CN111638327B - Treatment fluid and treatment method for colloidal gold immunochromatographic test paper gold-labeled pad and sample pad - Google Patents
Treatment fluid and treatment method for colloidal gold immunochromatographic test paper gold-labeled pad and sample pad Download PDFInfo
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Abstract
The invention discloses a treatment fluid and a treatment method of a colloidal gold immunochromatographic test paper gold-labeled pad and a sample pad, wherein the treatment fluid comprises disodium hydrogen phosphate, potassium dihydrogen phosphate, sucrose, tween-20 and a surfactant; the treatment method comprises material selection, pretreatment, wetting and drying. After the sample and/or buffer solution is added into the test strip treated by the treatment solution and the method, gold particles are rapidly and uniformly released, and the gold particles are completely released without any residue; the background is clean and white after detection, which is helpful for distinguishing and judging the negative and the positive; the positive detection rate of the product is improved; the stability of the gold-labeled conjugate is greatly improved, and the stability of the assembled test strip is correspondingly improved.
Description
Technical Field
The invention relates to a treatment fluid and a treatment method of a colloidal gold immunochromatography test paper gold label pad and a sample pad, belonging to the technical field of immunochromatography detection.
Background
The colloidal gold immunochromatography (ColloidalGoldImmunochromatographyAssay, GICA) is a rapid detection method for expressing antigen-antibody specific reaction through colloidal gold labeling and immunochromatography, and has the advantages of rapidness, simplicity, convenience, no need of special experimental equipment, reliable results and the like compared with the traditional molecular biological methods such as serological detection, PCR and the like.
The gold-labeled pad and the sample pad are important components of the colloidal gold immunochromatographic test paper, the sample pad is generally required to be treated by a sample pad treatment liquid before use, and because of different detection samples and projects, when the sample pad is not treated or is applied to detection by using a conventional sample pad treatment liquid, the problems are large, for example: false positive and false negative reactions occur, the negative-positive contrast is not high, and the release of gold particles in the gold-labeled binding pad cannot be effectively promoted. The gold-labeled pad has the main function of adsorbing colloidal gold particles marked with corresponding proteins, and a polyester film is a common material. The gold-labeled pad determines the activity (including sensitivity and specificity), release effect and long-term stability of the colloidal gold particles labeled with the corresponding proteins, and thus has a great influence on the effect of the colloidal gold immunochromatographic test paper. At present, the preparation method of the gold-labeled bonding pad is mainly to directly spray or soak marked concentrated gold particles on a polyester film. Or soaking the polyester film in conventional treating liquid, and spraying or soaking the marked concentrated gold particles on the polyester film. However, in the using process, the gold-labeled binding pad prepared by the method is slow and incomplete to release in the detecting process, and the accuracy, the sensitivity and the like of the detecting result are affected.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a treatment fluid of a colloidal gold immunochromatography test paper gold label pad and a sample pad;
the invention further provides a treatment method of the colloidal gold immunochromatographic test paper gold-labeled pad and the sample pad.
The aim of the invention is achieved by the following technical scheme: a colloidal gold immunochromatographic test paper gold-labeled pad and sample pad treatment fluid comprises the following raw materials:
disodium hydrogen phosphate: 0.5 to 1.0 percent of potassium dihydrogen phosphate: 0.05 to 0.1 percent of sucrose: 1-3%, tween-20: 0.5-1 mL/L, surfactant: 1-2 mL/L. When the treatment object is a gold mark pad, the surfactant is S6; when the object to be treated is a sample pad, the surfactant is a mixed solution of S6 and S9.
As a preferable scheme, the preparation method comprises the following raw materials:
disodium hydrogen phosphate: 0.58%, potassium dihydrogen phosphate: 0.052%, sucrose: 1.0%, tween-20:0.5mL/L, surfactant: 1.0mL/L. When the treatment object is a gold mark pad, the surfactant is S6; when the object to be treated is a sample pad, the surfactant is a mixed solution of S6 and S9.
A processing method of a colloidal gold immunochromatographic test paper gold-labeled pad and a sample pad comprises the following steps:
s1, selecting materials: selecting a common glass cellulose film and a polyester film which are not subjected to more or special treatment;
s2, pretreatment: placing the glass cellulose film in an oven for fully drying; soaking the polyester film in purified water for 1.5-2.5 h, throwing away water after soaking, and placing in an oven for full drying; wherein the drying temperature of the oven is 110-130 ℃;
s3, wetting: uniformly coating the treatment liquid on the pretreated glass cellulose film and polyester film by using a liquid transfer device, and just wetting;
s4, drying: and (3) transferring the wet glass cellulose film and polyester film to an oven, drying at 75-85 ℃ to obtain a sample pad, and using the glass cellulose film as a gold mark pad for later use.
Further, the polyester film in step S1 was changed twice during the soaking of the purified water.
A colloidal gold test strip is prepared by the following method:
s1, preparing a gold mark bonding pad: marking corresponding proteins by using the prepared colloidal gold solution, sealing, centrifuging and concentrating the marked proteins to obtain concentrated colloidal gold solution, spraying the concentrated colloidal gold solution onto the treated polyester film by using a colloid Jin Penjin machine, and drying by blowing at 37-40 ℃ to prepare the Cheng Jinbiao bonding pad;
s2, assembling: and combining the prepared gold-labeled binding pad and the glass cellulose membrane treated by the method into a colloidal gold test strip.
Further, the labeled corresponding protein in the step S1 is any one of staphylococcus aureus protein A, a mouse monoclonal antibody IgG for detecting human cardiac troponin, a mouse monoclonal antibody IgG for detecting canine distemper virus or an antigen expressed by escherichia coli.
Further, the centrifugation conditions in step S1 are: the rotating speed is 13000-17000 rpm, and the centrifugation time is 15-30 min.
Further, the concentrated colloidal gold solution in step S1 is 1/10 of the original volume.
The invention has the following advantages: the treatment fluid of the colloidal gold immunochromatographic test paper gold-labeled pad and the sample pad is provided, and the modified treatment fluid can effectively improve some physical or chemical properties of the sample pad and the gold-labeled pad; compared with the common treatment mode, the treatment method disclosed by the invention has the advantages that the colloidal gold immunochromatographic test paper is obviously improved in the following aspects:
1. after the sample and/or buffer solution are added, gold particles are rapidly and uniformly released, and the gold particles are completely released without any residue;
2. the background is clean and white after detection, which is helpful for distinguishing and judging the negative and the positive;
3. the positive detection rate of the product is improved;
4. the stability of the gold-labeled conjugate is greatly improved, and the stability of the assembled test strip is correspondingly improved.
Namely: the test strip improved by the method of the invention improves the detection effect and the storage stability of the product.
Detailed Description
The invention will be further described with reference to examples, but the scope of the invention is not limited to the following:
example 1: a colloidal gold immunochromatographic test paper gold-labeled pad and sample pad treatment fluid comprises the following raw materials:
disodium hydrogen phosphate: 0.58%, potassium dihydrogen phosphate: 0.052%, sucrose: 1.0%, tween-20:0.5mL/L, surfactant: 1.0mL/L. When the treatment object is a gold mark pad, the surfactant is S6; when the object to be treated is a sample pad, the surfactant is a mixed solution of S6 and S9.
Example 2: a colloidal gold immunochromatographic test paper gold-labeled pad and sample pad treatment fluid comprises the following raw materials:
disodium hydrogen phosphate: 0.8%, potassium dihydrogen phosphate: 0.08%, sucrose: 2.0%, tween-20:0.8mL/L, surfactant: 1.5mL/L. When the treatment object is a gold mark pad, the surfactant is S6; when the object to be treated is a sample pad, the surfactant is a mixed solution of S6 and S9.
Example 3: a colloidal gold immunochromatographic test paper gold-labeled pad and sample pad treatment fluid comprises the following raw materials:
disodium hydrogen phosphate: 1.0%, monobasic potassium phosphate: 0.1%, sucrose: 3.0%, tween-20:1.0mL/L, surfactant: 2.0mL/L. When the treatment object is a gold mark pad, the surfactant is S6; when the object to be treated is a sample pad, the surfactant is a mixed solution of S6 and S9.
Example 4: a processing method of a colloidal gold immunochromatographic test paper gold-labeled pad and a sample pad comprises the following steps:
s1, selecting materials: selecting an untreated ordinary type glass cellulose film and a polyester film;
s2, pretreatment: placing the glass cellulose film in an oven for fully drying; soaking the polyester film in purified water for 1.5h, changing water twice in the soaking process, throwing away water after soaking, and placing in an oven for full drying; wherein the drying temperature of the oven is 110 ℃;
s3, wetting: uniformly coating the treatment liquid in the embodiment 1 on the pretreated glass cellulose film and polyester film by using a liquid dispenser, and just wetting;
s4, drying: and (3) transferring the wet glass cellulose film and polyester film to an oven, drying at 75 ℃ to obtain a sample pad, and using the glass cellulose film and the polyester film as a gold mark pad.
Example 5: a processing method of a colloidal gold immunochromatographic test paper gold-labeled pad and a sample pad comprises the following steps:
s1, selecting materials: selecting a common glass cellulose film and a polyester film which are not specially treated;
s2, pretreatment: placing the glass cellulose film in an oven for fully drying; soaking the polyester film in purified water for 2.5 hours, changing water twice in the soaking process, throwing away water after soaking, and placing in an oven for full drying; wherein the drying temperature of the oven is 130 ℃;
s3, wetting: uniformly coating the treatment liquid in the embodiment 2 on the pretreated glass cellulose film and polyester film by using a liquid dispenser, and just wetting;
s4, drying: and (3) transferring the wet glass cellulose film and polyester film to an oven, drying at 85 ℃, taking the glass cellulose film as a sample pad for standby and taking the polyester film as a gold mark pad for standby.
Example 6: a processing method of a colloidal gold immunochromatographic test paper gold-labeled pad and a sample pad comprises the following steps:
s1, selecting materials: selecting an untreated ordinary type glass cellulose film and a polyester film;
s2, pretreatment: placing the glass cellulose film in an oven for fully drying; soaking the polyester film in purified water for 2 hours, changing water twice in the soaking process, throwing away water after soaking, and placing in an oven for full drying; wherein the drying temperature of the oven is 120 ℃;
s3, wetting: uniformly coating the treatment liquid in the embodiment 3 on the pretreated glass cellulose film and polyester film by using a liquid dispenser, and just wetting;
s4, drying: and (3) transferring the wet glass cellulose film and polyester film to an oven, drying at 80 ℃ to obtain a sample pad, and using the glass cellulose film and the polyester film as a gold mark pad.
Example 7: a colloidal gold test strip is prepared by the following method:
s1, preparing a gold mark bonding pad: labeling staphylococcus aureus protein A by using the prepared colloidal gold solution, sealing the labeled protein, centrifuging and concentrating to obtain a concentrated colloidal gold solution with the volume of 1/10, wherein the centrifuging conditions are as follows: the rotational speed was 12000rpm and the centrifugation time was 15min, and concentrated colloidal gold solution was sprayed onto the polyester film treated as described in example 4 using a colloid Jin Penjin machine, and air-dried at 37 ℃ to prepare Cheng Jinbiao conjugate pad;
s2, assembling: the gold-labeled binding pad prepared above and the glass cellulose membrane treated in example 4 are combined with nitrocellulose membrane coated with mycobacterium tuberculosis antigen (TB-SA) protein, absorbent paper and PVC bottom plate, and then assembled and cut into colloidal gold test strips, which can be used for detecting mycobacterium tuberculosis IgG antibodies in human blood samples.
Example 8: a colloidal gold test strip is prepared by the following method:
s1, preparing a gold mark bonding pad: labeling and detecting human cardiac troponin mouse monoclonal antibody IgG by using the prepared colloidal gold solution, and blocking, centrifuging and concentrating the labeled protein to obtain a concentrated colloidal gold solution with the volume of 1/10 of that of the original sample, wherein the centrifuging conditions are as follows: the rotational speed was 17000rpm and the centrifugation time was 30min, and concentrated colloidal gold solution was sprayed onto the polyester film treated as described in example 5 using a colloid Jin Penjin machine, and air-dried at 38 ℃ to prepare Cheng Jinbiao conjugate pad;
s2, assembling: the gold-labeled binding pad prepared in the above way and the glass cellulose membrane treated by the method described in the example 5 are combined and coated with nitrocellulose membrane coated with another monoclonal antibody of mouse anti-human cardiac troponin cTnI, water-absorbing paper and PVC bottom plate, and then assembled and cut into colloidal gold test strips, so that the gold-labeled binding pad can be used for detecting cardiac troponin cTnI in human blood samples.
Example 9: a colloidal gold test strip is prepared by the following method:
s1, preparing a gold mark bonding pad: the prepared colloidal gold solution is used for marking a mouse monoclonal antibody IgG for detecting canine distemper virus, and the marked protein is blocked, centrifuged and concentrated to obtain a concentrated colloidal gold solution with the volume of 1/10, wherein the centrifugation conditions are as follows: the rotational speed was 15000rpm and the centrifugation time was 20min, and concentrated colloidal gold solution was sprayed onto the polyester film treated as described in example 6 using a colloid Jin Penjin machine, and dried by air blow at 40 ℃ to prepare Cheng Jinbiao conjugate pad;
s2, assembling: the gold-labeled binding pad prepared in the above way and the glass cellulose membrane treated by the method described in the example 6 are combined and coated with nitrocellulose membrane, absorbent paper and PVC bottom plate of another monoclonal antibody of the mouse anti-canine distemper virus, and then assembled and cut into colloidal gold test strips, so that the gold-labeled binding pad can be used for detecting canine distemper virus in canine secretion.
The beneficial effects of the invention are illustrated by the following experiments:
1. selection of sample pad (glass cellulose membrane) and gold-labeled conjugate pad (polyester membrane):
sample pads (glass cellulose films) and gold-labeled conjugate pads (polyester films) of different types or kinds are generally available from suppliers, and common glass cellulose films and polyester films which are not subjected to more or special treatment by the suppliers can be selected.
2. Sample pad (glass cellulose membrane) treatment:
1. placing a glass cellulose film with the size of A4 paper in an oven to be fully dried at 120 ℃;
2. preparing a glass cellulose membrane treatment solution: weighing 5.8g of disodium hydrogen phosphate, 0.52g of potassium dihydrogen phosphate, adding purified water for dissolution, adding 1mL of surfactant S6 and 1mL of surfactant S9, and adding purified water to fix the volume to 1L after dissolution;
3. uniformly coating the treatment liquid on the glass cellulose films by using a liquid transfer device, and just wetting, wherein the amount of the treatment liquid used by each glass cellulose film is kept the same;
4. and (3) transferring the glass cellulose film containing the treatment liquid to an oven, and fully drying at 80 ℃ for later use.
3. Treatment of gold-labeled conjugate pad (polyester film):
1. spreading a piece of polyester film with the size of A4 paper in a container containing 1L of purified water, standing for 2 hours, changing water twice, and fully removing water-soluble chemical reagents and other substances attached to the surface of the polyester film;
2. taking out the polyester film, throwing off water by a spin dryer, and placing the polyester film in an oven to be fully dried at 120 ℃;
3. preparing a polyester film treatment liquid: 5.8g of disodium hydrogen phosphate, 0.52g of potassium dihydrogen phosphate and 10g of sucrose, adding purified water for dissolution, adding 0.5mL of tween-20 and 2mL of surfactant S6, and adding purified water to fix the volume to 1L after dissolution;
4. spreading the dried polyester film on a glass plate, uniformly coating the treatment liquid on the polyester film by using a liquid transfer device, and just wetting, wherein the amount of the treatment liquid used by each polyester film is kept the same;
5. and (3) transferring the polyester film containing the treatment liquid to an oven, and fully drying at 80 ℃ for later use.
3. Preparation of gold-labeled binding pad:
1. and (3) preparing a colloidal gold solution according to a trisodium citrate reduction method to obtain a colloidal gold solution with the size of 20-40nm, and marking the corresponding protein.
Examples: labeling staphylococcus aureus protein A (SPA), regulating the pH value of colloidal gold to 6.0-6.5 by using 0.2M potassium carbonate, then putting colloid Jin Fang on a magnetic stirrer, and slowly dropwise adding the SPA protein solution with the concentration of 1mg/mL into the colloidal gold solution until the sodium chloride test is stable.
Similarly, other corresponding antigen-antibody proteins, such as mouse corresponding monoclonal antibody IgG and related antigens expressed by escherichia coli, can be marked, and staphylococcus aureus protein A, human cardiac troponin detection mouse monoclonal antibody IgG and canine distemper virus detection mouse monoclonal antibody IgG are marked.
2. The marked protein is blocked by 10 percent of BSA or other macromolecular substances (such as PEG20000, PVA and the like), the final concentration of the BSA is between 0.5 and 1.0 percent, and the macromolecular substances are between 0.05 and 0.1 percent, so as to obtain a blocked colloidal gold solution;
3. centrifuging the closed colloidal gold solution at 15000rpm for 30min, and if the particle size is above 30nm, reducing the centrifuging time to 15-20min. Collecting the precipitate, redissolving the precipitate with supernatant, and concentrating to 1/10 of the original volume;
4. spraying the concentrated colloidal gold solution onto the treated polyester film by using a colloid Jin Penjin machine, and drying by blowing at 37-40 ℃ to prepare the gold mark bonding pad.
4. The sample pad (glass cellulose film) and the gold-labeled conjugate pad (polyester film) are combined into a colloidal gold test strip and compared with the untreated gold-labeled pad effect:
1. untreated sample pads (glass cellulose films) and gold-labeled conjugate pads (polyester films) refer to conventional treatments purchased from suppliers either untreated or soaked with tween-20 and then dried.
2. The method comprises the steps of spraying a marked and sealed gold-labeled conjugate on a polyester film, drying, assembling a sample pad, a marked nitrocellulose film, absorbent paper and the like on a PVC bottom plate according to different detection items to form a colloidal gold test strip, assembling a test strip for detecting a mycobacterium tuberculosis IgG antibody (a colloidal gold particle marked SPA protein, a nitrocellulose film coated with tuberculosis antigen TB-SA protein), a test strip for detecting human cardiac troponin (cTnI) (a colloidal gold particle marked mouse anti-human cardiac troponin cTnI monoclonal antibody, a nitrocellulose film coated with a mouse anti-human cardiac troponin cTnI monoclonal antibody) and a test strip for detecting a canine distemper virus (a colloidal gold particle marked mouse anti-canine distemper virus monoclonal antibody, a nitrocellulose film coated with a mouse anti-canine distemper virus monoclonal antibody), and comparing detection effects, wherein the method has the following advantages:
A. after the treated polyester film is sprayed with the colloidal gold with the sealed marks, the colloid Jin Yanse is bright, and the color is red and bright after being dried; after the polyester film which is processed by conventional treatment, i.e. is not treated or is soaked by tween-20 and then dried, is sprayed with the colloidal gold with sealed marks, the color of the colloidal gold is slightly purple, and the color of the dried colloidal gold is more purple or even is purple brown.
B. Comparison of test strip detection effects assembled into:
1) The test strip effect of the IgG antibody for detecting mycobacterium tuberculosis is compared with that of the test strip shown in Table 1:
table 1: test strip effect contrast for detecting mycobacterium tuberculosis IgG antibody
2) Comparison of the test strip effects for detecting human cardiac troponin (cTnI) is shown in table 2:
table 2: test strip effect comparison for detecting human cardiac troponin
3) The test strip effect comparison for detecting canine distemper virus is shown in table 3:
table 3: test strip effect comparison for detecting canine distemper virus
Conclusion: through the processing of the above processes, the prepared sample pad (glass cellulose film) and gold-labeled bonding pad (polyester film) have stable properties, and when a sample is added for detection, gold particles can be rapidly and completely released, so that the yin-yang contrast is increased, the background is good, and the stability and the detection effect of a product are improved.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art who is skilled in the art to which the present invention pertains will appreciate that the technical scheme and the inventive concept according to the present invention are equally substituted or changed within the scope of the present invention.
Claims (5)
1. The colloidal gold test strip is characterized by being prepared by the following steps:
A. preparing a gold mark pad: marking corresponding proteins by using the prepared colloidal gold solution, sealing, centrifuging and concentrating the marked proteins to obtain concentrated colloidal gold solution, spraying the concentrated colloidal gold solution onto the treated polyester film by using a colloid Jin Penjin machine, and drying by blowing at 37-40 ℃ to prepare Cheng Jinbiao pads; the protein is any one of staphylococcus aureus protein A, a mouse monoclonal antibody IgG for detecting human cardiac troponin, a mouse monoclonal antibody IgG for detecting canine distemper virus or an antigen expressed by escherichia coli;
B. and (3) assembling: combining the prepared gold-labeled pad, the treated glass cellulose membrane and other necessary components into a colloidal gold test strip;
the treatment method of the polyester film and the gold mark pad comprises the following steps:
s1, selecting materials: selecting a common glass cellulose film and a polyester film which are not subjected to more or special treatment, wherein the common glass cellulose film and the polyester film which are not subjected to more or special treatment are untreated common glass cellulose film and polyester film;
s2, pretreatment: placing the glass cellulose film in an oven for fully drying; soaking the polyester film in purified water for 1.5-2.5 h, throwing away water after soaking, and placing in an oven for full drying; wherein the drying temperature of the oven is 110-130 ℃;
s3, wetting: uniformly coating the treatment liquid on the pretreated glass cellulose film and polyester film by using a liquid transfer device, and just wetting;
the treatment fluid comprises the following raw materials: disodium hydrogen phosphate: 0.5 to 1.0 percent of potassium dihydrogen phosphate: 0.05 to 0.1 percent of sucrose: 1-3%, tween-20: 0.5-1 mL/L, surfactant: 1-2 mL/L, wherein when the treatment object is a gold mark pad, the surfactant is S6; when the treatment object is a sample pad, the surfactant is a mixed solution of S6 and S9;
s4, drying: and (3) transferring the wet glass cellulose film and polyester film to an oven, drying at 75-85 ℃ to obtain a sample pad, and using the glass cellulose film as a gold mark pad for later use.
2. The colloidal gold test strip according to claim 1, wherein the treatment fluid comprises the following raw materials:
disodium hydrogen phosphate: 0.58% potassium dihydrogen phosphate: 0.052% sucrose: 1.0 Percent, tween-20:0.5mL/L, surfactant: 1.0mL/L, wherein when the treatment object is a gold-labeled pad, the surfactant is S6; when the object to be treated is a sample pad, the surfactant is a mixed solution of S6 and S9.
3. The colloidal gold test strip according to claim 1, wherein the polyester film in step S2 is changed twice in the process of immersing in purified water.
4. The colloidal gold test strip according to claim 1, wherein the centrifugation conditions in step a are: the rotating speed is 13000-17000 rpm, and the centrifugation time is 15-30 min.
5. The colloidal gold test strip according to claim 1, wherein the concentrated colloidal gold solution in the step a is 1/10 of the original volume.
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