CN111239395A - Preparation method of HPV16 type E7 protein sandwich immunodetection kit - Google Patents
Preparation method of HPV16 type E7 protein sandwich immunodetection kit Download PDFInfo
- Publication number
- CN111239395A CN111239395A CN202010042572.1A CN202010042572A CN111239395A CN 111239395 A CN111239395 A CN 111239395A CN 202010042572 A CN202010042572 A CN 202010042572A CN 111239395 A CN111239395 A CN 111239395A
- Authority
- CN
- China
- Prior art keywords
- solution
- protein
- hpv16 type
- sandwich structure
- gold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000341655 Human papillomavirus type 16 Species 0.000 title claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 43
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 28
- 239000000758 substrate Substances 0.000 claims abstract description 16
- 238000003018 immunoassay Methods 0.000 claims abstract description 15
- 238000001069 Raman spectroscopy Methods 0.000 claims abstract description 14
- 239000013592 cell lysate Substances 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 90
- 239000002073 nanorod Substances 0.000 claims description 23
- CNSGPAMLXMBLNA-UHFFFAOYSA-N 2-amino-3,4,5,6-tetrafluorobenzoic acid Chemical group NC1=C(F)C(F)=C(F)C(F)=C1C(O)=O CNSGPAMLXMBLNA-UHFFFAOYSA-N 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- 238000010257 thawing Methods 0.000 claims description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 6
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims description 5
- 238000001237 Raman spectrum Methods 0.000 claims description 4
- 239000003607 modifier Substances 0.000 claims description 4
- 238000002331 protein detection Methods 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 230000009870 specific binding Effects 0.000 claims description 3
- 238000001228 spectrum Methods 0.000 claims description 3
- 108010087765 Antipain Proteins 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- 102000015833 Cystatin Human genes 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 2
- SDNYTAYICBFYFH-TUFLPTIASA-N antipain Chemical compound NC(N)=NCCC[C@@H](C=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SDNYTAYICBFYFH-TUFLPTIASA-N 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 108050004038 cystatin Proteins 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 230000010360 secondary oscillation Effects 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 239000011780 sodium chloride Substances 0.000 claims 1
- 125000003396 thiol group Chemical group [H]S* 0.000 claims 1
- 238000005336 cracking Methods 0.000 abstract 1
- 230000003287 optical effect Effects 0.000 abstract 1
- 241000701806 Human papillomavirus Species 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 206010008342 Cervix carcinoma Diseases 0.000 description 7
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 7
- 201000010881 cervical cancer Diseases 0.000 description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 6
- 230000003902 lesion Effects 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 208000009608 Papillomavirus Infections Diseases 0.000 description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 229910052709 silver Inorganic materials 0.000 description 3
- 239000004332 silver Substances 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 229910004042 HAuCl4 Inorganic materials 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000000405 serological effect Effects 0.000 description 2
- 229910001961 silver nitrate Inorganic materials 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- RYWSYCQQUDFMAU-UHFFFAOYSA-N Acetomenaphthone Chemical compound C1=CC=C2C(OC(=O)C)=CC(C)=C(OC(C)=O)C2=C1 RYWSYCQQUDFMAU-UHFFFAOYSA-N 0.000 description 1
- 206010059313 Anogenital warts Diseases 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- BCKXLBQYZLBQEK-KVVVOXFISA-M Sodium oleate Chemical compound [Na+].CCCCCCCC\C=C/CCCCCCCC([O-])=O BCKXLBQYZLBQEK-KVVVOXFISA-M 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 238000002573 colposcopy Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 208000021145 human papilloma virus infection Diseases 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229960003151 mercaptamine Drugs 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 102000027450 oncoproteins Human genes 0.000 description 1
- 108091008819 oncoproteins Proteins 0.000 description 1
- 238000009595 pap smear Methods 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57442—Specifically defined cancers of the uterus and endometrial
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Reproductive Health (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a preparation method of an HPV16 type E7 protein sandwich immunoassay kit, which comprises the following steps: (1) preparing a sandwich structure top layer solution; (2) the method comprises the steps of preparing a sandwich structure substrate solution, preparing a kit prepared by the method, and further comprising a human cervical exfoliated cell lysate, cracking the human cervical exfoliated cells in the use process, fixing HPV16 type E7 protein by the sandwich structure substrate solution, and then adding a sandwich structure top layer solution to form a sandwich structure, wherein when detection is carried out, the sandwich structure generates an optical signal under Raman laser to realize detection of HPV16 type E7 protein.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a preparation method of an HPV16 type E7 protein sandwich immunoassay kit.
Background
Cervical cancer is the second most common cancer diagnosis in women and is associated with high-risk human papillomavirus infection in 99.7% of cases, with about 520000 cervical cancers worldwide each year, with nearly 260000 deaths. There are over 100 different isolates of HPV, which have been broadly subdivided into high-risk and low-risk subtypes according to their association with cervical cancer or with benign cervical lesions or atypical hyperplasia. The low-risk HPV comprises HPV6, 11, 42, 43, 44 and the like, benign lesions such as external genital condyloma and the like often cause cervical intraepithelial low-grade lesions (CIN I), the high-risk HPV comprises HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, CP8304 and other subtypes, and is related to the occurrence of cervical cancer and cervical intraepithelial high-grade lesions (CIN II/III), particularly HPV16 and 18 types, wherein HPV16 accounts for more than 50 percent.
Human Papilloma Virus (HPV) is an epitheliotropic virus, consisting of 3 genetic regions including an Early Region (E Region), a Late Region (L Region), and a noncoding Region (UCR) or an Upstream Regulatory Region (URR). The E region is 7 genes of E6, E7, E1, E2, E3, E4 and E5 in sequence, and is involved in the replication, transcription and encoding of viral DNA, and maintenance of high copy number of viruses in cells, wherein E6 and E7 are main oncogenes of HPV, and are related to the transformation function of viral cells and carcinogenicity. The E6 and E7 proteins inactivate the tumor suppressor proteins p53 and pRB, respectively, releasing cell cycle control and inhibiting apoptosis. Thus, the best method for determining HPV status in a tumor is to measure the E6/E7 protein in tumor cells.
High-risk Human Papillomavirus (HPV) has been identified as a causative agent of cervical cancer, but more than 90% of HPV infections are transient and are cleared by the immune system within 2 years. When HPV virus is persistently infected, especially after HPV DNA and human cervical epithelial cell DNA are integrated, E6 and E7 genes can express a substance called mRNA in a large amount, so that oncogenic protein is produced in a large amount, and human cells are gradually cancerized.
Current detection methods for HPV infection are of type 3:
1. detection of HPV by conventional morphological methods
Including pap smear cytopathology tests, colposcopy, cervical biopsy histopathology, etc. Cervical cytology has high specificity but low sensitivity and is expensive. The cells are infected with HPV before they become diseased, so other methods must be incorporated.
2. Detection of HPV DNA
As mentioned above, the direct cause of cervical cancer upon persistent infection with high-risk HPV, detection of HPV DNA is important before cytological screening for normality. Currently, PCR is the most commonly used, followed by hybrid capture technology. The PCR sensitivity is high, a plurality of subtypes can be diagnosed at the same time, but false positive can occur, and the specificity is low; the hybrid capture technology has no false positive, but needs professional equipment and personnel, and cannot be popularized at present to limit the technology.
3. Serological and immunological methods
Mainly detecting the L1 antibody in the serum of a patient or detecting an antigen by using the antibody. However, the L1 antibody has a high titer several months to 1 year after HPV clearance, so detection of L1 antibody in serum is not known to be present infection or past infection. The detection of HPV E6-specific antibodies in serum by ELISA has not been studied in other HPV-associated cancers, and E6-specific antibodies can be detected in the serum of patients with oral cancer who are positive for HPV 16. The method of detecting an antigen with an antibody is mainly referred to as immunohistochemical method because of its long time and cumbersome operation. The use of serological and immunological methods for detection is therefore limited.
The above methods for detecting HPV play an important role in cervical cancer and precancerous lesion screening and precancerous prognosis, but have a common limitation: the operation procedure is complex, professional instruments and equipment are needed, the cost is high, and the popularization is not easy.
Disclosure of Invention
The invention provides a preparation method of an HPV16 type E7 protein sandwich immunoassay kit for overcoming the defects of an HPV detection method in the prior art, and the preparation method comprises the following steps:
(1) preparation of the Top layer solution of Sandwich Structure
Adding a Raman detection molecule solution into a gold-core silver-shell nanorod solution, gently shaking and centrifuging, then dispersing into deionized water, adding a functional modifier solution, gently shaking and centrifuging, then dispersing into an HPV16 type E7 protein detection antibody solution to obtain a mixed solution, storing the mixed solution for 12h, then centrifugally purifying with 1 XPBS buffer solution, and storing at 4 ℃ for 12h to obtain a sandwich structure top layer solution;
(2) preparation of Sandwich Structure substrate solution
Adding an HPV16 type E7 protein capture antibody solution into a gold-core silver-shell nanorod solution, preserving at 4 ℃ for 12 hours, adding a BSA solution into the preserved solution, and standing to obtain a sandwich structure substrate solution for later use;
further, the Raman detection molecule is 2-amino-3, 4,5, 6-tetrafluorobenzoic acid, and sulfydryl is introduced into the 2-amino-3, 4,5, 6-tetrafluorobenzoic acid through a NaSH-DMF system method;
the volume ratio of the Raman detection molecular solution to the gold-core silver-shell nanorod solution is 1: 500;
the functionalized modifier solution is a glutaraldehyde solution, and the concentration of the glutaraldehyde solution is 25% wt;
further, the HPV16 type E7 protein detection antibody is an HPV16 type E7 protein polyclonal antibody;
the HPV16 type E7 protein capture antibody is an HPV16 type E7 protein monoclonal antibody.
Further, the time for the mild shaking in the step (1) is 2 h; the centrifugation condition is 7000rpm, 10 min;
and (3) standing for 1h in the step (2).
The invention also provides an HPV16 type E7 protein sandwich immunoassay kit prepared by the method, and the kit also comprises human cervical exfoliated cell lysate;
further, the human cervical exfoliated cell lysate comprises: 0.05 to 1 percent of surfactant; 10 mM-1M buffer solution; 1mM to 10mM protease inhibitor.
Further, the surfactant is one or more than two of Tween 20, Triton-100 or sodium dodecyl sulfate; the buffer solution is phosphate-buffered saline, Tris-HCL or carbonate solution; the protease inhibitor is one or more than two of cystatin, PMSF or Antipain.
The invention further provides a use method of the HPV16 type E7 protein sandwich immunoassay kit, which comprises the following specific steps:
(a) immobilization of HPV16 type E7 protein
Obtaining sufficient cervical exfoliated cells of a human body, adding the cervical exfoliated cells into a human cervical exfoliated cell lysate, oscillating, freezing and thawing, oscillating again, centrifuging, collecting a supernatant, dropwise adding the supernatant into a gold-core silver-shell nanorod substrate solution, and culturing at room temperature;
(b) forming a sandwich structure
Dropwise adding the top layer solution of the gold-core silver-shell nanorod into a substrate solution of the gold-core silver-shell nanorod fixed with HPV16 type E7 protein, and forming a sandwich structure of detection antibody-antigen to be detected-capture antibody through specific binding;
(c) spectrum collection
Using Raman laser to irradiate the sandwich structure, and collecting a Raman spectrum;
further, the oscillation condition in the step (a) is vortex oscillation for 2-10 min;
the freezing and thawing condition is freezing and thawing for 1-3 times in a refrigerator at-20 ℃;
the secondary oscillation condition is vortex oscillation for 2-10 min;
the centrifugation condition is 13000rpm for 10-30 min;
the room temperature incubation time was 1 h.
Compared with the prior art, the invention has the advantages that:
the invention provides a preparation method of an HPV16 type E7 protein sandwich immunodetection kit, which adopts gold-core silver-shell nanoparticles to bond Raman detection molecules and detection antibodies to obtain a sandwich structure top layer solution, and adopts gold-core silver-shell nanoparticles to bond capture antibodies to obtain a sandwich structure substrate solution, and the preparation method is easy to operate and low in cost;
the invention also provides an HPV16 type E7 protein sandwich immunoassay kit, which comprises: the kit comprises a sandwich structure top layer solution, a sandwich structure substrate solution and a human cervical exfoliated cell lysate, wherein the top layer solution in the kit adopts 2-amino-3, 4,5, 6-tetrafluorobenzoic acid molecules as Raman detection molecules, and has a stronger function of strengthening Raman signals compared with P-ATP molecules commonly used in the prior art;
the invention further provides a use method of the HPV16 type E7 protein sandwich immunoassay kit, and the use method is simple and convenient to operate, high in sensitivity and capable of rapidly determining the target protein.
Drawings
FIG. 1 is a diagram showing the process of the formation of a sandwich immunoassay structure for HPV16 type E7 protein in this example;
FIG. 2 is a Raman spectrum of HPV16 type E7 protein.
Detailed Description
The objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.
Embodiments of the present invention will hereinafter be described with reference to the accompanying drawings, wherein like reference numerals denote like or similar parts, or like or similar steps.
Examples
The embodiment provides a preparation method of an HPV16 type E7 protein sandwich immunoassay kit, which comprises the following steps:
1. preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6 mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
3. Raman detection of molecular bonding
Introducing sulfydryl into 2 mu L of 5mM 2-amino-3, 4,5, 6-tetrafluorobenzoic acid by a NaSH-DMF system method, adding the sulfydryl into 1.0ml of gold-core silver-shell nanorod suspension to obtain a mixture, and gently shaking the mixture for 2h to obtain a 2-amino-3, 4,5, 6-tetrafluorobenzoic acid-labeled gold-core silver-shell nanorod solution.
4. Preparation of top layer structure with labeled detection antibody
2-amino-3, 4,5, 6-tetrafluorobenzoic acid thiol group was introduced by NaSH-DMF system method, added to 1.0mL of gold core silver shell nanorod solution, and the mixture was gently shaken for 2h to obtain vitamin K4 labeled gold core silver shell nanorod solution, which was then centrifuged at 7000rpm for 10min and dispersed in 1.0mL of deionized water. Next, 2. mu.L of a 25% wt glutaraldehyde solution was added to 1.0mL of 2-amino-3, 4,5, 6-tetrafluorobenzoic acid-labeled gold-core silver-shell nanorod solution, gently shaken for 1.5h, the product was centrifuged at 6000rpm for 10min, and then dispersed in 1mL of 9. mu.g/mL HPV16 type E7 protein polyclonal antibody solution, and the mixed solution was stored at 4 ℃ for 12h, then centrifuged and purified with 1.0mL of 1 XPBS buffer, and stored at 4 ℃ for use.
5. Preparation of substrate Structure with Capture antibody
Firstly, carrying out amino functionalization treatment on a gold-core silver-shell nanorod solution by using a 25mM cysteamine solution, then adding 2 mu L of 25% wt glutaraldehyde solution to functionalize the gold-core silver-shell nanorod solution, then adding 2mL of HPV16 type E7 protein monoclonal antibody solution with the concentration of 20 mu g/mL into the functionalized gold-core silver-shell nanorod solution, storing the solution at the temperature of 4 ℃ for 12 hours, then adding 2mL of BSA solution with the concentration of 1% into the stored solution, and standing the solution for 1 hour to block non-specific binding active sites.
6. Immobilization of HPV16 type E7 protein
Obtaining sufficient cervical exfoliated cells of a human body, adding the obtained cervical exfoliated cells into a human cervical exfoliated cell lysate, performing vortex oscillation for 2-10 min, putting the obtained product into a refrigerator with the temperature of-20 ℃ for freeze thawing for 1-3 times, performing oscillation for 2-10 min again, centrifuging for 10-30 min at the centrifugation speed of 13000rpm, and collecting supernatant;
wherein, the cervical exfoliated cell lysate entering the human body is 1% Triton-100 prepared by using 100mM Tris-HCL solution; adding a protease inhibitor PMSF with the final concentration of 10mM before use;
taking 200 mu L of supernatant, dripping the supernatant on a substrate with an HPV16 type E7 protein monoclonal antibody, and incubating for 1h at room temperature;
meanwhile, a blank control group is prepared, namely human cervical exfoliated cells are not added into the lysate, and the rest steps are unchanged.
7. Sandwich structure assembly of detection antibody-antigen to be detected-capture antibody
Dripping 200 mu L of 2-amino-3, 4,5, 6-tetrafluorobenzoic acid labeled gold core silver shell nanorod solution bonded with HPV16 type E7 protein polyclonal antibody into substrate solutions of an experimental group and a blank group, wherein the substrate solutions are fixed with HPV16 type E7 protein, and forming a sandwich structure of detection antibody-antigen to be detected-capture antibody after specific binding;
test examples
A Renishaw inVia confocal Raman spectrometer is adopted, a 20-time objective lens and 785nm laser are used as excitation sources, and Raman spectra of an experimental group and a blank group in the embodiment are collected on a Leica microscope. The spectrum is in the range of 800-1800cm < -1 >, the exposure time is 10s, the detection result is shown in figure 2, and the result shows that the sandwich immunoassay kit for the HPV16 type E7 protein prepared in the example can realize the rapid detection of the HPV16 type E7 protein.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (9)
1. A preparation method of an HPV16 type E7 protein sandwich immunoassay kit is characterized by comprising the following steps:
(1) preparation of the Top layer solution of Sandwich Structure
Adding a Raman detection molecule solution into a gold-core silver-shell nanorod solution, gently shaking and centrifuging, then dispersing into deionized water, adding a functional modifier solution, gently shaking and centrifuging, then dispersing into an HPV16 type E7 protein detection antibody solution to obtain a mixed solution, storing the mixed solution for 12h, then centrifugally purifying with 1 XPBS buffer solution, and storing at 4 ℃ for 12h to obtain a sandwich structure top layer solution;
(2) preparation of Sandwich Structure substrate solution
Adding an HPV16 type E7 protein capture antibody solution into a gold-core silver-shell nanorod solution, preserving at 4 ℃ for 12 hours, adding a BSA solution into the preserved solution, and standing to obtain a sandwich structure substrate solution for later use.
2. The method for preparing the kit according to claim 1, wherein the raman detection molecule is 2-amino-3, 4,5, 6-tetrafluorobenzoic acid, and the 2-amino-3, 4,5, 6-tetrafluorobenzoic acid is introduced with a thiol group by NaSH-DMF system method;
the volume ratio of the Raman detection molecular solution to the gold-core silver-shell nanorod solution is 1: 500;
the functionalized modifier solution is glutaraldehyde solution, and the concentration of the glutaraldehyde solution is 25% wt.
3. The method for preparing a kit according to claim 1, wherein the HPV16 type E7 protein detection antibody is an HPV16 type E7 protein polyclonal antibody;
the HPV16 type E7 protein capture antibody is an HPV16 type E7 protein monoclonal antibody.
4. The method for preparing a kit according to claim 1, wherein the time for the gentle shaking of step (1) is 2 hours; the centrifugation condition is 7000rpm, 10 min;
and (3) standing for 1h in the step (2).
5. An HPV16 type E7 protein sandwich immunoassay kit prepared by the method of claims 1-4, wherein the kit further comprises a human cervical exfoliated cell lysate.
6. The sandwich immunoassay kit of claim 5, wherein the human cervical exfoliated cell lysate comprises: 0.05 to 1 percent of surfactant; 10 mM-1M buffer solution; 1mM to 10mM protease inhibitor.
7. The sandwich immunoassay kit of claim 6, wherein the surfactant is one or more of Tween 20, Triton-100 or sodium dodecyl sulfate; the buffer solution is phosphate-buffer saline, Tris-HCL or carbonate solution; the protease inhibitor is one or more than two of cystatin, PMSF or Antipain.
8. A method of using the HPV16 type E7 protein sandwich immunoassay kit of claim 5, characterized in that the method of use comprises the following steps:
(a) immobilization of HPV16 type E7 protein
Obtaining sufficient cervical exfoliated cells of a human body, adding the cervical exfoliated cells into a human cervical exfoliated cell lysate, oscillating, freezing and thawing, oscillating again, centrifuging, collecting a supernatant, dropwise adding the supernatant into a gold-core silver-shell nanorod substrate solution, and culturing at room temperature;
(b) forming a sandwich structure
Dropwise adding the top layer solution of the gold-core silver-shell nanorod into a substrate solution of the gold-core silver-shell nanorod fixed with HPV16 type E7 protein, and forming a sandwich structure of detection antibody-antigen to be detected-capture antibody through specific binding;
(c) spectrum collection
The sandwich structure was illuminated with raman laser and raman spectra were collected.
9. The use method according to claim 8, wherein the shaking condition in the step (a) is vortex shaking for 2-10 min; the freezing and thawing condition is freezing and thawing for 1-3 times in a refrigerator at-20 ℃; the secondary oscillation condition is vortex oscillation for 2-10 min; the centrifugation condition is 13000rpm for 10-30 min; the room temperature incubation time was 1 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010042572.1A CN111239395A (en) | 2020-01-15 | 2020-01-15 | Preparation method of HPV16 type E7 protein sandwich immunodetection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010042572.1A CN111239395A (en) | 2020-01-15 | 2020-01-15 | Preparation method of HPV16 type E7 protein sandwich immunodetection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111239395A true CN111239395A (en) | 2020-06-05 |
Family
ID=70871140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010042572.1A Pending CN111239395A (en) | 2020-01-15 | 2020-01-15 | Preparation method of HPV16 type E7 protein sandwich immunodetection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111239395A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113820481A (en) * | 2020-06-19 | 2021-12-21 | 亳州市新健康科技有限公司 | Sandwich immunoassay kit |
| CN113820505A (en) * | 2020-06-19 | 2021-12-21 | 亳州市新健康科技有限公司 | Kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101504416A (en) * | 2008-05-20 | 2009-08-12 | 湖南工业大学 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
| WO2010109268A1 (en) * | 2009-03-23 | 2010-09-30 | Yeditepe Universitesi | Synthesis of oligonucleotide mediated gold core- silver shell nanoparticles |
| CN106610378A (en) * | 2016-02-26 | 2017-05-03 | 弗雷米德生物医药技术（天津）有限公司 | Detection kit for HPV16-E7 protein |
| CN108088994A (en) * | 2017-12-15 | 2018-05-29 | 南京医科大学第二附属医院 | A kind of hollow core shell nanoparticles and preparation method, test strips and test method |
| WO2018102982A1 (en) * | 2016-12-06 | 2018-06-14 | 亳州市新健康科技有限公司 | Poct fluorescence quantitative detection kit for hpv16 e7 protein and application thereof |
| CN108218983A (en) * | 2018-03-01 | 2018-06-29 | 珠海美华医疗科技有限公司 | Monoclonal antibody, Test paper and the detection kit of anti-HPV18 types E7 albumen |
| CN110618123A (en) * | 2019-09-11 | 2019-12-27 | 亳州市新健康科技有限公司 | Efficient surface-enhanced Raman scattering substrate material and preparation method thereof |
-
2020
- 2020-01-15 CN CN202010042572.1A patent/CN111239395A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101504416A (en) * | 2008-05-20 | 2009-08-12 | 湖南工业大学 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
| WO2010109268A1 (en) * | 2009-03-23 | 2010-09-30 | Yeditepe Universitesi | Synthesis of oligonucleotide mediated gold core- silver shell nanoparticles |
| CN106610378A (en) * | 2016-02-26 | 2017-05-03 | 弗雷米德生物医药技术（天津）有限公司 | Detection kit for HPV16-E7 protein |
| WO2018102982A1 (en) * | 2016-12-06 | 2018-06-14 | 亳州市新健康科技有限公司 | Poct fluorescence quantitative detection kit for hpv16 e7 protein and application thereof |
| CN108088994A (en) * | 2017-12-15 | 2018-05-29 | 南京医科大学第二附属医院 | A kind of hollow core shell nanoparticles and preparation method, test strips and test method |
| CN108218983A (en) * | 2018-03-01 | 2018-06-29 | 珠海美华医疗科技有限公司 | Monoclonal antibody, Test paper and the detection kit of anti-HPV18 types E7 albumen |
| CN110618123A (en) * | 2019-09-11 | 2019-12-27 | 亳州市新健康科技有限公司 | Efficient surface-enhanced Raman scattering substrate material and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| LEI WU等: "A SERS-based immunoassay with highly increased sensitivity using gold/silver core-shell nanorods", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 刘春竹等: "金纳米棒催化的鲁米诺-硝酸银化学发光及免疫分析应用", 《中国科学技术大学学报》 * |
| 周松伟: "HPV新型夹心酶联免疫检测法的建立及验证", 《标记免疫分析与临床》 * |
| 郭红燕等: "SERS标记的金纳米棒探针用于免疫检测", 《化学学报》 * |
| 韦超: "二元纳米离子-金属耦合体系的SERS研究及其在免疫分析中的应用", 《中国博士学位论文全文数据库（工程科技Ⅰ辑）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113820481A (en) * | 2020-06-19 | 2021-12-21 | 亳州市新健康科技有限公司 | Sandwich immunoassay kit |
| CN113820505A (en) * | 2020-06-19 | 2021-12-21 | 亳州市新健康科技有限公司 | Kit |
| CN113820502A (en) * | 2020-06-19 | 2021-12-21 | 亳州市新健康科技有限公司 | Sandwich immunoassay kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2300824B1 (en) | Detection of early stages and late stages hpv infection | |
| JP5819851B2 (en) | Cell-based high-throughput HPV immunoassay for the treatment and screening of HPV-related cancers | |
| US9995745B2 (en) | Method of efficient extraction of protein from cells | |
| US10031130B2 (en) | Device for high throughput detection of cervical disease | |
| CN101186636A (en) | Human papillomavirus coat protein L1 short peptide and its application | |
| JP2004505247A (en) | Detection of abnormalities leading to cervical malignancy | |
| Xiao et al. | Observations on the expression of human papillomavirus major capsid protein in HeLa cells | |
| JP4290118B2 (en) | Methods for distinguishing dysplasia from neoplastic or preneoplastic lesions | |
| CN111239395A (en) | Preparation method of HPV16 type E7 protein sandwich immunodetection kit | |
| CN103033625B (en) | Human bladder cancer cellular chemiluminescent detection kit and preparation method thereof | |
| Kong et al. | Analysis of the role of the human papillomavirus 16/18 E7 protein assay in screening for cervical intraepithelial neoplasia: a case control study | |
| Zheng et al. | High-risk human papillomavirus identification in precancerous cervical intraepithelial lesions | |
| CN105548539B (en) | A kind of indirect immunofluorescene assay method of Lassa virus IgG antibody | |
| Bhattarakosol et al. | Immunogold-agglutination assay for direct detection of HPV-16 E6 and L1 proteins from clinical specimens | |
| US8968995B2 (en) | Detection, screening, and diagnosis of HPV-associated cancers | |
| Faoro et al. | Detection of HPV E7 oncoviral protein in cervical lesions by a new antibody | |
| TWI497075B (en) | Detection of early stages and late stages hpv infection | |
| CN108218983A (en) | Monoclonal antibody, Test paper and the detection kit of anti-HPV18 types E7 albumen | |
| Chen et al. | Non-invasive urinary sediment double-immunostaining predicts BK polyomavirus associated-nephropathy in kidney transplant recipients | |
| El-Wakil et al. | High-risk human papillomavirus infection and cervical cytopathology: relationship with cervical nitric oxide levels | |
| Haltas et al. | The immunohistochemical detection of P16 and HPV L1 capsid protein on cell block sections from residual PapSpin liquid-based gynecology cytology specimens as a diagnostic and prognostic tool. | |
| JP2006521104A (en) | Method for early diagnosis of carcinoma and test kit for performing the same | |
| KR100671825B1 (en) | Cervical cancer diagnostic kit caused by HPV | |
| EP1936379A1 (en) | Method for the detection of active hpv infection in biological samples by a sandwich enzyme-linked immunosorbent assay and test kit for performing the method | |
| Khan et al. | Molecular Diagnosis of Human Papillomavirus: Current Trends and Recent Approaches in Molecular Diagnosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200605 |