CN107402300B - A kind of method of quick identification human parathyroid - Google Patents
A kind of method of quick identification human parathyroid Download PDFInfo
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- CN107402300B CN107402300B CN201710650822.8A CN201710650822A CN107402300B CN 107402300 B CN107402300 B CN 107402300B CN 201710650822 A CN201710650822 A CN 201710650822A CN 107402300 B CN107402300 B CN 107402300B
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention discloses a kind of method for quickly identifying human parathyroid, it is realized in a manner of PTH content in parathyroid hormone (PTH) fluorescence immune chromatography test paper measurement prepare liquid, the test paper detects people PTH by double antibody sandwich method and fluorescence immune chromatography technology, and the antibody is prepared using specific epitope peptide.The present invention can accurately detect the people PTH in determinand, easy to operate, quick, detection range is wide, it is specific it is high, sensitivity is good.
Description
Technical field
The invention belongs to field of immunodetection, and in particular to a method of quickly identify human parathyroid.
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 the function of adjusting and is kept serum calcium in normal level.How
It is a great problem that parathyroid gland is effectively avoided damage in surgical procedure.Protection technique includes the knowledge of parathyroid gland in parathyroidectomy
Not, fining parathyroid gland protection operating technology, three aspect of remedial parathyroid gland autotransplanting technology.The identification of parathyroid gland
It is a step of parathyroid gland protection most critical in art.Since parathyroid gland body is small in size, around have thyroid gland, fat, lymph
The tissue such as knot, thymus gland, muscle, recognizes in art and has difficulties.Even if the accuracy rate of experienced surgeon's naked eyes identification
Only nearly 70%, there is up to 9~19% parathyroid gland accident resection rate in thyroid gland art.
Frozen section is still the goldstandard for identifying parathyroid gland at present.But this needs cut-out Parathyroid Tissue, art
In it is waiting for a long time, expense is expensive, and there is also 1.3% error rate, the parathyroid hormone (PTH) of fine needle puncture tissue eluent is examined
It surveys, the method for identifying parathyroid gland in preoperative and art has document report.Multiple studies have shown that parathyroid gland puncturing tissue
The PTH value of eluent is apparently higher than non-Parathyroid Tissue, is detected by the PTH of fine needle puncture tissue eluent and is identified in art
Parathyroid Tissue, easy to operate, sensibility and specificity is high, and fool proof.The detection of PTH is generally by electrification at present
It learns to shine and carry out, need large-scale detection device, detection time needs half an hour or more, so to promote and apply in distress plus the shipping time
Degree.
Immunochromatography technique is a kind of unique immunoassay formats for coming across the initial stage eighties, it is usually with strip fibre
Dimension chromatographic material is solid phase, makes sample solution swimming on chromatography strip through capillary action, and make the determinand in sample simultaneously
The immune response that high specificity compatibility occurs with the receptor (such as antibody or antigen) for being directed to determinand on chromatographic material, chromatographed
Immune complex is enriched with or is trapped in the certain area (detection band) of chromatographic material in journey, and using by enzyme reaction or directly can
The marker (such as colloidal gold) of range estimation and obtain intuitive experimental result (such as showing different colors).And free label is then
Detection band is crossed, the purpose being automatically separated with binding label is reached.The common trace labelling particle of immunochromatography technique has glue
Body gold, latex, electroselenium, gelatin etc., wherein being colloidal gold with most successful marker.But colloidal gold immunochromatographimethod tries
Paper slip has the following deficiencies:
(1) colloid gold label process is Electrostatic Absorption process, is a kind of physical absorption, therefore stability is poor in the liquid phase,
The protein molecular often resulted on marked falls off once more.
(2) for testing result by showing that single aubergine item brings judgement, color is single, it is difficult to realize more inspections and connection
Inspection.
(3) only when gold particle gathers it is a certain amount of when, people's naked eyes are just it is observed that purplish red band, and the coloured panel
It is little with background contrasts, to limit detection sensitivity.
(4) different material matrix effects is obvious, and background interference is very big.
(5) detection sensitivity is lower.
(6) it cannot achieve quantitative detection.
Also occurs the detection method using quantum dot mark fast immune chromatographic test paper bar at present, but the method is not
It is able to achieve the quantitative detection to detectable substance.
In addition, detection PTH level method of immunity in, still lack can double-antibody sandwich measure in can Gao Ling
Quick, high specific detection combination, finds the more particularly suitable PTH epitope peptide with immunogenicity, prepares specificity
PTH antigen and antibody are also the emphasis for needing to solve.
Summary of the invention
The technical problem to be solved by the present invention is to overcome deficiencies of the prior art, provide it is a kind of highly sensitive and
High specific, in batch, batch between small, easy to operate, the at low cost quick identification human parathyroid of error method.
Specifically, a kind of method for quickly identifying human parathyroid is provided comprising following steps:
1) in vitro doubtful Parathyroid Tissue is sampled with syringe needle fine needle puncture, connection note in syringe needle rear after sampling
Emitter；
2) solution conduit containing PBS buffer solution is taken, balance to room temperature protrudes into the syringe after fine needle puncture in buffer,
Mixing is sufficiently washed by Smoking regime, and sample to be tested is made；
3) above-mentioned sample to be tested is added in the sample application zone of human parathyroid hormone (PTH) fluorescence immune chromatography test paper, in incubating
Change membrane chromatographic reaction in device；
4) by after reaction fluorescence immune chromatography test paper and calibration card insertion Portable fluorescence immune quantitative analyzer insert
Bayonet, 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；
5) 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 that puncturing object is Parathyroid Tissue；
As T/C < 0.2, result is feminine gender, shows to puncture the non-Parathyroid Tissue of object.
Preferably, the puncture time in step 1) is 3 times, syringe needle 26-gague, and syringe volume is 1ml.
Preferably, PBS buffer solution volume is 200 μ l in solution conduit in step 2), and the volume of sample to be tested is added in step 3)
For 60 μ l.
Preferably, the fluorescence immune chromatography test paper detects people by double antibody sandwich method and fluorescence immune chromatography technology
PTH, in which:
The double antibody sandwich method is used as detection antibody, institute using the first PTH monoclonal antibody for being marked with fluorescent microsphere
The first PTH monoclonal antibody is stated to prepare using one in people PTH epitope peptide (1) and (2) as antigen；And
The double antibody sandwich method is using the 2nd PTH monoclonal antibody as capture antibody, the second monoclonal antibody
It is prepared using another in people PTH epitope peptide (1) and (2) as antigen；
The people PTH epitope peptide (1) and (2) are respectively as follows:
(1): ERVEWLRKKLQ (SEQ ID NO:2)
(2): RKKEDNVLVESHEKSLGEAD (SEQ ID NO:3).
Further, the monoclonal antibody is the antigen being prepared by PTH epitope peptide and carrier protein couplet
It is prepared.
Preferably, the fluorescent material on the fluorescent microsphere is rare earth ion europium；
Preferably, the micro-sphere material of the fluorescent microsphere is polystyrene, polymethyl methacrylate or methacrylic acid
The copolymer of methyl esters.
Preferably, the test paper have bottom plate, and on which floor plate along using when chromatography direction successively with the side of contact
Formula is provided with sample pad, bonding pad, nitrocellulose filter, blotting paper, and the bonding pad, which is provided with the label, to be had
The first PTH monoclonal antibody, the nitrocellulose filter includes detection band and quality control band, and the detection band is coated with described the
Two PTH monoclonal antibodies, the quality control band, which is fixed with, to mark the first PTH monoclonal antibody having special with described
Property combine antiantibody.
Preferably, the bottom plate does not have photoluminescent property.
Preferably, the antiantibody is sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody.
Further, the present invention also provides a kind of methods for preparing the fluorescence immune chromatography test paper comprising following step
It is rapid:
1) the first PTH monoclonal antibody for being marked with fluorescent microsphere is provided；
2) bonding pad is provided, wherein being combined with the first PTH monoclonal of the mark fluorescent microballoon on the bonding pad
Antibody；
3) nitrocellulose filter is provided, wherein chromatography direction interval when on the nitrocellulose filter along use is fixed
2nd PTH monoclonal antibody and antiantibody, to be respectively formed detection band and quality control band；
4) on bottom plate along use when chromatography direction successively with the way of contact setting sample pad, bonding pad, described
Nitrocellulose filter, blotting paper, so that the fluorescence immune chromatography test paper be made.
The step 1) includes:
Fluorescent microsphere is washed using the MES activation buffer of pH7.2-7.6, carbodiimide (EDC) and N- hydroxyl amber is added
Amber acid imide (NHS) reacts at room temperature certain time, washs fluorescent microsphere, multiple with the phosphate buffer of 0.05M pH7.2-7.6
Labelled antibody is added after molten, reacts at room temperature 2 hours, the phosphate-buffered of the 0.05M pH7.2-7.6 containing 10%BSA is added
Liquid reacts at room temperature 30 minutes, fluorescent microsphere is washed, with containing 1%BSA, 0.1%Tween-20, the phosphorus of 0.05M pH7.2-7.6
Phthalate buffer is redissolved to original volume, is quantitatively sprayed on bonding pad, is protected from light 35-38 DEG C and is dried 1 hour, and desiccant is added and seals up for safekeeping
It is spare.
In the step 3), the detection band and Quality Control spaced 3mm to 8mm, the 2nd PTH monoclonal antibody and
The peridium concentration of the antiantibody is respectively 0.5~2mg/ml.
The present invention has the positive effect that compared with prior art:
(1) laboratory that sample is sent to corresponding unit need to be will test for the detection of PTH at present, it is time-consuming in testing instruments
15-20 minutes.It is examined even if method for quick identification of the invention uses, by fine-needle puncturing technique, Fast Detection Technique and portable
The analysis of formula instrument combines, and the entire detection process time is short, can make operative doctor that first can be rapidly completed without leaving operating room
The identification of gland by shape.
(2) people's PTH epitope peptide of the invention has good antigenicity, can with its antigen-immunized animal prepared
Generate the monoclonal antibody and polyclonal antibody of high degree of specificity.The antibody can be tied with the PTH in sample to high special
It closes.
(3) present invention combines fluorescence analysis with flash chromatography immunological technique, passes through people's PTH fluorescence immune chromatography
Test paper detects the people PTH in determinand, easy to operate, quick, in 5 minutes the quick PTH in detection puncturing tissue liquid, and
Detection range is wide, it is specific it is high, sensitivity is good, provide section that is quick, accurate and can be achieved in operating room for surgeon
Parathyroid gland discrimination method reduces the postoperative parathyroid function of patient and subtracts in order to preferably protect parathyroid gland in art
The incidence moved back shortens the hospital stays, reduces medical expense.
(4) present invention is touched during preparing the fluorescence immune chromatography test paper of the people PTH by largely testing
Rope optimizes the preparation condition of various aspects, when so that being detected with fluorescence immune chromatography test paper of the invention, fluorescence signal-to-background ratio
It greatly improves, to improve detection sensitivity and result credibility；In addition, the present invention also passes through detection band and the Quality Control of test paper
The variation of the fluorescence intensity ratio of band carrys out the content of PTH in response sample, this only examines or check detection band with traditional chromatographic technique
Absolute fluorescence intensity is compared, and is reduced the influence of external condition and background etc. to the full extent, is further improved testing result
Confidence level.
(5) the currently used main CLIA of PTH detection method, CLIA method accuracy and sensitivity are relatively high, but to instrument
The requirement of device equipment and operator are too high, and reagent cost is also more expensive.We develop fluorescence immune chromatography test paper bar have compared with
High sensitivity and specificity, error is small in batch, between criticizing, and is not significantly different, complies fully with CLIA method detection acquired results
Clinical application requirement, but it is easy to operate, it is small in size, easy to carry, it is easy to save.It can using the fluorescence immune chromatography test paper bar
It realizes quick, single part quantitative detection, is more suitable for the needs of clinical examination, while realizing the industrialization of detecting instrument and reagent,
Reach preferable Social benefit and economic benefit, is worth clinical application.
Detailed description of the invention
Fig. 1 is the diagrammatic cross-section of fluorescence immune chromatography test paper along its length in embodiment 4；
Fig. 2 is fluorescence immune chromatography test paper floor map in embodiment 4.
Appended drawing reference indicates in figure are as follows: 1- bottom plate, 2- nitrocellulose filter, and 3- blotting paper, 4- sample pad, 5- bonding pad,
6- quality control band, 7- detect band.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
Embodiment 1: people's PTH epitope peptide
People PTH described herein be it is known in the art, complete PTH is the single polypeptide chain containing 84 amino acid
It constitutes, molecular weight is about 9500 dalton.Its amino acid sequence be it is known in the art, can be in the specialized databases such as NCBI
It finds, particular sequence is as follows:
Mankind PTH (1-84):
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNV
LTKAKSQ (SEQ ID NO:1)
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) is one section of peptide fragment containing 11 amino acid of N-terminal 19-29 of people's PTH polypeptide, from
And constitute the epitope peptide (1) for containing 11 amino acid: ERVEWLRKKLQ (SEQ ID NO:2)
PTH epitope peptide (2) includes people PTH C-terminal the 52nd to the 71st peptide fragment, contains 21 amino to constitute
The epitope peptide (2) of acid: RKKEDNVLVESHEKSLGEAD (SEQ ID NO:3)
The characteristics of the two peptide fragments all have hydrophily, antigenicity by force and are readily synthesized.
Epitope peptide is prepared by American AB I431A type polypeptide automatic synthesizer, by Solid phase synthesis, and passes through
The synthesized epitope peptide sequence of polypeptide sequence measurement identification.The purity of peptide fragment can with thin-layer chromatography and high performance liquid chromatography into
Row evaluation, and measure the concentration of epitope peptide.
The preparation of embodiment 2:PTH antibody
The resulting PTH epitope peptide (1) of embodiment 1 and (2) are connect with carrier protein respectively immune with anti-to prepare
Animal is immunized using gained antigen (1) and (2) in former (1) and (2) respectively, to prepare the monoclonal of specificity using antigen (1)
Antibody and polyclonal antibody, and specific monoclonal antibody and polyclonal antibody are prepared using antigen (2).
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 (19-29), PTH
(51-71) 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 Na of 0.2mol/L2HPO481ml 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 CO237 DEG C after culture 10 days in incubator, 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
Hand over oncocyte 3 × 106A/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.
3. immune animal prepares polyclonal antibody:
3.1. male white big ear rabbit 2 is taken, weight about 2Kg is random to divide 3 groups, every group 2.With two kinds of PTH polypeptide antigens
Every group of rabbit is immunized respectively, using subcutaneous multiple spot skeptophylaxis method.1. fundamental immunity: every rabbit injection of BCG vaccine 2.5mg is held
Continuous 10d；2. Freund's complete adjuvant: injecting every group of rabbit respectively with 2 kinds of complex immunogens, 2mg/ rabbit (contains BSA and polypeptide respectively about
1mg), pure polypeptide immunogene PTH 1-84 injects 1mg/ rabbit, continues 28d；3. freund 's incomplete adjuvant is immune: 2 kinds of compounds
Immunogene injects 1.6mg/ rabbit of every group of rabbit (containing BSA and polypeptide respectively about 0.8mg), pure polypeptide immunogene PTH 1-84 note respectively
0.8mg/ rabbit is penetrated, 28d is continued；4. booster immunization: distinguishing booster immunization, agent with the aqua of 3 kinds of immunogene preparations weekly later
Amount is the same as 3..
3.2. it measures antibody titer: utilizing the polyclonal antibody (1) of PTH antigen (1) preparation with indirect elisa method measurement
Potency, antibody titer reaches 1:28000 or more as the result is shown.
It is also measured, is imitated using identical method using the potency of the polyclonal antibody (2) of PTH antigen (2) preparation
Valence also reaches 1:30000 or more.
3.3. take blood and separation serum: arteria carotis intubation takes blood, separates serum.
4. isolating and purifying antibody: after ammonium sulfate precipitation, then through Protein G (being purchased from sigma company) affinity purification.
5. being lyophilized after antibody packing, cryo-conservation.
Embodiment 3: the specificity identification of people PTH antibody (1) and (2)
It is detected with ELISA.It is respectively that detection is anti-with people PTH, Actin albumen, neuronspecific enolase NSE
Primordial covering elisa plate detects the special of prepared PTH monoclonal antibody (1) and (2) and different albumen by ELISA respectively
Property reaction, negative control is made with normal BALB/c mouse serum, PBS liquid makees blank control.
As a result: PTH monoclonal antibody (1) and (2) only reacts respectively with PTH for the positive (P/N > 2.1), and with Actin egg
White, neuronspecific enolase NSE reaction is feminine gender, illustrates that the monoclonal prepared using PTH epitope peptide of the invention is anti-
Body (1) and (2) are respectively provided with specificity.
Identification polyclonal antibody is carried out using method identical with above-mentioned identification monoclonal antibody specificity.
As the result is shown: PTH polyclonal antibody (1) and (2) are reacted respectively with PTH for positive (P/N > 2.1), and and Actin
Albumen, neuronspecific enolase NSE reaction are feminine gender, illustrate the polyclonal antibody of PTH epitope peptide preparation of the invention
(1) and (2) are respectively provided with specificity.
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, is punctured by first shape in syringe art
Gland body of gland, lymph node, musculature, adipose tissue, thyroid glands, thymic tissue sampling are stand-by.
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 invention, 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 (1-34) 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. preparation method:
3.1.1 antibody mark fluorescent microballoon:
Fluorescent microsphere is washed using the MES activation buffer of pH7.2-7.6, carbodiimide (EDC) and N- hydroxyl amber is added
Amber acid imide (NHS) reacts at room temperature certain time, washs fluorescent microsphere, multiple with the phosphate buffer of 0.05M pH7.2-7.6
Labelled antibody is added after molten, reacts at room temperature 2 hours, the phosphate-buffered of the 0.05M pH7.2-7.6 containing 10%BSA is added
Liquid reacts at room temperature 30 minutes, fluorescent microsphere is washed, with containing 1%BSA, 0.1%Tween-20, the phosphorus of 0.05M pH7.2-7.6
Phthalate buffer is redissolved to original volume, is quantitatively sprayed on bonding pad, is protected from light 35-38 DEG C and is dried 1 hour, and desiccant is added and seals up for safekeeping
It is spare；
3.1.2 fluorescence immune chromatography test paper bar assembling parts are handled:
(1) processing of sample pad
Sample is impregnated using the phosphate buffer of the 0.02M pH7.4 containing 1%BSA, 0.1%Triton100 to dry.
(2) preparation of nitrocellulose filter
Using the phosphate buffer of the 0.02M pH7.4 containing 1% sucrose, antibody and the dilution of Quality Control antibody will test respectively
To 1mg/ml, the two is sprayed on nitrocellulose filter with the interval of 0.5cm, addition desiccant is sealed up for safekeeping spare after drying；
3.1.3 the assembling of fluorescence immune chromatography test paper bar:
In humidity less than 35%, in the environment of stablizing 20-25 DEG C, upper nitrocellulose filter 2, knot are pasted on PVC bottom plate 1
Bonding pad 5, sample pad 4 and the blotting paper 3 for having closed fluorescent microsphere label form micro-filtration system, are cut into 0.3cm wide, loading is got stuck
In i.e. test strips (Fig. 1 and Fig. 2) is made.
4. detection method:
4.1 samplings: in vitro doubtful Parathyroid Tissue 3 sub-sampling of 26-gague syringe needle fine needle puncture takes
Syringe needle rear connects 1ml syringe after sample.
4.2 sample pretreatments: the solution conduit for containing 200 μ lPBS buffers is taken, balances to room temperature, institute is ensured before use
There is liquid all in the bottom of pipe.Syringe after fine needle puncture is protruded into buffer, is sufficiently washed by Smoking regime mixed
It is even, 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 that puncturing object is Parathyroid Tissue；
As T/C < 0.2, result is feminine gender, shows to puncture 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 unbonded detection
There is the aggregation of fluorescent microsphere in conjunction with the sheep anti-mouse igg at C line at C line in the part fluorescent microsphere of antibody.If C line does not have
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 R of the standard curve2It is 0.9996, it is linear preferable, meet 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 R2It is 0.9207, it is linearly poor relative to test group.
1 PTH standard items testing result of table
3.PTH immunochromatography fluorescent test paper strip performance evaluation:
(1) sensitivity: the zero-dose point mean value reading of test group is 14.26, and conversion is 0.18pg/ on standard curve
mL.The sample of 0.20pg/mL, 1pg/ml, 2pg/mL, 4pg/mL, 8pg/mL is taken to be detected, and dense using standard curve progress
Degree conversion, it is found that the PTH sample of this series of concentrations gradient can accurately detected.And control group using the same manner into
Row verifying, sensitivity are only capable of reaching 2.0pg/mL, and the two differs an order of magnitude.
(2) stability and precision: the corresponding CV value of the standard curve each group of test group is respectively less than 5% (table 1).It 4 DEG C, keeps away
When saving test strips detection in 6 months under the conditions of light, CV is respectively 4.94% and 5.26% in batch and between criticizing, the results showed that test paper
Item detects stability and precision is preferable.Control group is not much different in stability and precision with test group.
(3) specific: prepared PTH standard items and thyroxine standard items are carried out with the test strips of test group simultaneously
Detection, each concentration point cross reacting rate CR%=measured value thyroxine/measured value PTH × 100%, in above-mentioned concentration range
It is interior, it is respectively less than 0.1% with the cross reacting rate of thyroxine, illustrates the two no cross reaction, method specificity is relatively good.When adopting
It is more than 3% in the cross reacting rate of low concentration point and thyroxine when with control group test strips, specificity is obviously not as good as test
Group.
4. compared with CLIA method: parathyroid gland bodies of gland, lymph node are punctured to 60 patients, musculature, fatty group
It knits, thyroid glands dilution uses CLIA kit respectively and test group fluorescence immune chromatography test paper bar, control group immunochromatography
Test strips are detected, and taking serum PTH values upper limit 65pg/mL is the cut off value of this kit, and is analyzed 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.
2 different tissues eluent PTH testing result of table
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.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
SEQUENCE LISTING
<110>Jiangsu Inst of Atomic Medical Sciences
<120>a kind of method for quickly identifying human parathyroid
<130> 1
<140> 201710650822.8
<141> 2017-08-02
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 84
<212> PRT
<213>artificial sequence
<220>
<223>PTH overall length
<400> 1
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser
35 40 45
Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu
50 55 60
Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys
65 70 75 80
Ala Lys Ser Gln
<210> 2
<211> 11
<212> PRT
<213>artificial sequence
<220>
<223>PTH epitope peptide 1
<400> 2
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln
1 5 10
<210> 3
<211> 20
<212> PRT
<213>mankind
<220>
<223>PTH epitope peptide 2
<400> 3
Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu Lys Ser Leu
1 5 10 15
Gly Glu Ala Asp
20
Claims (9)
1. a kind of method of non-diagnostic, non-treatment purpose quick identification human parathyroid, which is characterized in that including following step
It is rapid:
1) in vitro doubtful Parathyroid Tissue is sampled with syringe needle fine needle puncture, connection injection in syringe needle rear after sampling
Device；
2) solution conduit containing PBS buffer solution is taken, balances to room temperature, the syringe after fine needle puncture is protruded into buffer, is passed through
Smoking regime sufficiently washs mixing, and sample to be tested is made；
3) above-mentioned sample to be tested is added in the sample application zone of human parathyroid hormone (PTH) fluorescence immune chromatography test paper, in incubator
Middle membrane chromatographic reaction；
4) by after reaction fluorescence immune chromatography test paper and calibration card insertion Portable fluorescence immune quantitative analyzer card inserting mouth,
Instrument is run, the automatic card reading of instrument provides quality control band C value and detection band T value in fluorescence immune chromatography test paper；
5) 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 that puncturing object is Parathyroid Tissue；
As T/C < 0.2, result is feminine gender, shows to puncture the non-Parathyroid Tissue of object；
The fluorescence immune chromatography test paper detects people PTH by double antibody sandwich method and fluorescence immune chromatography technology, in which:
The double antibody sandwich method is using being marked with the first PTH monoclonal antibody of fluorescent microsphere as detection antibody, and described the
One PTH monoclonal antibody is prepared using one in people PTH epitope peptide (1) and (2) as antigen；And
Using the 2nd PTH monoclonal antibody as capture antibody, the second monoclonal antibody utilizes the double antibody sandwich method
Another in people PTH epitope peptide (1) and (2) is prepared as antigen；
The people PTH epitope peptide (1) and (2) are respectively as follows:
(1): ERVEWLRKKLQ (SEQ ID NO:2)
(2): RKKEDNVLVESHEKSLGEAD (SEQ ID NO:3).
2. the method for non-diagnostic, non-treatment purpose quick identification human parathyroid according to claim 1, feature exist
In the puncture time in step 1) is 3 times, syringe needle 26-gague, and syringe volume is 1ml.
3. the method for non-diagnostic, non-treatment purpose quick identification human parathyroid according to claim 1, feature exist
In PBS buffer solution volume is 200 μ l in solution conduit in step 2), and the volume that sample to be tested is added in step 3) is 60 μ l.
4. the method for non-diagnostic, non-treatment purpose quick identification human parathyroid according to claim 1, feature exist
In the monoclonal antibody is prepared by PTH epitope peptide and the antigen that carrier protein couplet is prepared.
5. the method for non-diagnostic, non-treatment purpose quick identification human parathyroid according to claim 1, feature exist
In the fluorescent material on the fluorescent microsphere is rare earth ion europium.
6. the method for non-diagnostic, non-treatment purpose quick identification human parathyroid according to claim 1, feature exist
In the micro-sphere material of the fluorescent microsphere is the copolymer of polystyrene, polymethyl methacrylate or methyl methacrylate.
7. the method for non-diagnostic, non-treatment purpose quick identification human parathyroid according to claim 1, feature exist
In the test paper has bottom plate (1), and chromatography direction when on the bottom plate (1) along use is successively arranged with the way of contact
Have: sample pad (4), bonding pad (5), nitrocellulose filter (2), blotting paper (3), the bonding pad (5) are provided with the label
There is the first PTH monoclonal antibody of fluorescent microsphere, the nitrocellulose filter (2) includes detection band (7) and quality control band (6), institute
It states detection band (7) and is fixed with the 2nd PTH monoclonal antibody, the quality control band (6), which is coated with, to be had with the label
The antiantibody that first PTH monoclonal antibody specificity of microballoon combines.
8. the method for non-diagnostic, non-treatment purpose quick identification human parathyroid according to claim 7, feature exist
In the bottom plate (1) does not have photoluminescent property.
9. the method for non-diagnostic, non-treatment purpose quick identification human parathyroid according to claim 7, feature exist
In the antiantibody is sheep anti-mouse igg polyclonal antibody or rabbit anti-mouse igg polyclonal antibody.
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| CN108445237A (en) * | 2018-06-20 | 2018-08-24 | 广州市康润生物科技有限公司 | A kind of new application of PTH tachysynthesises detecting system |
| CN111334282B (en) * | 2020-03-18 | 2023-05-26 | 厦门稀土材料研究所 | PTH rare earth detection kit, PTH rare earth detection card, microsphere and preparation and detection methods thereof |
| CN113588602B (en) * | 2021-07-08 | 2024-06-04 | 合肥新唯基因科技有限公司 | Rapid parathyroid hormone detection method |
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| CN105167810B (en) * | 2015-09-25 | 2017-12-15 | 江苏省原子医学研究所 | Puncture outfit in parathyroid gland or lymph node art |
| CN205280727U (en) * | 2015-10-15 | 2016-06-01 | 厦门宝太生物科技有限公司 | Fluorescence immunity chromatography test paper |
| CN205333645U (en) * | 2015-12-30 | 2016-06-22 | 厦门依柯利斯医疗科技有限公司 | Lp -PLA2 quick quantitative detection test paper strip in people's serum |
| CN205958585U (en) * | 2016-08-17 | 2017-02-15 | 遂宁市中心医院 | Parathyrine detection test strip that determines nature |
| CN106771128A (en) * | 2016-12-15 | 2017-05-31 | 威海纽普生物技术有限公司 | Parathyroid hormone determines kit and preparation method |
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