CN109187975A - A kind of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip - Google Patents
A kind of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip Download PDFInfo
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- CN109187975A CN109187975A CN201810898334.3A CN201810898334A CN109187975A CN 109187975 A CN109187975 A CN 109187975A CN 201810898334 A CN201810898334 A CN 201810898334A CN 109187975 A CN109187975 A CN 109187975A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
Abstract
The present invention relates to a kind of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strips, including bottom plate and the sample pad being sequentially arranged on bottom plate, bonding pad, analyzing film and water absorption pad, the anti-human calcitonin detection antibody of colloid gold label and the anti-carcinoembryonic antigen detection antibody of colloid gold label are combined on bonding pad, the human calcitonin that analyzing film is equipped with detects band T1 line, carcinomebryonic antigen detection band T2 line and quality control band C line, calcitonin detection band T1 line, anti-human calcitonin capture antibody is coated on carcinomebryonic antigen detection band T2 line and quality control band C line respectively, anti-carcinoembryonic antigen captures antibody and sheep anti-mouse igg polyclonal antibody, the present invention also provides the preparation methods of above-mentioned medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip.The present invention is convenient to use, at low cost, specificity and high sensitivity.
Description
Technical field
The present invention relates to field of immunoassay detection, more particularly to a kind of medullary carcinoma of thyroid gland joint-detection colloid
Golden immuno-chromatographic test paper strip and preparation method thereof.
Background technique
Medullary carcinoma of thyroid gland (MTC) derives from parafollicular cell (also known as C cell) canceration, belongs to neuroendocrine
Cell.It is mainly characterized by secreting calcitonin and many kinds of substance includes carcinomebryonic antigen, and generates amyloid.Nineteen fifty-nine by
Hazand etc. is proposed first as an independent clinicopathologic pattern, morbidity, the unique feature of diagnosing and treating.Thyroid gland
Cephaloma is more rare, accounts for about the 3~10% of thyroid cancer, and it is pernicious to belong to moderate.Tumor invading blood vessel occurs DISTANT METASTASES IN and is up to
15%, common liver, lung, Bone tumour are patient's MTC underlying cause of death.MTC can be divided into sporadic and heredity two major classes, it is seen that
In any age, sporadic occurrence is in, old age is common, and familial age of onset is more early, men and women's morbidity no significant difference, medical history
Longer, moderate pernicious, lymphatic metastasis is common and relatively early occurs, and the rate of transform is high, and about 10% double Cervical Lymph Node Metastasis occurs,
First visit rate of cervical lymph node metastasis may be up to 70%, wherein 1/4 patient examines symptom as head, advanced stage can be transferred to through blood lung,
The distal organs such as liver, bone.Survival rate is slightly below papillary carcinoma, and prognosis is affected by many factors, II-a of MEN,muitiple endocrine neoplasms
Type prognosis is best.
C cell belongs to neuroendocrine cell, can synthesize secretion various biological active material, as calcitonin, cancer embryo are anti-
Original, corticotropin, histamine and vasoactive peptide etc., wherein calcitonin is the tumor-marker of cephaloma specificity
Object can be used as medullary carcinoma of thyroid gland diagnosis and judge the index of operative effect and postoperative recurrence.
Calcitonin is a kind of peptide hormone, and major function is to reduce blood calcium, and calcitonin increases to originating from thin by folliculus
The diagnosis of the medullary carcinoma of thyroid gland of born of the same parents judges that operative effect and observation postoperative recurrence etc. are significant.Calcitonin is MTC sensitivity
And the index of specificity, without stimulation, the obvious high expression of calcitonin then prompts the presence of MTC.Calcitonin
It horizontal tumor size with MTC and is positively correlated by stages, Calcitonin Level doubling time reflects lesion growth degree, with prognosis
It is related.
Be down to maintenance level within calcitonin postoperative 1 week or so, so can will at this time Calcitonin Level as a monitoring point, art
MTC patient's month after operation calcitonin level that precalcitonin level increases underwent operative treatment, which is down to, normally illustrates that tumour is cut
Except thorough, calcitonin is still higher than normal patient that residual tumor is more easy to recur more, and postoperative calcitonin level raising shows
Tumor recurrence or DISTANT METASTASES IN.If Follow-up After medullary carcinoma of thyroid gland patient has found that calcitonin persistently increases, residual tumor is prompted
Recur or have DISTANT METASTASES IN, should row neck portion B super, CT, MR etc. check that feasible PET-CT is checked when necessary, as can operative treatment person
It selects second operation or carries out the treatment such as radiotherapy or molecular targeted agents, largely reduce patient and toss about inspection
Pain, and save time and cost.But clinically it has also been found that there is quite a few MTC patient's Calcitonin Level not high, this portion
Divide patient that cannot judge recurrence and prognosis with calcitonin.By document verification and repetition test, discovery is equally secreted by C cell
Carcinomebryonic antigen and MTC lesion degree also correlation.
Carcinomebryonic antigen (CEA) is originally found in colon cancer and fetal gut tissue, and CEA raising is common in colorectal cancer, pancreas
Cancer, gastric cancer, breast cancer, medullary carcinoma of thyroid gland etc., but smoking, the gestational period and cardiovascular disease, diabetes, nonspecific colonitis
Etc. diseases can also increase, so CEA is not the specificity marker of malignant tumour, only there is auxiliary to be worth in diagnosis.
For MTC more than 50% there are the height expression of change of serum C EA, expression and calcitonin have synchronism.MTC patient's inspection
The change of serum C EA measured increases generally will not be obvious as tumor in digestive tract, and CEA raising more readily occurs in Tumor Differentiation and compares
In poor big case, it is prognosis that if the Calcitonin Level of MTC patient is stable, CEA, which is persistently increased and often illustrated tumor de-differentiation,
Bad mark.So CEA is very necessary in the tracking and monitoring of prognostic stage.
Since change of serum C EA half-life period is long compared with calcitonin, about 2-8 days, therefore when the postoperative change of serum C EA of MTC restores normal
Between will be longer.If tumor resection is thorough, calcitonin and general full recovery in CEA 3 months after surgery are normal, but change of serum C EA declines
Process compared with calcitonin lag.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of easy to operate, high sensitivities, the high and low cost of accuracy
Medullary carcinoma of thyroid gland calcitonin, carcinomebryonic antigen joint-detection colloidal gold immune chromatography test are realized to human calcitonin, carcinomebryonic antigen
Joint-detection while two kinds of ingredients.
The technical scheme to solve the above technical problems is that a kind of medullary carcinoma of thyroid gland joint-detection colloidal gold is exempted from
Epidemic disease chromatograph test strip, it is special including bottom plate and the sample pad, bonding pad, analyzing film and the water absorption pad that are sequentially arranged on the bottom plate
Sign is that the anticancer embryo of anti-human calcitonin detection antibody and colloid gold label that colloid gold label is combined on the bonding pad is anti-
Original detection antibody, the human calcitonin that the analyzing film is equipped with detect band T1 line, carcinomebryonic antigen detection band T2 line and quality control band C line,
It is coated with anti-human drop respectively on the calcitonin detection band T1 line, carcinomebryonic antigen detection band T2 line and the quality control band C line
Calcium element captures antibody, anti-carcinoembryonic antigen capture antibody and sheep anti-mouse igg polyclonal antibody, the amino of human calcitonin and carcinomebryonic antigen
Acid sequence is respectively shown in SEQ ID NO:1 and SEQ ID NO:4, and the anti-human calcitonin detection antibody is corresponding to have such as SEQ
The epitope of amino acid sequence shown in ID NO:2~SEQ ID NO:3 is any, the anti-human calcitonin capture antibody are corresponding
The epitope of amino acid sequence shown in remaining the next item down in SEQ ID NO:2~SEQ ID NO:3, the anti-carcinoembryonic antigen inspection
The corresponding epitope with the amino acid sequence as shown in SEQ ID NO:5~SEQ ID any one of NO:6 of antibody is surveyed, it is described anti-
Carcinomebryonic antigen capture antibody corresponds to the antigen of amino acid sequence shown in remaining the next item down in SEQ ID NO:5~SEQ ID NO:6
Epitope.
The testing principle of test strips: human calcitonin and cancer after sample to be tested is loaded onto sample pad, in sample
Embryonal antigen forms with the human calcitonin of the colloid gold label on bonding pad detection antibody, carcinomebryonic antigen detection antibody immune multiple respectively
Close object, due to capillary effect, immune complex to the swimming of water absorption pad direction, respectively be coated on human calcitonin detection band T1
Line and carcinomebryonic antigen detection are combined with the capture antibody on T2 line, gold mark human calcitonin detection antibody-human calcitonin antigen-people's drop
Calcium element capture antibody triplet compound is trapped on human calcitonin detection band T1 line, and gold mark carcinomebryonic antigen detects antibody-cancer
Embryonal antigen-carcinomebryonic antigen capture antibody triplet compound is trapped on carcinomebryonic antigen detection band T2 line, is progressively enriched with to be formed
The human calcitonin of deeper aubergine band, gold mark label detects antibody and carcinomebryonic antigen antibody and is coated in nature controlling line C line
Sheep anti-mouse igg polyclonal antibody occur immune response be trapped, be progressively enriched with and form deeper aubergine in nature controlling line C line
Band, extra unbonded material continue in swimming to water absorption pad, therefore the judgement of band all occur in detection band and quality control band
For positive findings；If not containing calcitonin and carcinomebryonic antigen in blood serum sample, when golden target detection antibody reaches detection band, not with
It is coated on the capture antibody that detection takes to be immunoreacted, therefore does not occur developed band at detection band, and gold mark label
Swimming and the corresponding sheep anti-mouse igg being coated at nature controlling line C line are more forward for human calcitonin and the continuation of carcinomebryonic antigen detection antibody
Clonal antibody occurs the immune response of specificity and is trapped, and is progressively enriched with the formation aubergine band in nature controlling line C line, therefore
There is band and detects band T1, T2 line to be to form band to be judged as negative findings in quality control band C line, if quality control band and wherein one inspection
Measuring tape colour developing, another detection band do not develop the color, then judging result is feminine gender.
Compared with prior art, advantages of the present invention are as follows: it is easy to use quickly to be used convenient for base or scene, it is all anti-
It can should be completed in 20min；It is at low cost, do not need special instrument and equipment；Have a wide range of application, applicable a variety of detector bars
Part；Multinomial detection, the more difficult acquisition of weakly positive sample can be carried out, multinomial detection can save sample, reduce cost；To temperature
Without particular/special requirement, it is not necessarily to freezen protective, stores convenient transportation, room temperature can be reserved for 24 months；Colloidal gold sheet is not needed as red
The step of chromogenic agents are added, eliminate enzyme target carcinogenicity substrate and terminate liquid, it is harmless to the human body；Detection specificity and spirit
Sensitivity is high.
The present invention also provides a kind of preparations of above-mentioned medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip
Method, comprising the following steps:
(1) anti-human calcitonin detection antibody, anti-human calcitonin capture antibody, anti-carcinoembryonic antigen detection antibody, anticancer are prepared
Embryonal antigen captures antibody and sheep anti-mouse igg polyclonal antibody,；
(2) anti-human calcitonin is detected antibody and anticancer embryo detection antibody to be marked with colloidal gold, and is sprayed on glass
On tunica fibrosa or polyester film, bonding pad is made；
(3) anti-human calcitonin antibody, anti-carcinoembryonic antigen capture antibody and sheep anti-mouse igg antibody is captured to be coated on respectively
Calcitonin detection band T1 line, the carcinomebryonic antigen of nitrocellulose filter detect and analysis are prepared on band T2 line and quality control band C line
Film；
(4) sample pad, bonding pad, analyzing film and water absorption pad are cut into suitably sized, and are successively overlapped on bottom plate, make
Obtain medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip.
Based on the above technical solution, the present invention can also be improved as follows:
Further, the material of the sample pad is glass fibre element film or polyester film.
Further, the sample pad be glass fibre membrane or polyester film after the immersion of sample pad treatment fluid in room epidemic disaster
Dry under conditions of not higher than 30% to be made, the sample pad treatment fluid includes 0.01MPBS buffer, 0.1wt%Tween-
20,1wt% bovine serum albumin and 10wt% sucrose.
Further, the specific steps of the step (2) are as follows: colloidal gold solution 100ml is taken, pH to 6.5~8.0 is adjusted, according to
The anti-human calcitonin detection antibody of 0.5~2mg of secondary addition and anti-carcinoembryonic antigen detect antibody, 1h are stirred at room temperature, with casein or BSA
Closing, 12000r/min are centrifuged 30min, abandon supernatant, are redissolved with colloidal gold working solution to 70mL, be sprayed on glass fibre membrane,
The bonding pad is dried to obtain under conditions of room temperature, relative humidity are not higher than 30%.
Further, the diameter of the colloidal gold is 25~35nm.
Further, the colloidal gold working solution include 1wt% bovine serum albumin(BSA), 0.1M Tris-Hcl buffer and
20wt% sucrose.
Further, the specific steps of the step (3) are as follows: catch the anti-human calcitonin capture antibody and anti-carcinoembryonic antigen
It obtains antibody and is diluted to 0.5~1.5mg/mL with coating buffer respectively, sheep anti-mouse igg polyclonal antibody is diluted to 1~2mg/
Then mL is sprayed at human calcitonin detection band T1 line, carcinomebryonic antigen detection band T2 line and quality control band C line respectively with 1~2uL/cm
On, it is dry under conditions of room temperature, relative humidity are not higher than 30%.
Further, the coating buffer is that borate, carbonate, phosphate, Tris- hydrochloric acid or Tris- phosphate are slow
The concentration of any one of fliud flushing, the coating buffer is 0.01M, and the pH value of the coating buffer is 6.5~7.5, more
Preferably, pH is 7.0~7.4.
The present invention also provides the application method of above-mentioned medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip, packets
Include following steps:
(1) sample treatment: taking serum, blood plasma or whole blood sample, dilutes 0~100 times with sample loading buffer；
(2) above-mentioned processed sample 80uL is taken to be added drop-wise in the sample pad of test strips, 5~20min of standing, observation T1,
T2 and C line；
(3) result judges: band is presented in T1, T2 line and C line, and result is the positive；T1, T2 line are presented without band, and C line is in
Existing band, result are feminine gender；Band is not presented for T line and C line, prompts test paper failure.
Detailed description of the invention
Fig. 1 is medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip structural schematic diagram of the present invention；
Fig. 2 is the different testing result schematic diagrames of the present invention, and wherein a is band schematic diagram, and b is positive findings figure, and c, d, e are
Recessive result figure, f and g indicate test strips fail result figure；
In attached drawing, parts list represented by the reference numerals are as follows:
1, sample pad, 2, bonding pad, 3, analyzing film, 4, human calcitonin detection band T1 line, 5, carcinomebryonic antigen detection band T2 line,
6, quality control band C line, 7, water absorption pad, 8, bottom plate.
Specific embodiment
Principles and features of the present invention are described below in conjunction with drawings and the specific embodiments, example is served only for solving
The present invention is released, is not intended to limit the scope of the present invention.
The preparation of embodiment 1 anti-calcitonin (CT), carcinomebryonic antigen (CEA) specific antibody
(1) the anti-calcitonin of people (CT), the preparation of carcinomebryonic antigen (CEA) epitope peptide
According to the database lookups such as NCBI human calcitonin (CT), the amino acid sequence of carcinomebryonic antigen (CEA), wherein calcium drops in people
The sequence amino acid sequence of element and carcinomebryonic antigen is respectively SEQ ID:1 and SEQ ID NO:4, and particular sequence is as follows:
Human calcitonin (CT): CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP (SEQ ID NO:1)；
Human cancer embryoantigen
(CEA):MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHL
FGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQF
RVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYK
CETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNIT
VNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVS
PRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASN
PPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDA
VAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPD
TPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVK
SITVSASGTSPGLSAGATVGIMIGVLVGVALI(SEQ ID NO:4)。
People's CT and CEA amino acid sequence is directed respectively into epitope design software, protein conformation external ammonia will be located at
Base acid sequence comes out, then CT and CEA amino acid sequence is directed respectively into DNASTAR software, passes through Epitope prediction work
Tool predicted, and and screened by WesternBlot and Elisa test, finally respectively obtain two kinds have it is good anti-
The epitope peptide of originality.Wherein two kinds of epitope peptides of CT are respectively two kinds of antigens of peptide fragment CT-1 and peptide fragment CT-2, CEA
Epitope peptide is respectively peptide fragment CEA-1 and peptide fragment CEA-2, and the amino acid sequence of peptide fragment CT-1 and peptide fragment CT-2 are respectively SEQ ID
NO:2 and SEQ ID NO:3, wherein CT-1 is people CT N-terminal the 1st to the 14th amino acids, amino acid sequence are as follows:
CSNLSTCVLGKLSQ (SEQ ID NO:2), CT-2 are people CT C-terminal the 18th to the 32nd amino acids, amino acid sequence
Are as follows: KLQTYPRTNTGSGTP (SEQ ID NO:3)；CEA-1 is the 35th, the end people CEAN to the 114th amino acids, amino acid
Sequence is KLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQAT PGPAYSGREIIYPN
ASLLIQNIIQ (SEQ ID NO:5), CEA-2 people CEA C-terminals the 502nd to the 593rd amino acids, amino acid sequence
Are as follows: KPSISSNNSKPVEDKDVAVFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLT LFNVTRNDARAYVCGIQ
NSVSANRSDPVTLDVLYGP (SEQ ID NO:6).
The characteristics of above-mentioned peptide fragment all has hydrophily, antigenicity by force and is readily synthesized, epitope peptide is prepared by
American AB I431A type polypeptide automatic synthesizer, by Solid phase synthesis, and the epitope synthesized by polypeptide sequence identification
The purity of peptide sequence, peptide fragment can be evaluated with thin-layer chromatography and high performance liquid chromatography, and measure the concentration of epitope peptide.
(2) preparation of antibody
Peptide fragment CT-1, CT-2, CEA-1, CEA-2 and carrier protein connection are prepared into immune antigen respectively, utilize gained
Animal is immunized in immunising antigen respectively, prepares the monoclonal antibody of specificity.
The preparation of antigen: peptide fragment CT-1, CT-2, CEA-1, CEA-2 are connect with carrier protein BSA respectively be prepared into it is immune
With antigen, taking pH is that the PBS and dimethyl sulfoxide (DMSO) each 0.25mL of 6.0 0.01mol/L dissolves 5mg BSA, takes 1.2mg
2- (N- morpholine) ethanesulfonic acid (MBS) is dissolved in 100mL DMSO, and MBS solution is added in BSA solution and is stirred at room temperature
30min, then supernatant is left and taken in 4 DEG C of 5000rpm centrifugations, and peptide fragment CT-1, CT-2, CEA-1 and CEA-2 are dissolved in pH respectively is
The PBS and DMSO of 7.2 0.01mol/L is amounted in 500 μ L, each peptide fragment solution is mixed with BSA-MBS solution respectively, room temperature is stirred
- 20 DEG C of spare, Na of PBS buffer solution 0.2mol/L are stored in after mixing 60min2HPO481ml adds 0.2mol/L's
NaH2PO419ml is formulated.
Immune animal prepares monoclonal antibody: immune by above-mentioned preparation is helped with isometric Freund completely respectively with antigen
After agent is sufficiently mixed, immune Balb/c mouse surveys serum titer after 4 weeks according to every 50 subcutaneous multi-point injection of μ g antigen of mouse,
The mouse for selecting immunoreactivity good again press after taking antigen and isometric incomplete Freund's adjuvant to be sufficiently mixed by booster immunization
According to every 25 subcutaneous multi-point injection of μ g antigen of mouse, the number of booster immunization is 6 times, and booster immunization twice, then takes before merging
Splenocyte is merged according to a conventional method with 50% PEG (MW4000) mediation with Sp2/0 myeloma cell and is put into CO2Culture
37 DEG C after culture 10 days in case, occurs biggish cell clone in culture hole, screened with indirect ELISA, to the primary dcreening operation positive
Culture hole utilize limiting dilution assay to carry out 4 time cloning cultures (even if screening after a large amount of schizogamies of cell), Zhi Houkuo
Increase cell, freeze, prepare ascites, Balb/c mouse is only handled with norphytane 0.6ml/, intraperitoneal inoculation hybridizes after a week
Oncocyte 3 × 106It is a/only, collect ascites after 10 days, measure antibody titer, with indirect ELISA method measurement BSA-CT-1 and
The antibody titer of BSA-CEA-1 preparation reaches 1:35000 or more, the Dan Ke prepared with BSA-CT-2 and BSA-CEA-2
Grand antibody titer is up to 1:33000 or more.
2 medullary carcinoma of thyroid gland human calcitonin of embodiment and the preparation of carcinomebryonic antigen joint-detection colloidal gold immuno-chromatography test paper strip
As shown in Figure 1, medullary carcinoma of thyroid gland human calcitonin and carcinomebryonic antigen joint-detection colloidal gold immuno-chromatography test paper strip
Including bottom plate 8 and the sample pad 1, bonding pad 2, analyzing film 3 and the water absorption pad 7 that are sequentially arranged on bottom plate 8, analyzing film is dropped equipped with people
Calcium element detection band T1 line, carcinomebryonic antigen detect band T2 line and quality control band C line, and human calcitonin detection antibody is coated on bonding pad 2
Antibody is detected with carcinomebryonic antigen, human calcitonin capture capture antibody, carcinomebryonic antigen detection are coated on calcitonin detection band T1 line
It is coated with carcinomebryonic antigen capture antibody on band T2 line, is coated with sheep anti-mouse igg antibody on quality control band C line, wherein human calcitonin is examined
It surveys antibody and human calcitonin capture antibody is respectively the immune monoclonal being prepared with antigen of peptide fragment CT-1 and CT-2 preparation
Antibody, carcinomebryonic antigen detection antibody and carcinomebryonic antigen capture antibody are respectively that the immune of peptide fragment CEA-1 and CEA-2 preparation uses antigen
The monoclonal antibody being prepared.
(1) preparation of bonding pad
The colloidal gold solution that diameter is 25~35nm is prepared with gold chloride-sodium citrate, 100ml colloidal gold liquid is taken to put
In beaker, with the K of 0.2M2CO3Being adjusted to pH is 6.5~8, then is separately added into 0.5~2mg human calcitonin detection antibody and cancer embryo
Antigen detects antibody, and half an hour is stirred at room temperature, and is closed with casein or BSA, and 12000r/min is centrifuged 30min, abandons supernatant, matches
Colloidal gold working solution is set, including including 1% bovine serum albumin(BSA), 0.lM Tr is-HCl buffer, 20% sucrose, pH value is
8.0, precipitating is redissolved to 70ml with colloidal gold working solution, the material of bonding pad is all-glass paper or polyester film, with preparing
Bonding pad treatment fluid impregnate all-glass paper or polyester film, dried in 37 DEG C of placements 17h, be 1.2 μ L/cm's according to package amount
The anti-carcinoembryonic antigen detection antibody that the anti-human calcitonin of colloid gold label detects antibody and colloid gold label is sprayed on pre- place by amount
It is dry under conditions of room temperature, relative humidity are not higher than 30% (generally 15%~30%) on the glass fibre membrane or polyester film of reason
It is dry, it is sealed spare.
(2) preparation of analyzing film
Analyzing film selects nitrocellulose filter (NC), human calcitonin is captured antibody, carcinomebryonic antigen captures antibody, adds respectively
Enter in the 10mM PB buffer that pH is 7.2 and be configured to the capture antibody-solutions that concentration is 0.4mg/mL, is distinguished with film gold spraying instrument is drawn
On the human calcitonin detection band T1 line and carcinomebryonic antigen detection band T2 line being sprayed on nitrocellulose membrane, quantity for spray is 1 μ L/cm, sheep
Anti- mouse IgG polyclonal antibody is added in the 10mM PB buffer that pH is 7.2 and is configured to the polyclonal antibody that concentration is 0.4mg/mL
It in solution, is sprayed on quality control band C line with film gold spraying instrument is drawn, quantity for spray is 1 μ L/cm, wherein human calcitonin detection band TI linear distance
Bonding pad 9mm, carcinomebryonic antigen detection are about 3mm at a distance from quality control band C line with T2 line, and quality control band C linear distance water absorption pad is about
7mm, then 37 DEG C of analyzing film drying, encapsulates spare.
(3) preparation of sample pad
The material of sample pad is glass fibre membrane, impregnates sample pad with prepared sample pad treatment fluid, does at 37 DEG C
Dry 17h obtains sample pad.Wherein sample pad buffer: the 10mM PB buffer that pH is 7.0~9.0,1%BSA, 10%~
20% sucrose, 0.1%Tween-20, sample pad can not also pre-process, and directly use.
(4) assembling of test strips
Sample pad, bonding pad, analyzing film and water absorption pad are successively cut into it is suitably sized, wherein width be 3~4mm,
Length is 7cm, and the spacing between T line, T2 line and C line is 3mm, and analyzing film is fitted in the centre of bottom plate, the left and right of analyzing film
Bonding pad and water absorption pad are posted in both ends respectively, and Chong Die with the both ends of analyzing film respectively, and bonding pad is pasted far from one end of analyzing film
Conjunction has sample pad.
The testing principle of test strips: human calcitonin and cancer after sample to be tested is loaded onto sample pad, in sample
Embryonal antigen forms with the human calcitonin of the colloid gold label on bonding pad detection antibody, carcinomebryonic antigen detection antibody immune multiple respectively
Close object, due to capillary effect, immune complex to the swimming of water absorption pad direction, respectively be coated on human calcitonin detection band T1
Line and carcinomebryonic antigen detection are combined with the capture antibody on T2 line, gold mark human calcitonin detection antibody-human calcitonin antigen-people's drop
Calcium element capture antibody triplet compound is trapped on human calcitonin detection band T1 line, and gold mark carcinomebryonic antigen detects antibody-cancer
Embryonal antigen-carcinomebryonic antigen capture antibody triplet compound is trapped on carcinomebryonic antigen detection band T2 line, is progressively enriched with to be formed
The human calcitonin of deeper aubergine band, gold mark label detects antibody and carcinomebryonic antigen antibody and is coated in nature controlling line C line
Sheep anti-mouse igg polyclonal antibody occur immune response be trapped, be progressively enriched with and form deeper aubergine in nature controlling line C line
Band, extra unbonded material continue in swimming to water absorption pad, therefore the judgement of band all occur in detection band and nature controlling line
For positive findings；If not containing calcitonin and carcinomebryonic antigen in blood serum sample, when golden target detection antibody reaches detection band, not with
It is coated on the capture antibody that detection takes to be immunoreacted, therefore does not occur developed band at detection band, and gold mark label
Swimming and the corresponding sheep anti-mouse igg being coated at nature controlling line C line are more forward for human calcitonin and the continuation of carcinomebryonic antigen detection antibody
Clonal antibody occurs the immune response of specificity and is trapped, and is progressively enriched with the formation aubergine band in nature controlling line C line, therefore
Only occur band in nature controlling line C line and is judged as negative findings, if quality control band develops the color with a wherein detection band, another detection
Band does not develop the color, then judging result is feminine gender, and different testing results is as shown in Figure 2.
1 test strips Performance Evaluation of test example
Inaccuracy is assessed between test strips performance includes stability, batch interior inaccuracy and criticizes.
(1) 4 DEG C of stability test
The small aluminium foil bag of test strips is sealed, 2~28 DEG C of warehouses are placed, monthly takes out and uses quality-control product and yin and yang attribute reference material
The withinrun precision of each 10 parts of yin and yang attribute recombination rate judges the stability of test paper.The results show that Quality Control product examine after 12 months
It surveys result and meets expection, the yin and yang attribute coincidence rate of each yin and yang attribute reference material is 100%.The results show that each yin after 18 months
The yin and yang attribute coincidence rate of positive reference product is still 100%.Testing result is shown after 24 months, does not occur false negative.In summary
As a result illustrate that test paper is stored at 2~28 DEG C, be stable in 2 years.
(2) within-assay
The gold label test strip of each batch is taken, respectively with high, medium and low three kinds of positive serums and each portion of negative serum,
Every part of serum uses gold label test strip and latex test strips to repeat detection 10 times respectively, the results are shown in Table 1 all positive serums
Testing result is the positive, and the testing result of all negative serums is feminine gender, is prompted in colloidal gold strip of the invention batch
Accuracy complies with standard.
Table 1
(3) between-batch precision
1~3 each three batch of test strips is detected with high, medium and low three kinds of positive serums and negative serum, every part of serum inspection
10 are surveyed, as a result, the results are shown in Table 2, the testing result of each three different batches is consistent for observation after 10min, illustrates essence between criticizing
Exactness complies with standard.
Table 2
The detection of 2 test strips of test example and clinical performance assessment
The test strips prepared with embodiment 2 detect the patient made a definite diagnosis and normal human serum and blood plasma each 100, wherein detecting
Sensitivity is that test strips detect percentage shared by number positive in all positive clinical case loads, and detection specificity is all
Test strips detect percentage shared by negative number in clinical negative case number, and testing result is as shown in table 3.
Table 3
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
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Claims (10)
1. a kind of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip, including bottom plate (8) and it is sequentially arranged at described
Sample pad (1), bonding pad (2), analyzing film (3) and water absorption pad (7) on bottom plate (8), which is characterized in that the bonding pad (2)
On be combined with colloid gold label anti-human calcitonin detection antibody and colloid gold label anti-carcinoembryonic antigen detection antibody, described point
It analyses film (3) and is equipped with human calcitonin detection band T1 line (4), carcinomebryonic antigen detection band T2 line (5) and quality control band C line (6), the drop
It is coated with respectively on calcium element detection band T1 line (4), carcinomebryonic antigen detection band T2 line (5) and the quality control band C line (6) anti-human
Calcitonin captures antibody, anti-carcinoembryonic antigen capture antibody and sheep anti-mouse igg polyclonal antibody, the ammonia of human calcitonin and carcinomebryonic antigen
Base acid sequence is respectively shown in SEQ ID NO:1 and SEQ ID NO:4, and the anti-human calcitonin detection antibody is corresponding to be had such as
The epitope of amino acid sequence shown in SEQ ID NO:2~SEQ ID NO:3 is any, the anti-human calcitonin capture antibody pair
Answer the epitope of amino acid sequence shown in remaining the next item down in SEQ ID NO:2~SEQ ID NO:3, the anti-carcinoembryonic antigen
The corresponding epitope with the amino acid sequence as shown in SEQ ID NO:5~SEQ ID any one of NO:6 of antibody is detected, it is described
Anti-carcinoembryonic antigen capture antibody correspond to resisting for amino acid sequence shown in remaining the next item down in SEQ ID NO:5~SEQ ID NO:6
Former epitope.
2. a kind of preparation side of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip as described in claim 1
Method, which comprises the following steps:
(1) it is anti-that anti-human calcitonin detection antibody, anti-human calcitonin capture antibody, anti-carcinoembryonic antigen detection antibody, anticancer embryo are prepared
Original capture antibody and sheep anti-mouse igg polyclonal antibody；
(2) anti-human calcitonin is detected antibody and anticancer embryo detection antibody to be marked with colloidal gold, and is sprayed on glass fibre
On film or polyester film, bonding pad is made；
(3) anti-human calcitonin is captured into antibody, anti-carcinoembryonic antigen capture antibody and sheep anti-mouse igg antibody and is coated on nitric acid respectively
Calcitonin detection band T1 line, the carcinomebryonic antigen of cellulose membrane detect and analyzing film are prepared on band T2 line and quality control band C line；
(4) sample pad, bonding pad, analyzing film and water absorption pad are cut into suitably sized, and are successively overlapped on bottom plate, first is made
Shape gland cephaloma joint-detection colloidal gold immuno-chromatography test paper strip.
3. the preparation method of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip according to claim 2,
It is characterized in that, the material of the sample pad is glass fibre element film or polyester film.
4. the preparation method of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip according to claim 3,
It is characterized in that, the sample pad is that glass fibre membrane or polyester film be not high in room epidemic disaster after the immersion of sample pad treatment fluid
Dry under conditions of 30% to be made, the sample pad treatment fluid includes 0.01M PBS buffer solution, 0.1wt%Tween-20,
1wt% bovine serum albumin and 10wt% sucrose.
5. the preparation method of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip according to claim 2,
It is characterized in that, the specific steps of the step (2) are as follows: take colloidal gold solution 100ml, adjust pH to 6.5~8.0, successively plus
Enter the anti-human calcitonin detection antibody of 0.5~2mg and anti-carcinoembryonic antigen detection antibody, 1h is stirred at room temperature, is sealed with casein or BSA
It closing, 12000r/min is centrifuged 30min, and supernatant is abandoned, is redissolved with colloidal gold working solution to 70mL, is sprayed on glass fibre membrane, in
Room temperature, relative humidity are dried to obtain the bonding pad under conditions of being not higher than 30%.
6. the preparation method of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip according to claim 5,
It is characterized in that, the diameter of the colloidal gold is 25~35nm.
7. the preparation method of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip according to claim 5,
It is characterized in that, the colloidal gold working solution includes 1wt% bovine serum albumin(BSA), 0.1M Tris-Hcl buffer and 20wt%
Sucrose.
8. the preparation method of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip according to claim 2,
It is characterized in that, the specific steps of the step (3) are as follows: capture the anti-human calcitonin capture antibody and anti-carcinoembryonic antigen anti-
Body is diluted to 0.5~1.5mg/mL with coating buffer respectively, sheep anti-mouse igg polyclonal antibody is diluted to 1~2mg/mL, so
It is sprayed at respectively with 1~2uL/cm on human calcitonin detection band T1 line, carcinomebryonic antigen detection band T2 line and quality control band C line afterwards, in
Room temperature, relative humidity are dry under conditions of being not higher than 30%.
9. the preparation method of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip according to claim 8,
It is characterized in that, the coating buffer is borate, carbonate, phosphate, Tris- hydrochloric acid or Tris- phosphate buffer
Any one of, the pH value of the coating buffer is 6.5~7.5, and the concentration of the coating buffer is 0.01M.
10. the preparation method of medullary carcinoma of thyroid gland joint-detection colloidal gold immuno-chromatography test paper strip according to claim 9,
It is characterized in that, the pH value of the coating buffer is 7.0~7.4.
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| CN114280312A (en) * | 2020-09-27 | 2022-04-05 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
| CN114280312B (en) * | 2020-09-27 | 2023-09-15 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
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