CN110054663A - CKAP4 epitope peptide, antigen, monoclonal antibody and its application and CKAP4 test strip - Google Patents
CKAP4 epitope peptide, antigen, monoclonal antibody and its application and CKAP4 test strip Download PDFInfo
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Abstract
The present invention relates to immunohistochemistries and proteomic techniques field, and in particular to CKAP4 epitope peptide, antigen, monoclonal antibody and its application and CKAP4 test strip.CKAP4 epitope peptide is polypeptide A or polypeptide B, and the amino acid sequence of polypeptide A is RSHQDFSRQREEL, and the amino acid sequence of polypeptide B is DSHGPKEDGG.Corresponding antigen and monoclonal antibody are prepared for using polypeptide A and polypeptide B as source respectively, it can be specifically in conjunction with CKAP4, obtained monoclonal antibody can also be used for the detection of CKAP4, and made CKAP4 test strip, it can be achieved that in serum CKAP4 quick detection.
Description
Technical field
The present invention relates to immunohistochemistries and proteomic techniques field, and in particular to CKAP4 epitope peptide, antigen,
Monoclonal antibody and its application and CKAP4 test strip.
Background technique
Lung cancer morbidity rate has ranked first in male malignancy, also increases rapidly in women disease incidence, and lung cancer becomes danger
A kind of principal disease of evil life and health.The state of an illness has been developed to middle and advanced stage when most of patients is made a definite diagnosis, and misses the treatment of disease most
Good period, treatment difficulty further increase.Therefore, effective tumor markers are found for the mechanism study of canceration, disease
Early diagnosis and the screening of people at highest risk become the task of top priority of lung cancer research.
Clinically commonly the method for early diagnosis lung cancer mainly has traditional x-ray rabat, Sputum and fibre at present
The inspection methods such as bronchoscope are tieed up, being applied to the screening (especially in people at highest risk) of lung cancer, there is no reductions to be screened crowd
The death rate of lung cancer, simultaneously because detection sensitivity is poor, painful, patient tolerability are poor, cannot early stage dynamic monitoring etc. and
It is restricted.
Lung cancer marker be during tumorigenesis by tumor cells expression, be distributed in tissue, blood and body fluid
In macromolecular substances, there is tissue specificity, type and the concentration variation of distribution and the tumours of Affected individuals origin and raw
Length is closely related, because it has many advantages, such as that molecular diagnosis, parting can be carried out by stages to lung cancer from molecular biology direction, in recent years
It is judged to be clinically widely used in lung cancer early diagnosis, clinical classification and prognosis.Therefore study and find the special of lung cancer
Property marker, establish detection method that is economic, being more suitable for early diagnosis, great work will undoubtedly be played to the preventing and controlling of lung cancer
With.
CKAP4, also known as P63, cytoskeleton related membrane protein 4 (CKAP4), molecular weight 63KD are a kind of II type cross-film eggs
It is white, it is connected between endoplasmic reticulum and micro-pipe.It recent studies have shown that, in I-IV phase of lung cancer, serum cancer embryonic antigen in serum, carefully
Born of the same parents' Keratin 19 segment and the sensibility of SCC antigen are respectively 30%-52%, 17%-82% and 24%-39%.Research points out,
The sensibility of CKAP4 is 81% in training group in serum, is 69% in validation group, is above the serodiagnosis of current clinical application
The sensibility of marker.
Currently, the development of CKAP4 quick diagnosis reagent kit is in the starting stage, wherein the specificity for obtaining anti-CKAP4 is more
It is the key technology for preparing CKAP4 quick diagnosis reagent kit that peptide or antibody and the suitable method of use, which are applied to detection,.It is anti-
Former epitope, also known as antigenic determinant are the antigenic bases of antigen molecule.Therefore, CKAP4 epitope is the discovery that its Dan Ke
The important foundation of the research and development of grand antibody and quick diagnosis reagent kit has had not yet to see the relevant text of research CKAP4 epitope
Offer disclosure.
Although epitope can use bioinformatics software to predict at present, however, more and more studying table
Bright, the error probability of software prediction epitope is higher, and the prediction of software counterwork original epitope is just for purpose antigen albumen
Overall length really often has specificity during application, needs to test confirmation.
Summary of the invention
The present invention studies the epitope of CKAP4, it was found that 2 epitope peptides；
In addition, the present invention is also prepared for CKAP4 antigen on the basis of above-mentioned epitope peptide；
The present invention be also prepared can the above-mentioned antigen epitope polypeptide of specific recognition and carrier conjugation be prepared it is anti-
Former monoclonal antibody；
Another object of the present invention is to specify that said monoclonal antibody can be used for preparing detection CKAP4 kit/examination
Paper slip；
Finally, the present invention also establishes the Test paper that can quickly detect CKAP4 on the basis of said monoclonal antibody
Item.
CKAP4 epitope peptide of the invention adopts the following technical scheme that a kind of CKAP4 epitope peptide, the antigen
Epitope peptide is polypeptide A or polypeptide B, and the amino acid sequence of the polypeptide A is RSHQDFSRQREEL, the amino acid sequence of the polypeptide B
It is classified as DSHGPKEDGG.
A kind of CKAP4 antigen, the CKAP4 antigen are CKAP4-A antigen, and the CKAP4-A antigen is by making polypeptide A
(amino acid sequence are as follows: RSHQDFSRQREEL) and carrier protein couplet are prepared.Carrier protein can be selected from KLH, and (keyhole blood is blue
Albumen), BSA (bovine serum albumin(BSA)), ovalbumin OVA etc..Preferably, the carrier protein of the CKAP4-A antigen is BSA.
A kind of CKAP4 antigen, the CKAP4 antigen are CKAP4-B antigen, and the CKAP4-B antigen is by making polypeptide B
(amino acid sequence are as follows: DSHGPKEDGG) and carrier protein couplet are prepared.Preferably, the carrier of the CKAP4-B antigen
Albumen is BSA.
A kind of CKAP4 monoclonal antibody, the CKAP4 monoclonal antibody be from the above mentioned CKAP4-A antigen preparation and
At monoclonal antibody (derive from polypeptide A).
A kind of CKAP4 monoclonal antibody, the CKAP4 monoclonal antibody are prepared by CKAP4-B antigen as described above
Made of monoclonal antibody (derive from polypeptide B).
Two kinds of monoclonal antibodies as described above are preparing the application in CKAP4 diagnostic kit.
Two kinds of monoclonal antibodies as described above are in the application for preparing CKAP4 test strip.
A kind of CKAP4 test strip, labelled antibody and the coated antibody of the test strips are difference anti-from CKAP4
The monoclonal antibody of former epitope peptide, the labelled antibodies of the test strips be in above two monoclonal antibody any one (i.e.
From polypeptide A or polypeptide B).
A kind of CKAP4 test strip, labelled antibody and the coated antibody of the test strips are difference anti-from CKAP4
The monoclonal antibody of former epitope peptide, the coated antibodies of the test strips be in above two monoclonal antibody any one (i.e.
From polypeptide A or polypeptide B).
Wherein, the labelled antibody of above-mentioned test strips refers to and the marks such as fluorescent microsphere/colloidal gold/nanometer magnetic bead/quantum dot
The antibody that object (preferably time-resolved fluorescence microballoon, such as Eu- carboxyl fluorescent microsphere) combines；Coated antibody, which refers to, is coated in nitric acid fibre
Tie up the antibody on plain film or other solid dielectrics as detection line.Preferably, the labelled antibody of the CKAP4 test strip is
From the monoclonal antibody of polypeptide A, coated antibody is the monoclonal antibody from polypeptide B；Or the labelled antibody is next
Derived from the monoclonal antibody of polypeptide B, the coated antibody is the monoclonal antibody from polypeptide A, to guarantee the test strips
Testing result specificity, accuracy and validity.
The beneficial effects of the present invention are: epitope peptide polypeptide A and polypeptide B of the invention all has good antigenicity,
Being used to be immunized animal for the antigen that polypeptide A and polypeptide B and carrier protein couplet are prepared respectively can be specific to generating
Good monoclonal antibody, and then it is used to prepare kit/test strips of detection CKAP4.
The present invention provides one kind can quickly (5min) detection serum in CKAP4 test strips, detection efficiency is higher, linearly
Range 5-100ng/ml, compared with the existing technology in CKAP4 monoclonal antibody have a broader range of linearity, specific good, energy
Enough rapid assisted diagnosis state of an illness in time, monitor prognosis.CKAP4 test strip of the invention and Immunohistochemical Method correlation
Good, detection effect is reliable.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is the structural schematic diagram of CKAP4 test strips of the invention；
Fig. 2 is the result schematic diagram of the CKAP4 test strips qualitative detection；
Fig. 3 is the canonical plotting of test strips 1
In Fig. 1: 1- sample pad；2- bonding pad；3- nitrocellulose filter；4-T line；5-C line；6- water absorption pad；7- bottom plate.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment one
1. the screening of epitope:
NCBI, which is consulted, obtains people CKAP4 amino acid sequence, with DNAssist, DNA STAR, BcePred Prediction
The softwares such as Server analyze CKAP4 protein B cell epitope.The antigen that will be analyzed through software when carrying out Characterization of antigenic epitopes
Epitope peptide is docked with EGF-R ELISA EGFR, chooses polypeptide of the scoring functions greater than 130 points as candidate antigens
Epitope peptide, and further screened by experiment.Through lot of experiment validation, finally screen more to two CKAP4 epitopes
Peptide:
The amino acid sequence of polypeptide A: RSHQDFSRQREEL
The amino acid sequence of polypeptide B: DSHGPKEDGG
Chemical synthesis preparation, such as solid-phase synthesis can be used in polypeptide A and polypeptide B.Details are not described herein again.
The preparation and identification of 2.CKAP4 antigen:
The preparation of 2.1 CKAP4 antigens, the specific steps are as follows:
(1) BSA solid 4mg is weighed, the dissolution of 400 μ L MES buffers (0.1M, pH 5.0) is placed in, prepares 10mg/mL
BSA solution；
(2) weigh polypeptide A (by Shanghai Qiangyao Biotechnology Co., Ltd. according to the amino acid sequence synthesis polypeptide of offer),
Polypeptide B (by Shanghai Qiangyao Biotechnology Co., Ltd. according to the amino acid sequence synthesis polypeptide of offer) each 2mg, is dissolved separately in
In 500 μ L MES buffers (0.1M, pH 5.0), CKAP4 (A), CKAP4 (B) solution is made；It is taken obtained by step (1) respectively
200 μ L of BSA solution be placed in the CKAP4 (A) of 4mg/mL, CKAP4 (B) solution, whirlpool mixes；
(3) EDC is balanced into room temperature, weighs EDC 20mg, is dissolved in 2mL purified water, prepares the EDC solution of 10mg/mL；
(4) EDC solution of above-mentioned preparation is taken into 100 μ L, is respectively placed in CKAP4 (A) and BSA, CKAP4 (B) and BSA at once
Mixed liquor in, 25 DEG C of shaking tables vibrate 2h, finally prepare CKAP4-A (CKAP4 (A)-BSA), CKAP4-B (CKAP4 (B)-BSA)
Two kinds of comlete antigens, -20 DEG C of preservations；
(5) part steps (4) obtained coupled product is taken, is dialysed using the phosphate buffer of pH 7.2, every 4h is more
Dialyzate is changed, product identification is taken out after replacement 4 times.
2.2 pairs of obtained antigens detect
2.2.1 under micro ultraviolet specrophotometer respectively to CKAP4 (A), CKAP4 (B), CKAP4-A, CKAP4-B and
BSA carries out uv scan, compares the scanning spectra variation before being coupled, after coupling, preliminary judgement polypeptide A or polypeptide B and carries
Whether body protein (BSA) is coupled.The sample for choosing polypeptide A or polypeptide B and carrier conjugation carries out PAGE gel electrophoresis.
2.2.2 SDS-PAGE electroresis appraisal (5% concentration glue, 8% separation gel), the specific steps are as follows:
(1) related reagent and instrument and equipment needed for getting out SDS-PAGE experiment；
(2) purified water rinses electrophoresis plate, and plate folder is clamped electrophoresis plate, hunted leak using purified water；After anhydrous leaching, purifying is poured out
Water, strip blotting paper remove remaining purified water；
(3) it (is selected according to sample molecule size) according to formula preparative separation glue, it is along electrophoresis plate side gap that it is slow
Inject bottom end, prevent separation gel in there are bubbles, under room temperature, purified water water seal about 1h；
(4) after above-mentioned gelling is solid, the purified water of falling fluid-tight, blotting paper removes purified water against side；
(5) 5% concentration glue of preparation, is slowly injected into separation gel top against side gap, to avoid comb teeth lower end bubble from depositing
, cleaning comb is kept, slowly insertion concentration glue (depth about 1cm), it is stand-by after to be solidified；
(6) comb is slowly extracted, is assembled on electrophoresis equipment, is poured into 1 × running buffer solution, utilize homemade elongated pipette tips
Sample-adding, 6 hole μ L/ of point sample amount；
(7) power on, when bubbling from bottom to top in electrophoretic buffer, electrophoresis starts, when blue dyeing band will
When migrating out electrophoresis plate, power supply is closed；
(8) electrophoresis plate is taken out, goes board falling to press from both sides, electrophoresis plate is opened, after marking sample hole location, is slowly withdrawn gel, is purified
Water rinses removal of impurities three times, is dipped in dyeing liquor, and decolorization swinging table dyes about 40min；
(9) after dyeing, purified water rinses stained gel, then is placed in destainer, is decolourized 2h with decolorization swinging table, on time
Old destainer is replaced, gel background is observed, is cleaned up if its bleach, lain low and taken pictures in lamp box.
3. mouse immune program
BALB/c female mice 6 are taken, about 6~8 week old, wherein CKAP4-A, CKAP4-B are each 3 immune, using subcutaneous
Multiple spot inoculation.It is immunized according to following scheme:
Head exempts to mix in equal volume using Freund's complete adjuvant (FCA) with CKAP4-A/CKAP4-B；
Two exempt from, three exempt to mix in equal volume using incomplete Freund's adjuvant (FIA) with CKAP4-A/CKAP4-B；
If three exempt from rear mice serum antibody titer < 1:104, immune to continue, until antibody titer > 1:104；
First 3 days of fusion carries out booster immunization, does not add adjuvant.
According to above scheme to mouse immune CKAP4-A/CKAP4-B.Highest potency is small in two immune groups of selection
Mouse continues to test in next step.
Specific immunization protocol is as shown in table 1 below:
Table 1
The preparation of 4.CKAP4 monoclonal antibody
For the hybridoma for obtaining specific recognition CKAP4 antigen, infinite multiplication, cell fusion is first carried out, with laggard
Row Cell-cloned.Through 2-4 time cloning, the list of stably excreting CKAP4 antibody is finally screened after measuring with indirect ELISA
Clonal cell line induces method preparation monoclonal antibody using mouse peritoneal inoculation.Using caprylic acid --- sulfuric acid is purified by the precipitation method
Ascites monoclonal antibody.
4.1 cell fusion
Spleen cell and (myeloma cell line) solution of SP 2/0 are mixed in 1:6 ratio, vortex is uniform, 1600rpm from
Core cell 5min, blotting paper remove moisture content, and slight wobble disperses precipitating.Under 37 DEG C of water bath conditions, 1mL preheating is added dropwise
PEG2000 (operation needs to complete in about 1min), timing 1min promotes fusion reaction.At once the 1640 culture medium of preheating is added, it will
Fused cell slow-speed of revolution centrifugation, abandons waste liquid, washs 2 times, slightly dispels cell with HAT culture solution.It is ready to be covered with feeder layer thin
96 orifice plates of born of the same parents, are added dropwise the fused cell dispelled, and 100 holes μ L/ are put into CO2Incubator culture, is changed during culture according to the time
Liquid.
4.2.1 positive monoclonal cell screening and cloning
For the hybridoma for obtaining specific recognition CKAP4 antigen, infinite multiplication, after fused, further cell is needed
Cloning.The positive hybridoma cell of indirect elisa method initial survey also can cause cell because of factors such as cell mutations after cultivation
The ability of secretion CKAP4 antibody is lost, usually need to finally screen to stablize and divide after 2~4 time clonings, indirect ELISA measurement
Secrete the monoclonal cell strain of CKAP4 antibody.This research carries out positive cell clone using limiting dilution assay, will be single after about 10d
It clones hole supernatant to measure using indirect elisa method, the hole for therefrom choosing high OD value carries out cloning (the immune preceding blood of detection respectively again
The OD450 value of (N) and Post-immunisation serum (P) clearly, with the inverse of the multiple of maximum dilution containing antibody serum for evaluation index.When immune
(result is the positive, specific data to OD value (N)/Post-immunisation serum OD value of preceding serum when)≤2.1 P (potency 1:64000)
Referring to the following table 2), it repeats 2~3 times, selecting clone for several times is that positive monoclonal cell strain does alternative cell strain.It will filter out
Cell strain expand culture, and pass on and freeze in time, freeze 15d, 30d, 60d respectively, recover after 90d, by right
Cell Trypan Blue, cell count detect cell viability, with the energy of indirect ELISA method detection cell secreting specificity antibody
Power, thus the stability for the cell strain that judgement filters out.Finally screen to 8 plants of cell strains, number be respectively as follows: 2A2,3C2,
4D5,4E2,5C4,6E1,7F2,8D5, it is spare.
2 indirect elisa method of table measures serum antibody
4.2.2 monoclonal cell strain hypotype measures
Primary operational is as follows to be identified respectively to the supernatant of stable cell line secretion:
(1) each component in kit is pipetted, 30min, balance to room temperature are placed.
(2) concentration washing lotion is diluted to required concentration, takes out ELISA Plate, it is respectively that every plant of monoclonal antibody is dilute with Sample dilution
It releases, 100 holes μ L/, sample diluting liquid marks in detail on ELISA Plate as compareing, sticks tight sealing plate film, 37 DEG C of reaction 30min.
(3) prepare 6 kinds of HRP ELIAS secondary antibodies, including IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, 100 are distinguished after dilution
The hole μ L/ marks on ELISA Plate in detail, sticks tight sealing plate film, 37 DEG C of reaction 30min.
Monoclonal antibody hypotype measurement result, see the table below shown in 3:
Table 3
| Antibody number | 2A2 | 3C2 | 4D5 | 4E2 | 5C4 | 6E1 | 7F2 | 8D5 |
| Hypotype type | IgG2a | IgG1 | IgG1 | IgG1 | IgG2b | IgG1 | IgG1 | IgG1 |
It is found that 2A2 is IgG2a, 5C4 is the hypotype of the monoclonal antibody generated by 8 strain of hybridoma of measurement
IgG2b, other 6 plants are IgG1, and the monoclonal antibody of 6 plants of IgG1 can be used as the raw material of lower step pairing screening.
The preparation of 4.3 monoclonal antibody ascites
Method preparation monoclonal antibody is induced using mouse peritoneal inoculation, the specific steps are as follows:
(1) the injecting fluid paraffin in mouse peritoneal, 0.5mL/ only, make male mice (about 12 week old) sensitization, induce abdomen
Water.
(2) positive hybridoma cell strain (being in logarithmic phase) is collected, mouse peritoneal is injected respectively, 3 mouse/
Every plant, 0.2mL/ is only.
(3) after 7-10d, the mouse of the obvious protrusion of abdomen and execution are filtered out, with syringe collecting ascites.4 DEG C of ascites are quiet
6h or more is set, 4000rpm is centrifuged 5min, removes upper layer grease, and it is ascites that intermediate clarification part, which is slowly sucked out,.
The purifying and performance detection of 4.4 monoclonal antibodies:
4.4.1 ascites monoclonal antibody is purified using caprylic acid-ammonium sulfate precipitation method.By the micro ultraviolet spectrometry of each monoclonal antibody
Then photometric determination concentration passes through SDS-PAGE electrophoresis technique determining each monoclonal antibody purity after purification.6 μ L/ of sample after processing
Hole, 3 μ L/ hole Marker, using 8% separation gel, 5% concentration glue carry out electrophoresis, dyed, decolourize after observe, packing label, ﹣
20 DEG C of preservations, measure the protein concentration of 6 plants of monoclonal antibodies after purification, and antibody concentration more than 5mg/ml, meets method and establishes to measurement
The requirement of concentration.Specific data are as shown in table 4 below:
Table 4
| Antibody number | 3C2 | 4D5 | 4E2 | 6E1 | 7F2 | 8D5 |
| Concentration (mg/ml) | 6.23 | 5.31 | 5.34 | 7.42 | 8.68 | 9.07 |
4.4.2 using 8% separation gel, 5% concentration glue, SDS-PAGE identification is carried out to monoclonal antibody after purification.Knot
Fruit shows that 6 plants of monoclonal antibody bands are all near 150 to 170KD, and molecular weight and expection are in the same size, and purity is preferable.
The identification of 4.5 monoclonal antibody specificities
4.5.1 by CKAP4 (A)-BSA, CKAP4 (B)-BSA, BSA respectively as envelope antigen, to thin from 6 plants of hybridomas
The monoclonal antibody that born of the same parents (3C2,4D5,4E2,6E1,7F2 and 8D5) are purified into is respectively adopted indirect ELISA method and identifies,
Parallel testing 3 times, while blank control and negative control are set.
The cross reaction of monoclonal antibody and protein carrier is as shown in table 5 below:
Table 5
Note :+,-it is respectively positive, feminine gender, value > 0.105 OD is the positive, otherwise is negative.
Conclusion: as can be known from the above table, the hole for being coated with BSA is feminine gender, and be coated with CKAP4 (A)-BSA hole and 3C2,6E1 and
The monoclonal antibody of 7F2 is positive, negative with 4D5,4E2 and 8D5；Be coated with CKAP4 (B)-BSA hole and 4D5,
4E2 and 8D5 are positive, negative with the monoclonal antibody of 3C2,6E1 and 7F2, illustrate that obtained monoclonal antibody is to be directed to
The specific antibody of CKAP4, rather than it is directed to the antibody of carrier B SA；Wherein 3C2,6E1,7F2 are completely anti-for CKAP4-A
Former specific antibody, 4D5,4E2 and 8D5 are the specific antibody of CKAP4-B comlete antigen.
4.5.2 verify whether 6 plants of CKAP4 monoclonal antibodies have nonspecific reaction:
It is immunoreacted with the antibody being purified and CEA antigen and CA125 antigen serum, the results showed that purifying is single
It is anti-that there is specificity, it was demonstrated that screened CKAP4 epitope peptide (polypeptide A and polypeptide B) has potential antigenic determinant characteristic.
Concrete outcome see the table below 6:
Table 6
Conclusion: for 6 plants of monoclonal antibodies to interference antigen (CEA antigen, CA125 antigen) without idiosyncrasy, effect is preferable.
4.6 ELISA methods screening pairing monoclonal antibody
By the CKAP4 monoclonal antibody of preparation, HRP mark is carried out to gained monoclonal antibody using the Over-voltage protection of improvement
Note.The method detected using double-antibody sandwich elisa is to sample with the standard items of preparation with reference to this laboratory testing system
This, carries out pairing screening (removal self pair) to obtained monoclonal antibody and HRP label monoclonal antibody, is judged by the OD value height of measurement anti-
Whether body matches, and selects OD value high as more excellent pairing.Key step is as follows:
(1) using CKAP4 monoclonal antibody as coating, package amount: the hole 500ng/, sample size: 20 holes μ L/；
(2) HRP labelled antibody 1/2000 is diluted with enzyme mark dilution, 50 holes μ L/；
The more excellent pairing screened see the table below shown in 7:
Table 7
The preparation of two: CKAP4 test strip of embodiment (referring to Figure of description Fig. 1)
The preparation of 1.1 bonding pads 2
1.1.1 CKAP4 labelled antibody-fluorescent microsphere compound preparation
(1) fluorescent microsphere 15uL is taken, is added in 600uL 50mmol/L borate buffer, vortex mixes；
(2) EDC of 100ug is added, 25-30 DEG C of shaking table vibrates 30min, 14000r/min, be centrifuged 20min；
(3) precipitating is resuspended in borate buffer, cleans 1 time, and centrifugal synchronous is rapid (2)；
(4) precipitating is resuspended using 1mL 50mmol/L borate buffer, 100ug labelled antibody is added (with fluorescent microsphere shape
Can be monoclonal antibody or polyclonal antibody at the antibody of compound), vortex mixes；
(5) it is added 50uL 10%BSA (can play the role of preventing fluorescent microsphere and labelled antibody from separating), 25-30 DEG C
Oscillation closing 1h, 14000r/min, are centrifuged 15min；Precipitating 1mL protection liquid (Tris-Hcl of 0.05M pH=8.0,0.5%
(volume fraction) TWEEN-20 and 0.1%BSA, the concentration of above-mentioned each component are final concentration, and the concentration of BSA is quality percentage
Number) it dispels, ultrasonic 30s, 4 DEG C save backup；
(6) 25-30 DEG C of oscillation is coupled 2h, and centrifugal synchronous is rapid (2), and 50uL 10%BSA, 25-30 DEG C of oscillation 1h is added,
14000r/min is centrifuged 15min；Precipitating protects liquid (Tris-Hcl of 0.05M pH=8.0,0.5% (volume fraction) with 1mL
TWEEN-20 and 0.1%BSA, the concentration of above-mentioned each component are final concentration, and the concentration of BSA is mass percent) it dispels, ultrasound
30s, 4 DEG C save backup.It protects the specific preparation method of liquid as follows: taking pH=8.0 0.2M Tris-Hcl 25mL, TWEEN-
20 0.5mL, BSA 0.1g, purified water 75mL are uniformly mixed.
1.1.2 CKAP4 labelled antibody-fluorescent microsphere compound is fixed on glass fibre membrane and bonding pad 2 is made
Glass fibre membrane (long 10cm, wide 7mm) is utilized into treatment fluid (0.01M pH=8.0 Tris-Hcl, 0.1% (body
Fraction) TWEEN-20,0.1% (mass fraction) BSA and 0.1% (mass fraction) sucrose, the concentration of above-mentioned each component is
Final concentration) processing, 37 DEG C of drying, then by the one fluorescent microsphere solution of CKAP4 labelled antibody prepared, ((40uL/cm) is applied to
Glass fibre membrane, it is desirable that solution is uniformly filled in glass fibre membrane, and 2,4 DEG C of aluminium foil bags of bonding pad are made (containing drying in 37 DEG C of drying
Agent) sealing it is spare.The specific preparation method for the treatment of fluid is as follows: taking pH=8.0 0.2M Tris-Hcl 10mL, TWEEN-20
0.1mL, BSA 0.1g, sucrose 5g, purified water 75mL are uniformly mixed.
1.2 are coated with T line and C line on nitrocellulose filter
The intermediate adhesive label of bottom plate 7 (PVC board) is thrown off, NC film (nitrocellulose filter 3) is close to the lower end of upper end paster.It draws
Before line, adjust the scribing position (X, Y, Z) of XYZ three-dimensional point film gold spraying instrument, draw sample amount etc., after adjustment, simulation scribing line makes matter
Line C line 5 and detection line T line 4 are controlled behind 3 middle position of NC film, sheep anti-mouse igg (1mg/mL) is used as nature controlling line C line 5, will be wrapped
By antibody, (1.5mg/mL, coated antibody refer to the antibody being coated on nitrocellulose filter as detection line T line, can be Dan Ke
Grand antibody or polyclonal antibody) it is used as detection line T line 4, it uniformly crosses in NC film 3, after scribing line, 37 DEG C of drying 2h, sealing
It is spare in valve bag.
The assembling of 1.3 test strips
Bottom plate 7 (PVC board) upper end one and the sticky paper patch of lower end two are thrown off, by water absorption pad 6, to have spread CKAP4 label anti-
The bonding pad 2 of one fluorescent microsphere of body, sample pad 1 successively overlap assembling, and each overlapping region is 1mm by the automatic cutting machine of micro computer
Cover start, assembled coating plate is put into material placement platform, is chopped into the test strips of wide 3.9mm, 4 DEG C of aluminium foil bags are close
(structure of assembled test strips is referring to Figure of description Fig. 1) for envelope preservation.
The application method of 1.4 test strips
After test strips restore room temperature, sample to be examined (serum sample) is added in sample-adding hole site, the hole 70uL/, timing
5min；
1.4.1 quantitative detection: test strips are moved in fluorescence immune chromatography readout instrument and are detected, and record T line and C line fluorescent value,
It is calculated, can be directly read in institute's test sample sheet according to the standard curve being pre-set in fluorescence immune chromatography readout instrument
The concentration of CKAP4.
1.4.2 qualitative detection: test strips are moved under ultraviolet lamp and observed, can direct interpretation result: C line is with or without fluorescent brightness
Occur, T line unstressed configuration brightness, determines that result is invalid；C and T line shows fluorescent brightness, determines that result is the positive；C line unstressed configuration
Brightness occurs, and T line has fluorescent brightness, determines test strips result for negative (referring to Figure of description Fig. 2).
The performance verification of test strips obtained by three present invention of embodiment
The foundation of 1.1 standard curves:
Configure a series of CKAP4 titer of concentration gradients: 0,0.39,0.78,1.56,3.125,6.25,12.5,25,
50,100,150,200,300,400 (ng/ml), takes 70uL to be added drop-wise on immuno-chromatographic test paper strip, and fluorescence immunoassay layer is used after 5min
The fluorescence intensity that readout instrument reads T line and C line is analysed, is respectively done the fluorescence intensity of T/C and corresponding CKAP4 concentration of standard solution quasi-
Curve is closed, fluorescence intensity formula corresponding with concentration is obtained.
The detection of 1.2 accuracies
1.2.1 it criticizes interior accuracy detection: repeating detection intermediate value quality-control product (50ng/ with 50 CKAP4 test strips of batch
ML), using the coefficient of variation of T line fluorescence intensityBatch interior accuracy is calculated.
1.2.2 an accuracy is criticized: using the CKAP4 detection examination of 3 batches (every batch of 20) prepared by the same way
Paper slip detects intermediate value quality-control product (50ng/mL), using the coefficient of variation of T line fluorescence intensityEssence between criticizing is calculated
Close property.
2. the test strips for carrying out above-mentioned test (1.1-1.3):
Test strips 1: used labelled antibody is monoclonal antibody obtained by embodiment one when test strips production
6E1, coated antibody are monoclonal antibody 4D5 obtained by embodiment one, and referring to table 6, (other monoclonal antibodies in table 6 can also
For making test strips, and coated antibody and labelled antibody can be interchanged), remaining making step with embodiment two (test strips 1
Canonical plotting is referring to Figure of description Fig. 3)；
Test strips 2: the two kinds of CKAP4 antibody samples provided with Shanghai Shuo Bo Biotechnology Co., Ltd (therefrom match by selection
To best antibody) respectively as labelled antibody and coated antibody, remaining making step is the same as embodiment two；
3. testing result: specific testing result see the table below 8
Table 8
| Test strips | Accuracy in batch | Accuracy between batch | The range of linearity (gradient dilution quality-control product) | R2 |
| Test strips 1 | CV=14.12 | CV=14.88 | 5-100ng/ml | 0.9877 |
| Test strips 2 | CV=10.37 | CV=12.69 | 10-95ng/ml | 0.9631 |
Example IV methodology compares
This method (is detected) the attached 45 parts of patients with lung cancer of institute's Immunohistochemical Method Parallel testing of He Zheng great mono- using test strips 1
Clinical blood sample, for coincidence rate up to 91%, concrete outcome is detailed in the following table 9
Table 9
By upper table 9 it is found that detection method and Immunohistochemical Method of the invention has good correlation, testing result can
It leans on, can be used for the quick detection of CKAP4.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
<110>Henan Biology Engineering Research Center Co., Ltd
Henan Biology Engineering Research Center
<120>CKAP4 epitope peptide, antigen, monoclonal antibody and its application and CKAP4 test strip
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Arg Ser His Gln Asp Phe Ser Arg Gln Arg Glu Glu Leu
1 5 10
<210> 2
<211> 10
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Asp Ser His Gly Pro Lys Glu Asp Gly Gly
1 5 10
Claims (9)
1. a kind of CKAP4 epitope peptide, which is characterized in that the epitope peptide be polypeptide A or polypeptide B, the polypeptide A
Amino acid sequence is RSHQDFSRQREEL, and the amino acid sequence of the polypeptide B is DSHGPKEDGG.
2. a kind of CKAP4 antigen, which is characterized in that the CKAP4 antigen is CKAP4-A antigen, and the CKAP4-A antigen passes through
Polypeptide A and carrier conjugation as described in claim 1 is prepared.
3. a kind of CKAP4 antigen, which is characterized in that the CKAP4 antigen is CKAP4-B antigen, and the CKAP4-B antigen passes through
Polypeptide B and carrier protein couplet as described in claim 1 is prepared.
4. a kind of CKAP4 monoclonal antibody, which is characterized in that the CKAP4 monoclonal antibody is by as claimed in claim 2
The monoclonal antibody that CKAP4 antigen is prepared.
5. a kind of CKAP4 monoclonal antibody, which is characterized in that the CKAP4 monoclonal antibody is by as claimed in claim 3
The monoclonal antibody that CKAP4 antigen is prepared.
6. as monoclonal antibody described in claim 5 or 6 is preparing the application in CKAP4 diagnostic kit.
7. as monoclonal antibody described in claim 5 or 6 is preparing the application in CKAP4 test strip.
8. a kind of CKAP4 test strip, the labelled antibody and coated antibody of the test strips are from CKAP4 not synantigen
The monoclonal antibody of epitope peptide, which is characterized in that the labelled antibody of the test strips is monoclonal described in claim 4 or 5
Antibody.
9. a kind of CKAP4 test strip, the labelled antibody and coated antibody of the test strips are from CKAP4 not synantigen
The monoclonal antibody of epitope peptide, which is characterized in that the coated antibody of the test strips is monoclonal described in claim 4 or 5
Antibody.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107709351A (en) * | 2015-06-17 | 2018-02-16 | 株式会社医学生物学研究所 | Cytotoxic t cell epitope peptide for sars and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| CHAVDA ET AL: "Antiproliferative factor (APF) binds specifically to sites within the cytoskeleton-associated protein 4 (CKAP4) extracellular domain", 《BMC BIOCHEMISTRY》 * |
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