CN109188000A - A kind of test strips and preparation method thereof of portable inspectiont human parathyroid hormone - Google Patents
A kind of test strips and preparation method thereof of portable inspectiont human parathyroid hormone Download PDFInfo
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- CN109188000A CN109188000A CN201810898371.4A CN201810898371A CN109188000A CN 109188000 A CN109188000 A CN 109188000A CN 201810898371 A CN201810898371 A CN 201810898371A CN 109188000 A CN109188000 A CN 109188000A
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- pad
- antibody
- pth
- nitrocellulose filter
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
-
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
Abstract
The invention discloses a kind of test strips and preparation method thereof of portable inspectiont human parathyroid hormone, nitrocellulose filter pad including the bar shaped being transversely and horizontally set, the nature controlling line of longitudinal gap and the detection line for being respectively arranged with bar shaped and bar shaped that are parallel to each other on the nitrocellulose membrane pad, wherein the nitrocellulose filter pad abuts the water absorption pad for the bar shaped for having one to be transversely and horizontally set close to nature controlling line one end, the nitrocellulose filter pad abuts the bonding pad for the bar shaped for having one to be transversely and horizontally set close to one end of detection line, the bonding pad abuts the sample pad for having a bar shaped being transversely and horizontally set far from one end of nitrocellulose filter pad；Containing the anti-human PTH antibody marked by colloidal gold or fluorescent microsphere in the bonding pad, the detection line is coated with the detection antibody that can be specifically bound with PTH, and nature controlling line is coated with can be with the Quality Control antibody in conjunction with free anti-human PTH antibody specificity.Its detection structure accuracy is high.
Description
Technical field
The present invention relates to a kind of biochemical diagnosis test strips more particularly to a kind of test paper of portable inspectiont parathyroid hormone
Item and preparation method thereof.
Background technique
Thyroid cancer is the most common malignant tumour of incidence, and most fast pernicious swollen of disease incidence growth rate in recent years
One of tumor, thyroid cancer have become the fifth-largest common cancer of women.The treatment of thyroid cancer is mainly controlled with surgical operation
Based on treatment, row thyroid gland cut entirely it is postoperative should determine parathyroid function situation as early as possible, take specific aim measure carry out prevention and treatment first shape
Other adenasthenia.If unexpected when operation cut off parathyroid gland, the blood calcium concentration of patient can be substantially reduced, and caused brothers and taken out
It jerks or even threat to life.The incidence of the temporary hypocalcemia of document report L thyroxine tab is 0.3~86%, permanent
Hypocalcemia is 0~13%.PTH is secreted by chief cell, is had and is adjusted and keep serum calcium in normal level
Effect.How effectively to avoid damage to parathyroid gland in the course of surgery is a great problem.Protection technique includes in parathyroidectomy
The identification of parathyroid gland, fining parathyroid gland protection operating technology, three aspect of remedial parathyroid gland autotransplanting technology.First
The identification of gland is a step of parathyroid gland protection most critical in art by shape.Since parathyroid gland body is small in size, around have first shape
The tissue such as gland, fat, lymph node, thymus gland, muscle, recognizes in art and has difficulties.Even if experienced surgeon's naked eyes are known
Other accuracy rate also only has nearly 70%, there is up to 9~19% parathyroid gland accident resection rate in thyroid gland art.Pathology at present
It is still the goldstandard for identifying parathyroid gland.But this needs cut-out Parathyroid Tissue, and waiting for a long time in art, expense is expensive,
There is also 1.3% error rate, the parathyroid hormone (PTH) of fine needle puncture tissue eluent is detected, for reflecting in preoperative and art
The method of other parathyroid gland has document report.Multiple studies have shown that the PTH value of parathyroid gland puncturing tissue eluent is obviously high
In non-Parathyroid Tissue, Parathyroid Tissue, operation letter are identified in art by the PTH detection of fine needle puncture tissue eluent
Single, sensibility and specificity is high, and fool proof.The detection of PTH is carried out generally by electrochemical luminescence at present, needs large-scale
Detection device, detection time need half an hour or more, so to promote and apply difficult plus the shipping time.
Immunochromatographic method (Immunochromatography) is a kind of quick diagnosis technology risen in recent years, former
Reason is that special antibody is first fixed on to a certain zone of nitrocellulose filter or cellulose acetate film, when dry cellulose one
After sample (tissue fluid or blood) is immersed at end, due to capillarity, sample will be moved forward along the film, when being moved to fixation
When having the region of antibody, corresponding antigen is specifically bound with the antibody in sample, and type is at macroscopic detection band.
The existing common trace labelling particle of immuno-chromatographic test paper strip product has nanogold, nanometer selenium, colored latex etc., wherein receiving
Meter Jin is most widely used, and nanogold is also referred to as colloidal gold.
Immunochromatography technique spy using colloidal gold as trace labelling object is known as immune colloidal gold technique
(Immunecolloidal gold technique, GICT).Colloidal gold be by gold chloride (HAuCl4) reducing agent such as white phosphorus,
Under the effects of ascorbic acid, sodium citrate, tannic acid, polymerization becomes the gold particle of particular size, and since electrostatic interaction becomes one
It plants stable colloidal state and gains the name.Colloidal gold is negatively charged under mild alkaline conditions, can be with the positive charge group of protein molecule
Type is at firm connection, due to this convolution electrostatical binding, so not influencing the biological nature of protein.Colloidal gold in addition to egg
It, can also be in conjunction with many other large biological molecules, such as SPA, PHA, ConA other than white matter combines.According to some objects of colloidal gold
Rationality shape, such as high electron density, granular size, type shape and color reaction, in addition the immune and biological characteristics of conjugate, thus
Colloidal gold is set to be widely used in the fields such as immunology, histology, pathology and cell biology.
With existing Hemagglutination Method, radioimmunoassay (RIA), exempt from compared with the method for putting (IMA) and science of law luminescence method detect, immunochromatographic method
The advantages of detection, specifically includes that
(1) easy to use quickly to use convenient for base and use with scene, all reactions can be completed in 20 minutes；
(2) at low cost, do not need special instrument and equipment；
(3) have a wide range of application, be suitable for a variety of testing conditions；
(4) can carry out multinomial detection, the more difficult acquisition of weakly positive sample, multinomial detection can with economization sample, reduce at
This；
(5) marker is stablized, and label sample stores 2 years at 4 DEG C or more, no signal relaxation phenomenon；
(6) colloidal gold sheet does not need that chromogenic agents are added, eliminates enzyme target carcinogenicity substrate and terminate liquid as red
The step of, it is harmless to the human body.
Summary of the invention
In order to solve the above-mentioned technical problem, quickly puncturing tissue can be eluted one of the objects of the present invention is to provide a kind of
The PTH value of liquid carries out quantitative analysis test strips, and high sensitivity can be accurately to reflect in clinical thyroid cancer row operation consent or art
Other parathyroid gland.
To achieve the goals above, the technical scheme is that a kind of examination of portable inspectiont human parathyroid hormone
Paper slip, which is characterized in that longitudinal on the nitrocellulose membrane pad including the nitrocellulose filter pad for the bar shaped being transversely and horizontally set
The nature controlling line of the detection line for being respectively arranged with a bar shaped and a bar shaped for being spaced and being parallel to each other, the detection line and Quality Control
The both ends of line are concordant with the two sides of the nitrocellulose filter pad respectively, wherein the nitrocellulose filter pad is close to nature controlling line
One end abuts the water absorption pad for having a bar shaped being transversely and horizontally set, and the nitrocellulose filter pad is abutted close to one end of detection line
There is the bonding pad of a bar shaped being transversely and horizontally set, the bonding pad is abutted far from one end of nitrocellulose filter pad a transverse direction
The sample pad of horizontally disposed bar shaped, the sample pad, bonding pad, the two sides of nitrocellulose filter pad and water absorption pad are flat
Together；
Containing the anti-human PTH antibody marked by colloidal gold or fluorescent microsphere in the bonding pad, the detection line is coated with
The detection antibody that can be specifically bound with PTH, the nature controlling line is coated with can be in conjunction with free anti-human PTH antibody specificity
Quality Control antibody.
The beneficial effect of above-mentioned technical proposal is: anti-using the anti-human PTH for being marked with nanometer fluorescent microspheres or colloidal gold
Immune response occurs between PTH in body and sample and generates the compound of the anti-human PTH antibody-PTH of nanometer fluorescent microspheres/colloidal gold-
Body, and mobile to water absorption pad side under the action of siphon, when reaching detection line another site of PTH with detect in conjunction with antibody,
The complex of the anti-human PTH antibody-PTH- detection antibody of formation nanometer fluorescent microspheres/colloidal gold-, and free nanometer fluorescent microspheres
Or the anti-human PTH antibody of colloid gold label is continued to move along to nature controlling line, and occurs to be immunized with the Quality Control antibody on nature controlling line
Reaction forms the compound of the anti-human PTH antibody-Quality Control antibody of nanometer fluorescent microspheres/colloidal gold-, wherein fluorescent nanometer microsphere band
There is fluorescence, the depth that can detecte the fluorescence in detection line and nature controlling line by fluorescence detection device judges that fluorescence nano is micro-
The content of ball, and colloidal gold has Chinese red, the lines of Chinese red can be respectively formed in detection line and nature controlling line, according to Exocarpium Citri Rubrum
The depth of color line color be can determine whether wherein PTH content number.
Sample pad described in above-mentioned technical proposal be by treatment fluid handle glass fibre membrane in room temperature relative humidity be 15-
Obtained from drying and be sealed under conditions of 30%, wherein the treatment fluid includes 0.01M PBS, 0.1wt%Tween-
20,1wt% bovine serum albumin(BSA), 10wt% sucrose.
The beneficial effect of above-mentioned technical proposal is: the detection of sample pad PTH suitable for body fluid/serum/plasma.
Sample pad described in above-mentioned technical proposal includes through red blood cell monoclonal antibody treated fiber glass film and blood filter membrane, institute
It states blood filter membrane one end to abut with the bonding pad corresponding end, the blood filter membrane is arranged in far from bonding pad in the fiber glass film
One end, and the fiber glass film is covered close to one end of the blood filter membrane in the blood filter membrane upper end or the fiber glass
Film one end is abutted with the bonding pad corresponding end, and the described one end of fiber glass film far from bonding pad is arranged in the blood filter membrane,
And the blood filter membrane is covered close to one end of the fiber glass film in the fiber glass film upper end, and the sample pad is placed in
Room temperature relative humidity is dried and is sealed spare under conditions of being 15-30%.Wherein, whether fiber glass film upper or
Blood filter membrane is in upper, at the two overlapping the preferred 2-4mm of length, and whole blood sample is added dropwise in blood filter membrane or fibre far from bonding pad
It ties up on glass film.
The beneficial effect of above-mentioned technical proposal is: the detection of sample pad PTH suitable for whole blood sample, wherein red
Cell monoclonal antibodies treated fiber glass film and blood filter membrane can retain the ingredients such as red blood cell, blood platelet in whole blood.
Detection antibody described in above-mentioned technical proposal is PTH antibody, and the Quality Control antibody is sheep anti-mouse igg.
The beneficial effect of above-mentioned technical proposal is: being easy, high specificity.
Water absorption pad described in above-mentioned technical proposal is blotting paper.
The beneficial effect of above-mentioned technical proposal is: being easy, soaking effect is good.
It further include the bottom plate of a bar shaped, the sample pad, bonding pad, nitrocellulose filter and suction in above-mentioned technical proposal
Water cushion is successively laid on the bottom plate upper end along floor length direction.
The beneficial effect of above-mentioned technical proposal is: so that test strips facilitate taking and placing and are not easily susceptible to pollute when in use,
Stablize Each part.
The second object of the present invention is to provide a kind of method for preparing above-mentioned test strips, and its step are as follows: by sample pad,
Bonding pad, nitrocellulose filter pad and water absorption pad are successively adhered to the bottom plate upper end along floor length direction, and sample pad with
Bonding pad side close to each other mutually abuts, and the bonding pad is mutually abutted with nitrocellulose filter pad one end close to each other, institute
State mutual the abutting of nitrocellulose filter pad one end close to each other with water absorption pad.
The beneficial effect of above-mentioned technical proposal is: so that sample pad, bonding pad, nitrocellulose filter pad and water absorption pad are pressed
Sequence is connected on the bottom plate, and installation is simple.
In above-mentioned technical proposal before the nitrocellulose filter pad is adhered to the bottom plate upper end, PTH antibody is used
The antibody-solutions that the phosphate buffered saline that concentration is 0.01M and pH is 7.4 is 0.5-2mg/ml at concentration, by it with 1.0-
The quantity for spray of 2.0uL/cm uses spray film instrument to be crossed on nitrocellulose filter pad as detection line, while by IgG concentration
For 0.01M and antibody-solutions that phosphate buffered saline that pH is 7.4 is 1-2mg/mL at concentration, by it with 1.0-2.0uL/
The quantity for spray of cm uses spray film instrument to be crossed on nitrocellulose filter pad as nature controlling line, will be coated with PTH antibody and sheep anti mouse
The nitrocellulose filter pad of IgG is placed in room temperature and relative humidity humidity to dry and being sealed spare under conditions of 15-30%.
The beneficial effect of above-mentioned technical proposal is: will test antibody and Quality Control antibody is delineated in nitrocellulose filter pad
On, the fluorescent nanometer microsphere with PTH/colloid gold particle is retained using detection antibody, and the fluorescence nano without containing PTH is micro-
Ball/colloidal gold in conjunction with Quality Control antibody,.
When anti-human PTH antibody on bonding pad described in above-mentioned technical proposal is by colloid gold label, the system of the bonding pad
Preparation Method are as follows: take colloidal gold stoste and be 0.2M with concentration K2C03 solution be adjusted to pH6.25-6.5, add label
PTH antibody, wherein the ratio between dosage and the dosage of colloidal gold stoste of the PTH antibody of label are 1-4ug:20mL, room
Temperature stirring 1 hour, is closed with casein or BSA, and 12000r/min is centrifuged 30 minutes, abandons supernatant, multiple with colloidal gold working solution
It is molten to the 70% of colloidal gold stoste volume, be then equably sprayed on glass fibre membrane with metal spraying machine, in room temperature relative humidity
It is dry under conditions of not higher than 30%, it is sealed spare；
When anti-human PTH antibody on the bonding pad is marked by fluorescent microsphere, the bonding pad is the nitre through Seal treatment
Acid cellulose film, preparation method are: nitrocellulose filter being placed in and is spat containing 1.5wt%NaCl, 5wt%BSA, 0.5wt%
10min is impregnated in the 100mMPBS buffer of temperature -20 and 5wt% sucrose, takes out, drains away the water, 50 DEG C are dried overnight；Pass through
The airjet spray head of Bigot instrument, by the anti-human PTH antibody marked containing fluorescent microsphere according to 1 μ L/cm amount ullrasonic spraying extremely
On nitrocellulose filter after Seal treatment, 60 DEG C are dried overnight.
The beneficial effect of above-mentioned technical proposal is: method is simple.
Colloidal gold stoste described in above-mentioned technical proposal be using the preparation of gold chloride-sodium citrate diameter 30 ±
The colloidal gold solution of 5nm；The colloidal gold working solution include 1wt% bovine serum albumin(BSA), 0.1M Tris-HCL buffer and
20wt% sucrose, and pH is 8.0.
The beneficial effect of above-mentioned technical proposal is: preparation method is simple.
The preparation method of anti-human PTH antibody containing fluorescent microsphere label described in above-mentioned technical proposal is using pH7.2-
7.6 MES activation buffer washs fluorescent microsphere, and carbodiimide (EDC) and n-hydroxysuccinimide (NHS), room temperature is added
Certain time is reacted, fluorescent microsphere is washed, it is anti-that anti-human PTH is added after being redissolved with the phosphate buffer of 0.05M pH7.2-7.6
Body reacts at room temperature 2 hours, and the phosphate buffer of the 0.05M pH7.2-7.6 containing 10%BSA is added, and reacts at room temperature 30 points
Clock washs fluorescent microsphere, and with 1%BSA is contained, the phosphate buffer of 0.1%Tween-20,0.05M pH7.2-7.6 redissolve
To original volume.
The beneficial effect of above-mentioned technical proposal is: preparation method is simple.
Fluorescent molecule in fluorescent microsphere described in above-mentioned technical proposal is the chelating of europium in lanthanide series, terbium, samarium or dysprosium
One kind of object.
The beneficial effect of above-mentioned technical proposal is that such fluorescent material can decay at any time, can utilize the original of its decaying
Reason treats detection substance using time-resolved fluorescence photometer and carries out quantitative detection.
Detailed description of the invention
Fig. 1 is the structure diagram of test strips of the present invention.
In figure: 1 sample pad, 2 bonding pads, 3 nitrocellulose filter pads, 4 detection lines, 5 nature controlling lines, 6 water absorption pads, 7 bottom plates.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
Embodiment 1
The preparation of people's PTH epitope peptide
Heretofore described people PTH be it is known in the art, complete PTH is single more containing 84 amino acid
Peptide chain is constituted, and molecular weight is about 9500 dalton.Its amino acid sequence be it is known in the art, can be in expert datas such as NCBI
It is found in library, particular sequence is as follows:
Mankind PTH (1-84): SEQ ID NO.1:Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
Val His Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser Gln
Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys Ala Lys Ser Gln；
The present inventor gropes by a large amount of theoretical research and experiment, finally screens to obtain two kinds with good
Antigenic epitope peptide.PTH epitope peptide (1) includes people PTH N-terminal the 1st to the 6th peptide fragment, is contained to constitute
The epitope peptide (1) of 6 amino acid: SEQ ID NO.2:SerVal Ser Glu Ile Gln；PTH epitope peptide (2)
Comprising the 52nd to the 71st peptide fragment of people PTH C-terminal, to constitute the epitope peptide (2) for containing 21 amino acid: SEQ ID
NO.3:Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu Lys Ser Leu Gly Glu
The characteristics of Ala Asp, the two peptide fragments all have hydrophily, antigenicity by force and are readily synthesized.
Two epitope peptides are prepared by American AB I431A type polypeptide automatic synthesizer, are closed by solid phase method
At, and the epitope peptide sequence synthesized by polypeptide sequence measurement identification, the purity of peptide fragment can with thin-layer chromatography and efficiently
Liquid chromatogram is evaluated, and measures the concentration of epitope peptide.
Embodiment 2
The preparation of PTH antibody
The resulting PTH epitope peptide (1) of embodiment 1 and PTH epitope peptide (2) are connect with carrier protein respectively with
Immune antigen (1) and (2) are prepared, animal is immunized respectively using gained antigen (1) and (2), to be prepared using antigen (1) special
Anisotropic monoclonal antibody and polyclonal antibody, and specific monoclonal antibody and Anti-TNF-α are prepared using antigen (2)
Body.
1. the preparation of antigen: PTH peptide fragment being connect with carrier protein BSA respectively and is prepared into PTH antigen.Take pH6.0
0.01mol/L PBS and dimethyl sulfoxide (DMSO) each 0.25mL dissolve 5mg BSA, and MBS 1.2mg is taken to be dissolved in 100 blood DMSO
In.MBS is added in BSA liquid, 30rnin is stirred at room temperature, upper liquid is left and taken in 4 DEG C of 5000r/min centrifugations.Take PTH (1-6), PTH
(64-84) is dissolved in 0.01mol/L pH7.2PBS and DMSO respectively and is total in about 500 μ L.BSA-MBS is mixed with each polypeptide liquid respectively
Close, be stirred at room temperature after 60min be stored in -20 DEG C it is spare.
In the present embodiment, the formula of PBS buffer solution are as follows: the Na2HPO4 81ml of 0.2mol/L adds 0.2mol/L's
Na2HPO419ml is mixed.
2. immune animal prepares monoclonal antibody:
2.1. take the PTH antigen (immunogene) of above-mentioned preparation respectively with isometric Freund's complete adjuvant (purchased from Shang Haiyuan
Poly- biotech firm) be sufficiently mixed after, be immunized Balb/c mouse, 50 μ g antigens/only, subcutaneous multi-point injection.Serum effect is surveyed after 4 weeks
Valence selects the good mouse of immunoreactivity booster immunization again: after taking antigen and isometric incomplete Freund's adjuvant to be sufficiently mixed,
Only, subcutaneous multi-point injection, the number of booster immunization is 6 times to 25 μ g/ of antigen dose, merges preceding continuous booster immunization twice, later
Extracting spleen cell is merged with 50%PEG (MW4000) mediation according to a conventional method with Sp2/0 myeloma cell, and is cultivated.Fusion
After be put into CO2 incubator 37 DEG C of cultures 10 days after, appearance biggish cell clone hole in.It is screened with indirect ELISA, it is right
The hole of the primary dcreening operation positive carries out 4 time cloning cultures (even if a large amount of schizogamies of cell after screening) using limiting dilution assay, it
Afterwards amplifying cells, freeze, prepare ascites.
2.2. Balb/c mouse is only handled with norphytane (being purchased from sigma company) 0.6mL/, intraperitoneal inoculation is miscellaneous after a week
Friendship oncocyte 3 × 106/only, ascites is collected after 10 days.
2.3. antibody titer is measured: the monoclonal antibody (1) with indirect ELISA method measurement using PTH antigen (1) preparation
Potency, the potency of monoclonal antibody reaches 1:35000 or more as the result is shown.
It is also measured, is imitated using identical method using the potency of the monoclonal antibody (2) of PTH antigen (2) preparation
Valence also reaches 1:33000 or more.
Embodiment 4: for detecting the preparation of the fluorescence immune chromatography test paper of people PTH in determinand
1. research object: sample comes from 60 surgery patients of Jiang Yuan hospital, with resisting for vacuum blood collection tube acquisition vein
The vein anticoagulated whole blood centrifugation of solidifying whole blood or serum, acquisition obtains plasma sample；Parathyroid gland is punctured in syringe art
Body of gland, lymph node, musculature, adipose tissue, thyroid glands, thymic tissue sampling are spare.
2. reagent and instrument:
Immune chromatography test paper is divided into test group and control group, and wherein the labelled antibody of test group is to utilize this in embodiment 2
The monoclonal antibody of epitope peptide (1) preparation of preparation, detection antibody are to be prepared in embodiment 2 using epitope peptide (2) of the invention
Monoclonal antibody.And control group as labelled antibody and is directed to using the current commercialized PTH antibody for PTH overall length
The antibody of PTH (1-6) epitope is as detection antibody.Quality Control antibody (sheep anti-mouse igg), fluorescent microsphere, fluorescence in immunochromatography
Detector comes from Wuxi City Jiang Yuan industry Technology and Trade Co., Ltd, nitrocellulose filter (Merck-Millipore company of the U.S.), sample
Pad, bonding pad, bottom plate, blotting paper buying are in the outstanding company in Shanghai, CLIA detection kit, Roche Holding Ag's product, other reagents
It is pure for domestic analysis.
3. the preparation of test strips
The preparation of 3.1 bonding pads
The bonding pad of nanometer fluorescent microspheres label: the bonding pad is the nitrocellulose filter through Seal treatment, preparation
Method is: nitrocellulose filter is placed in containing 1.5wt%NaCl, 5wt%BSA, 0.5wt% Tween-20 and 5wt% sucrose
10min is impregnated in 100mMPBS buffer, is taken out, is drained away the water, 50 DEG C are dried overnight；It is sprayed by the airjet of Bigot instrument
Head, will be containing the nanometer fluorescent microspheres that PTH epitope peptide (1) corresponding antibody occasionally marks according to the amount ullrasonic spraying of 1 μ L/cm
On to dried glass fibre element film, 60 DEG C are dried overnight.
Wherein, the preparation method for the fluorescent nanometer microsphere that can decay is: the corresponding antibody of the mouse TH epitope peptide (1)
The preparation method of the nanometer fluorescent microspheres of label be using EDC/NHS method by the corresponding antibody of TH epitope peptide (1) with contain
Fluorescent molecule COOH activation polystyrene PS or polymethyl benzene ethylene PVT) made from fluorescent microsphere be coupled.Wherein
It is A1 using polystyrene PS as fluorescent microsphere made from material, wherein fluorescent molecule is europium in lanthanide series, terbium, samarium or dysprosium
One kind of chelate, and the commercially available nanometer fluorescent microspheres that can not decay are A2
The bonding pad of colloid gold label: the K for taking colloidal gold stoste and being 0.2M with concentration2C03Solution is adjusted to pH6.25-
6.5, add the corresponding antibody of PTH epitope peptide (1) of label, wherein the PTH epitope peptide (1) of label is right
The ratio between dosage of dosage of the antibody answered and colloidal gold stoste is 1-4ug:20mL, is stirred at room temperature 1 hour, with casein or
BSA closing, 12000r/min are centrifuged 30 minutes, are abandoned supernatant, are redissolved with colloidal gold working solution to the 70% of colloidal gold stoste volume,
Then it is equably sprayed on glass fibre membrane with metal spraying machine, it is dry under conditions of room temperature relative humidity is not higher than 30%, it is close
Envelope saves backup.
Wherein, the ratio between dosage of dosage Yu colloidal gold stoste of the corresponding antibody of PTH epitope peptide (1) of A3 is 1ug；
The ratio between dosage and the dosage of colloidal gold stoste of the corresponding antibody of PTH epitope peptide (1) of 20mL, A4 are 1ug:8mL；A5's
The ratio between dosage and the dosage of colloidal gold stoste of the corresponding antibody of PTH epitope peptide (1) are 1ug:5mL.
The preparation of 3.2 sample pads
Body fluid type sample pad: the sample pad be by treatment fluid handle glass fibre membrane in room temperature relative humidity be 15-
It is dry under conditions of 30%, obtained from being sealed, wherein the treatment fluid include 0.01MPBS, 0.1wt%Tween-20,
1wt% bovine serum albumin(BSA), 10wt% sucrose.
Body fluid type sample pad C1 is dry under conditions of relative humidity is 15% in room temperature, and C2 is in relative humidity in room temperature
Dry under conditions of 22%, C3 is dry under conditions of relative humidity is 30% in room temperature.
Whole blood type sample pad: the sample pad includes through red blood cell monoclonal antibody treated fiber glass film and blood filter membrane group
At the blood filter membrane is covered in the fiber glass film lower end, to be filtered to the haemocyte in whole blood sample；Wherein,
There is 2-4mm overlapping at fiber glass film and the edge of blood filter membrane, and it is 15-30%'s that obtained sample, which is padded on room temperature relative humidity,
Under the conditions of dry and be sealed and obtain.
Whole blood type sample pad C4 is dry under conditions of relative humidity is 15% in room temperature, and C5 is in relative humidity in room temperature
Dry under conditions of 22%, C6 is dry under conditions of relative humidity is 30% in room temperature.
The preparation of 3.3 nitrocellulose filter pads
The detection antibody of the nitrocellulose filter pad be PTH epitope peptide (2) corresponding antibody, use concentration for
The antibody-solutions that the phosphate buffered saline that 0.01M and pH are 7.4 is 0.5-2mg/mL at concentration, by it with 1.0-2.0uL/
The quantity for spray of cm uses spray film instrument to be crossed on nitrocellulose filter pad as detection line, while by sheep anti mouse polyclonal antibody
The antibody-solutions that the phosphate buffered saline that with the concentration containing 1% be 0.01M and pH is 7.4 is 1-2mg/mL at concentration, will
It uses spray film instrument to be crossed on nitrocellulose filter pad as nature controlling line using the quantity for spray of 1.0-2.0uL/cm, will be resulting
Nitrocellulose filter is padded on dry under conditions of room temperature relative humidity humidity is 15-30%, is sealed spare, is specifically shown in Table 1.
Table 1 is that the nitrocellulose filter of three kinds of different ratios pads preparation parameter specific value
The present invention provides four kinds of test strips, and one is be used to detect in serum/plasma/body fluid with colloid gold label
The test strips of PTH content, by A3/A4/A5, B1/B2/B3, C1/C2/C3 is formed at random, and one is the use with colloid gold label
The test strips of PTH content in detection whole blood, by A3/A4/A5, B1/B2/B3, C4/C5/C6 is formed at random, and one is with glimmering
Light nanosphere label for detecting the test strips of PTH content in serum/plasma/body fluid, by A1/A2, B1/B2/B3,
C1/C2/C3 is formed at random, one is with fluorescent nanometer microsphere mark for detecting the test strips of PTH content in whole blood, by
A1/A2, B1/B2/B3, C4/C5/C6 are formed at random, wherein every kind of test strips are by bonding pad, sample pad and nitrocellulose
One is selected in film pad to be combined.
Wherein, three kinds of group merging are specifically chosen in every kind of test strips herein and is assembled into test strips conduct in conjunction with water absorption pad
Test group, the test strips for being used to detect the PTH content in serum/plasma/body fluid of colloid gold label, A3B1C1, A4B2C2,
A5B3C3；The test strips for being used to detect PTH content in whole blood of colloid gold label, A3B1C4, A4B2C5, A5B3C6；Use fluorescence
The test strips for being used to detect the PTH content in serum/plasma/body fluid of nanosphere label, A1B1C1, A2B2C2, A1B3C3；
The test strips for being used to detect PTH content in whole blood marked with fluorescent nanometer microsphere, A1B1C4, A2B2C5, A1B3C6.
Control group, ibid, difference are that the antibody on the bonding pad uses the commercialized PTH for PTH overall length
Antibody is as labelled antibody.
4. detection method:
4.1 acquire the anticoagulated whole blood or serum of vein with vacuum blood collection tube；The vein anticoagulated whole blood centrifugation of acquisition obtains
Obtain plasma sample.With in syringe art puncture parathyroid gland body of gland, lymph node, musculature, adipose tissue, thyroid glands,
Thymic tissue sampling is spare.
The pretreatment of 4.2 tissue samples: the solution conduit for containing 200 μ LPBS buffers, balance to room temperature, using preceding true are taken
All liq is protected all in the bottom of pipe.Syringe after fine needle puncture is protruded into buffer, is sufficiently washed by Smoking regime
Mixing is washed, tissue fluid sample to be tested is made.
4.3 sample-addings: above-mentioned 60 μ L of sample to be tested is added in the sample application zone of PTH fluorescence immune chromatography test paper, in incubator
Membrane chromatographic reacts 5 minutes.
4.4 detection: by after reaction fluorescence immune chromatography test paper and calibration card insertion Portable fluorescence immune quantitative analysis
The card inserting mouth of instrument, runs instrument, and the automatic card reading of instrument provides quality control band C value and detection band T value in fluorescence immune chromatography test paper.
The judgement of 4.5 results:
As C < 10000, expression result is invalid detection, needs to re-replace test paper detection；
As T/C >=0.2, result is the positive, shows to contain parathyroid hormone in sample, or puncturing object is by first shape
Glandular tissue；
As T/C < 0.2, result is feminine gender, shows not containing perhaps micro parathyroid hormone in sample or punctures
The non-Parathyroid Tissue of object.
5. the drafting of standard curve: PTH standard items are made 6 different concentration, respectively 0 μ g/L, 10pg/mL,
50pg/mL, 100pg/mL, 200pg/mL, 300pg/mL, each concentration do 5 Duplicate Samples.
6. fluorescence immune chromatography test paper bar performance test: (1) sensitivity: 10 blank samples of measurement are averaged (x) and mark
Quasi- poor (s) calculates x ± s, finds corresponding dosage on standard curve with this numerical value.(2) test strips are protected from light condition at 4 DEG C
The fluorescence immune chromatography test paper bar of the PTH of same batch and different batches is extracted in lower preservation respectively after 6 months, dense with 100pg/mL
The standard items of degree are tested, and crowd interior and difference between batch CV is calculated.(3) standard items are made corresponding with PTH standard curve 6
A concentration carries out specific detection.
7. compared with Electrochemiluminescince (CLIA) detection kit: being operated in strict accordance with CLIA detection kit specification
It is required that carrying out Parallel testing to the puncture parathyroid gland sample of 60 patients simultaneously respectively with PTH fluorescence detection test.
8. statistical procedures: being analyzed with 19.0 statistical software of SPSS data, chi-square criterion is used between group, P value is less than
0.05 is statistically significant.Correlation is carried out with paired-samples T-test and otherness compares.
As a result with analysis:
1. testing result interpretation: when detection, since chromatography application fluids move forward.If the content of PTH is very few in sample,
PTH in sample forms compound C1 in conjunction with the fluorescent microsphere in conjunction with labelled antibody on bonding pad then corresponding less, bonding pad
In labelled antibody largely combined with the Quality Control antibody on C line, therefore T line will be more many than C line fluorescence or can't detect completely
Fluorescence, result are feminine gender；If the concentration of PTH is larger in sample, compound C1 is accordingly more, with the detection antibody knot at T line
Merge a large amount of formation antibody-antigen-antibody compound C2, and PTH is higher in sample, the colour developing of T line is deeper, and result is the positive.
Either positive or negative findings because the fluorescent microsphere of murine antibody label is excessively coated with, thus always have not
In conjunction with the part fluorescent microsphere for detecting antibody in conjunction with the sheep anti-mouse igg at C line, occurs the aggregation of fluorescent microsphere at C line.Such as
Fruit C line does not have a fluorescent bands, no matter T line whether there is or not fluorescent bands, it is as a result invalid.
2. Specification Curve of Increasing: according to statistical method, using test sample fluorescence value signal as ordinate, PTH standard items
Concentration is abscissa (table 1 is the data of test group, that is, utilizes the antibody of epitope of the present invention), establishes equation and is fitted to mark
Directrix curve.The R2 of the standard curve is 0.9996, linear preferable, meets the requirement of quantitative detection.And control group is (i.e. using commercially available
PTH full length antibody and PTH 1-34 epitope antibodies) standard curve R2 be 0.9207, it is linearly poor relative to test group.
3.PTH immunochromatography fluorescent test paper strip performance evaluation: (1) sensitivity: the zero-dose point mean value of test group, which is read, is
14.26, conversion is 0.18pg/mL on standard curve.Take the sample of 0.20pg/mL, 1pg/mL, 2pg/mL, 4pg/mL, 8pg/mL
Product are detected, and carry out concentration conversion using standard curve, it is found that the PTH sample of this series of concentrations gradient can be accurate
It detected.And control group is verified using the same manner, sensitivity is only capable of reaching 2.0pg/mL, and the two differs a quantity
Grade.(2) stability and precision: the corresponding CV value of the standard curve each group of test group is respectively less than 5% (table 1).4 DEG C, it is protected from light item
When saving test strips detection in 6 months under part, CV is respectively 4.94% and 5.26% in batch and between criticizing, the results showed that test strips inspection
It surveys stability and precision is preferable.Control group is not much different in stability and precision with test group.(3) specific: will to match
The PTH standard items made are detected with the test strips of test group simultaneously with thyroxine standard items, each concentration point cross reaction
Rate CR%=measured value thyroxine/measured value PTH × 100%, the cross reaction in above-mentioned concentration range, with thyroxine
Rate is respectively less than 0.1%, illustrates the two no cross reaction, and method specificity is relatively good.When using control group test strips, low dense
Degree point and the cross reacting rate of thyroxine are more than 3%, and specificity is obviously not so good as test group.
4. compared with CLIA method: used respectively the serum of 60 patients, blood plasma, the dilution of puncturing tissue and whole blood
CLIA kit is detected with test group fluorescence immune chromatography test paper bar, control group immuno-chromatographic test paper strip, takes serum PTH values
Upper limit 65pg/mL is the cut off value of this kit, and analyzes data.
The result shows that the related coefficient of test group immuno-chromatographic test paper strip and CLIA kit is 0.9951, correlation is bent
Line is Y=0.9793X+1.8376, and wherein Y is the PTH concentration (pg/ that fluorescence immunoassay test strip of the invention detects
ML), X is the PTH concentration (pg/mL) that CLIA kit detects.It can be seen that the correlation of the two is fine.Furthermore as the result is shown just
The PTH concentration of normal parathyroid gland and non-Parathyroid Tissue has significant difference (table 2), and less than 0.001, correlation has P value
Statistical significance.And the related coefficient of control group immuno-chromatographic test paper strip and CLIA kit is 0.9176, correlation is compared to examination
It is poor to test group.
By above embodiments and investigation result it is found that the monoclonal antibody of two PTH epitope peptides preparation through the invention
Energy specific recognition PTH, can be in short time accurate quantitative analysis using its fluorescence immune chromatography POCT quantitative detecting method prepared
PTH is not significantly different with CLIA method detection acquired results, complies fully with clinical application requirement, easy to operate, small in size, just
In carrying, it is easy to save, is worth clinical application.
Wherein, in 6-8cm, width is controlled in 2-4cm for the total length control of the test strips.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Sequence table
<110>Shi Kangpei medical science and technology (Wuhan) Co., Ltd
<120>a kind of test strips and preparation method thereof of portable inspectiont human parathyroid hormone
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Claims (12)
1. a kind of test strips of portable inspectiont human parathyroid hormone, which is characterized in that including the bar shaped being transversely and horizontally set
Nitrocellulose filter pad (3), longitudinal gap and what is be parallel to each other be respectively arranged with an item on the nitrocellulose membrane pad (3)
The both ends of the detection line (4) of shape and the nature controlling line (5) of a bar shaped, the detection line (4) and nature controlling line (5) respectively with the nitre
The two sides of acid cellulose film pad (3) are concordant, wherein the nitrocellulose filter pad (3) is abutted close to nature controlling line (5) one end to be had
The water absorption pad (6) of one bar shaped being transversely and horizontally set, the nitrocellulose filter pad (3) abut close to the one end of detection line (4)
There is the bonding pad (2) of a bar shaped being transversely and horizontally set, the bonding pad (2) is supported far from the one end of nitrocellulose filter pad (3)
It is connected to the sample pad (1) for the bar shaped that one is transversely and horizontally set, the sample pad (1), bonding pad (2), nitrocellulose filter pad (3)
It is concordant with the two sides of water absorption pad (6)；
Contain the anti-human PTH antibody marked by colloidal gold or fluorescent microsphere, detection line (4) coating in the bonding pad (2)
There is the detection antibody that can be specifically bound with PTH, the nature controlling line (5) is coated with can be with free anti-human PTH antibody specificity
In conjunction with Quality Control antibody.
2. the test strips of portable inspectiont human parathyroid hormone according to claim 1, which is characterized in that the sample
Pad (1) is that the glass fibre membrane handled by treatment fluid is dried and is sealed under conditions of room temperature relative humidity is 15-30%
Obtained from, wherein the treatment fluid includes 0.01M PBS, 0.1wt%Tween-20,1wt% bovine serum albumin(BSA), 10wt%
Sucrose.
3. the test strips of portable inspectiont human parathyroid hormone according to claim 1, which is characterized in that the sample
Pad (1) includes through red blood cell monoclonal antibody treated fiber glass film and blood filter membrane, described blood filter membrane one end and the bonding pad pair
Abutting should be held, the one end of the blood filter membrane far from bonding pad is arranged in the fiber glass film, and the fiber glass film is close
One end of the blood filter membrane, which is covered, to be supported in the blood filter membrane upper end, or described fiber glass film one end with the bonding pad corresponding end
It connects, the described one end of fiber glass film far from bonding pad is arranged in the blood filter membrane, and the blood filter membrane is close to the fiber glass
One end of fine film is covered in the fiber glass film upper end, and it is 15-30%'s that the sample pad (1), which is placed in room temperature relative humidity,
Under the conditions of dry and be sealed spare.
4. the test strips of portable inspectiont human parathyroid hormone according to claim 2 or 3, which is characterized in that described
Detection antibody is PTH antibody, and the Quality Control antibody is sheep anti-mouse igg.
5. the test strips of portable inspectiont human parathyroid hormone according to claim 4, which is characterized in that the water suction
Pad is blotting paper.
6. the test strips of portable inspectiont human parathyroid hormone according to claim 5, which is characterized in that further include one
The bottom plate (7) of a bar shaped, the sample pad (1), bonding pad (2), nitrocellulose filter pad (3) and water absorption pad (6) are along bottom plate (7)
Length direction is successively laid on the bottom plate (7) upper end.
7. a kind of preparation method of test strips as claimed in claim 6, which is characterized in that steps are as follows: by sample pad (1),
Bonding pad (2), nitrocellulose filter pad (3) and water absorption pad (6) are successively adhered to the bottom plate (7) along bottom plate (7) length direction
Upper end, and sample pad (1) is mutually abutted with bonding pad (2) side close to each other, the bonding pad (2) and nitrocellulose filter pad
(3) one end close to each other mutually abuts, the nitrocellulose filter pad (3) one end close to each other with water absorption pad (6) it is mutual
It abuts.
8. the preparation method of test strips according to claim 7, which is characterized in that in the nitrocellulose filter pad (3)
Be adhered to before the bottom plate (7) upper end, be 0.01M with concentration by PTH antibody and phosphate buffered saline that pH is 7.4 at
Concentration is the antibody-solutions of 0.5-2mg/ml, uses spray film instrument in nitrocellulose filter pad with the quantity for spray of 1.0-2.0uL/cm it
(3) scribing line is carried out on as detection line (4), while being 0.01M with concentration by IgG and phosphate buffered saline that pH is 7.4
It is used spray film instrument in nitrocellulose filter pad by the antibody-solutions for being 1-2mg/mL at concentration with the quantity for spray of 1.0-2.0uL/cm
(3) scribing line is carried out on as nature controlling line (5), and the nitrocellulose filter pad (3) for being coated with PTH antibody and sheep anti-mouse igg is placed in room
Temperature and relative humidity humidity are dried and are sealed spare under conditions of being 15-30%.
9. the preparation method of test strips according to claim 7, which is characterized in that the anti-human PTH on the bonding pad (2)
When antibody is by colloid gold label, the bonding pad (2) the preparation method comprises the following steps: taking colloidal gold stoste and being 0.2M's with concentration
K2C03 solution is adjusted to pH6.25-6.5, adds the PTH antibody of label, wherein the dosage of the PTH antibody of label
It is 1-4ug:20mL with the ratio between the dosage of colloidal gold stoste, is stirred at room temperature 1 hour, is closed with casein or BSA, 12000r/
Min is centrifuged 30 minutes, is abandoned supernatant, is redissolved with colloidal gold working solution to the 70% of colloidal gold stoste volume, then equal with metal spraying machine
It is sprayed on glass fibre membrane evenly, it is dry under conditions of room temperature relative humidity is not higher than 30%, it is sealed spare；
When anti-human PTH antibody on the bonding pad (2) is marked by fluorescent microsphere, the bonding pad (2) is through Seal treatment
Nitrocellulose filter, preparation method are: nitrocellulose filter is placed in containing 1.5wt%NaCl, 5wt%BSA, 0.5wt%
10min is impregnated in the 100mMPBS buffer of Tween-20 and 5wt% sucrose, takes out, drains away the water, 50 DEG C are dried overnight；Pass through
The airjet spray head of Bigot instrument, by the anti-human PTH antibody marked containing fluorescent microsphere according to 1 μ L/cm amount ullrasonic spraying extremely
On nitrocellulose filter after Seal treatment, 60 DEG C are dried overnight.
10. the preparation method of test strips according to claim 9, which is characterized in that the colloidal gold stoste is using chlorine
The colloidal gold solution of auric acid-sodium citrate preparation 30 ± 5nm of diameter；The colloidal gold working solution includes 1wt% ox blood
Pure albumen, 0.1MTris-HCL buffer and 20wt% sucrose, and pH is 8.0.
11. the preparation method of test strips according to claim 9, which is characterized in that it is described containing fluorescent microsphere label
The preparation method of anti-human PTH antibody is to wash fluorescent microsphere using the MES activation buffer of pH7.2-7.6, and carbodiimide is added
(EDC) and n-hydroxysuccinimide (NHS), room temperature reaction certain time wash fluorescent microsphere, with 0.05M pH7.2-7.6
Phosphate buffer redissolve after be added anti-human PTH antibody, react at room temperature 2 hours, the 0.05M containing 10%BSA be added
The phosphate buffer of pH7.2-7.6 reacts at room temperature 30 minutes, fluorescent microsphere is washed, with containing 1%BSA, 0.1%Tween-
The phosphate buffer of 20,0.05M pH7.2-7.6 is redissolved to original volume.
12. the preparation method of test strips according to claim 11, which is characterized in that the fluorescence point in the fluorescent microsphere
Son is one kind of the chelate of europium in lanthanide series, terbium, samarium or dysprosium.
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| CN111334282A (en) * | 2020-03-18 | 2020-06-26 | 厦门稀土材料研究所 | PTH rare earth detection kit, detection card, microspheres thereof, preparation method and detection method |
| CN112881714A (en) * | 2021-02-01 | 2021-06-01 | 德林医疗科技(武汉)有限公司 | Colloidal gold test strip for detecting human parathyroid hormone |
| CN114184796A (en) * | 2021-12-03 | 2022-03-15 | 广州达泰生物工程技术有限公司 | Kit and method for quantitatively detecting full-segment parathyroid hormone |
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