CN109900905A - A kind of colloidal gold test strip for detecting carcinoembryonic antigen and preparation method - Google Patents
A kind of colloidal gold test strip for detecting carcinoembryonic antigen and preparation method Download PDFInfo
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- CN109900905A CN109900905A CN201910148524.8A CN201910148524A CN109900905A CN 109900905 A CN109900905 A CN 109900905A CN 201910148524 A CN201910148524 A CN 201910148524A CN 109900905 A CN109900905 A CN 109900905A
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- colloidal gold
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- nitrocellulose membrane
- carcinoembryonic antigen
- test strip
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 238000012360 testing method Methods 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 title claims abstract 9
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 title claims abstract 9
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 26
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 26
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 14
- 108020004414 DNA Proteins 0.000 claims abstract description 13
- 102000053602 DNA Human genes 0.000 claims abstract description 13
- 108091023037 Aptamer Proteins 0.000 claims abstract description 7
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 4
- 230000000295 complement effect Effects 0.000 claims abstract description 4
- 239000012528 membrane Substances 0.000 claims abstract 11
- 230000002745 absorbent Effects 0.000 claims abstract 6
- 239000002250 absorbent Substances 0.000 claims abstract 6
- 239000000084 colloidal system Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 239000003365 glass fiber Substances 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 3
- 239000010931 gold Substances 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 4
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- 230000000903 blocking effect Effects 0.000 claims 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims 1
- 239000011259 mixed solution Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 239000000427 antigen Substances 0.000 description 32
- 108091007433 antigens Proteins 0.000 description 32
- 102000036639 antigens Human genes 0.000 description 32
- 239000000523 sample Substances 0.000 description 23
- 238000000034 method Methods 0.000 description 6
- 229960002685 biotin Drugs 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 208000006990 cholangiocarcinoma Diseases 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于疾病检测、免疫传感技术领域，涉及一种检测癌胚抗原的胶体金试纸条及制备方法。本发明所述的胶体金试纸条包括样品垫、底板、硝酸纤维素膜、结合垫以及吸水纸；所述底板上粘贴有硝酸纤维素膜，所述硝酸纤维素膜的一端粘贴有结合垫，另一端粘贴有吸水纸，所述结合垫上粘贴有样品垫；所述硝酸纤维素膜上设置有测试带和控制带，所述测试带内包被有癌胚抗原单克隆抗体，所述控制带内包被有ssDNA，所述ssDNA是与癌胚抗原适配体互补的单链DNA；所述结合垫包被有胶体金标记链霉亲和素。本发明制备的一种检测癌胚抗原的胶体金试纸条具有方便快捷，操作简单，耗时较短，便于临床推广应用等优点。
The invention belongs to the technical fields of disease detection and immune sensing, and relates to a colloidal gold test strip for detecting carcinoembryonic antigen and a preparation method. The colloidal gold test strip of the present invention includes a sample pad, a bottom plate, a nitrocellulose membrane, a binding pad and a absorbent paper; the bottom plate is pasted with a nitrocellulose membrane, and one end of the nitrocellulose membrane is pasted with a binding pad , the other end is pasted with absorbent paper, and the binding pad is pasted with a sample pad; the nitrocellulose membrane is provided with a test strip and a control strip, the test strip is coated with carcinoembryonic antigen monoclonal antibody, and the control strip is The inside is coated with ssDNA, and the ssDNA is a single-stranded DNA complementary to the carcinoembryonic antigen aptamer; the binding pad is coated with colloidal gold-labeled streptavidin. The colloidal gold test strip for detecting carcinoembryonic antigen prepared by the invention has the advantages of convenience and quickness, simple operation, short time consumption, convenient clinical popularization and application, and the like.
Description
Technical field
The invention belongs to disease detections, immune sensing technical field, and in particular to a kind of colloidal gold for detecting carcinomebryonic antigen
Test strips and preparation method.
Background technique
Cancer has become one of most destructive disease in the world, causes more than 1,000 ten thousand new cases and 760 every year
Ten thousand deaths.Carcinoembryonic Antigen CEA is a kind of tumor associated antigen, carcinomebryonic antigen content apparent increase be common in colon cancer,
Gastric cancer, lung cancer, cholangiocarcinoma；In liver cancer, breast cancer, oophoroma, Pancreas cancer patients body, carcinomebryonic antigen content also has raising.Cause
This, the content for measuring carcinomebryonic antigen is significant to the prevention and diagnosis of these diseases.
And carcinomebryonic antigen is generally detected using enzyme-linked immunoassay kit in the prior art, although enzyme-linked immunization is examined
The accuracy of survey is higher, but this method detection process is cumbersome, takes a long time, and sample needs batch detection, be not suitable for and
When detect.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, provide a kind of colloidal gold strip for detecting carcinomebryonic antigen and
Preparation method.
In order to solve the above-mentioned technical problem, this invention takes following technical measures:
Firstly, prepare Colloidal gold conjugated streptavidin probe, method the following steps are included:
(1) it prepares colloidal gold solution: the chlorauric acid solution of 100mL0.01% being added in round-bottomed flask, magnetic agitation adds
Heat is extremely boiled；Addition 5mL1% sodium citrate in solution is stated then up, after solution becomes claret, is continued to boil 10min, be stopped
It only heats, continues to stir 15min, obtain colloidal gold solution, 4 DEG C are kept in dark place.
(2) 0.1molL is added into the colloidal gold solution of 1mL-1K2CO3(5 μ L) is mixed, and the strepto- of 8 μ g is added after 5min
Avidin, room temperature shake 30min.10%BSA (100 μ L) is added and closes 30min, 4 DEG C of centrifugations (10 × 103Rpm, 10min), it obtains
To Colloidal gold conjugated streptavidin probe.Clear liquid is abandoned, with 100 μ L borate buffer (0.2mol L-1, pH9.0) and colloid is resuspended
Golden labelled streptavidin probe.
Secondly, a kind of colloidal gold strip for detecting carcinomebryonic antigen includes sample pad, bottom plate, nitrocellulose filter, combination
Pad and blotting paper, preparation method includes the following steps:
(1) nitrocellulose filter is pasted onto the middle part of the bottom plate；
(2) blotting paper is pasted on the bottom plate of side of the nitrocellulose filter, the blotting paper with it is described
There is the overlapping of 2mm between nitrocellulose filter；
(3) bonding pad is pasted on the bottom plate of the other side of the nitrocellulose filter, the bonding pad and institute
State the overlapping for having 2mm between nitrocellulose filter；The bonding pad is coated with Colloidal gold conjugated streptavidin；
(4) sample pad is pasted on the bottom plate of the other side of the bonding pad, the sample pad and the combination
There is the overlapping of 2mm between pad；The sample pad is immersed in by glass fibre containing 1%BSA, 2%Triton, 2%
In the solution of PEG4000,20mMTris-Ac and 50mM NaAc, formed after drying.
Preferably, it is provided with calibration tape and control band on the nitrocellulose filter, is control close to one end of blotting paper
Band is calibration tape close to one end of bonding pad, and the calibration tape endoperidium has Cea Monoclonal Antibodies, and the control is in
It is coated with ssDNA, the ssDNA is the single stranded DNA complementary with carcinomebryonic antigen aptamers.
Compared with prior art, the advantages and beneficial effects of the present invention are:
1) present invention detects carcinomebryonic antigen using test strips method, easy to be quick, can observe with the naked eye detection in 5 minutes
As a result.
2) a kind of colloidal gold strip for detecting carcinomebryonic antigen of the present invention has very high specificity, non-purpose egg
It is white that interference is not generated to detection signal.
3) a kind of colloidal gold strip for detecting carcinomebryonic antigen of the present invention, it is easy to operate, it can be used for colon cancer, stomach
The early diagnosis of cancer, lung cancer and cholangiocarcinoma, medical field with good application prospect.
Detailed description of the invention
Fig. 1 is a kind of colloidal gold strip structural schematic diagram for detecting carcinomebryonic antigen of the present invention.
Fig. 2 is a kind of colloidal gold strip detection schematic diagram for detecting carcinomebryonic antigen of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Whole description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
The embodiment of the present invention, all other reality obtained by those of ordinary skill in the art without making creative efforts
Example is applied, shall fall within the protection scope of the present invention.
Embodiment 1
Prepare a kind of colloidal gold strip for detecting carcinomebryonic antigen
A, Colloidal gold conjugated streptavidin probe is prepared
(1) chlorauric acid solution of 100mL 0.01% is added in round-bottomed flask, magnetic agitation is heated to boiling；Then to
1% sodium citrate of 5mL is added in above-mentioned solution, after solution becomes claret, continues to boil 10min, stops heating, continue to stir
15min is mixed, colloidal gold solution is obtained, 4 DEG C are kept in dark place.
(2) 0.1molL is added into the colloidal gold solution of 1mL-1K2CO3(5 μ L) is mixed, and the strepto- of 8 μ g is added after 5min
Avidin, room temperature shake 30min.10%BSA (100 μ L) is added and closes 30min, 4 DEG C of centrifugations (10 × 103Rpm, 10min), it obtains
To Colloidal gold conjugated streptavidin probe.Clear liquid is abandoned, with 100 μ L borate buffer (0.2mol L-1, pH9.0) and colloid is resuspended
Golden labelled streptavidin probe.
B, it is a kind of detect carcinomebryonic antigen colloidal gold strip include sample pad, bottom plate, nitrocellulose filter, bonding pad with
And blotting paper, structure is as shown in Figure 1, preparation method is as follows:
(1) nitrocellulose filter is pasted onto the middle part of the bottom plate；
(2) blotting paper is pasted on the bottom plate of side of the nitrocellulose filter, the blotting paper with it is described
There is the overlapping of 2mm between nitrocellulose filter；
(3) bonding pad is pasted on the bottom plate of the other side of the nitrocellulose filter, the bonding pad and institute
State the overlapping for having 2mm between nitrocellulose filter；The bonding pad is coated with Colloidal gold conjugated streptavidin；
(4) sample pad is pasted on the bottom plate of the other side of the bonding pad, the sample pad and the combination
There is the overlapping of 2mm between pad；The sample pad is immersed in by glass fibre containing 1%BSA, 2%Triton, 2%
In the solution of PEG4000,20mMTris-Ac and 50mM NaAc, formed after drying.
It is provided with calibration tape and control band on the nitrocellulose filter, is control band close to one end of blotting paper, it is close
One end of bonding pad is calibration tape, and the calibration tape endoperidium has Cea Monoclonal Antibodies, and the control band endoperidium has
SsDNA, the ssDNA are the single stranded DNAs complementary with carcinomebryonic antigen aptamers.
Wherein, a kind of testing principle for the colloidal gold strip detecting carcinomebryonic antigen, as shown in Figure 2: by cancer embryo to be measured
Antigenic solution sample in conjunction with biotinylated aptamers after be added drop-wise to sample pad, by with the colloid gold label chain on bonding pad
The specific binding of mould Avidin, conjugate chromatography is to being coated with Cea Monoclonal Antibodies region (on nitrocellulose filter
Calibration tape) after, carcinomebryonic antigen-aptamers-biotin and Colloidal gold conjugated streptavidin compound are by carcinomebryonic antigen Dan Ke
Grand antibody capture, carcinomebryonic antigen concentration is higher in sample, and calibration tape red is deeper；When being free of carcinomebryonic antigen in sample, biology
The couplings of Colloidal gold conjugated streptavidin will not be tested with coated carcinomebryonic antigen list in elementization aptamers and bonding pad
Clonal antibody capture, and can be captured by the ssDNA that control takes, the aobvious red of control band, calibration tape does not develop the color.
Embodiment 2
A kind of colloidal gold strip detecting carcinomebryonic antigen of the present invention, detection method are as follows:
Biotinylated carcinomebryonic antigen aptamers and carcinomebryonic antigen solution example to be measured are uniformly mixed, are reacted at room temperature
After 30min, 4 DEG C are centrifuged 3 times (12 × 103Rmp, 10min), supernatant is abandoned, with 100 μ L borate buffer (0.2mol L-1,
PH9.0 carcinomebryonic antigen-aptamers-biotin) is resuspended.60 μ L of carcinomebryonic antigen-aptamers-biotin solution is taken, sample is added drop-wise to
On pad.After 5min, observation experiment result.
The experimental results showed that the red of calibration tape is gradually deepened with the increase of carcinomebryonic antigen concentration in sample.Work as sample
In be free of carcinomebryonic antigen when, only control band colour developing, test zone do not develop the color.
Embodiment 3
A kind of colloidal gold strip detecting carcinomebryonic antigen of the present invention, method for detecting specificity are as follows:
According to the detection method of above-mentioned carcinomebryonic antigen, use respectively alpha-fetoprotein, CA125 and human serum albumins as pair
According to the specificity of analysis present invention detection carcinomebryonic antigen.Testing result shows reference protein (alpha-fetoprotein, CA125 respectively
And human serum albumins) preparation albumen-aptamers-biotin solution when being added drop-wise in the sample pad of test strips of the present invention, survey
Test strip does not develop the color, only the colour developing of control band.And carcinomebryonic antigen-aptamers-the biotin solution for using carcinomebryonic antigen to prepare, use meat
Eye is in calibration tape it is seen that apparent red stripes.Illustrate that test strips specificity is good, testing result is not by other albumen
Interference.
Claims (4)
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| CN111077313A (en) * | 2019-12-23 | 2020-04-28 | 佛山科学技术学院 | A kind of colloidal gold test strip for detecting Staphylococcus aureus and preparation method |
| CN111398587A (en) * | 2020-04-02 | 2020-07-10 | 安徽科技学院 | A kind of colloidal gold lateral chromatography test strip for detecting cervical cancer and preparation method thereof |
| CN112198316A (en) * | 2020-10-28 | 2021-01-08 | 北京安必奇生物科技有限公司 | Aptamer colloidal gold test paper for detecting novel coronavirus N protein and preparation method thereof |
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Application publication date: 20190618 |