CN109387627A - A kind of Reagent Protocol of screening for cancer and early diagnosis based on placenta sample chondroitin sulfate A (CSA) - Google Patents
A kind of Reagent Protocol of screening for cancer and early diagnosis based on placenta sample chondroitin sulfate A (CSA) Download PDFInfo
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- CN109387627A CN109387627A CN201811203459.6A CN201811203459A CN109387627A CN 109387627 A CN109387627 A CN 109387627A CN 201811203459 A CN201811203459 A CN 201811203459A CN 109387627 A CN109387627 A CN 109387627A
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Abstract
The present invention relates to the reagents and method of a kind of screening for cancer based on placenta sample chondroitin sulfate A (CSA) and early diagnosis, it specifically discloses a kind of for detecting the ELISA kit of tumour, it includes the capture albumen for combining with placenta sample chondroitin sulfate A (CSA) (pl-CSA), preferably, the capture albumen is selected from Infected With Plasmodium erythrocyte surface antigen (VAR2CSA, rVAR2 minimum combination peptide fragment), it is highly preferred that the minimum combines peptide section sequence as shown in SEQ ID No.1.Present invention firstly discovers that pl-CSA can be detected in a variety of body fluid, this provides foundation for qualitative or quantitative Biological Detection pl-CSA in vitro, and method detection of the invention limits low, reproducible, specific height.
Description
Technical field
The present invention relates to field of biological technology detection, and in particular to a kind of cancer sieve based on placenta sample chondroitin sulfate A (CSA)
Look into and early diagnose ELISA method and its kit.
Background technique
Cancer is serious public health problem in a kind of global range, derived from the uncontrollable misgrowth of cancer cell and is turned
It moves.According to statistics in the 2012 of international cancer tissue, about 14,100,000 newly-increased cases of cancer, was caused about every year
8200000 patients are dead, and because increased trend year by year is presented in living-pattern preservation, as diet is unbalance, does not get enough athletic exercise and high
Age fertility etc.[1-3].Regrettably, the screening and early diagnosis of cancer are the sciences problems not yet overcome, cases of cancer quilt at present
It was found that when have generally entered middle and advanced stage, at this time cancer cell extensively transfer or overpreading, common drug be difficult to inhibit
Growth of cancer cells, chemotherapy become main mode, and most chemotherapeutics inhibits while eliminating or inhibiting cancer cell
The growth and update of normal cell, so that cancer becomes incurable disease.The early stage of cancer may be the window phase for the treatment of, therefore
There is an urgent need to explore the method for a kind of more effective screening for cancer and early diagnosis.
Recent molecular marker research provides a kind of new visual angle, such as glycosaminoglycan for cancer mechanism[4], blood
Pipe generates element[5]Deng.But reliable screening and diagnostic method are also lacked based on these biomarkers.Enzyme-linked immunosorbent assay
(enzyme-linked immuno-sorbent assay, ELISA) originates in 1975, anti-for detecting trichina at that time
Body, to DIAGNOSIS OF TRICHINOSIS WITH[6].Due to the spies such as the sensibility of ELISA method is high, specificity is good, easy to operate, high-throughput
Point[7,8], first have been widely used for bacterium[9], virus[7]And helminth[10]Etc. related diseases screening, diagnosis and immune school superintendent
It surveys.However such a method is but seldom applied and promotes in the screening of cancer and early diagnosis, most important reason
It is a lack of a kind of cancer cell specificity expression, and the biological markers being widely present in kinds cancer tissue.
In having been found that cancer markers, placenta sample chondroitin sulfate A (CSA) (placental like chondroitin
Sulfate A, pl-CSA) it is a kind of characteristic glycosaminoglycan, there is characteristic disaccharide unit[11], in cancerous tissue
Specific expressed, wide expression that is interesting is it in kinds cancer tissue may play crucial work in cancer development process
With being positively correlated property of grade malignancy with tumour[12].But pl-CSA is initially as Infected With Plasmodium red blood cell between placental villi
A kind of receptor of gap aggregation is found, and can be specifically bound with Infected With Plasmodium erythrocyte surface antigen VAR2CSA.
The combination of VAR2CSA and pl-CSA has compatibility, the specificity of height, and the affinity KD of minimum bonding unit is about 15Nm[13], enable VAR2CSA to become a species specific capture albumen of pl-CSA, purifying pl-CSA, or in its other party
Face is applied, but lacks the development and application of this respect reagent and method.It is immunized with the pl-CSA of purifying, preparation is anti-
Body, from immunological monitoring methods are further established, the solarization for cancer is produced and early diagnosis will have extensively and have very much prospect
Application value.
ELISA method has the characteristics that high susceptibility, high degree of specificity, easy to operate, high flux property[7,8], by
It is widely used in bacterium[9], virus[7]And helminth[10]Etc. related diseases screening, diagnosis and Immunological Surveillance.However such one
Kind method, which lacks, to be seldom applied in the screening of cancer and early diagnosis, and it is special that most important reason is a lack of a kind of cancer cell
Opposite sex expression, and the biological markers being widely present in kinds cancer tissue.
The present invention is directed to this sciences problems, and selection is expressed in Cancer Tissue-Specific, and wide in kinds cancer tissue
The marker pl-CSA of general property expression, but whether it can discharge into biofluid, such as culture supernatant or serum, the prior art
In do not report, it is unclear that whether can develop ELISA detection method, detect and released in cancer cell itself and biofluid
The pl-CSA content put, so that it is determined that the presence of cancer cell, achievees the purpose that screening for cancer and early diagnosis.
Bibliography
1.Torre LA,Bray F,Siegel RL,Ferlay J,Lortet-Tieulent J,Jemal A:Global
cancer statistics,2012.CA:a cancer journal for clinicians 2015,65(2):87-108.
2.Siegel R,Ma J,Zou Z,Jemal A:Cancer statistics,2014.CA:a cancer
journal for clinicians 2014,64(1):9-29.
3.Siegel RL,Miller KD,Jemal A:Cancer statistics,2016.CA:a cancer
journal for clinicians 2016,66(1):7-30.
4.Gama CI,Tully SE,Sotogaku N,Clark PM,Rawat M,Vaidehi N,Goddard WA,
3rd,Nishi A,Hsieh-Wilson LC:Sulfation patterns of glycosaminoglycans encode
molecular recognition and activity.Nature chemical biology 2006,2(9):467-473.
5.Keskin D,Kim J,Cooke VG,Wu CC,Sugimoto H,Gu C,De Palma M,Kalluri R,
LeBleu VS:Targeting vascular pericytes in hypoxic tumors increases lung
metastasis via angiopoietin-2.Cell reports 2015,10(7):1066-1081.
6.Ruitenberg EJ,Ljungstrom I,Steerenberg PA,Buys J:Application of
immunofluorescence and immunoenzyme methods in the serodiagnosis of
Trichinella spiralis infection.Annals of the New York Academy of Sciences
1975,254:296-303.
7.Qiu X,Wong G,Audet J,Bello A,Fernando L,Alimonti JB,Fausther-
Bovendo H,Wei H,Aviles J,Hiatt E et al:Reversion of advanced Ebola virus
disease in nonhuman primates with ZMapp.Nature 2014,514(7520):47-53.
8.Dwyer DS,Bradley RJ,Urquhart CK,Kearney JF:Naturally occurring
anti-idiotypic antibodies in myasthenia gravis patients.Nature 1983,301
(5901):611-614.
9.Wadhwa A,Johonson RE,Eda K,Waters WR,Palmer MV,Bannantine JP,Eda S:
Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in
cattle and deer.BMC veterinary research 2014,10:147.
10.Hosseininejad M:Evaluation of an indirect ELISA using a tachyzoite
surface antigen SAG1for diagnosis of Toxoplasma gondii infection in
cats.Experimental parasitology 2012,132(4):556-560.
11.Mikami T,Kitagawa H:Biosynthesis and function of chondroitin
sulfate.Biochimica et biophysica acta 2013,1830(10):4719-4733.
12.Salanti A,Clausen TM,Agerbaek MO,Al Nakouzi N,Dahlback M,Oo HZ,Lee
S,Gustavsson T,Rich JR,Hedberg BJ et al:Targeting Human Cancer by a
Glycosaminoglycan Binding Malaria Protein.Cancer cell 2015,28(4):500-514.
13.Clausen TM,Christoffersen S,Dahlback M,Langkilde AE,Jensen KE,
Resende M,Agerbaek MO,Andersen D,Berisha B,Ditlev SB et al:Structural and
functional insight into how the Plasmodium falciparum VAR2CSA protein
mediates binding to chondroitin sulfate A in placental malaria.The Journal of
biological chemistry 2012,287(28):23332-23345.
Summary of the invention
More difficult in order to solve early diagnosis cancer, verification method is few etc., and problems, the present invention confirm raw in blood etc. for the first time
Pl-CSA can be detected in thing liquid body, and demonstrate the generation that tumour is suffered from by the horizontal confirmation subject of analysis pl-CSA,
Development or rehabilitation situation.The present invention is using the Infected With Plasmodium erythrocyte surface antigen combination peptide fragment and pl-CSA for capturing pl-CSA
Antibody detects subject as detection reagent.Confirm pl-CSA and its antibody can by the vitro detection of body fluid,
Carry out diagnosing tumor and the tumor development monitoring of early stage.
Specifically, first aspect of the present invention provide provide it is a kind of for detecting the ELISA kit of tumour comprising
For detecting the capture albumen of placenta sample chondroitin sulfate A (CSA) (pl-CSA).
In technical solution in the present invention, the ELISA kit includes stationary phase carrier, and the stationary phase carrier is preferred
For biocompatible materials such as 96 hole micro plates, micro cup, nanometer rods, nanoparticles, if its can will capture proteopexy in
Fixed phase surface is without destroying bioactivity.
In the inventive solutions, the capture albumen is selected from Infected With Plasmodium erythrocyte surface antigen
The minimum combination peptide fragment of (VAR2CSA, rVAR2), sequence as shown in SEQ ID No.1,
EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID No.1。
In the above-mentioned methods, the method is non-diagnostic and therapeutical uses.
It in the inventive solutions, further include detection antibody in kit；The detection antibody is placenta sample sulphur
The antibody of sour Chondroitin A, it is therefore preferable to monoclonal antibody, the polyclonal antibody, multi-specific antibody of placenta sample chondroitin sulfate A (CSA)
(for example, bispecific antibody) and antibody fragment, as long as it can show required antigen-binding activity.
The placenta sample chondroitin sulfate A (CSA) is prepared by the following: commercially available acquisition, chemical synthesis process, chemistry point
It is made from the method in purification process, such as CN201710966913.2.
The detection antibody by obtaining antibody after organism is immunized with placenta sample chondroitin sulfate A (CSA), by miscellaneous
Tumor method is handed over to obtain antibody or obtain antibody by gene engineering method.
It in the inventive solutions, further include enzyme labelled antibody in kit, the enzyme labelled antibody is anti-detection antibody
Enzyme target antibody.
In the inventive solutions, the enzyme in the enzyme labelled antibody is selected from horseradish peroxidase, alkaline phosphatase
Enzymes, the gold colloids such as enzyme (ALP), beta galactose enzyme etc., but it is not limited to above-mentioned marker.
In the inventive solutions, using horseradish peroxidase, 3 are selected from as chromogenic substrate,
3', 5,5'- tetramethyl benzidines, o-phenylenediamine etc..Using ALP, p-nitrophenyl phosphorus is selected from as chromogenic substrate
Acid esters etc..When using beta galactose enzyme, as chromogenic substrate, selected from O-Nitrophenylfluorone-β-D- galactopyranoside etc..
It in the inventive solutions, further include cleaning solution, sample diluting liquid, developing solution, terminate liquid, mark in kit
Quasi- reference protein.
In the inventive solutions, the detection of pl-CSA is limited to 310ng/ml or more, preferably 1ug/ml or more.
In the inventive solutions, examined sample is cell pyrolysis liquid, cell culture fluid, blood, serum, blood
Slurry.
In the inventive solutions, the tumour includes oophoroma, embryonal-cell lipoma, lung cancer, liver cancer, breast cancer, bone
Marrow cancer, interstitial tissue[of testis] tumor, prostate cancer, cancer of pancreas, cervical carcinoma, colon cancer.
Another aspect of the invention provides the minimum combination peptide fragment of parasitized erythrocyte surface antigen and/or resisting for pl-CSA
Purposes of the body in the reagent of preparation detection tumor screening, early diagnosis of tumor, tumour progression or tumor recovering, the infection
The sequence of the minimum combination peptide fragment of erythrocyte surface antigen is as shown in SEQ ID No.1.
Heretofore described reagent is that EIA enzyme immunoassay (ELISA) uses reagent, chemiluminescent enzyme immunoassay method
(CLEIA) immune with reagent, chemiluminescent immunoassay (CLIA) reagent, fluorescence anti-body method (FAT) reagent, luciferase
Measuring method (FEIA) reagent, electrochemiluminescent immunoassay method (ECLIA) reagent, radioimmunoassay (RIA) examination
Agent, immunochromatographic method reagent, agglutination reagent, competition law reagent, colloidal gold strip, the examination of nanometer detection reagent colloid
Agent, ELISA reagent etc., still, however it is not limited to these methods.In the above-mentioned technical solutions, the EIA enzyme immunoassay is selected from ELISA
Direct method, ELISA indirect method, ELISA sandwich method, preferably ELISA sandwich method.
Detection of the heretofore described reagent for subject's blood or body fluid.
In the inventive solutions, the pl-CSA is placenta sample chondroitin sulfate A (CSA).
Another aspect of the invention provides the ELISA kit for being previously used for detection tumour as tumor screening, tumour
The purposes of early diagnosis, the detection reagent of tumour progression or tumor recovering or tool.
Another aspect of the invention provides a kind of method of ELISA detection placenta sample chondroitin sulfate A (CSA), wherein with malaria original
For the minimum combination peptide fragment of insect infection erythrocyte surface antigen (VAR2CSA, rVAR2) as capture albumen, Infected With Plasmodium is red thin
The minimum combination peptide section sequence of cellular surface antigen (VAR2CSA, rVAR2) as shown in SEQ ID No.1,
EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID No.1。
In the above-mentioned methods, the method is non-diagnostic and therapeutical uses.
In the above-mentioned methods, the ELISA is Direct ELISA, indirect ELISA or sandwich method ELISA.
Another aspect of the present invention provides a kind of tumor screening based on placenta sample chondroitin sulfate A (CSA), tumour early stage examines
Disconnected, tumour progression or tumor recovering detection method, the method are with the minimum combination peptide fragment of parasitized erythrocyte surface antigen
Sequence as detection reagent, the method for pl-CSA in vitro detection subject's body is described with parasitized erythrocyte surface antigen
Sequence is as shown in SEQ ID No.1.
In the above method, the vitro detection is with EIA enzyme immunoassay (ELISA) method, chemiluminescent enzyme immunoassay method
(CLEIA), chemiluminescent immunoassay (CLIA), fluorescence anti-body method (FAT), fluorescence enzyme immunoassay (FEIA), electrification
Learn luminescence immunoassay (ECLIA), radioimmunoassay (RIA), immunochromatographic method, agglutination, competition law, colloidal gold examination
Paper slip, nanometer detection reagent colloid method are detected, still, however it is not limited to these methods.In the above-mentioned technical solutions, described
EIA enzyme immunoassay is selected from ELISA direct method, ELISA indirect method, ELISA sandwich method, preferably ELISA sandwich method.
In the above method, the sample of vitro detection is cell pyrolysis liquid, cell culture fluid, blood, serum, the blood of subject
Slurry.
Preferably, it is detected using aforementioned ELISA kit.
In the present invention, the detection antibody is the antibody or antibody fragment with object to be detected.
In the present invention, the enzyme labelled antibody is the secondary antibody with markd detection antibody.
In the inventive solutions, the tumor screening, early diagnosis of tumor, tumour progression or tumor recovering
Detection method or reagent, refer to detected in sample to be tested placenta sample chondroitin sulfate A (CSA) then sample to be tested subject suffer from it is swollen
Tumor.
Beneficial effect
1) present invention firstly discovers that pl-CSA can be detected in a variety of body fluid, this is qualitatively or quantitatively gives birth in vitro
Object detection pl-CSA provides foundation.
2) present invention, which is demonstrated, can judge that subject suffers from tumor presence, examines for infantile tumour by vitro detection pl-CSA
It is disconnected to provide new method.
3) method of the invention is simple, while detecting low, reproducible, the specific height of limit.
Detailed description of the invention
Fig. 1 is technical schematic diagram of the invention.
Fig. 2 is sensitivity and repeated testing result.Wherein A is sensitivity experiments as a result, B is repeated experimental result.
Fig. 3 is the level that tumor cells expression pl-CSA is detected using the method for the present invention.
Fig. 4 is that model mouse and clinical case sample verify testing result.Wherein A is model mouse experimental result, and B is clinical disease
Example experimental result.
Specific embodiment
The preparation of embodiment 1.ELISA capture albumen
The minimum combination peptide fragment EDVKDINFDTKEKFLAGCLIVSFHEGKC of chemical synthesis VAR2CSA, is named as pl-
Capture albumen of the CSA-BP as ELISA method.
The pl-CSA that embodiment 2. purifies prepares
Pl-CSA can be prepared by commercially available or chemical method, and the present invention is using the first patented method preparation in this laboratory
pl-CAS.Preparation method is referring to application number: the preparation in 201710966913.2.It is specific as follows,
Chromatographic purifying is carried out to placenta sample chondroitin sulfate A (CSA) or derivatives thereof in the method for affinity chromatography, wherein affine layer
Analysis column material is the conjugate for recombinating Infected With Plasmodium erythrocyte surface antigen albumen and affinity chromatography matrices, the chromatographic purifying
Method is that placenta sample chondroitin sulfate A (CSA) or derivatives thereof crude product is splined on affinity column, and is washed with cleaning solution
It is flowed out to no impurity, then is eluted with eluent and collect placenta sample chondroitin sulfate A (CSA) sterling or derivatives thereof sterling.Institute
The sequence of recombinant plasmodium parasitized erythrocyte surface antigen protein is stated as shown in SEQ ID No.2.
SEQ ID No.2
LENYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITH
SSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQK
KLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDIN
FDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGK
LFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYL
RFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQ
IYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADK
APWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSAR
TMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDGNDVTFFNLFEQWNKEIQYQIEQYMTNANISCIDEKEVLDSVS
DEGTPKVRGGYEDGRNNNTDQGTNCKEKCKCYKLWIEKINDQWGKQKDNYNKFRSKQIYDANKGSQNKKVVSLSNFL
FFSCWEEYIQKYFNGDWSKIKNIGSDTFEFLIKKCGNNSAHGEEIFNEKLKNAEKKCKENESTDTNINKSETSCDLN
ATNYIRGCQSKTYDGKIFPGKGGEKQWICKDTIIHGDTNGACIPPRTQNLCVGELWDKSYGGRSNIKNDTKELLKEK
The preparation of embodiment 3.pl-CSA antibody
1) Balb/c mouse immune
50 μ g immunogene pl-CSA are dissolved in 200 μ L PBS solutions, mix with isometric complete Freund's adjuvant, trace antigen
Emulsifier unit fully emulsified 1 hour.Then six week old female Balb/c mouse of the nape of the neck multi-point injection.Initial immunity uses Freund
Freund's complete adjuvant, and be repeated once, booster immunization is incomplete Freund's adjuvant, and the immunization interval phase is 2 weeks, fusion first three days abdominal cavity note
It penetrates 100 μ g pl-CSA immunogenes and is dissolved in 200 μ L PBS solutions, adjuvant is not added.7 days to 10 days mouse tails after third time is immune
Portion's blood sampling, about 15 μ L of every mouse carry out bioactivity.
2) titration
Titration is using indirect non-competing ELISA method.1 μ g/ml pl-CSA is dissolved in coating buffer, enzyme is added
In target, 100 holes μ L/, 4 DEG C overnight.PBST board-washing three times after, every hole 200ml 2%BSA confining liquid, 37 DEG C react 1 hour.
Three times, 100 μ L antibody serum dilute solutions are added in every hole to PBST board-washing.37 DEG C reaction 1 hour after, PBST board-washing three times, every hole
100 μ L sheep anti mouse ELIAS secondary antibodies are added, 37 DEG C are reacted 1 hour.PBST board-washing six times, substrate developing solution is added and reacts 15 minutes.
50 μ L 2M sulfuric acid are added and terminate reaction.Absorption value is measured at 450 nm.
3) cell fusion
Immunized mice blood is acquired, 4 DEG C of 1200rpm are centrifuged 30 minutes, collect serum, this serum had both been pl-CSA Anti-TNF-α
Body.
Under aseptic conditions, the spleen for taking out antibody serum high-titer Balb/c mouse is put into and fills RPMI-1640 culture
The homogenizer of liquid, spleen is lightly ground, and cell suspension is sucked out, and is centrifuged impurity elimination, and RPMI-1640 culture solution washes twice.It takes pair
The myeloma cell SP2/0 of number growth, 1200rpm are centrifuged 5 minutes, abandon supernatant, with counting after RPMI-1640 culture solution suspension cell
Number, takes required cell number.SP2/0 myeloma cell and splenocyte are mixed in 1:10 ratio, in 50ml centrifuge tube
It is interior to be washed 1 time, 1200rpm with RPMI-1640 culture solution, it is centrifuged 8 minutes, abandons supernatant, exhaust residual liquid, in case it is dense to influence PEG
Degree.Gently attack centrifuge tube bottom, loosens cell precipitation slightly.At 37 DEG C, the 1m L 50%PEG of preheating is added in 30 seconds,
It is stirring while adding.After 90 seconds, it is slowly added to preheating RPMI-1640 culture solution and terminates PEG effect.1200rpm is centrifuged 5 minutes, is abandoned
Supernatant is added 10m L RPMI-1640 culture solution and cell is resuspended, is sufficiently mixed with 90m L semisolid HAT culture solution.Using 20m
L syringe draws semisolid culturemedium, is transferred to 6 porocyte culture plates, every hole 1.5m L, and 37 DEG C, 5%CO2Incubator culture
14 days.
4) screening of hybridoma
Grow macroscopic white point after 14 days, in culture plate, each white point and be a strain of hybridoma strain.It will be each
White point is transferred to 96 porocyte culture plates in time.It observes under inverted microscope, when cell will be filled with the visual field, measures on cell
Clearly.First using indirect non-competing ELISA method, 2.2.3.2 is seen.Every hole adds 100 μ L cell conditioned mediums to measure.Positive hole is taken out,
Indirect competitive ELISA method is done again.1 μ g/m L pl-CSA is dissolved in coating buffer, is added in ELISA Plate, 100 holes μ L/, 4
DEG C overnight.PBST board-washing three times after, every 200 μ L 1.5%OVA confining liquid of hole, 37 DEG C react 1 hour.PBST board-washing three times, often
50 μ L cell conditioned mediums and 50 μ L PL-CSA titers are added in hole.After 37 DEG C are reacted 1 hour, three times, every hole is added PBST board-washing
100 μ L sheep anti mouse ELIAS secondary antibodies, 37 DEG C are reacted 1 hour.PBST board-washing six times, substrate developing solution is added and reacts 15 minutes.It is added
50 μ L 2M sulfuric acid terminate reaction.Absorption value is measured at 450 nm, there will be the cell strain of inhibition to screen pl-CSA.
5) preparation of monoclonal antibody
Monoclonal antibody is prepared using mouse ascites.Incomplete Freund's adjuvant intraperitoneal injection of mice, 0.4m L/, after 3 days
Every mouse peritoneal injects hybridoma, after 7~12 days, extracts ascites when mouse web portion expansion is obvious.3000rpm/min
Centrifugation 10 minutes discards top fat, collect it is intermediate clarify ascites, -20 DEG C freeze it is spare.
6) purifying and identification of monoclonal antibody
The purifying of monoclonal antibody is purified using saturated ammonium sulfate method and Protein A method.
Steps are as follows: saturated ammonium sulfate method: the mouse ascites for taking 10m L to handle well move into beaker, under magnetic stirring,
Saturated ammonium sulfate solution 5.0m L is slowly added dropwise；Continue after stirring 30min；10000r/min is centrifuged 15min；Liquid is discarded supernatant,
Sediment is resuspended with 1/3 saturation degree ammonium sulfate, and after stirring action 30min, 10000r/min is centrifuged 15min；It discards supernatant
Liquid, sediment are dissolved in 1.5m L pure water, are packed into bag filter, and dialysis for 24 hours, removes salt ion, and every 6h changes water.After dialysis is good, very
Vacuum freecing-dry, -20 DEG C save backup.
Protein A method: being diluted to 50m L with PBS solution for 5m L mouse ascites, after 0.45 μm of membrane filtration on
Protein A column.Flow velocity is 1m L/min；Wash 20m L again with PBS solution, flow velocity is 1m L/min；It is slow with p H4.0 citric acid
Fliud flushing elution, flow velocity are 1m L/min, collect eluting peak, wash 20m L with stream of pure water, then rinse 5m L with 0.3%Na N3PBS
And save, flow velocity is 2m L/min, and pillar is placed in 4 DEG C and saves.Eluent is packed into bag filter, and dialysis for 24 hours, removes salt ion,
Every 6h changes water.After dialysis is good, vacuum freeze drying, -20 DEG C are saved backup.
The purity of monoclonal antibody uses PAGE gel electrophoresis technique determining.Step are as follows: 10% separation gel is injected into glass
In glass interlayer, top is sealed with pure water, after glue polymerization to be separated, removes pure water, 5% concentration glue of injection, insertion point sample comb.It will be single
Isometric 2 × SDS-PAGE sample treatment liquid is added to final concentration of 5 μ g/m L, 100 DEG C of water-bath 5min, after cooling in clonal antibody
It is spare.After being loaded 10 μ L, then starting voltage 80V is separated with 120V voltage, reaches bottom sides to bromophenol blue indicator
Stop electrophoresis when edge.Then stripping glue dyes, and is destained overnight after 0.5h with destainer, gel imager is taken pictures.
7) specific assay of monoclonal antibody
By pl-CSA and its analogue CSB, CSC as Competitive assays original.Competition original is first subjected to gradient dilution, respectively
Take 50 μ L to mix with equivalent antibody, be added and be coated and in the elisa plate closed, 37 DEG C of incubation 1h, remaining step together between
Connect non-competing ELISA method.Using each Competitive assays original content as abscissa, with inhibiting rate (each concentration standard Competitive assays foramen primum OD
It is worth and is not added the percentage of Competitive assays foramen primum OD value) it is that ordinate draws standard curve.With each curve inhibiting rate be 50% when
The concentration of corresponding Competitive assays original is calculated as IC50.Compared with the IC50 value of the IC50 value of pl-CSA and other structures analog
Percentage is come the cross reacting rate both measured.
The optimum diluting multiple of embodiment 4.pl-CSA antibody
Different batches antibody extension rate has differences, and every batch of is needed to detect.This part selects polyclonal antibody and list
Clonal antibody is all feasible, preferably monoclonal antibody.
The measurement of Pl-CSA antibody extension rate uses matrix method, and both 96 hole micro plates were coated with pl-CSA-BP, and concentration is big
It is diluted in the carbonate buffer solution of 50mM pH 9.6 in 5 μ g/ml, 20 μ g/ml of preferred concentration, every 200 μ l of hole, 4 DEG C of incubations
Overnight, PBST board-washing is three times.37 DEG C of BAS of 2% are closed 2 hours.A series of pl-CSA of concentration gradients, including 3.91 μ g/
ml,7.81μg/ml,15.63μg/ml,31.25μg/m,62.50μg/m,125.00μg/ml,250.00μg/ml,and
500.00 μ g/ml, 12 multiple holes of each concentration, 37 DEG C are incubated for 2 hours, PBST board-washing 3 times, every time 5 minutes.Pl-CSA antibody,
Including 1:100,1:1 000,1:10 000,1:100 000,3 multiple holes of each concentration, 37 DEG C are incubated for 2 hours, if nonimmune mouse
Serum is negative control, ibid PBST board-washing 3 times.After 37 DEG C are reacted 1 hour, three times, 100 μ L goat-antis are added in every hole to PBST board-washing
Mouse ELIAS secondary antibody (1:10 000), 37 DEG C are reacted 1 hour.PBST board-washing six times, tmb substrate developing solution is added and reacts 15 minutes.
50 μ L 2M sulfuric acid are added and terminate reaction.Absorption value is measured at 450 nm.
The P/N value of experimental antibodies should be higher than that national standard (national standard is >=2.1), and this detection optimum diluting multiple
For 1:1000.
The optimum diluting multiple of 1. batch antibody of table detects.
The verifying of embodiment 5.ELISA sensitivity
The pl-CSA-BP (20 μ g/ml) of preferred concentration is diluted in the carbonate buffer solution of 50mM pH 9.6, every hole 200
μ l is coated with 96 hole micro plates, and 4 DEG C of overnight incubations, PBST board-washing is three times.37 DEG C of BAS of 2% are closed 2 hours.A series of concentration ladders
The pl-CSA of degree, including 0.31,0.61 μ g/ml, 1.22 μ g/ml, 2.44 μ g/ml, 4.88 μ g/ml, 9.77 μ g/ml, 19.53 μ
g/ml,39.06μg/ml,78.13μg/ml,156.25μg/ml,312.50μg/ml,625.00μg/ml,1 250.00μg/ml,
2 500.00 μ g/ml and 5 000.00 μ g/ml, 3 multiple holes of each concentration, 37 DEG C are incubated for 2 hours, and PBST board-washing 3 times, every time 5
Minute.The optimal dilution 1:1 000 of pl-CSA antibody, if nonimmune mouse serum be negative control, 37 DEG C be incubated for 2 hours, ibid
PBST board-washing 3 times.After 37 DEG C are reacted 1 hour, three times, 100 μ L sheep anti mouse ELIAS secondary antibody (1:10 are added in every hole to PBST board-washing
000) it, reacts 1 hour for 37 DEG C.PBST board-washing six times, tmb substrate developing solution is added and reacts 15 minutes.It is whole that 50 μ L 2M sulfuric acid are added
Only react.Absorption value is measured at 450 nm.Experimental result A referring to fig. 2.
The sensitivity of ELISA detection method of the present invention is 310ng/ml, and most suitable detection range is in 3.91 μ g/ml to 500.00
μg/ml。
The verifying of embodiment 6.ELISA repeatability
Such as the experimental procedure in previous embodiment 5, selection pl-CSA concentration is 3.91 μ g/ml, 7.81 μ g/ml, 15.63 μ
G/ml, 31.25 μ g/ml, 62.50 μ g/ml, 125.00 μ g/ml, 250.00 μ g/ml, 500.00 μ g/ml are standard series concentration
Carry out repeated verifying.Experimental result B referring to fig. 2.
Most suitable detection range has preferable repeatability in 1.00 μ g/ml to 500.00 μ g/ml within this range.
Embodiment 7.ELISA specificity verification
According to the step in above-mentioned experiment, CSB (500 μ g/ml) and CSC (500 μ g/ml) are detected, experiment reliable value uses
P/N≥2.1.Using the congener CSB and CSC of pl-CSA, the OD450nm of the two is similar to negative serum detected value, and P/N is about etc.
In 1, and do not have substantive antagonism to the specific binding of pl-CSA.Experimental result confirms that method of the invention is special
It is anisotropic good.
Embodiment 8 is directed to the detection of cell lysate and supernatant
15 kinds of cells, including 10 kinds of cancer cells and 4 kinds of normal cells, DMEM/DF12 culture medium supplement 10% are selected
FBS, 37 DEG C 5% of CO2 incubator grows to the fusion rate of 80-90% to cell, washed 2 times with PBS, replaces fresh no blood
Clear culture medium continues culture for 24 hours, collects culture supernatant；1000rpm is centrifuged 10min, collects supernatant for detecting；Cultivate cell
It is washed 2 times with PBS, 0.25% digest without EDTA pancreatin separates cell, collects cell suspension, it is thin that 1000rpm is centrifuged 5min collection
Born of the same parents, cell are resuspended in PBS, the power ultrasonic 20sec (ultrasonic 5sec, stopping 5sec) of condition of ice bath 50%, 1000rpm centrifugation
5min, collecting supernatant is cell pyrolysis liquid, for detecting.It is detected using this ELISA method, the same ELISA of detection method
Repeatability detection.The pl-CSA of cancer cell expression can be accurately detected by studying, to distinguish normal cell and cancer is thin
Born of the same parents.Specifying information is referring to table 1.As a result referring to Fig. 3.Experimental result confirms the culture supernatant of cancer cell, can examine in lysate
Pl-CSA is measured, and normal cell can't detect.It with this proves that normal cell can be identified in the method for used ELISA and cancer is thin
Born of the same parents carry out screening and early diagnosis and treatment effectiveness evaluation using biofluid or tumor tissue cell to realize.
Cell selected by table 1.
Embodiment 9 is directed to the detection of animal model sample
Select 2 kinds of cancer model mouse serum, i.e. oophoroma (10) and suede cancer (10), choriocarcinoma cell JEG3 and oophoroma
Cell SKOV3 (FBS of supplement 10%) in DMEM/DF12 culture medium, 37 DEG C 5% of CO2 incubator is grown to cell
The fusion rate of 80-90%, 0.25% digest without EDTA pancreatin separate cell, collect cell suspension, and 1000rpm is centrifuged 5min and receives
Collect cell, cell is resuspended in PBS.106A cell mouse tail vein whole body plants tumor or subcutaneously plants tumor, and measurement is subcutaneous swollen weekly
Tumor tissue size；Using Fluc gene, intraperitoneal injection fluorescein, by small animal imaging monitor interior tumor cell growth,
After tumour growth 26 days, mouse is put to death in anesthesia eyeball blood sampling.Blood 1000rpm is centrifuged 20min, and collecting supernatant is both serum, uses
In detection.Application verification of the invention is carried out by ELISA method, experimental program is with repeatability verifying, and experimental result is referring to figure
4A, the experimental result illustrate that method of the invention can accurately detect there is free pl-CSA in cancer model mouse serum,
To realize difference cancer model mouse and normal health mouse serum.
Embodiment 10 is directed to the detection of Patient Sample A
The present invention selects 2 kinds of clinical cancer cases, both cervical carcinoma (7) and oophoroma (7), and tumor blood sample comes from
Shenzhen South Mountain hospital and BeiJing University ShenZhen Hospital have passed through Ethic review and informed consent, 4 DEG C of blood sample fortune
Laboratory is gone back to, 1000rpm is centrifuged 20min, and collecting supernatant is both serum, for detecting.It is detected by ELISA method, it is complete
At clinical sample application verification, B result can accurately detect the pl-CSA in cases of cancer serum to experimental result referring to fig. 4, from
And distinguish cancer patient and healthy population serum.
SEQUENCE LISTING
<110>Shenzhen Institutes of Advanced Technology, Chinese Academy of Science
<120>a kind of Reagent Protocol of screening for cancer and early diagnosis based on placenta sample chondroitin sulfate A (CSA)
<130> CP11801303C
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> PRT
<213>artificial sequence
<400> 1
Glu Asp Val Lys Asp Ile Asn Phe Asp Thr Lys Glu Lys Phe Leu Ala
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Gly Cys Leu Ile Val Ser Phe His Glu Gly Lys Cys
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<210> 2
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<212> PRT
<213>artificial sequence
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Leu Glu Asn Tyr Ile Lys Gly Asp Pro Tyr Phe Ala Glu Tyr Ala Thr
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Lys Leu Ser Phe Ile Leu Asn Pro Ser Asp Ala Asn Asn Pro Ser Gly
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Glu Thr Ala Asn His Asn Asp Glu Ala Cys Asn Cys Asn Glu Ser Gly
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Ile Ser Ser Val Gly Gln Ala Gln Thr Ser Gly Pro Ser Ser Asn Lys
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Thr Cys Ile Thr His Ser Ser Ile Lys Thr Asn Lys Lys Lys Glu Cys
65 70 75 80
Lys Asp Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Lys Ile
85 90 95
Cys Val Ile Glu Asp Thr Ser Leu Ser Gly Val Asp Asn Cys Cys Cys
100 105 110
Gln Asp Leu Leu Gly Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Arg
115 120 125
Gly Ser Ser Ser Asn Asp Ser Cys Asp Asn Lys Asn Gln Asp Glu Cys
130 135 140
Gln Lys Lys Leu Glu Lys Val Phe Ala Ser Leu Thr Asn Gly Tyr Lys
145 150 155 160
Cys Asp Lys Cys Lys Ser Gly Thr Ser Arg Ser Lys Lys Lys Trp Ile
165 170 175
Trp Lys Lys Ser Ser Gly Asn Glu Glu Gly Leu Gln Glu Glu Tyr Ala
180 185 190
Asn Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Tyr Leu Gly Asn
195 200 205
Leu Pro Lys Leu Glu Asn Val Cys Glu Asp Val Lys Asp Ile Asn Phe
210 215 220
Asp Thr Lys Glu Lys Phe Leu Ala Gly Cys Leu Ile Val Ser Phe His
225 230 235 240
Glu Gly Lys Asn Leu Lys Lys Arg Tyr Pro Gln Asn Lys Asn Ser Gly
245 250 255
Asn Lys Glu Asn Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr
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Gly Asp Leu Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys
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Asp Leu Glu Leu Asn Leu Gln Asn Asn Phe Gly Lys Leu Phe Gly Lys
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Tyr Ile Lys Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser
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Leu Asp Glu Leu Arg Glu Ser Trp Trp Asn Thr Asn Lys Lys Tyr Ile
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Ala Asp Gly Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro
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Thr Ile Asp Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val
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Glu Asn Phe Cys Glu Gln Arg Gln Ala Lys Val Lys Asp Val Ile Thr
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Asn Cys Lys Ser Cys Lys Glu Ser Gly Asn Lys Cys Lys Thr Glu Cys
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Lys Thr Lys Cys Lys Asp Glu Cys Glu Lys Tyr Lys Lys Phe Ile Glu
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Ala Cys Gly Thr Ala Gly Gly Gly Ile Gly Thr Ala Gly Ser Pro Trp
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Ser Lys Arg Trp Asp Gln Ile Tyr Lys Arg Tyr Ser Lys His Ile Glu
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Asp Ala Lys Arg Asn Arg Lys Ala Gly Thr Lys Asn Cys Gly Thr Ser
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Ser Thr Thr Asn Ala Ala Ala Ser Thr Asp Glu Asn Lys Cys Val Gln
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Ser Asp Ile Asp Ser Phe Phe Lys His Leu Ile Asp Ile Gly Leu Thr
500 505 510
Thr Pro Ser Ser Tyr Leu Ser Asn Val Leu Asp Asp Asn Ile Cys Gly
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Ala Asp Lys Ala Pro Trp Thr Thr Tyr Thr Thr Tyr Thr Thr Thr Glu
530 535 540
Lys Cys Asn Lys Glu Arg Asp Lys Ser Lys Ser Gln Ser Ser Asp Thr
545 550 555 560
Leu Val Val Val Asn Val Pro Ser Pro Leu Gly Asn Thr Pro Tyr Arg
565 570 575
Tyr Lys Tyr Ala Cys Gln Cys Lys Ile Pro Thr Asn Glu Glu Thr Cys
580 585 590
Asp Asp Arg Lys Glu Tyr Met Asn Gln Trp Ser Cys Gly Ser Ala Arg
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Thr Met Lys Arg Gly Tyr Lys Asn Asp Asn Tyr Glu Leu Cys Lys Tyr
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Asn Gly Val Asp Val Lys Pro Thr Thr Val Arg Ser Asn Ser Ser Lys
625 630 635 640
Leu Asp Gly Asn Asp Val Thr Phe Phe Asn Leu Phe Glu Gln Trp Asn
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Lys Glu Ile Gln Tyr Gln Ile Glu Gln Tyr Met Thr Asn Ala Asn Ile
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Ser Cys Ile Asp Glu Lys Glu Val Leu Asp Ser Val Ser Asp Glu Gly
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Thr Pro Lys Val Arg Gly Gly Tyr Glu Asp Gly Arg Asn Asn Asn Thr
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Asp Gln Gly Thr Asn Cys Lys Glu Lys Cys Lys Cys Tyr Lys Leu Trp
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Ile Glu Lys Ile Asn Asp Gln Trp Gly Lys Gln Lys Asp Asn Tyr Asn
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Lys Phe Arg Ser Lys Gln Ile Tyr Asp Ala Asn Lys Gly Ser Gln Asn
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Ile Gly Ser Asp Thr Phe Glu Phe Leu Ile Lys Lys Cys Gly Asn Asn
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Ser Ala His Gly Glu Glu Ile Phe Asn Glu Lys Leu Lys Asn Ala Glu
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820 825 830
Glu Thr Ser Cys Asp Leu Asn Ala Thr Asn Tyr Ile Arg Gly Cys Gln
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Ser Lys Thr Tyr Asp Gly Lys Ile Phe Pro Gly Lys Gly Gly Glu Lys
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Gln Trp Ile Cys Lys Asp Thr Ile Ile His Gly Asp Thr Asn Gly Ala
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Cys Ile Pro Pro Arg Thr Gln Asn Leu Cys Val Gly Glu Leu Trp Asp
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Lys Ser Tyr Gly Gly Arg Ser Asn Ile Lys Asn Asp Thr Lys Glu Leu
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915
Claims (13)
1. a kind of for detecting the ELISA kit of tumour comprising for being combined with placenta sample chondroitin sulfate A (CSA) (pl-CSA)
Capture albumen, it is preferable that the capture albumen is selected from Infected With Plasmodium erythrocyte surface antigen (VAR2CSA, rVAR2)
Minimum combines peptide fragment, it is highly preferred that the minimum combines peptide section sequence as shown in SEQ ID No.1,
EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID No.1。
2. ELISA kit according to claim 1, which is characterized in that further include detection antibody in kit；Described
Detect the antibody that antibody is placenta sample chondroitin sulfate A (CSA), it is therefore preferable to the monoclonal antibody of placenta sample chondroitin sulfate A (CSA), more grams
Grand antibody, multi-specificity antibody and antibody fragment.
3. -2 described in any item ELISA kits according to claim 1, which is characterized in that further include that enzyme mark is anti-in kit
Body, the enzyme labelled antibody are the antibody of anti-detection enzyme-labeled antibody；
Preferably, the enzyme in the enzyme labelled antibody be selected from horseradish peroxidase, alkaline phosphatase (ALP), beta galactose enzyme,
Gold colloid,
It is highly preferred that joining as chromogenic substrate selected from 3,3', 5,5'- tetramethyls using horseradish peroxidase
Aniline, o-phenylenediamine；Using ALP, p-nitrophenyl phosphate is selected from as chromogenic substrate；Using beta galactose
When enzyme, as chromogenic substrate, it is selected from O-Nitrophenylfluorone-β-D- galactopyranoside.
4. ELISA kit according to claim 1-3, which is characterized in that further include in kit cleaning solution,
Sample diluting liquid, developing solution, terminate liquid, standard control albumen.
5. ELISA kit according to claim 1-4, which is characterized in that lowest detection is limited to 310ng/ml
More than, preferably 1ug/ml or more.
6. the described in any item ELISA kits of claim 1-5 as tumor screening, early diagnosis of tumor, tumour progression or
The detection reagent of tumor recovering or the purposes of tool.
7. a kind of ELISA method for detecting placenta sample chondroitin sulfate A (CSA), wherein most with Infected With Plasmodium erythrocyte surface antigen
Small combination peptide fragment is as capture albumen, the minimum combination peptide section sequence such as SEQ ID of Infected With Plasmodium erythrocyte surface antigen
Shown in No.1,
EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID No.1；
Preferably, the ELISA is Direct ELISA, indirect ELISA or sandwich method ELISA；
It is highly preferred that when for sandwich method ELISA with placenta sample chondroitin sulfate A (CSA) antibody be detection antibody.
8. the minimum combination peptide fragment of parasitized erythrocyte surface antigen and/or the antibody of placenta sample chondroitin sulfate A (CSA) are detected in preparation
Subject suffers from the purposes in the reagent of tumor presence, the sequence of the minimum combination peptide fragment of the parasitized erythrocyte surface antigen
As shown in SEQ ID No.1；
Preferably, examined sample is biological fluid,
More preferably cell pyrolysis liquid, cell culture fluid, blood, serum, blood plasma；
The tumor presence of suffering from includes tumor screening, early diagnosis of tumor, tumour progression or tumor recovering.
9. purposes according to claim 6, which is characterized in that wherein the reagent is EIA enzyme immunoassay reagent, changes
It is immune to learn fluorescence enzyme immunoassay method reagent, chemiluminescent immunoassay reagent, fluorescence anti-body method reagent, luciferase
Measuring method reagent, electrochemiluminescent immunoassay method reagent, radioimmunoassay reagent, immunochromatographic method examination
Agent, agglutination reagent, competition law reagent, colloidal gold strip, nanometer detection reagent colloidal agent, ELISA reagent；
Preferably, the EIA enzyme immunoassay be selected from ELISA direct method, ELISA indirect method, ELISA sandwich method,
More preferably ELISA sandwich method.
10. a kind of be used for tumor screening, early diagnosis of tumor, tumour progression or tumor recovering based on placenta sample chondroitin sulfate A (CSA)
Detection method, the method using the specific binding peptide fragment of placenta sample chondroitin sulfate A (CSA) as detection reagent, vitro detection by
The method of pl-CSA in examination person's body,
Preferably, the specific binding peptide fragment of the placenta sample chondroitin sulfate A (CSA) is the most brief summary of parasitized erythrocyte surface antigen
Peptide fragment is closed, sequence is as shown in SEQ ID No.1.
11. detection method according to claim 10, which is characterized in that the vitro detection is with EIA enzyme immunoassay
Method, chemiluminescent enzyme immunoassay method, chemiluminescent immunoassay, fluorescence anti-body method, fluorescence enzyme immunoassay, electrochemistry
Luminescence immunoassay, radioimmunoassay, immunochromatographic method, agglutination, competition law, colloidal gold strip, nanometer detection
Reagent colloid method is detected,
Preferably, the EIA enzyme immunoassay be selected from ELISA direct method, ELISA indirect method, ELISA sandwich method, more preferably
ELISA sandwich method；
It is highly preferred that being detected using any one of the claim 1-5 ELISA kit.
12. detection method described in 0 or 11 according to claim 1, which is characterized in that the sample of vitro detection is the life of subject
Object liquid, it is preferable that biological fluid is selected from cell pyrolysis liquid, cell culture fluid, blood, serum, blood plasma.
13. according to the described in any item purposes of claim 8-12 or method, which is characterized in that the tumour be selected from oophoroma,
Embryonal-cell lipoma, lung cancer, liver cancer, breast cancer, bone marrow cancer, interstitial tissue[of testis] tumor, prostate cancer, cancer of pancreas, cervical carcinoma, colon cancer.
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| WO2022016752A1 (en) * | 2020-07-21 | 2022-01-27 | 深圳先进技术研究院 | Endometriosis lesion-targeted nano-delivery system, and preparation method therefor and use thereof |
| CN113740521A (en) * | 2021-08-27 | 2021-12-03 | 安徽贝铭生物科技有限公司 | Application of preparation for detecting carcinoembryonic chondroitin sulfate in urine in preparation of preparation and kit for diagnosing malignant tumor of urinary system |
| CN116874578A (en) * | 2022-12-09 | 2023-10-13 | 中山大学肿瘤防治中心(中山大学附属肿瘤医院 中山大学肿瘤研究所) | VAR2CSA recombinant protein and preparation method and application thereof |
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