CN107402300A - A kind of method of quick discriminating human parathyroid - Google Patents
A kind of method of quick discriminating human parathyroid Download PDFInfo
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- CN107402300A CN107402300A CN201710650822.8A CN201710650822A CN107402300A CN 107402300 A CN107402300 A CN 107402300A CN 201710650822 A CN201710650822 A CN 201710650822A CN 107402300 A CN107402300 A CN 107402300A
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Abstract
The present invention discloses a kind of method of quick discriminating human parathyroid, it is determined in prepare liquid by parathyroid hormone (PTH) fluorescence immune chromatography test paper and realized in a manner of PTH contents, for the test paper by double antibody sandwich method and fluorescence immune chromatography technology for detection people PTH, the antibody is prepared using specific epitope peptide.The present invention can detect the people PTH in determinand exactly, 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 kind of method of quick discriminating human parathyroid.
Background technology
Thyroid cancer is the most common malignant tumour of incidence, and most fast pernicious swollen of incidence of disease growth rate in recent years
One of knurl, 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 prevent and treat first shape
Other adenasthenia.If unexpected during operation cut off parathyroid gland, the blood calcium concentration of patient can be reduced substantially, trigger brothers to take out
Jerk, 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 that to adjust 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, the parathyroid gland that becomes more meticulous protection operating technology, the aspect of remedial parathyroid gland autotransplanting technology three.The identification of parathyroid gland
It is a step of parathyroid gland protection most critical in art.There are thyroid gland, fat, lymph due to parathyroid gland body small volume, around it
Knot, thymus gland, muscle etc. are organized, and are recognized and are had difficulties in art.Even if the accuracy rate of experienced surgeon naked eyes identification
Only nearly 70%, up to 9~19% parathyroid gland accident resection rate in thyroid gland art be present.
Frozen section is still the goldstandard for differentiating parathyroid gland at present.But this needs cut-out Parathyroid Tissue, art
Middle stand-by period length, expense is expensive, is also examined in the presence of 1.3% error rate, the parathyroid hormone (PTH) of FNA tissue eluent
Survey, the method for differentiating parathyroid gland in preoperative and art, existing document report.Multiple studies have shown that parathyroid gland puncturing tissue
The PTH values of eluent are detected by the PTH of FNA tissue eluent and differentiated in art apparently higher than non-Parathyroid Tissue
Parathyroid Tissue, simple to operate, Sensitivity and Specificity is high, and fool proof.PTH detection is generally by electrification at present
Learn it is luminous carry out, need large-scale detection device, detection time needs more than half an hour plus the shipping time, and such popularization and application are in distress
Degree.
Immunochromatography technique is a kind of unique immunoassay formats for coming across the initial stage eighties, and it is generally fine with strip
Dimension chromatographic material is solid phase, makes sample solution swimming on chromatography strip by capillarity, and make the determinand in sample simultaneously
The immune response of highly-specific highly compatibility occurs for the acceptor (such as antibody or antigen) with being directed to determinand on chromatographic material, chromatographs
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 label (such as collaurum) of range estimation and obtain intuitively experimental result (such as showing different colors).And free label is then
Detection band is crossed, reaches the purpose being automatically separated with binding label.The common trace labelling particle of immunochromatography technique has glue
Body gold, latex, electroselenium, gelatin etc., wherein being collaurum with most successful label.But colloidal gold immunochromatographimethod tries
Following defect be present in paper slip：
(1) colloid gold label process is Electrostatic Absorption process, is a kind of physical absorption, therefore less stable in the liquid phase,
The protein molecular often resulted on marked comes off once more.
(2) testing result brings judgement by showing single aubergine bar, and color is single, it is difficult to realizes more inspections and connection
Inspection.
(3) only when gold grain 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, so as to limit detection sensitivity.
(4) different matrix of materials effects is obvious, and ambient interferences are very big.
(5) detection sensitivity is relatively low.
(6) quantitative detection can not be realized.
Also occurs the detection method using quantum dot mark fast immune chromatographic test paper bar at present, but the method is not
The quantitative detection to detectable substance can be realized.
In addition, in the horizontal method of immunity of detection PTH, still lacking can Gao Ling in double-antibody sandwich measure
The combination of quick, high specific detection, find the more particularly suitable PTH epitope peptides with immunogenicity, prepare it is specific
PTH antigens and antibody are also the emphasis for needing to solve.
The content of the invention
The technical problem to be solved in the present invention is to overcome deficiencies of the prior art, there is provided a kind of high sensitivity and
High specific, in batch, batch between error is small, simple to operate, cost is low quick discriminating human parathyroid method.
Specifically, there is provided a kind of method of quick discriminating human parathyroid, it comprises the following steps：
1) in vitro doubtful Parathyroid Tissue is sampled with syringe needle FNA, connection note in syringe needle rear after sampling
Emitter；
2) solution conduit containing PBS is taken, is balanced to room temperature, the syringe after FNA is stretched into buffer solution,
Mixing is fully washed by Smoking regime, testing sample is made；
3) above-mentioned testing sample is added in the sample application zone of human parathyroid hormone (PTH) fluorescence immune chromatography test paper, in incubating
Change membrane chromatographic in device to react；
4) reacted fluorescence immune chromatography test paper and calibration card are inserted into inserting for Portable fluorescence immune quantitative analyzer
Bayonet socket, runs instrument, and the automatic Card Reader of instrument provides quality control band C values and detection band T values in fluorescence immune chromatography test paper；
5) C is worked as<When 10000, it is that invalid detection detects, it is necessary to re-replace test paper to represent result；
It is as a result the positive as T/C >=0.2, shows that it is Parathyroid Tissue to puncture thing；
Work as T/C<It is as a result feminine gender when 0.2, shows to puncture the non-Parathyroid Tissue of thing.
Preferably, the puncture time in step 1) is 3 times, syringe needle 26-gague, and syringe volume is 1ml.
Preferably, PBS volume is 200 μ l in solution conduit in step 2), and step 3) adds the volume of testing sample
For 60 μ l.
Preferably, the fluorescence immune chromatography test paper passes through double antibody sandwich method and fluorescence immune chromatography technology for detection people
PTH, wherein：
The double antibody sandwich method is used as detection antibody, institute using the first PTH monoclonal antibodies for being marked with fluorescent microsphere
The first PTH monoclonal antibodies are stated to prepare by the use of one in people PTH epitope peptides (1) and (2) as antigen；And
The double antibody sandwich method is using the 2nd PTH monoclonal antibodies as capture antibody, the second monoclonal antibody
Prepared by the use of another in people PTH epitope peptides (1) and (2) as antigen；
The people PTH epitope peptides (1) and (2) are respectively：
(1)：ERVEWLRKKLQ(SEQ ID NO：2)
(2)：RKKEDNVLVESHEKSLGEAD(SEQ ID NO：3).
Further, the monoclonal antibody is the antigen being prepared by PTH epitope peptides 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 has a bottom plate, and on which floor plate along using when chromatography direction successively with the side of contact
Formula is provided with：Sample pad, pad, nitrocellulose filter, blotting paper, the pad, which is provided with the mark, to be had
The first PTH monoclonal antibodies, the nitrocellulose filter includes detection band and quality control band, and the detection band is coated with described
Two PTH monoclonal antibodies, the quality control band, which is fixed with, to mark the first PTH monoclonal antibodies 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 method for preparing the fluorescence immune chromatography test paper, and it includes following step
Suddenly：
1) the first PTH monoclonal antibodies for being marked with fluorescent microsphere are provided；
2) pad is provided, wherein being combined with the first PTH monoclonals of the mark fluorescent microballoon on the pad
Antibody；
3) nitrocellulose filter is provided, wherein being fixed on the nitrocellulose filter along chromatography direction interval when using
2nd PTH monoclonal antibodies and antiantibody, to form detection band and quality control band respectively；
4) the sample pad, pad, described is being set with the way of contact successively along chromatography direction when using on bottom plate
Nitrocellulose filter, blotting paper, so as to which the fluorescence immune chromatography test paper be made.
The step 1) includes：
Fluorescent microsphere is washed using pH7.2-7.6 MES activation buffers, adds carbodiimide (EDC) and N- hydroxyl ambers
Amber acid imide (NHS), certain time is reacted at room temperature, wash fluorescent microsphere, answered with 0.05M pH7.2-7.6 phosphate buffer
Labelled antibody is added after molten, is reacted at room temperature 2 hours, adds the phosphate-buffered of the 0.05M pH7.2-7.6 containing 10%BSA
Liquid, react at room temperature 30 minutes, fluorescent microsphere is washed, with containing 1%BSA, 0.1%Tween-20,0.05M pH7.2-7.6 phosphorus
Phthalate buffer is redissolved to original volume, is quantitatively sprayed on pad, and 35-38 DEG C of lucifuge is dried 1 hour, is added drier and is sealed up for safekeeping
It is standby.
In the step 3), the detection band and Quality Control spaced 3mm to 8mm, the 2nd PTH monoclonal antibodies and
The coating concentration of the antiantibody is respectively 0.5~2mg/ml.
The present invention has the positive effect that compared with prior art：
(1) detection sample need to be sent to the laboratory of corresponding unit by the detection for PTH at present, be taken in testing instruments
15-20 minutes.Examined even if the method for quick identification of the present invention uses, by fine-needle puncturing technique, Fast Detection Technique and portable
Formula Instrumental Analysis is combined, and the whole detection process time is short, and operative doctor can be made to leave operating room with regard to that can be rapidly completed first
The discriminating of gland by shape.
(2) people's PTH epitope peptides of the invention have good antigenicity, can with the antigen-immunized animal of its preparation
Produce the monoclonal antibody and polyclonal antibody of high degree of specificity.The antibody can be tied high special with the PTH in sample
Close.
(3) fluorescence analysis are combined by the present invention with flash chromatography immunological technique, pass through people's PTH fluorescence immune chromatographies
People PTH, easy to operate, quick, the PTH in 5 minutes in quick detection puncturing tissue liquid in test paper detection determinand, and
Detection range is wide, it is specific it is high, sensitivity is good, is provided quickly, accurately and the section that can be achieved with operating room for surgeon
Parathyroid gland discrimination method, in order to which preferably protection parathyroid gland, the reduction postoperative parathyroid function of patient subtract in art
The incidence moved back, shorten the hospital stays, reduce medical expense.
(4) present invention is touched during the fluorescence immune chromatography test paper of the people PTH is prepared by largely testing
Rope, optimize the preparation condition of each side so that when being detected with the fluorescence immune chromatography test paper of the present invention, fluorescence signal-to-background ratio
Greatly improve, so as to improve detection sensitivity and credible result degree；In addition, detection band and Quality Control of the present invention also by test paper
The change of the fluorescence intensity ratio of band carrys out the content of PTH in response sample, and 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 increased testing result
Confidence level.
(5) the currently used main CLIA of PTH detection methods, CLIA methods accuracy and sensitivity are all higher, but to instrument
The requirement of device equipment and operating personnel 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, batch in, batch between error it is small, with CLIA methods detection acquired results be not significantly different, comply fully with
Clinical practice requirement, but it is simple to operate, small volume, it is easy to carry, is easy to preserve.Can using the fluorescence immune chromatography test paper bar
Realize that quick, single part is quantitatively detected, be more suitable for the needs of clinical examination, while realize the industrialization of detecting instrument and reagent,
Reach preferable Social benefit and economic benefit, clinic popularization and application.
Brief description of the drawings
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.
Reference is expressed as in figure：1- bottom plates, 2- nitrocellulose filters, 3- blotting papers, 4- sample pads, 5- pads,
6- quality control bands, 7- detection bands.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
Embodiment 1：People's PTH epitope peptides
People PTH specifically described herein is known in the art, and complete PTH is the single polypeptide chain containing 84 amino acid
Form, 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
Find, particular sequence is as follows：
Mankind PTH (1-84)：
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNV
LTKAKSQ(SEQ ID NO：1)
The present inventor gropes by substantial amounts of theoretical research and experiment, and final screening, which obtains two kinds, to be had well
Antigenic epitope peptide.
PTH epitope peptides (1) are one section of peptide fragments containing 11 amino acid of the N-terminal 19-29 positions of people's PTH polypeptides, from
And form the epitope peptide (1) containing 11 amino acid：ERVEWLRKKLQ(SEQ ID NO：2)
PTH epitope peptides (2) include the peptide fragment of people PTH C-terminals the 52nd to the 71st, so as to form containing 21 amino
The epitope peptide (2) of acid：RKKEDNVLVESHEKSLGEAD(SEQ ID NO：3)
The characteristics of the two peptide fragments are respectively provided with hydrophily, antigenicity by force and are readily synthesized.
Epitope peptide is prepared by the more automatic peptide synthesizers of American AB I431A types, by Solid phase synthesis, and passes through
The synthesized epitope peptide sequence of peptide sequence measure identification.The purity of peptide fragment can be entered with thin-layer chromatography and high performance liquid chromatography
Row evaluation, and determine the concentration of epitope peptide.
Embodiment 2：The preparation of PTH antibody
The PTH epitope peptides (1) of the gained of embodiment 1 and (2) are connected with carrier protein respectively immune with anti-to prepare
Former (1) and (2), animal is immunized respectively using gained antigen (1) and (2), so as to prepare specific monoclonal using antigen (1)
Antibody and polyclonal antibody, and prepare specific monoclonal antibody and polyclonal antibody using antigen (2).
1. the preparation of antigen：PTH peptide fragments are connected with carrier protein BSA respectively and are prepared into PTH antigens.Take pH6.0
0.01mol/L PBS and dimethyl sulfoxide (DMSO) (DMSO) each 0.25mL dissolve 5mg BSA, take MBS 1.2mg 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 altogether in about 500 μ L respectively.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 standby.
In the present embodiment, the formula of PBS is：0.2mol/L Na2HPO481ml adds 0.2mol/L's
Na2HPO419ml is mixed.
2. immune animal prepares monoclonal antibody：
2.1. the PTH antigens (immunogene) of above-mentioned preparation are taken (to be purchased from Shang Haiyuan with isometric Freund's complete adjuvant respectively
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
Valency, select the good mouse of immunoreactivity booster immunization again：After taking antigen to be sufficiently mixed with isometric incomplete Freund's adjuvant,
Only, subcutaneous multi-point injection, the number of booster immunization is 6 times to the μ g/ of antigen dose 25, before fusion continuous booster immunization twice, afterwards
Extracting spleen cell is merged with 50%PEG (MW4000) mediations according to a conventional method with Sp2/0 myeloma cell, and is cultivated.Fusion
After be put into CO2In incubator after 37 DEG C of cultures 10 days, appearance larger cell clone hole in.Screened with indirect ELISA, it is right
The positive hole of primary dcreening operation 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 are only handled with norphytane (being purchased from sigma companies) 0.6ml/, pneumoretroperitoneum inoculation in one week is miscellaneous
Hand over oncocyte 3 × 106Individual/only, collect ascites after 10 days.
2.3. antibody titer is determined：The monoclonal antibody (1) prepared with indirect ELISA method measure using PTH antigens (1)
Potency, as a result show that the potency of monoclonal antibody reaches 1:More than 35000.
The potency of the monoclonal antibody (2) prepared using PTH antigens (2) is also measured using identical method, and it is imitated
Valency also reaches 1:More than 33000.
3. immune animal prepares polyclonal antibody：
3.1. male white big ear rabbit 2 is taken, body weight about 2Kg, divides 3 groups, every group 2 at random.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：Every group of rabbit is injected respectively with 2 kinds of complex immunogens, and 2mg/ rabbit is (each about containing BSA and polypeptide
1mg), pure polypeptide immunogene PTH 1-84 inject 1mg/ rabbit, continue 28d；3. freund 's incomplete adjuvant is immunized：2 kinds of compounds
Immunogene injects 1.6mg/ rabbit of every group of rabbit (containing BSA and each about 0.8mg of polypeptide), pure polypeptide immunogene PTH 1-84 notes respectively
0.8mg/ rabbit is penetrated, continues 28d；4. booster immunization：The aqua prepared later with 3 kinds of immunogenes weekly distinguishes booster immunization, agent
Amount is the same as 3..
3.2. antibody titer is determined：The polyclonal antibody (1) prepared with indirect elisa method measure using PTH antigens (1)
Potency, as a result show that antibody titer reaches 1:More than 28000.
The potency of the polyclonal antibody (2) prepared using PTH antigens (2) is also measured using identical method, and it is imitated
Valency also reaches 1:More than 30000.
3.3. blood and separation serum are taken：Arteria carotis intubation takes blood, separates serum.
4. isolate and purify antibody：After ammonium sulfate precipitation, then through Protein G (being purchased from sigma companies) affinity purification.
5. freezed after antibody packing, Cord blood.
Embodiment 3：People PTH antibody (1) and the specificity identification of (2)
Detected with ELISA.It is anti-as detection using people PTH, Actin albumen, neuronspecific enolase NSE respectively
Primordial covering elisa plate, detect the special of prepared PTH monoclonal antibodies (1) and (2) and different albumen respectively by ELISA
Property reaction, negative control is made with normal BALB/c mouse serum, PBS liquid makees blank control.
As a result：PTH monoclonal antibodies (1) and (2) respectively only and PTH reactions are positive (P/N ＞ 2.1), and with Actin eggs
In vain, neuronspecific enolase NSE reactions are feminine gender, illustrate to resist using monoclonal prepared by the PTH epitope peptides of the present invention
Body (1) and (2) have specificity respectively.
Identification polyclonal antibody is carried out using with above-mentioned identification monoclonal antibody specificity identical method.
As a result show：PTH polyclonal antibodies (1) and (2) they are respectively positive (P/N ＞ 2.1) with PTH reactions, and and Actin
Albumen, neuronspecific enolase NSE reactions are feminine gender, illustrate polyclonal antibody prepared by the PTH epitope peptides of the present invention
(1) and (2) have specificity respectively.
Embodiment 4：For detecting the preparation of people PTH fluorescence immune chromatography test paper in determinand
1. research object：Sample comes from 60 surgery patients of Jiang Yuan hospitals, is punctured with syringe art by first shape
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 the labelled antibody of wherein 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 the epitope peptide (2) of the present invention
Monoclonal antibody.And control group as labelled antibody and is directed to using the current commercialized PTH antibody for PTH total lengths
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 companies of the U.S.), sample
Pad, pad, bottom plate, blotting paper are purchased in the outstanding company in Shanghai, CLIA detection kits, Roche Holding Ag's product, other reagents
It is pure for domestic analysis.
3. preparation method：
3.1.1 antibody labeling fluorescent microsphere：
Fluorescent microsphere is washed using pH7.2-7.6 MES activation buffers, adds carbodiimide (EDC) and N- hydroxyl ambers
Amber acid imide (NHS), certain time is reacted at room temperature, wash fluorescent microsphere, answered with 0.05M pH7.2-7.6 phosphate buffer
Labelled antibody is added after molten, is reacted at room temperature 2 hours, adds the phosphate-buffered of the 0.05M pH7.2-7.6 containing 10%BSA
Liquid, react at room temperature 30 minutes, fluorescent microsphere is washed, with containing 1%BSA, 0.1%Tween-20,0.05M pH7.2-7.6 phosphorus
Phthalate buffer is redissolved to original volume, is quantitatively sprayed on pad, and 35-38 DEG C of lucifuge is dried 1 hour, is added drier and is sealed up for safekeeping
It is standby；
3.1.2 fluorescence immune chromatography test paper bar assembling parts are handled：
(1) processing of sample pad
Dried using the phosphate buffer immersion sample of the 0.02M pH7.4 containing 1%BSA, 0.1%Triton100.
(2) preparation of nitrocellulose filter
, respectively will detection antibody and the dilution of Quality Control antibody using the phosphate buffer of the 0.02M pH7.4 containing 1% sucrose
To 1mg/ml, the two is sprayed on nitrocellulose filter with 0.5cm interval, addition drier is sealed up for safekeeping standby after drying；
3.1.3 the assembling of fluorescence immune chromatography test paper bar：
It is less than 35%, in the environment of stable 20-25 DEG C in humidity, nitrocellulose filter 2, knot is pasted on PVC bottom plates 1
Closed the pad 5 of fluorescent microsphere mark, sample pad 4 and blotting paper 3 form micro-filtration system, cut into that 0.3cm is wide, and loading is got stuck
In i.e. test strips (Fig. 1 and Fig. 2) are made.
4. detection method：
4.1 sampling：By the in vitro doubtful Parathyroid Tissue sub-sampling of 26-gague syringe needles FNA 3, take
Syringe needle rear connects 1ml syringes after sample.
4.2 sample pretreatment：A solution conduit for containing 200 μ lPBS buffer solutions is taken, balances to room temperature, institute is ensured before use
There is bottom of the liquid all in pipe.Syringe after FNA is stretched into buffer solution, fully washed by Smoking regime mixed
It is even, testing sample is made.
4.3 sample-adding：The above-mentioned μ l of testing sample 60 are added in the sample application zone of PTH fluorescence immune chromatography test papers, in incubator
Membrane chromatographic reacts 5 minutes.
4.4 detection：By reacted 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 Reader of instrument provides quality control band C values and detection band T values in fluorescence immune chromatography test paper.
4.5 results judge：
Work as C<When 10000, it is that invalid detection detects, it is necessary to re-replace test paper to represent result；
It is as a result the positive as T/C >=0.2, shows that it is Parathyroid Tissue to puncture thing；
Work as T/C<It is as a result feminine gender when 0.2, shows to puncture the non-Parathyroid Tissue of thing.
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 are determined, averages (x) and marks
Accurate poor (s), x ± s is calculated, corresponding dosage is found on standard curve with this numerical value.(2) test strips are at 4 DEG C, lucifuge condition
The PTH of same batch and different batches fluorescence immune chromatography test paper bar is extracted in lower preservation respectively after 6 months, dense with 100pg/mL
The standard items of degree are tested, and calculate crowd interior and difference between batch CV.(3) 6 corresponding with PTH standard curves are made in standard items
Individual concentration, carry out specific detection.
7. compared with Electrochemiluminescince (CLIA) detection kit：Operated in strict accordance with CLIA detection kits specification
It is required that Parallel testing is carried out to the puncture parathyroid gland sample of 60 patients simultaneously respectively with PTH fluorescence detection tests.
8. statistical procedures：Data are analyzed with the statistical softwares of SPSS 19.0, chi-square criterion is used between group, P values are 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：During detection, because chromatography application fluids move forward.If PTH content is very few in sample,
PTH in sample and then corresponding less, the pad that marks the fluorescent microsphere of antibody binding to combine to form compound C1 on pad
In labelled antibody largely combined with the Quality Control antibody on C lines, therefore T lines will be more shallow than C line fluorescence many or can't detect completely
Fluorescence, it is as a result feminine gender；If PTH concentration is larger in sample, compound C1 is accordingly more, with the detection antibody knot at T lines
Merge a large amount of formation antibody-antigen-antibody compound C2, and PTH is higher in sample, and the colour developing of T lines is deeper, is as a result the positive.
Either positive or negative findings, because the fluorescent microsphere of murine antibody mark is excessively coated with, thus always there is uncombined detection
The part fluorescent microsphere of antibody is combined with the sheep anti-mouse igg at C lines, occurs the aggregation of fluorescent microsphere at C lines.If C lines do not have
There are fluorescent bands, no matter T lines whether there is fluorescent bands, as a result invalid.
2. Specification Curve of Increasing：According to statistical method, to detect fluorescent 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 curve2For 0.9996, it is linear preferably, meet the requirement of quantitative detection.And control group is (i.e. using commercially available
PTH full length antibodies and PTH 1-34 epitope antibodies) standard curve R2It is linearly poor relative to test group for 0.9207.
The PTH standard items testing results of table 1
3.PTH immunochromatography fluorescent test paper strip performance evaluations：
(1) sensitivity：The zero-dose point average reading of test group is 14.26, and conversion is 0.18pg/ on standard curve
mL.0.20pg/mL, 1pg/ml, 2pg/mL, 4pg/mL, 8pg/mL sample is taken to be detected, and it is dense using standard curve progress
Degree conversion, it is found that the PTH samples of this series concentration gradient can be detected accurately.And control group is entered using the same manner
Row checking, sensitivity are only capable of reaching 2.0pg/mL, the two difference an order of magnitude.
(2) stability and precision：CV values corresponding to the standard curve each group of test group are respectively less than 5% (table 1).4 DEG C, keep away
When the ELISA test strip of 6 months is preserved under optical condition, batch in and batch between CV be respectively 4.94% and 5.26%, the results showed that test paper
Bar detects stability and precision is preferable.Control group is more or less the same in stability and precision with test group.
(3) it is specific：Carried out the PTH standard items prepared and thyroxine standard items while with the test strips of test group
Detection, each concentration point cross reacting rate CR%=measured values thyroxine/measured value PTH × 100%, in above-mentioned concentration range
It is interior, 0.1% is respectively less than with the cross reacting rate of thyroxine, illustrates both no cross reactions, method specificity is relatively good.When adopting
During with control group test strips, in the cross reacting rate of low concentration point and thyroxine more than 3%, specificity is substantially not as experiment
Group.
4. the comparison with CLIA methods：60 patients are punctured with parathyroid gland body of gland, lymph nodes, musculature, fatty group
Knit, thyroid glands dilution uses CLIA kits and test group fluorescence immune chromatography test paper bar, control group immunochromatography respectively
Test strips are detected, and take serum PTH values upper limit 65pg/mL to be the cut off values of this kit, and analyze data.
As a result show, the coefficient correlation of test group immuno-chromatographic test paper strip and CLIA kits is 0.9951, and correlation is bent
Line is Y=0.9793X+1.8376, and the fluorescence immunoassay test strip that wherein Y is the present invention detects obtained PTH concentration (pg/
Ml), X is the PTH concentration (pg/ml) that CLIA kits detect to obtain.It can be seen that both correlations are fine.In addition result is shown just
The PTH concentration of normal parathyroid gland and non-Parathyroid Tissue has significant difference (table 2), and P values are less than 0.001, and correlation has
Statistical significance.And the coefficient correlation of control group immuno-chromatographic test paper strip and CLIA kits is 0.9176, correlation is compared to examination
It is poor to test group.
The different tissues eluent PTH testing results of table 2
From above example and result is investigated, the monoclonal antibody prepared by two PTH epitope peptides of the present invention
Energy specific recognition PTH, can be in short time accurate quantitative analysis using fluorescence immune chromatography POCT quantitative detecting methods of its preparation
PTH, it is not significantly different with CLIA methods detection acquired results, complies fully with clinical practice requirement, simple to operate, small volume, just
In carrying, it is easy to preserve, clinic popularization and application.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With.It can be applied to various suitable the field of the invention completely., can be easily for those skilled in the art
Realize other modification.Therefore it is of the invention and unlimited under the universal limited without departing substantially from claim and equivalency range
In specific details and shown here as the legend with description.
Claims (10)
- A kind of 1. method of quick discriminating human parathyroid, it is characterised in that comprise the following steps：1) in vitro doubtful Parathyroid Tissue is sampled with syringe needle FNA, connection injection in syringe needle rear after sampling Device；2) solution conduit containing PBS is taken, is balanced to room temperature, the syringe after FNA is stretched into buffer solution, passed through Smoking regime fully washs mixing, and testing sample is made；3) above-mentioned testing sample is added in the sample application zone of human parathyroid hormone (PTH) fluorescence immune chromatography test paper, in incubator Middle membrane chromatographic reaction；4) reacted fluorescence immune chromatography test paper and calibration card are inserted to the card inserting mouth of Portable fluorescence immune quantitative analyzer, Instrument is run, the automatic Card Reader of instrument provides quality control band C values and detection band T values in fluorescence immune chromatography test paper；5) C is worked as<When 10000, it is that invalid detection detects, it is necessary to re-replace test paper to represent result；It is as a result the positive as T/C >=0.2, shows that it is Parathyroid Tissue to puncture thing；Work as T/C<It is as a result feminine gender when 0.2, shows to puncture the non-Parathyroid Tissue of thing.
- 2. the method for quick discriminating human parathyroid according to claim 1, it is characterised in that the puncture time in step 1) Number is 3 times, syringe needle 26-gague, and syringe volume is 1ml.
- 3. the method for quick discriminating human parathyroid according to claim 1, it is characterised in that in step 2) in solution conduit PBS volume is 200 μ l, and the volume that step 3) adds testing sample is 60 μ l.
- 4. the method for quick discriminating human parathyroid according to claim 1, it is characterised in that the fluorescence immune chromatography Test paper by double antibody sandwich method and fluorescence immune chromatography technology for detection people PTH, wherein：The double antibody sandwich method is using being marked with the first PTH monoclonal antibodies of fluorescent microsphere as detection antibody, and described the One PTH monoclonal antibodies are prepared by the use of one in people PTH epitope peptides (1) and (2) as antigen；AndThe double antibody sandwich method is utilized using the 2nd PTH monoclonal antibodies as capture antibody, the second monoclonal antibody Another in people PTH epitope peptides (1) and (2) prepares as antigen；The people PTH epitope peptides (1) and (2) are respectively：(1)：ERVEWLRKKLQ(SEQ ID NO：2)(2)：RKKEDNVLVESHEKSLGEAD(SEQ ID NO：3).
- 5. the method for quick discriminating human parathyroid according to claim 1, it is characterised in that the monoclonal antibody is It is prepared by PTH epitope peptides and the antigen that carrier protein couplet is prepared.
- 6. the method for quick discriminating human parathyroid according to claim 1, it is characterised in that on the fluorescent microsphere Fluorescent material is rare earth ion europium.
- 7. the method for quick discriminating human parathyroid according to claim 1, it is characterised in that the fluorescent microsphere it is micro- Ball material is the copolymer of polystyrene, polymethyl methacrylate or methyl methacrylate.
- 8. the method for quick discriminating human parathyroid according to claim 1, it is characterised in that the test paper has bottom plate (1) it is being provided with, and with the way of contact successively along chromatography direction when using on the bottom plate (1)：Sample pad (4), pad (5), nitrocellulose filter (2), blotting paper (3), the pad (5) are provided with the first PTH for marking and having Monoclonal antibody, the nitrocellulose filter (2) include detection band (7) and quality control band (6), and the detection band (7) is fixed The 2nd PTH monoclonal antibodies are stated, the quality control band (6), which is coated with, to mark the first PTH monoclonals having with described The antiantibody that antibody specificity combines.
- 9. the method for quick discriminating human parathyroid according to claim 8, it is characterised in that the bottom plate (1) does not have There is photoluminescent property.
- 10. the method for quick discriminating human parathyroid according to claim 8, it is characterised in that the antiantibody is sheep Anti- mouse IgG polyclonal antibodies or rabbit anti-mouse igg polyclonal antibody.
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| CN107402300B (en) | 2019-01-11 |
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