CN105785044A - Bladder cancer detection kit and method and application of human complement factor H related protein therein - Google Patents

Bladder cancer detection kit and method and application of human complement factor H related protein therein Download PDF

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Publication number
CN105785044A
CN105785044A CN201610224922.XA CN201610224922A CN105785044A CN 105785044 A CN105785044 A CN 105785044A CN 201610224922 A CN201610224922 A CN 201610224922A CN 105785044 A CN105785044 A CN 105785044A
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bladder cancer
detection kit
antibody
cancer detection
detection
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CN105785044B (en
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邢志祺
齐颖颖
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Hebei Tewente Biological Technology Development Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

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  • Urology & Nephrology (AREA)
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Abstract

The invention relates to a bladder cancer detection kit and method. A monoclonal antibody and a polyclonal antibody which are formed through the human complement factor H are involved. The invention further relates to application of human complement factor H related protein. BTA is selected as a marker for detecting bladder cancer, three different monoclonal antibodies obtained from polypeptide immune are mixed to be used as a coating antibody, the sensitivity is high, and specificity is high. The coating antibody and a detection antibody are obtained from antigen immune of different sources, and then detection accuracy is improved. The bladder cancer detection kit and method are high in detection precision, good in specificity, high in patient compliance and capable of achieving detection conveniently.

Description

Bladder cancer detection kit, detection method and human complement factor H associated protein are at it In application
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of bladder cancer detection kit, detection method and the mankind and mend The application wherein of body factor H associated protein.
Background technology
CFH associated protein (human complement factor H related protein, HCFHrp), it is also called bladder tumor antigen (Bladder tumor antigen, BTA), complement factor H (Complement Factor H) it is its concrete composition, complement factor H is produced by cancer cell and macrophage, is not normal epidermis cell Produce, tumor cell secretion endogenous basement proteins and basement membrane surface protein receptor binding, and it is broken to discharge proteolytic enzyme Bad basement membrane, basement membrane fragment (collagen fragment, glycoprotein and proteoglycan etc.) enters intravesical and is polymerized to polymer composite i.e. BTA.Size is 16～165kd, discharges with urine, can detect as bladder cancer antigen.
BTA plays the part of the negative feedback of key controlling complement activation alternative route when, and this alternative route is permissible Cracking foreign cell (by membrane attack complex cell lysis).After BTA conjugated complement factor C3b, film can be suppressed to attack multiple The formation of compound, thus stop tumor cell lysis.The generation of BTA gives interior tumor cell one selective growth advantage, Cell evasion host immune system is allowed to monitor.Briefly, in regulation and control in BTA helps neoplastic cells escape human body, it is to avoid apoptosis.
Early diagnosis and recurrence monitoring are the important contents of bladder cancer diagnosis and treatment.Diagnostic measures mainly has urine de-the most clinically Fall cytolgical examination, cystoscopy and biopsy.Urine sediment checks that specificity is high, but sensitivity is low, especially leakiness Inspection C1 level tumor (BTA90%).And cystoscope is invasive inspection, it is impossible to early diagnosis and patient compliance are poor.Therefore BTA is subject to Increasing concern.Urine sediment checks, indwelling twenty-four-hour urine, and the urine taking off face deposition does microscopy, even Continuous doing three days takes a block organization with pincers under bladder cancer biopsy usually cystoscope and does pathological examination.Sealing wax section microscopy urine comes off Cytolgical examination is mainly used in upper urinary tract cell carcinoma, and the most important clinical characters of bladder transitional cell carcinoma is easily to recur and along with multiple Send out number of times increase tumor and be more prone to pernicious.Therefore the recurrence of suitable molecular biology mark prediction bladder transitional cell carcinoma is selected, can Take effective remedy measures in early days, reduce tumour progression chance.
The examination of tumor of bladder is ultrasonic, ultrasonic is found to have thing, is pelvic cavity CT, if to determine whether cancer, does Cystoscope takes biopsy (being not do urine sediment) first ultrasonic (noinvasive is the most cheap), and CT(costliness is penetrated again the most again Line), finally it is only cystoscope (invasive is also expensive).
Present stage, urgently develop a kind of can as a kind of detection method of bladder cancer early diagnosis fast qualitative again, And method can be commonly used to masses.
Summary of the invention
The technical problem to be solved is to provide that a kind of accuracy of detection is high, specificity is good, patient compliance is high, Bladder cancer detection kit easy to use, bladder cancer detection method, provide human complement factor H associated protein in preparation simultaneously Application in described bladder cancer detection kit.
The technical scheme is that a kind of bladder cancer detection kit, including polyclonal antibody, utilize human complement because of Sub-H construction recombination plasmid pET32a-CFH, with e. coli bl21 (DE3) as expressive host, then uses KTA system to carry out Protein purification, obtains BTA prokaryotic expression protein, immunity New Zealand white rabbit, obtains polyclonal antibody.
Further, during described construction recombination plasmid, forward primer sequence is SEQ ID No. 1: Agacttctagcaaagattat, reverse primer sequences is SEQ ID No. 2:tctttttgcacaagttggat.
Further, described polyclonal antibody carries out horseradish peroxidase labelling and is used as enzyme labelled antibody, polyclonal antibody Concentration is 7.5 μ g/mL.
Further, also including monoclonal antibody, choose three sections of synthesis polypeptide in BTA aminoacid sequence, polypeptide is respectively The immunity female 6 week old healthy mices of BALB/c, obtain three monoclonal antibodies 1B9,5F6 and 6D2, and sequence is respectively SEQ ID No. 3:tgsws dqtypegtqa iykcr, SEQ ID No. 4:qkrpcghp gdtpfgtftl tggnvfeyg and SEQ ID No. 5:fvc nsgykiegde emhcsddgfw.
Further, the mass ratio of monoclonal antibody is 1B9:5F6:6D2=2:1:3, as coated antibody, is coated Buffer is 0.01M CB, the carbonate buffer solution of the most conventional use of pH9.6, and being coated concentration is 5 μ g/mL.
The present invention also provides for a kind of utilizing described test kit to carry out the method detected, and uses double crush syndrome detection side Method.
Present invention simultaneously provides human complement factor H associated protein answering in preparing described bladder cancer detection kit With.
Beneficial effect: the present invention chooses the mark that BTA is detection bladder cancer, for 3 different polypeptide immune gained lists Resist to mix the coated antibody as the present invention, highly sensitive, high specificity；Coated antibody is separate sources with detection antibody Antigen immune gained, and then promote detection accuracy.
Accuracy of detection of the present invention is high, specificity is good, patient compliance is high, easy to detect.
Accompanying drawing explanation
Fig. 1 is the standard curve of test kit in embodiment.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, implement below in conjunction with the present invention Detailed description of the invention in example, is clearly and completely described the technical scheme in the embodiment of the present invention, it is clear that described Embodiment be only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, ability The every other embodiment that territory those of ordinary skill is obtained under not making creative work premise, broadly falls into the present invention and protects The scope protected.
Embodiment 1
A kind of bladder cancer detection kit, including polyclonal antibody, utilizes human complement factor H construction recombination plasmid pET32a- CFH, with BL21 (DE3) as expressive host, carries out protein expression, uses KTA system to carry out protein purification, obtains BTA protokaryon table Reach albumen, immunity New Zealand white rabbit, obtain polyclonal antibody.
During described construction recombination plasmid, with GenBank numbering CAA68704.1 as template, use Primer Premier 5 Design primer: forward primer is agacttctagcaaagattat, and reverse primer is Tctttttgcacaagttggat.
Described polyclonal antibody carries out horseradish peroxidase labelling and is used as enzyme labelled antibody, and Anti-TNF-α bulk concentration is 7.5 μ g/mL。
This test kit also includes monoclonal antibody, chooses three sections of synthesis polypeptide in BTA aminoacid sequence, and polypeptide is exempted from respectively The female 6 week old healthy mices of epidemic disease BALB/c, obtain three monoclonal antibodies 1B9,5F6 and 6D2.
The ratio of monoclonal antibody is 1B9:5F6:6D2=2:1:3, and as coated antibody, being coated buffer is 0.01M CB, being coated concentration is 5 μ g/mL.
This test kit detection bladder cancer uses double crush syndrome detection method, sets up double antibodies sandwich detection system: anti- 3 monoclonal antibodies of 3 sections of polypeptide immune gained of BTA mix (1B9:5F6:6D2=2:1:3), by a certain percentage for conduct Coated antibody, being coated buffer is 0.01M CB, and it is coated concentration is 5 μ g/mL；Gained polyclonal antibody carries out Radix Cochleariae officinalis peroxidating Enzyme labelling is used as enzyme labelled antibody, and its concentration is 7.5 μ g/mL, and its detection line is 0.5-16U/mL.
Embodiment 2
Sample is detected with the test kit of embodiment 1.
(1) drafting of standard curve
Taking out being coated plate in test kit, in recovering room temperature 30min, take 10 detection holes, every hole adds the standard of 100 microlitres Product, be specially with normal urine as blank, be separately added into 0 U/ml, 0.5U/ml, 1 U/ml, 2 U/ml, 4 U/ml, 8 U/ml, The BTA standard substance of 16 U/ml, 32U/ml, in 37 degree of incubation 40min, clean detection plate 3 times, and every hole adds 100 microlitres Enzyme labelled antibody, in 37 degree of incubation 25min, cleans detection plate 5 times, and every hole adds the nitrite ion of 100 microlitres, in 37 degree of incubators Hatching 10min, every hole adds the stop buffer of 50 microlitres, testing result at microplate reader 450nm, draws standard curve.It is specifically shown in Fig. 1.
(2) detection of sample
Taking out being coated plate in test kit, in recovering room temperature 30min, take 10 detection holes, every hole adds the sample of 100 microlitres, Its sample is that 10 positive samples of hospital's randomization detect plate 3 times in 37 degree of incubation 40min, cleaning, and it is micro-that every hole adds 100 The enzyme labelled antibody risen, in 37 degree of incubation 25min, cleans detection plate 5 times, and every hole adds the nitrite ion of 100 microlitres, in 37 degree Incubation 10min, every hole adds the stop buffer of 50 microlitres, testing result at microplate reader 450nm.Then with standard curve pair According to, draw BTA content.Certain commercial reagent box product on the market is used to carry out detection comparison.The results are shown in Table 1.
Embodiment 3
Hebei hospital journals monitoring
2014.12-2015.6 totally 66 example patients go to a doctor to certain hospital outpatient because of hematuria (53 example) or other main suits.
This organizes 66 examples, male 50 examples, female 16 example, age 20-88 year.
All patients carry out conventional preoperative planning, and the imaging examination such as B ultrasonic, CT or CTU after being admitted to hospital.
The urine specimen of all patients is left and taken before cystoscopy, carries out the detection of BTA test kit of the present invention.
Positive 35 examples: include urothelial tumor 25 example (bladder cancer 16 example, tumor of renal pelvis 5 example, tumor of ureter 4 example)； Urothelium papillary hyperplasia accompanies different increasing 3 example, ureteral calculus 3 example, urinary system infection 2 example, chyluria 1 example, cystectomy art Rear hydronephrosis 1 example；This test kit detects in strong positive.
Weak positive 8 examples: including bladder cancer 1 example, adenocarcinoma of bladder 1 example, carcinoma of prostate 1 example, glandular cystitis 1 example, on bladder Skin papillary hyperplasia accompanies different increasing 1 example, ureteral calculus 1 example, cyst of kidney 1 example, prostatic hyperplasia 1 example；This test kit detects in weak Positive.
Negative 23 examples: include bladder cancer 5 example, carcinoma of renal pelvis 1 example, clear cell carcinoma of kidney 4 example, carcinoma of prostate 1 example, urothelium Papillary hyperplasia accompanies different increasing 2 example, cyst of kidney 2 example, urethral caruncle 1 example, prostatic hyperplasia 3 example, ureteral calculus 1 example, bladder cancer Postoperative check no abnormality seen 3 example；The detection of this test kit is negative.
SEQUENCE LISTING
<110>Hebei Te Wente biotechnology Development Co., Ltd
<120>bladder cancer detection kit, detection method and the application wherein of human complement factor H associated protein
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
agacttctag caaagattat 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
tctttttgca caagttggat 20
<210> 3
<211> 20
<212> Protein
<213>mice
<400> 3
tgswsdqtyp egtqaiykcr 20
<210> 4
<211> 27
<212> Protein
<213>mice
<400> 4
qkrpcghpgd tpfgtftltg gnvfeyg 27
<210> 5
<211> 23
<212> Protein
<213>mice
<400> 5
fvcnsgykie gdeemhcsdd gfw 23

Claims (7)

1. a bladder cancer detection kit, it is characterised in that include polyclonal antibody, utilizes human complement factor H to build weight Group plasmid pET32a-CFH, with e. coli bl21 as expressive host, then carries out protein purification, obtains BTA prokaryotic expression egg In vain, immunity New Zealand white rabbit, obtain polyclonal antibody.
Bladder cancer detection kit the most according to claim 1, it is characterised in that during construction recombination plasmid, forward primer Sequence is SEQ ID No. 1, and reverse primer sequences is SEQ ID No. 2.
Bladder cancer detection kit the most according to claim 1, it is characterised in that described polyclonal antibody carries out Radix Cochleariae officinalis mistake Oxidase label is used as enzyme labelled antibody, and Anti-TNF-α bulk concentration is 7.5 μ g/mL.
Bladder cancer detection kit the most according to claim 1, it is characterised in that also include monoclonal antibody, choose BTA Three sections of synthesis polypeptide in aminoacid sequence, the polypeptide immunity female healthy mice of BALB/c respectively, obtain three monoclonal antibodies 1B9,5F6 and 6D2, sequence is respectively SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.
Bladder cancer detection kit the most according to claim 2, it is characterised in that the mass ratio of monoclonal antibody is 1B9:5F6:6D2=2:1:3, as coated antibody, being coated buffer is 0.01M CB, and being coated concentration is 5 μ g/mL.
6. utilize claim 1-5 any one test kit to carry out the method detected, use double crush syndrome detection side Method.
7. human complement factor H associated protein answering in bladder cancer detection kit described in preparation any one of claim 1-5 With.
CN201610224922.XA 2016-04-12 2016-04-12 Carcinoma of urinary bladder detection kit, the application of detection method and human complement factor H GAP-associated protein GAPs wherein Active CN105785044B (en)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN106519031A (en) * 2016-12-26 2017-03-22 广州泰诺迪生物科技有限公司 CFH antibody related with alternative pathway
CN107300621A (en) * 2017-06-23 2017-10-27 石家庄洹众生物科技有限公司 Quantitatively detect the detection reagent card and system of human complement factor H GAP-associated protein GAPs
CN111579786A (en) * 2020-05-27 2020-08-25 郑州大学第一附属医院 Test strip for screening early esophageal squamous carcinoma of high risk group
CN115261401A (en) * 2022-08-25 2022-11-01 杭州博岳生物技术有限公司 Method for developing bladder tumor antigen monoclonal antibody by using yeast cell surface display technology

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WO2009113674A1 (en) * 2008-03-14 2009-09-17 学校法人北里研究所 Diagnosis of bladder carcinoma
CN101900733A (en) * 2010-08-13 2010-12-01 中南大学 Tumor marker colloidal gold immunochromatographic assay quantitative detection test paper and method for preparing same
AU2012216822A1 (en) * 2012-09-13 2014-03-27 Randox Laboratories Ltd Bladder cancer
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106519031A (en) * 2016-12-26 2017-03-22 广州泰诺迪生物科技有限公司 CFH antibody related with alternative pathway
CN106519031B (en) * 2016-12-26 2022-03-22 广州泰诺迪生物科技有限公司 CFH antibody related to alternative pathway
CN107300621A (en) * 2017-06-23 2017-10-27 石家庄洹众生物科技有限公司 Quantitatively detect the detection reagent card and system of human complement factor H GAP-associated protein GAPs
CN111579786A (en) * 2020-05-27 2020-08-25 郑州大学第一附属医院 Test strip for screening early esophageal squamous carcinoma of high risk group
CN112415197A (en) * 2020-05-27 2021-02-26 郑州大学第一附属医院 Application of tumor markers in test paper strip for early screening of esophageal squamous cell carcinoma
CN115261401A (en) * 2022-08-25 2022-11-01 杭州博岳生物技术有限公司 Method for developing bladder tumor antigen monoclonal antibody by using yeast cell surface display technology
CN115261401B (en) * 2022-08-25 2023-10-20 杭州博岳生物技术有限公司 Method for developing bladder tumor antigen monoclonal antibody by using yeast cell surface display technology

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