CN109387627B - Reagent method for screening and early diagnosis of cancer based on placenta-like chondroitin sulfate A - Google Patents

Abstract

The invention relates to a reagent and a method for placenta-like chondroitin sulfate A-based cancer screening and early diagnosis, and particularly discloses an ELISA kit for detecting tumors, which comprises a capture protein for binding with placenta-like chondroitin sulfate A (pl-CSA), wherein the capture protein is preferably selected from a minimal binding peptide fragment of plasmodium infected erythrocyte surface antigen (VAR2CSA, rVAR2), and more preferably, the sequence of the minimal binding peptide fragment is shown as SEQ ID No. 1. The invention discovers for the first time that the pl-CSA can be detected in various body fluids, which provides basis for qualitatively or quantitatively detecting the pl-CSA in vitro biologically.

Description

Reagent method for screening and early diagnosis of cancer based on placenta-like chondroitin sulfate A

Technical Field

The invention relates to the field of biotechnology detection, in particular to an ELISA method for cancer screening and early diagnosis based on placenta-like chondroitin sulfate A and a kit thereof.

Background

Cancer is a serious public health problem worldwide, arising from uncontrolled abnormal growth and metastasis of cancer cells. According to the statistics of 2012 of the international cancer organization, about 1410 ten thousand new cancer cases are generated each year, about 820 ten thousand patients die, and the change of life style shows the trend of increasing year by year, such as unbalanced diet, lack of exercise, and advanced childbearing^[1-3]. Unfortunately, the screening and early diagnosis of cancer are still insurmountable scientific problems, cancer cases are usually found to enter the middle and late stages, cancer cells have metastasis widely or wild growth, common drugs are difficult to inhibit the growth of cancer cells, chemotherapy is the main mode, and most of chemotherapeutic drugs inhibit the growth and renewal of normal cells while eliminating or inhibiting the cancer cells, so that the cancer becomes an incurable disease. Early stages of cancer may be the window of treatment, and there is an urgent need to explore a more effective method for cancer screening and early diagnosis.

Recent molecular marker studies have provided a new view to the mechanism of carcinogenesis, such as glycosaminoglycans^[4]Angiogenin^[5]And the like. There is a lack of reliable screening and diagnostic methods based on these biomarkers. Enzyme-linked immunosorbent assay (ELISA) was initiated in 1975 and was used for the detection of trichina antibodies for the diagnosis of trichinosis^[6]. Due to the characteristics of high sensitivity, good specificity, simple operation, high flux and the like of the ELISA method^[7,8]Has been widely used for bacteria^[9]Virus, virus^[7]And parasites^[10]And the like, and the screening, diagnosis and immunological monitoring of relevant diseases. However, such a method is rarely applied and popularized in cancer screening and early diagnosis, and the main reason is the lack of a cancer cell-specific expression and a wide variety of biological markers in various cancer tissues.

Among the cancer markers that have been found, placental-like chondroitin sulfate a (pl-CSA) is a characteristic glycosaminoglycan with characteristic disaccharide units^[11]Specifically expressed in cancer tissues, interestingly expressed widely in various cancer tissues, may play a key role in the progression of cancer, and is positively correlated with the malignancy of the tumor^[12]. However, pl-CSA was originally discovered as a receptor for plasmodium-infected erythrocytes to aggregate in the interstitial space of placental villi, and was able to specifically bind to the plasmodium-infected erythrocyte surface antigen VAR2 CSA. The binding of VAR2CSA and pl-CSA has high affinity and specificity, and the affinity KD of the minimum binding unit is about 15Nm^[13]So that VAR2CSA can become a specific capture protein of pl-CSA for purification of pl-CSA, or can be used in other aspects, but lacks development and application of reagents and methods in this aspect. The purified pl-CSA is used for immunization, an antibody is prepared, and an immunological monitoring method is further established, so that the pCSA has wide and very promising application value for the solarization and early diagnosis of cancer.

The ELISA method has the characteristics of high sensitivity, high specificity, simple operation, high flux and the like^[7,8]Has been widely used for bacteria^[9]Virus, virus^[7]And parasites^[10]And the like, and the screening, diagnosis and immunological monitoring of relevant diseases. However, such a method has been rarely used for screening and early diagnosis of cancer, and the main reason is the lack of a biological marker that is expressed specifically by cancer cells and is widely present in various cancer tissues.

Aiming at the scientific problem, the invention selects the marker pl-CSA which is specifically expressed in cancer tissues and widely expressed in various cancer tissues, but whether the marker pl-CSA can be released into biological liquid such as culture supernatant or serum is not reported in the prior art, and whether an ELISA detection method can be developed to detect the cancer cells and the content of the pl-CSA released in the biological liquid so as to determine the existence of the cancer cells and achieve the purposes of cancer screening and early diagnosis is unclear.

Reference to the literature

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.

Disclosure of Invention

In order to solve the problems that early diagnosis of cancer is difficult, the verification method is few and the like, the invention firstly verifies that pl-CSA can be detected in biological fluid such as blood and the like, and verifies that the occurrence, development or rehabilitation condition of the tumor of a subject is verified by analyzing the level of the pl-CSA. The invention adopts plasmodium-infected erythrocyte surface antigen binding peptide segment for capturing pl-CSA and pl-CSA antibody as detection reagents to detect a subject. It is proved that pl-CSA and the antibody thereof can be used for early tumor diagnosis and tumor development monitoring through in vitro detection of body fluid.

Specifically, the first aspect of the present invention provides an ELISA kit for detecting tumors, which comprises a capture protein for detecting placental-like chondroitin sulfate a (pl-CSA).

In the technical scheme of the invention, the ELISA kit comprises a stationary phase carrier, wherein the stationary phase carrier is preferably a biocompatible material such as a 96-hole microplate, a microcup, a nanorod, a nanoparticle and the like, as long as the stationary phase carrier can fix the capture protein on the surface of the stationary phase without destroying the biological activity.

In the technical scheme of the invention, the capture protein is selected from the minimal binding peptide segment of plasmodium infected erythrocyte surface antigen (VAR2CSA, rVAR2), the sequence of the minimal binding peptide segment is shown as SEQ ID No.1,

EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID No.1。

in the above methods, the methods are of non-diagnostic and therapeutic use.

In the technical scheme of the invention, the kit also comprises a detection antibody; the detection antibody is an antibody to placental-like chondroitin sulfate a, preferably a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., bispecific antibody), and an antibody fragment of placental-like chondroitin sulfate a, as long as it can exhibit the desired antigen-binding activity.

The placenta-like chondroitin sulfate A is obtained by the following method: commercially available, chemical synthesis methods, chemical separation and purification methods, such as the method in CN 201710966913.2.

The detection antibody is obtained by immunizing an organism with placenta-like chondroitin sulfate A, obtaining the antibody by a hybridoma method or obtaining the antibody by a genetic engineering method.

In the technical scheme of the invention, the kit also comprises an enzyme-labeled antibody, wherein the enzyme-labeled antibody is an antibody for resisting enzyme labeling of a detection antibody.

In the technical scheme of the invention, the enzyme in the enzyme labeled antibody is selected from enzymes such as horseradish peroxidase, alkaline phosphatase (ALP) and beta-galactosidase, gold colloid and the like, but is not limited to the markers.

In the technical scheme of the invention, in the case of using horseradish peroxidase, the chromogenic substrate is selected from 3, 3', 5, 5' -tetramethyl benzidine, o-phenylenediamine and the like. In the case of ALP, p-nitrophenyl phosphate or the like is used as a chromogenic substrate. When beta-galactosidase is used, the chromogenic substrate is selected from o-nitrophenyl-beta-D-galactopyranoside and the like.

In the technical scheme of the invention, the kit also comprises a washing solution, a sample diluent, a developing solution, a stopping solution and a standard control protein.

In the technical scheme of the invention, the detection limit of the pl-CSA is more than 310ng/ml, and preferably more than 1 ug/ml.

In the technical scheme of the invention, the detected sample is cell lysate, cell culture solution, blood, serum and plasma.

In the technical scheme of the invention, the tumor comprises ovarian cancer, liposarcoma, lung cancer, liver cancer, breast cancer, bone marrow cancer, testicular interstitial tumor, prostate cancer, pancreatic cancer, cervical cancer and colon cancer.

In another aspect, the present invention provides a use of an antibody against a minimal binding peptide fragment and/or pl-CSA that infects an erythrocyte surface antigen, wherein the sequence of the minimal binding peptide fragment is shown in SEQ ID No.1, in the preparation of a reagent for detecting tumor screening, early diagnosis, progression or rehabilitation.

The reagent of the present invention is a reagent for enzyme immunoassay (ELISA), a reagent for chemiluminescent enzyme immunoassay (CLEIA), a reagent for chemiluminescent immunoassay (CLIA), a reagent for fluorescent antibody method (FAT), a reagent for Fluorescent Enzyme Immunoassay (FEIA), a reagent for electrochemiluminescent immunoassay (ECLIA), a reagent for Radioimmunoassay (RIA), a reagent for immunochromatography, a reagent for agglutination method, a reagent for competition method, a colloidal gold test strip, a nano detection reagent colloidal reagent, an ELISA reagent, etc., but is not limited to these methods. In the above technical solution, the enzyme immunoassay is selected from the group consisting of an ELISA direct method, an ELISA indirect method, and an ELISA sandwich method, preferably an ELISA sandwich method.

The reagents described in the present invention are useful for the detection of blood or body fluids of a subject.

In the technical scheme of the invention, the pl-CSA is placenta-like chondroitin sulfate A.

In another aspect, the invention provides the use of the ELISA kit for detecting tumor as a detection reagent or tool for tumor screening, early diagnosis of tumor, tumor progression or tumor rehabilitation.

In another aspect of the present invention, there is provided a method for detecting placenta-like chondroitin sulfate A by ELISA, wherein the minimal binding peptide fragment of plasmodium-infected erythrocyte surface antigen (VAR2CSA, rVAR2) is used as capture protein, the sequence of the minimal binding peptide fragment of plasmodium-infected erythrocyte surface antigen (VAR2CSA, rVAR2) is shown in SEQ ID No.1,

EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID No.1。

in the above methods, the methods are of non-diagnostic and therapeutic use.

In the above method, the ELISA is a direct ELISA, an indirect ELISA or a sandwich ELISA.

In still another aspect, the present invention provides a detection method for tumor screening, early tumor diagnosis, tumor progression or tumor rehabilitation based on placenta-like chondroitin sulfate a, wherein the method is a method for in vitro detection of pl-CSA in a subject by using a sequence of a minimal binding peptide segment of an infected erythrocyte surface antigen as a detection reagent, and the sequence of the infected erythrocyte surface antigen is shown as SEQ ID No. 1.

In the above-mentioned method, the in vitro detection is carried out by an enzyme-linked immunosorbent assay (ELISA), a chemiluminescent enzyme immunoassay (CLEIA), a chemiluminescent immunoassay (CLIA), a fluorescent antibody method (FAT), a Fluorescent Enzyme Immunoassay (FEIA), an electrochemiluminescent immunoassay (ECLIA), a Radioimmunoassay (RIA), an immunochromatography, an agglutination method, a competition method, a colloidal gold strip, or a nano-detection reagent colloid method, but the method is not limited to these methods. In the above technical solution, the enzyme immunoassay is selected from the group consisting of an ELISA direct method, an ELISA indirect method, and an ELISA sandwich method, preferably an ELISA sandwich method.

In the above method, the sample for in vitro detection is cell lysate, cell culture fluid, blood, serum, or plasma of the subject.

Preferably, the detection is carried out using the ELISA kit described above.

In the present invention, the detection antibody is an antibody or an antibody fragment to the analyte.

In the present invention, the enzyme-labeled antibody is a secondary antibody with a labeled detection antibody.

In the technical scheme of the invention, the detection method or reagent for tumor screening, early diagnosis of tumor, tumor progression or tumor rehabilitation refers to that a sample subject to be detected suffers from tumor when placenta-like chondroitin sulfate A is detected in the sample to be detected.

Advantageous effects

1) The invention discovers that the pl-CSA can be detected in various body fluids for the first time, and provides basis for qualitatively or quantitatively detecting the pl-CSA in vitro biologically.

2) The invention verifies that the tumor suffering condition of a subject can be judged by in vitro detection of pl-CSA, and provides a new method for early tumor diagnosis.

3) The method is simple, and has low detection limit, good repeatability and high specificity.

Drawings

Fig. 1 is a technical schematic diagram of the present invention.

FIG. 2 shows the results of the sensitivity and reproducibility measurements. Wherein A is a sensitivity experiment result, and B is a repeatability experiment result.

FIG. 3 shows the detection of the expression level of pl-CSA by tumor cells using the method of the present invention.

FIG. 4 is the test results of the model mouse and clinical case sample. Wherein A is the experimental result of the model mouse, and B is the experimental result of clinical cases.

Detailed Description

Example 1 preparation of ELISA Capture proteins

The minimal binding peptide segment EDVKDINFDTKEKFLAGCLIVSFHEGKC of VAR2CSA was chemically synthesized and named pl-CSA-BP as capture protein for ELISA method.

Example 2 preparation of purified pl-CSA

pl-CSA can be prepared commercially or chemically, and the present invention uses the laboratory patented earlier method to prepare pl-CAS. See application No.: 201710966913.2, respectively. Specifically, as follows, the following description will be given,

the method comprises the steps of loading a placenta-like chondroitin sulfate A or a derivative crude product thereof on an affinity chromatography column, washing the affinity chromatography column by using a washing solution until no impurities flow out, eluting by using an eluent and collecting a placenta-like chondroitin sulfate A pure product or a derivative pure product thereof. The sequence of the recombinant plasmodium infected erythrocyte surface antigen protein is shown as SEQ ID No. 2.

SEQ ID No.2

LENYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDGNDVTFFNLFEQWNKEIQYQIEQYMTNANISCIDEKEVLDSVSDEGTPKVRGGYEDGRNNNTDQGTNCKEKCKCYKLWIEKINDQWGKQKDNYNKFRSKQIYDANKGSQNKKVVSLSNFLFFSCWEEYIQKYFNGDWSKIKNIGSDTFEFLIKKCGNNSAHGEEIFNEKLKNAEKKCKENESTDTNINKSETSCDLNATNYIRGCQSKTYDGKIFPGKGGEKQWICKDTIIHGDTNGACIPPRTQNLCVGELWDKSYGGRSNIKNDTKELLKEK

Example 3 preparation of pl-CSA antibodies

1) Balb/c mouse immunization

Mu.g of immunogen pl-CSA was dissolved in 200. mu.L of PBS solution, mixed with an equal volume of complete Freund's adjuvant, and emulsified well in a microantigen emulsifying apparatus for 1 hour. Six-week-old female Balb/c mice were then injected dorsally and at multiple points. The Freund complete adjuvant is adopted for the primary immunization and repeated once, the boosting immunization is Freund incomplete adjuvant, the immunization interval period is 2 weeks, 100 mu g pl-CSA immunogen is intraperitoneally injected in 200 mu L PBS solution three days before fusion, and no adjuvant is added. Mice were bled from their tails 7 to 10 days after the third immunization, approximately 15 μ L per mouse, and titer was determined.

2) Potency assay

The titer was determined by indirect non-competitive ELISA. Mu.g/ml pl-CSA was dissolved in coating buffer and added to the microplate at 100. mu.L/well overnight at 4 ℃. After PBST washing three times, each well was washed with 200ml of 2% BSA blocking solution and reacted at 37 ℃ for 1 hour. The plates were washed three times with PBST and 100. mu.L of antibody serum dilution was added to each well. After 1 hour reaction at 37 ℃, the plates were washed three times with PBST, 100. mu.L of goat anti-mouse enzyme-labeled secondary antibody was added to each well, and the reaction was carried out at 37 ℃ for 1 hour. PBST plates were washed six times, and substrate developing solution was added to react for 15 minutes. The reaction was stopped by adding 50. mu.L of 2M sulfuric acid. The absorbance was measured at 450 nm.

3) Cell fusion

The immunized mouse blood was collected, centrifuged at 1200rpm at 4 ℃ for 30 minutes, and serum, which was the pl-CSA polyclonal antibody, was collected.

Under the aseptic condition, taking out the spleen of a Balb/c mouse with high titer of antibody serum, putting the spleen into a homogenizer containing RPMI-1640 culture solution, lightly grinding the spleen, sucking out cell suspension, centrifuging to remove impurities, and washing the RPMI-1640 culture solution twice. Taking logarithmic growth myeloma cells SP2/0, centrifuging at 1200rpm for 5 minutes, discarding the supernatant, suspending the cells with RPMI-1640 culture solution, counting, and taking the required number of cells. SP2/0 myeloma cells and splenocytes were mixed together at a ratio of 1:10, washed 1 time with RPMI-1640 medium in a 50ml centrifuge tube at 1200rpm for 8 minutes, the supernatant was discarded, and the residual liquid was aspirated to avoid affecting the PEG concentration. Lightly flick the bottom of the tube to loosen the cell pellet. Preheated 1m L50% PEG was added over 30 seconds at 37 ℃ with stirring. After 90 seconds, pre-warmed RPMI-1640 medium was slowly added to stop the PEG effect. 1200rpm, 5 minutes centrifugation, abandoning the supernatant, adding 10 ml RPMI-1640 culture solution to resuspend the cells, and mixing well with 90m L semisolid HAT culture solution. The semi-solid medium was aspirated by 20m L syringe and transferred to 6-well cell culture plates at 1.5m L, 37 ℃ in 5% CO per well₂The incubator is used for 14 days.

4) Screening of hybridoma cells

After 14 days, macroscopic white spots, each white spot and a hybridoma cell line, grew out of the culture plate. Each white dot was transferred to a 96-well cell culture plate in time. The cell supernatants were measured when the cells were about to fill the field of view under an inverted microscope. First, an indirect non-competitive ELISA was used, see 2.2.3.2. Add 100. mu.L of cell supernatant per well. The positive wells were removed and subjected to indirect competitive ELISA. Mu.g/ml pl-CSA was dissolved in coating buffer and added to the microplate at 100. mu.L/well overnight at 4 ℃. After three PBST plate washes, 200. mu.L of 1.5% OVA blocking solution per well was reacted at 37 ℃ for 1 hour. The plates were washed three times with PBST, and 50. mu.L of cell supernatant and 50. mu.L of PL-CSA standard solution were added to each well. After 1 hour reaction at 37 ℃, the plates were washed three times with PBST, 100. mu.L of goat anti-mouse enzyme-labeled secondary antibody was added to each well, and the reaction was carried out at 37 ℃ for 1 hour. PBST plates were washed six times, and substrate developing solution was added to react for 15 minutes. The reaction was stopped by adding 50. mu.L of 2M sulfuric acid. The absorbance was measured at 450nm, and cell lines showing inhibition of pl-CSA were selected.

5) Preparation of monoclonal antibodies

Monoclonal antibodies were prepared using mouse ascites fluid. Intraperitoneal injection of mice with incomplete Freund's adjuvant is carried out, 0.4m L per mouse is used, each mouse is subjected to intraperitoneal injection of hybridoma cells after 3 days, and ascites is extracted when abdominal distension of the mouse is obvious after 7-12 days. Centrifuging at 3000rpm/min for 10min, discarding upper part fat, collecting middle clear ascites, and freezing at-20 deg.C.

6) Purification and characterization of monoclonal antibodies

The monoclonal antibody is purified by a saturated ammonium sulfate method and a Protein A method.

The method comprises the following steps: saturated ammonium sulfate method: 10m L processed mouse ascites is taken and transferred into a beaker, and a saturated ammonium sulfate solution 5.0m L is slowly dropped under magnetic stirring; stirring for 30 min; centrifuging at 10000r/min for 15 min; discarding supernatant, resuspending the precipitate with 1/3 saturated ammonium sulfate solution, stirring for 30min, centrifuging at 10000r/min for 15 min; discarding supernatant, dissolving precipitate in 1.5m L pure water, placing into dialysis bag, dialyzing for 24 hr, removing salt ions, and changing water every 6 hr. After dialysis, vacuum freeze-drying and storing at-20 ℃ for later use.

Protein A method: ascites from 5m L mice was diluted with PBS solution to 50m L, filtered through a 0.45 μm filter and applied to a Protein A column. The flow rate was 1m L/min; rewashing with PBS solution 20m L at a flow rate of 1m L/min; eluting with p H4.0.0 citric acid buffer solution at flow rate of 1m L/min, collecting peak, washing with pure water 20m L, washing with 0.3% Na N3PBS 5m L, and storing at flow rate of 2m L/min, and storing at 4 deg.C. Loading the eluate into dialysis bag, dialyzing for 24 hr to remove salt ions, and changing water every 6 hr. After dialysis, vacuum freeze-drying and storing at-20 ℃ for later use.

The purity of the monoclonal antibody was determined by SDS-PAGE gel electrophoresis. The method comprises the following steps: injecting 10% separating gel into the glass interlayer, sealing the upper part with pure water, pouring out pure water after the separating gel is polymerized, injecting 5% concentrated gel, and inserting into a sample application comb. Adding the monoclonal antibody into the equal volume of 2 xSDS-PAGE sample treatment solution until the final concentration is 5 mug/m L, carrying out water bath at 100 ℃ for 5min, and cooling for later use. After loading 10. mu.L, the voltage was initially 80V, and separation was performed using 120V, and electrophoresis was stopped when the bromophenol blue indicator reached the bottom edge. Then stripping and dyeing, decoloring with a decoloring solution after 0.5h overnight, and taking a picture by a gel imager.

7) Determination of the specificity of monoclonal antibodies

pl-CSA and its structural analogue CSB, CSC were used as competitive inhibitors. The competitive antigen is firstly diluted in a gradient way, 50 mu L of the competitive antigen is mixed with the same amount of antibody, the mixture is added into an ELISA plate which is coated and sealed, the temperature is incubated for 1h at 37 ℃, and the rest steps are the same as the indirect non-competitive ELISA method. The concentration of each competitive inhibition antigen is used as an abscissa, and the inhibition rate (percentage of the OD value of each concentration standard competitive inhibition antigen to the OD value of the non-competitive inhibition antigen) is used as an ordinate to draw a standard curve. The concentration of the competitive inhibitor corresponding to the 50% inhibition ratio of each curve was taken as IC 50. The cross-reactivity of pl-CSA was measured as a percentage of its IC50 value compared to the IC50 value of other structural analogs.

Example 4 optimal dilution factor for pl-CSA antibody

Different batches of antibodies have different dilution times and need to be detected in each batch. This partial selection for both polyclonal and monoclonal antibodies is possible, with monoclonal antibodies being preferred.

Determination of dilution factor of Pl-CSA antibody Using a matrix method, a 96-well microplate was coated with pL-CSA-BP at a concentration of greater than 5. mu.g/ml, preferably at a concentration of 20. mu.g/ml, diluted in 50mM carbonate buffer pH 9.6, 200. mu.l per well, incubated overnight at 4 ℃ and washed three times with PBST. 2% BAS was blocked at 37 ℃ for 2 hours. A series of concentration gradients of pl-CSA comprising 3.91. mu.g/ml, 7.81. mu.g/ml, 15.63. mu.g/ml, 31.25. mu.g/m, 62.50. mu.g/m, 125.00. mu.g/ml, 250.00. mu.g/ml, and 500.00. mu.g/ml, 12 replicates per concentration, incubation at 37 ℃ for 2 hours, PBST plate wash 3 times, 5 minutes each. pL-CSA antibody, including 1:100,1: 1000, 1: 10000, 1: 100000, each concentration of 3 multiple wells, 37 degrees C were incubated for 2 hours, in the non immune mouse serum as negative control, PBST washing plate 3 times. After reaction at 37 ℃ for 1 hour, the plate was washed three times with PBST, 100. mu.L of goat anti-mouse enzyme-labeled secondary antibody (1: 10000) was added to each well, and the reaction was carried out at 37 ℃ for 1 hour. PBST washing plate six times, adding TMB substrate color solution to react for 15 minutes. The reaction was stopped by adding 50. mu.L of 2M sulfuric acid. The absorbance was measured at 450 nm.

The P/N value of the experimental antibody is higher than the national standard (the national standard is more than or equal to 2.1), and the optimal dilution factor of the detection is 1: 1000.

Table 1 optimal dilution fold detection of this batch of antibody.

Example 5 ELISA sensitivity verification

A preferred concentration of pl-CSA-BP (20. mu.g/ml) was diluted in 50mM carbonate buffer pH 9.6, coated in 96-well microtiter plates at 200. mu.l per well, incubated overnight at 4 ℃ and washed three times with PBST. 2% BAS was blocked at 37 ℃ for 2 hours. A series of concentration gradients of pl-CSA comprising 0.31, 0.61. mu.g/ml, 1.22. mu.g/ml, 2.44. mu.g/ml, 4.88. mu.g/ml, 9.77. mu.g/ml, 19.53. mu.g/ml, 39.06. mu.g/ml, 78.13. mu.g/ml, 156.25. mu.g/ml, 312.50. mu.g/ml, 625.00. mu.g/ml, 1250.00. mu.g/ml, 2500.00. mu.g/ml, and 5000.00. mu.g/ml, 3 replicates per concentration, incubation at 37 ℃ for 2 hours, PBST plate washes 3 times, 5 minutes each. The pl-CSA antibody was optimally diluted 1:1000, and incubated at 37 ℃ for 2 hours with non-immune mouse serum as a negative control, and washed 3 times with PBST as above. After reaction at 37 ℃ for 1 hour, the plate was washed three times with PBST, 100. mu.L of goat anti-mouse enzyme-labeled secondary antibody (1: 10000) was added to each well, and the reaction was carried out at 37 ℃ for 1 hour. PBST washing plate six times, adding TMB substrate color solution to react for 15 minutes. The reaction was stopped by adding 50. mu.L of 2M sulfuric acid. The absorbance was measured at 450 nm. See fig. 2A for experimental results.

The sensitivity of the ELISA detection method is 310ng/ml, and the optimal detection range is 3.91 mu g/ml to 500.00 mu g/ml.

Example 6 ELISA reproducibility verification

Repeatability tests were carried out as described in example 5 above using pl-CSA concentrations of 3.91. mu.g/ml, 7.81. mu.g/ml, 15.63. mu.g/ml, 31.25. mu.g/ml, 62.50. mu.g/ml, 125.00. mu.g/ml, 250.00. mu.g/ml, 500.00. mu.g/ml for the standard series of concentrations. See fig. 2B for experimental results.

The optimal detection range is 1.00 mu g/ml to 500.00 mu g/ml, and the repeatability is better in the range.

Example 7 ELISA specificity verification

According to the steps in the above experiment, CSB (500. mu.g/ml) and CSC (500. mu.g/ml) were detected, and the reliable value of the experiment was P/N ≥ 2.1. Using analogues of pl-CSA, CSB and CSC, both had OD450nm similar to negative serum measurements, with P/N equal to about 1 and no substantial antagonism of specific binding of pl-CSA. Experimental results prove that the method has good specificity.

Example 8 detection of cell lysates and supernatants

Selecting 15 cells comprising 10 cancer cells and 4 normal cells, supplementing 10% FBS with DMEM/DF12 culture medium, culturing in a CO2 incubator with the temperature of 37 ℃ and the concentration of 5%, washing with PBS for 2 times when the cells grow to the fusion rate of 80-90%, replacing fresh serum-free culture medium, continuously culturing for 24h, and collecting culture supernatant; centrifuging at 1000rpm for 10min, and collecting supernatant for detection; the cultured cells are washed for 2 times by PBS, 0.25 percent of EDTA-free pancreatin is used for digesting and separating the cells, cell suspension is collected, the cells are collected by centrifugation at 1000rpm for 5min, the cells are suspended in the PBS, power ultrasonic waves with ice bath conditions of 50 percent are carried out for 20sec (ultrasonic wave 5sec and stop for 5sec), centrifugation is carried out for 5min at 1000rpm, and supernatant is collected, namely cell lysate which is used for detection. The ELISA method is used for detection, and the detection method is the same as ELISA repeated detection. The pl-CSA expressed by the cancer cells can be accurately detected through research, so that normal cells and cancer cells can be distinguished. See table 1 for specific information. The results are shown in FIG. 3. Experimental results prove that pl-CSA can be detected in both the culture supernatant and the lysate of cancer cells, but normal cells cannot be detected. Thus, the method proves that normal cells and cancer cells can be identified by using an ELISA method, so that screening and early diagnosis and treatment effect evaluation by using biological fluid or tumor tissue cells are realized.

TABLE 1 cells selected

Example 9 testing on animal model samples

2 cancer model mouse sera, namely ovarian cancer (10 cases) and choriocarcinoma (10 cases), choriocarcinoma cell JEG3 and ovarian cancer cell SKOV3 were selected in DMEM/DF12 medium (supplemented with 10% FBS), 5% CO2 incubator at 37 ℃ until the cells were grown to 80-90% confluency, 0.25% EDTA-free pancreatin digested the detached cells, cell suspension was collected, centrifuged at 1000rpm for 5min to collect the cells, and the cells were resuspended in PBS. 10⁶Carrying out systemic tumor implantation or subcutaneous tumor implantation on the tail vein of the individual cell mouse, and measuring the size of subcutaneous tumor tissue every week; the growth of tumor cells in vivo is monitored by using Fluc gene, injecting fluorescein into abdominal cavity and imaging the small animals, and after the tumor grows for 26 days, blood is collected by anesthetizing eyeballs and the mice are sacrificed. The blood is centrifuged at 1000rpm for 20min, and the supernatant, namely serum, is collected for detection. The application verification of the invention is carried out by an ELISA method, the experimental scheme and the repeatability verification are carried out, and the experimental result is shown in figure 4A, which shows that the method can accurately detect the existence of free pl-CSA in the serum of the cancer model mouse, thereby realizing the distinction between the serum of the cancer model mouse and the serum of the normal healthy mouse.

Example 10 detection of patient samples

The invention selects 2 clinical cancer cases, namely cervical cancer (7 cases) and ovarian cancer (7 cases), tumor blood samples come from Shenzhen Nanshan Hospital and Shenzhen Hospital of Beijing university, through ethical examination and informed consent of patients, the blood samples are transported back to a laboratory at 4 ℃, centrifuged at 1000rpm for 20min, and supernatant is collected, namely serum, for detection. The detection is carried out by an ELISA method, the application verification of clinical samples is completed, and the experiment result is shown in the result of FIG. 4B, so that the pl-CSA in the serum of cancer cases can be accurately detected, and the serum of cancer patients and the serum of healthy people can be distinguished.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> a reagent method for cancer screening and early diagnosis based on placenta-like chondroitin sulfate A

<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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Ile Ser Ser Val Gly Gln Ala Gln Thr Ser Gly Pro Ser Ser Asn Lys

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Lys Asp Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Lys Ile

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Cys Val Ile Glu Asp Thr Ser Leu Ser Gly Val Asp Asn Cys Cys Cys

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Gln Asp Leu Leu Gly Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Arg

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Gly Ser Ser Ser Asn Asp Ser Cys Asp Asn Lys Asn Gln Asp Glu Cys

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Gln Lys Lys Leu Glu Lys Val Phe Ala Ser Leu Thr Asn Gly Tyr Lys

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Cys Asp Lys Cys Lys Ser Gly Thr Ser Arg Ser Lys Lys Lys Trp Ile

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Trp Lys Lys Ser Ser Gly Asn Glu Glu Gly Leu Gln Glu Glu Tyr Ala

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Asn Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Tyr Leu Gly Asn

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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

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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

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Lys Cys Asn Lys Glu Arg Asp Lys Ser Lys Ser Gln Ser Ser Asp Thr

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Leu Val Val Val Asn Val Pro Ser Pro Leu Gly Asn Thr Pro Tyr Arg

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Tyr Lys Tyr Ala Cys Gln Cys Lys Ile Pro Thr Asn Glu Glu Thr Cys

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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

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Leu Asp Gly Asn Asp Val Thr Phe Phe Asn Leu Phe Glu Gln Trp 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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Lys Lys Val Val Ser Leu Ser Asn Phe Leu Phe Phe Ser Cys Trp Glu

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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 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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Claims (26)

1. An ELISA kit for detecting tumor, which comprises a capture protein for binding with placenta-like chondroitin sulfate A (pl-CSA), wherein the capture protein is selected from a minimal binding peptide fragment of plasmodium infected erythrocyte surface antigen, the sequence of the minimal binding peptide fragment is shown as SEQ ID No.1, and the sequence of the SEQ ID No.1 is EDVKDINFDTKEKFLAGCLIVSFHEGKC.

2. The ELISA kit of claim 1, further comprising a detection antibody; the detection antibody is an antibody of placenta-like chondroitin sulfate A.

3. The ELISA kit of claim 2, wherein the detection antibody is a monoclonal antibody, a polyclonal antibody, a multispecific antibody, or an antibody fragment of placental-like chondroitin sulfate A.

4. The ELISA kit of any one of claims 1-3, wherein the kit further comprises an enzyme-labeled antibody, wherein the enzyme-labeled antibody is an antibody against the enzyme label of the detection antibody.

5. The ELISA kit of claim 4, wherein the enzyme in the enzyme-labeled antibody is selected from the group consisting of horseradish peroxidase, alkaline phosphatase (ALP), β -galactosidase, and gold colloid.

6. The ELISA kit according to claim 5, wherein in the case of using horseradish peroxidase, the chromogenic substrate is selected from the group consisting of 3, 3', 5, 5' -tetramethylbenzidine, o-phenylenediamine; in the case of ALP, p-nitrophenyl phosphate is chosen as chromogenic substrate; when beta-galactosidase is used, it is selected from the group consisting of o-nitrophenyl-beta-D-galactopyranosides as chromogenic substrate.

7. The ELISA kit of any one of claims 1-3, wherein the kit further comprises a washing solution, a sample diluent, a color development solution, a stop solution, and a standard control protein.

8. The ELISA kit of any one of claims 1 to 3 wherein the minimum detection limit is 310ng/mL or greater.

9. Use of the ELISA kit of any of claims 1-8 for the preparation of a detection reagent for tumor screening, early diagnosis of tumors, tumor progression or tumor rehabilitation.

10. An ELISA method for detecting placenta-like chondroitin sulfate A is characterized in that the minimum binding peptide fragment of plasmodium infected erythrocyte surface antigen is used as a capture protein, the sequence of the minimum binding peptide fragment of the plasmodium infected erythrocyte surface antigen is shown as SEQ ID No.1, and the sequence of the SEQ ID No.1 is EDVKDINFDTKEKFLAGCLIVSFHEGKC.

11. The ELISA method of detecting placental-like chondroitin sulfate A according to claim 10, wherein the ELISA method is a direct ELISA, an indirect ELISA or a sandwich ELISA.

12. The ELISA method for detecting placental-like chondroitin sulfate A according to claim 10, wherein the placental-like chondroitin sulfate A antibody is used as the detection antibody in the sandwich ELISA.

13. Use of the minimal binding peptide fragment of plasmodium infected erythrocyte surface antigen in preparing a reagent for detecting the tumor suffering condition of a subject, wherein the sequence of the minimal binding peptide fragment of the plasmodium infected erythrocyte surface antigen is shown as SEQ ID No. 1.

14. Use according to claim 13, characterized in that the sample to be tested is a biological fluid.

15. Use according to claim 13, characterized in that the sample to be tested is a cell lysate, a cell culture fluid, blood, serum, plasma;

the tumor-suffering condition comprises tumor screening, tumor early diagnosis, tumor progression or tumor rehabilitation.

16. The use according to claim 13, wherein the reagent is a reagent for enzyme immunoassay.

17. The use according to claim 13, wherein the reagent is a chemiluminescent enzyme immunoassay reagent, a fluorescent enzyme immunoassay reagent, or a radioimmunoassay reagent.

18. The use according to claim 13, wherein the reagent is a chemiluminescent immunoassay reagent or a fluorescent antibody reagent.

19. The use according to claim 13, wherein the reagent is a reagent for electrochemiluminescence immunoassay.

20. The use of claim 13, wherein the reagent is an immunochromatographic reagent.

21. The use according to claim 13, wherein the reagent is an agglutination reagent or a competition reagent.

22. The use of claim 13, wherein the reagent is a colloidal gold test strip.

23. The use according to claim 13, wherein the reagent is a nano detection reagent colloidal reagent.

24. The use according to claim 13, wherein the reagent is an ELISA reagent.

25. Use according to claim 16, wherein the enzyme immunoassay is selected from the group consisting of ELISA direct, ELISA indirect, ELISA sandwich.

26. Use according to claim 25, wherein the enzyme immunoassay is selected from ELISA sandwich.

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