gold: diluting an antibody protein which can perform antigen-antibody reaction with coronavirus S protein to 10 mu g/mL, marking the antibody protein on colloidal gold particles with the particle size of 30-80nm, adding 1% BSA9000r/min for centrifugation for 5min after marking, dissolving the colloidal gold precipitate with 150mM NaCl, 25mM Tris-HCl and pH 9.0 buffer solution after discarding the supernatant, adding 1 (w/v)% tween 20 for dissolving and uniformly mixing, dropwise adding the obtained colloidal gold solution to a glass cellulose membrane, and drying to obtain a marker binding pad;
the fourth group is the process of marking colloidal gold of the invention: diluting an antibody protein which can have antigen-antibody reaction with coronavirus S protein to 10 mu g/mL, marking the diluted antibody protein on colloidal gold particles with the particle size of 30-80nm, adding 1% BSA9000r/min after marking, centrifuging for 5min, discarding supernatant, dissolving colloidal gold precipitate with 150mM NaCl, 25mM Tris-HCl and pH 9.0 buffer solution, adding 0.04 (w/v)% HPMC and 1 (w/v)% tween 20, dissolving and uniformly mixing to obtain the colloidal gold-labeled solution composition, dropwise adding the colloidal gold-labeled solution composition to a glass cellulose membrane, and drying to obtain the label binding pad.
To investigate the effect of the addition of HPMC and tween 20 to the colloidal gold-labeled solution composition of the present invention on sensitivity, comparison was made between the addition and non-addition. And (3) drying and then assembling to form an immunochromatography device, dripping the coronavirus S protein quality control product subjected to gradient dilution onto the marker binding pads prepared from the first group to the fourth group during detection, and recording the gold card reading value of the colloid, wherein the larger the numerical value is, the stronger the color development is, and the higher the sensitivity is. Specific results are shown in table 2, the colloidal gold-labeled solution composition of the present invention significantly improved the detection sensitivity.
Table 2, test results of example 2
| Quality control product | 1μg/mL | 100ng/mL | 10ng/mL | 1ng/mL | 0.1ng/mL | 0.01ng/mL |
| First group | L8 | L7-L8 | L6 | L4 | L0-L1 | L0 |
| Second group | L8 | L7-L8 | L6-L7 | L4-L5 | L0-L1 | L0 |
| Third group | L8 | L7-L8 | L6-L7 | L4-L5 | L0-L1 | L0 |
| Fourth group | L8 | L7-L8 | L7 | L5-L6 | L1 | L0 |
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. A colloidal gold-labeling solution composition characterized by: the method comprises the steps of taking a buffer solution with the pH value of 8.0-9.5 as a solvent, wherein the buffer solution contains colloidal gold particles marked with antibodies capable of immunoreacting with protein to be detected, hydroxypropyl methylcellulose and a nonionic surfactant, wherein the mass volume percentage concentration of the hydroxypropyl methylcellulose in the colloidal gold marking solution composition is 0.03-0.05%.
2. The colloidal gold-labeling solution composition of claim 1, further comprising: the non-ionic surfactant is Tween 20, Tween 40, Pluronic F-127, Triton X-405 or Triton X-100.
3. The colloidal gold-labeling solution composition of claim 1, further comprising: the mass volume percentage concentration of the nonionic surfactant in the colloidal gold-labeled solution composition is 0.8-1.2%.
4. The colloidal gold-labeling solution composition of claim 1, further comprising: also contains inert protein.
5. The colloidal gold-labeling solution composition of claim 4, further comprising: the inert protein is BSA.
6. The colloidal gold-labeling solution composition of claim 1, further comprising: the buffer solution is Tris-HCl buffer solution, phosphate buffer solution or boric acid buffer solution.
7. The colloidal gold-labeling solution composition of claim 6, further comprising: the concentration of the buffer solution is 10-50 mM.
8. The colloidal gold-labeling solution composition of claim 1, further comprising: the particle size of the colloidal gold particles is 30-80 nm.
9. A colloidal gold label conjugate pad, comprising: prepared by dropping the colloidal gold-labeled solution composition according to any one of claims 1 to 8 onto a base film and drying.
10. The colloidal gold label conjugate pad of claim 9, wherein: the basement membrane is a glass fiber membrane or a nitrocellulose membrane.
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Patent Citations (9)
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| CN104991058A (en) * | 2015-07-06 | 2015-10-21 | 宋晓峰 | Preparation method for gold labeled immucochromatographic test strip jointly marked through colloidal gold and latex microsphere |
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