CN101726601A - Double antibody identification based quantitative detection method of MG7-Ag contained in serum - Google Patents
Double antibody identification based quantitative detection method of MG7-Ag contained in serum Download PDFInfo
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Abstract
The invention relates to a detection method of MG7-Ag contained in serum, belonging to the technical field of biology. The detection method comprises the following steps of: accurately quantitating and detecting the content of the trace antigen MG7-Ag contained in the serum by combining a real-time fluorescence quantitative immune PCR detection method on the basis of antigen signal transformation through the identification and the capture of the MG7-Ag by double antibodies; combining a real-time fluorescence quantitative PCR technology with a specific antigen and antibody reaction system to establish a real-time fluorescence quantitative immune PCR technology, wherein the real-time fluorescence quantitative immune PCR technology identifies the MG7-Ag contained in the serum through the specificity of the double antibodies, then respectively combines biotinylated MG7 with biotinylated DNA through streptavidin, converts a content signal of the MG7-Ag contained in the serum into a nucleic acid signal and then enters a real-time fluorescence quantitative PCR expanding way, thereby accurately detecting the trace antigen MG7-Ag contained in the serum.
Description
Technical field
The invention belongs to biological technical field, relate to the detection method of MG7-Ag in the serum, particularly a kind of quantitative detecting method based on MG7-Ag in the serum of double antibody identification.
Background technology
Malignant tumour is that current serious influences human health, threatens one of principal disease of human life.The research of malignant tumour and application direction are from the treatment being the main control combination that turns to.The early diagnosis of tumour is particularly important, and it has determined the success or failure of oncotherapy.Cancer of the stomach is one of common great malignant tumour of China, and its M ﹠ M is all continuing rising.Being used for the most reliable approach of cancer of the stomach early screening at present is that endoscopy is in conjunction with the biological tissue pathologic finding.But, be mixed in for minority that form in the normal tissue cell is not true to type stomach cancer cell, differentiation difference or undifferentiated stomach cancer cell, cancer of the stomach tumour that lymphatic metastasis takes place differentiates that difficulty is very big; And the inspection of tumor markers is a kind of efficient and simple method.
The discovery and the application of tumor markers (Cancer biomarker) are the methods of a system, from detecting of people at highest risk's examination, early-stage cancer, arrive diagnosis and location, reaction and the prognosis observation of body to treating, until the detection of cancer return, tumor markers has been brought into play important effect.The tumor markers of at present clinical cancer of the stomach commonly used has CEA, CA19-9, CA72-4 etc., is subjected to CEA, CA19-9 and the specific restriction of CA72-4 mark, can detect most patients of above mark, and its state of an illness has entered middle and advanced stage.
People's latest finds such as Fan Daiming a kind of cancer of the stomach specific antigen MG7-Ag (Lee CH, Lum JH, Fan DM et al.Proteomics.2005; 5 (4): 1160-1166), and at this antigen preparation mouse monoclonal antibody MG7 (Fan DM, Zhang XY, Chen XT et al.Journal of Gastroener-ology and Hepatology.2005; 20 (3): 360-365).Find that in Histological research MG7-Ag positive rate in cancer of the stomach reaches more than 90%, in normal gastric mucosa, do not express.The MG7-Ag expression is progressive and increases progressively in atrophic gastritis, dysplasia and stomach organization, atrophic gastritis and dysplasia patient to the MG7-Ag positive follows up a case by regular visits to simultaneously, it changes the cancer rate and significantly increases (Liu J, Hu JL, FanDM et al.Int J Clin Pract.2002; 56 (3): 169-172).Country's 973 projects (G1998051203) confirm by strict " double blinding " prediction retrospective study, the gastric epithelial intestinesization are given birth to, dysplasia is the MG7-Ag positive cases and compares with negative expresser; lesion growth is dangerous to increase more than 25～40 times; development takes place for prompting MG7-Ag and cancer of the stomach and differentiation has good correlativity, have important use to be worth for the high-risk individuality that detects cancer of the stomach.
Existing detection by quantitative for MG7-Ag mainly is by enzyme linked immunological (ELISA) method, resists steps such as reacting, develop the color, read plate comprising envelope antigen, antigen-antibody reaction, two.Because MG7-Ag belongs to micro-antigen in the serum, content is very little, and enzyme-linked immunoassay method susceptibility only reaches the ng level, and precision is not high, occurs error easily, can't realize accurately quantitatively.
The real-time fluorescence quantitative PCR technology comes across 1996, and the core of this technology is to add fluorophor in the PCR reaction system, utilizes the fluorescence signal accumulation, monitors whole PCR process in real time, unknown template is carried out the method for quantitative test by typical curve.Early stage in the PCR reaction, the level that produces fluorescence can not be distinguished significantly with background; The generation of fluorescence then enters the index amplification phase, and fluorescence level is exponential increase thereupon; Fluorescence level reaches capacity when PCR arrives plateau at last, and enlarges system because round pcr is an index, and the index of slight error amplifies makes the end-product amount have bigger dispersion.And the real-time fluorescence quantitative PCR technology has solved the limitation that conventional P CR can only end point determination effectively, has realized that each takes turns the intensity that circulation all detects the first order fluorescence signal.Setting the check point fluorescent value is fluorescence threshold in the index amplification stage, when fluorescence signal intensity arrives the fluorescence threshold that sets, writes down fluorescence signal automatically and reaches the period (Ct value) that fluorescence threshold experiences.
Verified, there is linear relationship in the logarithm of the Ct value of each template and the initial copy number of this template, and initial copy number is many more, and the Ct value is more little.Therefore utilize the standard items of known initial copy number can make typical curve unknown sample is carried out quantitative measurement.Because when PCR circulates in the period that arrives Ct value place, just entered the real index amplification phase, this moment, slight error was not amplified as yet, so the reappearance of Ct value is fabulous, be amplification in same template different time amplification or the same asynchronism(-nization) pipe, it is constant obtaining the Ct value.Based on above two big characteristics, real-time fluorescence quantitative PCR is the outer typical curve quantitative methods of a kind of employing.
Owing to traditional quantivative approach all is to detect after PCR arrives plateau, and PCR increases arrival during plateau through exponential phase, and detection reappearance extreme difference can't directly be extrapolated the starting template amount from terminal point product amount.Mark can partly be eliminated the quantitative inaccuracy that causes of end-product in adding.But the interior mark that in testing sample, adds known initial copy number, then PCR reacts and becomes double PCR, have interference and competition between two kinds of templates in the double PCR reaction, especially when the initial copy number of two kinds of templates differed bigger, this competition can show more significantly.Because the initial copy number of testing sample is unknown, so the known template that can't add suitable quantity is as interior mark.Also this reason just, though traditional quantivative approach add in mark, but still be a kind of sxemiquantitative, rough quantitative methods.External standard method quantitatively and the conclusion of the methodology of inner mark method ration after relatively be: internal standard method is insecure as quantitative or semiquantitative means, and the quantivative approach of outer typical curve be a kind of accurately, trustworthy scientific approach.
Along with constantly popularizing of real-time fluorescence quantitative PCR technology, this technology has been widely used in key areas such as basic scientific research, clinical diagnosis, disease research and medicament research and development.Mainly concentrate on the following aspects: 1, the absolute quantitation analysis of DNA or RNA comprises the detection of pathogenic microorganism or viral level, the detection of genetically modified animals and plants transgenosis copy number, the detection of RNAi gene inactivation rate etc.2, gene expression difference analysis, for example relatively pass through the differential expression (as drug treating, physical treatment, chemical treatment etc.) of specific gene between the different disposal sample, specific gene is in the conclusive evidence of the differential expression of phase and cDNA chip or difference display result simultaneously not.3, Genotyping, for example SNP detects, and detection etc. methylates.But the real-time fluorescence quantitative PCR technology only limits to nucleic acid quantification.
Application number is that 200910021300.7 Chinese invention patent discloses MG7-Ag detection of antigens method in a kind of serum, this method is based on the MG7-Ag in the antigen-antibody reaction seizure serum, and then biotinylation DNA and biotinylation two resistive connections are closed by streptavidin, protein signal is changed into nucleic acid signal, carry out Real-time pcr amplification amplifying signal then, by GCAA MG7Ag in the quantitative serum of real time fluorescent quantitative immuno-PCR.But still there is certain defective in said method, when MG7-Ag in the serum is caught in antigen-antibody reaction in the serum a large amount of non-specific materials can influence cancer of the stomach tumor associated antigen bag by efficient; Through repeatedly conversion of signals and after PCR amplifies, each link all can directly influence the specificity and the stability of method on this basis.
Summary of the invention
The object of the invention is to provide a kind of quantitative detecting method based on MG7-Ag in the serum of double antibody identification, improves the specificity and the accuracy of the MG7-Ag detection by quantitative relevant with cancer of the stomach of the trace in the serum.
The present invention is achieved through the following technical solutions:
1) bag quilt: be cushioned liquid with monoclonal antibody MGb2 concentration dilution to 10 μ g/ml with bag, join then in the realtime-PCR reaction tube, each reaction tube adds 50 μ l, hatches behind the 1h for 37 ℃ and washs with PBS-T liquid;
2) sealing: each reaction tube adds 5mg/ml BSA as confining liquid, hatches behind the 1h with the washing of PBS-T liquid for 37 ℃;
3) antigen-antibody reaction: after sealing is finished, add standard items or the test sample of 50ml in the corresponding reaction tube, 4 ℃ are spent the night, and discard liquid, then with the washing of PBS-T liquid;
Described standard items are the MG7-Ag standard items of gradient concentration dilution; Described test sample is a test serum;
4) biotinylated MG7 antibodies: after antigen-antibody reaction was finished, each reaction tube added the biotinylated MG7 antibody of 50ml, hatched behind the 1h with the washing of PBS-T liquid for 37 ℃;
5) streptavidin combination: after biotinylated MG7 antibodies was finished, each reaction tube added the streptavidin of 50ml, hatches 30min for 37 ℃, the washing of PBS-T liquid;
6) biotinylated DNA reporter molecules combination: streptavidin is in conjunction with after finishing, and each reaction tube adds the biotinylation DNA of 50ml, hatches behind the 30min with the washing of PBS-T liquid for 37 ℃;
7) real-time fluorescence quantitative PCR reacts: each reaction tube gained biotinylation DNA is that template is carried out the real-time fluorescence quantitative PCR reaction respectively after finishing with washing;
The PCR reaction conditions is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 45s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s; Extended 10 minutes in 72 ℃ after 35～40 loop ends;
Elongating temperature with pcr amplification biotinylation DNA locates to read the fluoroscopic examination amount for 72 ℃, obtains the pcr amplification curve of every hole correspondence;
8) drawing standard curve: according to one group of pcr amplification curve of standard QC correspondence, set the fluoroscopic examination threshold value, obtain one group of period of the corresponding standard QC at this fluoroscopic examination threshold value place; Standard items concentration according to each standard QC reaches the corresponding relation of the period of fluoroscopic examination threshold value with it, sets up typical curve;
9) determine the period of test sample: one group of pcr amplification curve according to test sample pipe correspondence obtains the period that every hole test sample reaches fluoroscopic examination threshold value place;
10) determine the MG7-Ag content of test sample:, determine its MG7-Ag content according to the period of every hole test sample according to typical curve.
Described biotinylation DNA is biotin labeled non-specific sequence DNA.
The fluoroscopic examination threshold value that sets is in the index stage of pcr amplification curve.
Described standard items are the MG7-Ag of purifying.
Described standard items are the lysate of the stomach cancer cell of surface expression MG7-Ag.
Compared with prior art, beneficial technical effects of the present invention:
1, the quantitative detecting method based on MG7-Ag in the serum of double antibody identification provided by the invention, described double antibody is to adopt the specific seizure of double antibody sandwich method MG7-Ag, one of double antibody be at bag by the MGb2 monoclonal antibody in stage, two be biotinylated MG7 antibody; The principal feature of double antibody identification is the two-fold identification to antigen, improves the specificity of antigen-antibody reaction, guarantees the specificity and the susceptibility of follow-up expansion signal;
Utilization of the present invention be connected in MGb2 antibody on the solid phase carrier (realtime-PCR reaction tube) and biotinylated MG7 antibody respectively with sample on the detected antigen molecule two antigenic determinants combine, form solid matrix antibody-antigen-enzyme labelled antibody immune complex; Because the amount of insolubilized antibody and enzyme labelled antibody is excessive with respect to determined antigen in the reactive system, so the formation amount of compound is directly proportional with the content of determined antigen, can determine determined antigen content;
On the basis that forms immune complex, combine with biotinylated DNA reporter molecules, the antigenic content signal is converted into the DNA original copy number that can enter the index amplification by streptavidin; Detect by the real-time fluorescence quantitative PCR that can accurately detect original copy number again, as reference, measure the MG7-Ag in the serum accurately with the typical curve of standard items amplification;
Therefore, immunoreactive design of the present invention has guaranteed the specificity that antigen is caught, and cleverly antigen signals conversion has been guaranteed the susceptibility and the specificity that detect.
2, the present invention is based on consideration to the purity of the specific recognition of MG7-Ag and antibody for the selection of double antibody, and the concrete bag of selecting is caught GCAA, biotinylated MG7 antibody submission MG7-Ag cont signal by the MGb2 monoclonal antibody;
Compare as the basis of submission MG7-Ag cont signal with the antigen-antibody immune complex that MG7-Ag constitutes with adopting MG7, the basis of the biotinylated MG7 antibody of the present invention submission antigenic content signal is that the MGb2 monoclonal antibody is caught GCAA, excluded the competition bag quilt of nonspecific material and MG7-Ag to a great extent, and MG7 antibody and the non-specific material cross-immune reaction that may exist;
Because the at present clinical antibody that is used to detect tumor markers is cancer of the stomach specific antibody preferably not still; And the applicant is the specific cancer of the stomach associated antibodies of two plant heights through experiment confirm MGb2 and MG7 for many years, has been guaranteed the hypersensitivity and the high specific of this experimental technique by their combinations of pairs of actual detected explanation of the present invention;
And MGb2 and MG7 be monoclonal antibody, can guarantee its quality and purity like this, and help the standardization that MG7-Ag detects; The MGb2 monoclonal antibody specifically referring to:
(1)Zhang?F，Ren?G，Lu?Y，Fan?D.Identification?of?TRAK1(Traffickingprotein，kinesin-binding?1)as?MGb2-Ag：a?novel?cancer?biomarker.Cancer?Lett.2009?18；274(2)：250-8.；
(2)Li?S，Zhang?XY，Chen?XT，Fan?DM，Zhan?SY，Chen?LJ，Shu?YH，ZhangJL.Specific?targeting?of?mitomycin?C?to?tumors?by?anti-gastric?cancer?monoclonalantibody.Chin?Med?J(Engl).1991；104(5)：358-62；
In addition, detection method provided by the invention can accurately detect MG7-Ag, and the improvement of the step of its concrete operations has following useful effect:
3, because the present invention's antigen to be detected is the very little cancer of the stomach tumor associated antigen MG7-Ag of content in the serum, if directly adopt the test serum direct coated, in fact the bag that adheres to is non-material-specific by the composition major part, a large amount of non-specific material even can compete micro-GCAA bag by the site, thus the susceptibility and the specificity of detection method influenced; And the difference of non-special composition also directly influences the pattern detection result in the serum specimen, has brought uncertainty to experimental result;
Compare with the method that adopts antigen (test serum) direct coated, the present invention adopts monoclonal antibody MGb2 direct coated realtime-PCR reaction tube, GCAA is understood specific identification with the MGb2 monoclonal antibody that adheres to like this, after washing, exclude non-specific material, avoided itself and biotinylated MG7 non-specific binding afterwards and the error of the Detection of antigen amount that causes;
In addition, if before carrying out realtime-PCR amplification, need the antigen-antibody immunoreactant changed over to and carry out subsequent detection in the PCR pipe, this step is subjected to operating instrument and operator's influence very big, can make detection method produce error, and PCR method is very responsive, and slight error will cause result's very big-difference; The present invention directly is coated on the realtime-PCR tube wall with the MGb2 monoclonal antibody, and subsequent reactions is finished in pipe successively, has avoided personal error, has improved the stable and repeatable of method greatly.
4, by the protein molecular signal be converted into enter behind the dna molecular signal PCR expansion approach be adopt realtime-PCR detect in the middle of very important signal step of converting;
Biotinylation two is anti-as comparing in conjunction with the bridge of MG7 and streptavidin with adopting, because it generally is polyclonal antibody that biotinylation two resists, its specificity and susceptibility all can not show a candle to monoclonal antibody, although MG7 monoclonal antibody susceptibility and specificity height, but, can reduce the susceptibility and the specificity of whole experiment owing to the two anti-specificitys and the not high reason of tiring; And adopt biotinylation MG7 antibody to combine with GCAA, and reduce biotinylation two resistive connections to close this link of step, overcome the influence that this experimental result produces because two resistances are verified, also simplified the experimental implementation step simultaneously.
5, not only have high specific and hypersensitivity based on the quantitative detecting method of MG7-Ag in the serum of double antibody identification but also improved the stability of experiment and repeated;
Because the simplification of design of the antibody of capture antigen and operation steps makes in the middle of the actual detected that repeatedly the stability and the repeatability of inspection all are improved, can monitor MG7-Ag before the cancer of the stomach art, postoperative, the expression difference among the precancerous lesion and the crowd that haves a medical check-up;
Testing result shows that the Mg7-Ag content in cancer patient's serum exceeds more than 10 times than normal population mean value.
6, compare with the detection method of antigen direct coated, because it needs direct coated blood sample to be measured, because sample difference to be checked must be finished by applying unit oneself; And the present invention adopts the monoclonal antibody of bag quilt and sealing step to be finished by production division's standardization, has further improved the versatility and the repeatability of experimental technique, and commensurate's testing result comparability is not stronger; Simultaneously, reduced user's operation steps, operability is stronger, is beneficial to promoting the use of of detection method.
Description of drawings
Fig. 1 is a real time fluorescent quantitative immuno-PCR reaction process synoptic diagram;
Fig. 2 is the real-time fluorescence PCR amplification curve diagram of standard items;
Fig. 3 is the typical curve of drawing according to the Ct value of the concentration of standard items and amplification;
Fig. 4 is the real-time fluorescence PCR amplification curve diagram of product to be tested.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further details.
The present invention realizes that in conjunction with real time fluorescent quantitative immuno-PCR detection method accurate quantification detects trace antigen MG7-Ag content in the serum by identification, the seizure of double antibody to MG7-Ag on the basis of antigen signals conversion.
The real-time fluorescence quantitative PCR technology is combined with specific antigen-antibody reaction system, set up the real time fluorescent quantitative immune PCR technique.This technology is by MG7-Ag in the specific identification serum of double antibody, then with streptavidin respectively in conjunction with biotinylation MG7 and biotinylated DNA, after the signal of the MG7-Ag content in the serum is converted into nucleic acid signal, enter real-time fluorescence quantitative PCR expansion approach, thereby accurately detect trace antigen MG7-Ag in the serum, its reaction process as shown in Figure 1.
According to the standard items and the corresponding relation that reaches the period (Ct value) of setting the fluoroscopic examination threshold value of variable concentrations, set up typical curve, then according to the Ct value of detected sample, on typical curve, determine the content of the MG7-Ag of detected sample.
The quantitative detecting method material requested of setting up mainly comprises: confining liquid, MGb2 monoclonal antibody, biotinylated MG7 antibody, streptavidin, biotinylated DNA, real time-PCR kit.Be specially:
Bag is cushioned liquid: pH9.6,0.05M carbonate buffer solution;
Confining liquid: 5mg/ml bovine serum albumin(BSA) (BSA);
Dilution: the 0.15M PBS damping fluid that contains 1mg/ml BSA;
Lavation buffer solution: the pH7.4, the 0.15M phosphate buffer (PBS-T liquid) that contain 0.05%Tween-20;
MGb2 monoclonal antibody: be mouse monoclonal antibody, open source literature: Li S, Zhang XY, Zhang SY, Chen XT, Chen LJ, Shu YH, Zhang JL, Fan DM.Preparation ofantigastric cancer monoclonal antibody MGb2-mitomycin C conjugate withimproved antitumor activity.Bioconjug Chem.1990; 1 (4): 245-50; Laboratory purifying preparation;
MG7 antibody: be mouse monoclonal antibody, open source literature: Fan DM, Zhang XY, ChenXT et al.Journal of Gastroenerology and Hepatology.2005; 20 (3): 360-365, the preparation of laboratory purifying;
Biotinylation MG7 antibody: adopt photobiotin method mark MG7 antibody, and with desalination pillar purifying, adopt ultraviolet spectrophotometer quantitative and calculate labelled antibody concentration then;
Streptavidin, real time-PCR kit all are obtained commercially.Wherein, the streptavidin that present embodiment adopts is available commercially from Thermo company; Real time-PCR kit is available commercially from TaKaRa company;
Biotinylated DNA: described dna sequence dna is non-specific sequence, carries out biotin labeling according to the routine techniques means.Present embodiment adopts photobiotin method marker DNA, and uses the ethanol precipitation purifying; Adopt ultraviolet spectrophotometer quantitatively and calculate the marker DNA molal quantity.
Described standard items adopt the MG7-Ag that determines content: such as the MG7-Ag of the definite purifying of concentration; The perhaps lysate of the stomach cancer cell of surface expression MG7-Ag, as stomach cancer cell SGC7901, MKN45, KATOIII clone etc.; Perhaps other contain the standard items of the preparations such as tissue, body fluid, cell of MG7-Ag.
With the MG7-Ag of purifying as standard items, the concentration content of trace antigen MG7-Ag in the detection by quantitative serum; As standard items, the content of trace antigen MG7-Ag can be represented with the cell number of stomach cancer cell SGC7901 in the detection by quantitative serum with stomach cancer cell SGC7901.
When embodiment adopted the standard items of MG7-Ag of purifying of gradient concentration dilution, its purification process was as follows:
1) preparation of tissue homogenate: fresh stomach organization with physiological saline flush away bloodstain and dirt, is removed coating or connective tissue in 4 ℃ of water-baths or ice bath, the tissue of cleaning is cut into 0.3～0.5cm
3Fritter adds an amount of physiological saline, packs into to smash to pieces and makes tissue homogenate in the machine barrel.Tissue homogenate is removed cell and fragment of tissue behind the centrifugal 10min of 3000r/min, supernatant retains stand-by.
Perhaps clasmatosis: collect stomach cancer cell line SGC7901 or MKN45, after the cell count, cell was put in the liquid nitrogen 10 minutes, take out 37 ℃ of thawings three frozen-thawed cells of repeatable operation then; Remove cell fragment, behind the centrifugal 10min of 3000r/min, supernatant retains stand-by.
2) MG7-Ag slightly carries: saturated ammonium sulfate saltout tissue homogenate or lysis supernatant, and make the ammonium sulfate saturation degree reach 50%, precipitate 4 ℃ and spend the night, use pH7.2,0.15mol/L PBS dialysis three times is crossed the DEAE gel column and is slightly carried, and MG7-Ag is carried out preliminary purification.
3) affinitive layer purification MG7-Ag: preparation MG7 affinity column, with MG7-Ag crude extract pH7.2,0.15mol/L PBS dialysed overnight, last sample is after pH7.2, the MG7 affinity column of 0.15mol/L PBS balance, flow velocity 0.5ml/ minute; Be washed till eluent OD280≤0.02 with 0.15mol/L PBS stream then, use pH2.4,0.1mol/L glycocoll-HCl wash-out again, eluent goes to pH7.2 immediately, and dialysed overnight promptly and is measured its protein concentration, purity and activity among the 0.15mol/LPBS.
Embodiment
As standard items, the MG7-Ag content in the test serum of detection by quantitative specifically may further comprise the steps with tumor cell lysate:
1) bag quilt: be cushioned liquid with monoclonal antibody MGb2 concentration dilution to 10 μ g/ml with bag, add then in the realtime-PCR reaction tube, every pipe adds 50 μ l, hatches 1h for 37 ℃, the washing of PBS-T liquid;
2) sealing: every pipe adds 5mg/ml BSA as confining liquid, hatches 1h for 37 ℃, the washing of PBS-T liquid;
3) antigen-antibody reaction: after sealing is finished, add the standard items of 50 μ l in the standard items reaction tube, as the standard control that detects test serum, specifically with stomach cancer cell MKN45 lysate as standard items:
To after the MKN45 cell count clasmatosis be prepared cell lysate, then with 5 * 10
6, 5 * 10
5, 5 * 10
4, 5 * 10
3, 5 * 10
2, 5 * 10
1, the dilution of 5,0 gradient concentration, the reaction tube of the corresponding different labels of each concentration adds 50 μ l cell lysates, as standard control;
Test serum adds respectively in the test serum reaction tube of different labels, every pipe 50 μ l; Test sample is the test serum of preparation: extract 2ml blood, room temperature left standstill 30 minutes, centrifugal 20 minutes of 3000rpm, draw supernatant put 4 ℃ stand-by; Every pipe adds the test serum of 50 μ l preparation;
Add behind standard items or the test serum 4 ℃ in the reaction tube and spend the night, discard liquid, the washing of PBS-T liquid;
4) biotinylated MG7 antibodies: after antigen-antibody reaction was finished, every pipe all added the biotinylated MG7 antibody of 50 μ l, hatches 1h for 37 ℃, the washing of PBS-T liquid;
5) streptavidin combination: after biotinylated MG7 antibodies was finished, each realtime-PCR reaction tube all added the streptavidin of 50 μ l, hatched 30min for 37 ℃, the washing of PBS-T liquid;
6) biotinylated DNA reporter molecules combination: each realtime-PCR reaction tube all adds the biotinylation DNA of 50 μ l, hatches 30min for 37 ℃, the washing of PBS-T liquid;
Be specially: as the DNA reporter molecules, it is carried out joining in the realtime-PCR reaction tube behind the biotinylation with the cDNA of ROBO1 gene (NM_002941); It carries out the fragment that pcr amplification obtains the 248bp size, and the primer of amplification is to being:
Forward primer P:gctctagagc atccctgtct taactg 26;
Reverse primer R:gctctagagc aaacgcttct caacat 26;
7) real-time fluorescence quantitative PCR reaction: with gained biotinylation DNA is that template is carried out the real-time fluorescence quantitative PCR reaction respectively, and wherein the PCR reaction conditions is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 45s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s; In 72 ℃ of extensions 10 minutes, locate to read the fluoroscopic examination amount for 72 ℃ after 38 loop ends, obtain the pcr amplification curve of every hole correspondence with the elongating temperature of pcr amplification biotinylation DNA.
8) drawing standard curve: according to one group of pcr amplification curve of standard items correspondence, set the fluoroscopic examination threshold value, obtain one group of period of the corresponding standard items at this fluoroscopic examination threshold value place; Standard items concentration according to each standard QC reaches the corresponding relation of the period of fluoroscopic examination threshold value with it, sets up typical curve;
9) determine the period of test sample: one group of pcr amplification curve according to the test sample correspondence obtains the period that every hole test sample reaches described fluoroscopic examination threshold value place;
10) determine the MG7-Ag content of test sample:, determine its MG7-Ag content according to the period of every hole test sample according to typical curve.
According to above-mentioned steps 8), 9), 10), present embodiment obtains standard items group amplification curve fluoroscopic examination threshold setting as shown in Figure 2, makes its corresponding amplification curve be in the index amplification stage; The concrete fluoroscopic examination threshold value of setting of present embodiment is 0.020;
Fig. 3 is the typical curve of drawing according to the testing result of standard items, and the detection numerical value of standard items is as shown in table 1; The amplification curve of test sample group as shown in Figure 4; The detected value that obtains the test sample group according to typical curve is as shown in table 2.
The standard items that obtain variable concentrations from standard items group amplification curve reach the period (Ct value) of setting the fluoroscopic examination threshold value; The logarithm value of the concentration of the MG7-Ag of standard items and Ct value are linear, and typical curve as shown in Figure 3, horizontal ordinate are that Ct value, ordinate are the logarithm value of the concentration content of antigen MG7-Ag.The test sample that obtains variable concentrations from one group of amplification curve of test sample reaches the period (Ct value) of setting the fluoroscopic examination threshold value; According to typical curve, determine the concentration content of the MG7-Ag of testing sample according to detected sample gained Ct value.
The real-time fluorescence PCR testing result of table 1 MG7-Ag standard items group
| The standard items sequence number | The Ct value | ????MG7-Ag | The standard items sequence number | The Ct value | ????MG-7Ag |
| ??D1 | ?17.224 | ????5×10 6 | ??E1 | ?17.233 | ????5×10 6 |
| ??D2 | ?13.172 | ????5×10 5 | ??E2 | ?15.248 | ????5×10 5 |
| ??D3 | ?12.811 | ????5×10 4 | ??E3 | ?15.510 | ????5×10 4 |
| ??D4 | ?15.379 | ????5×10 3 | ??E4 | ?16.058 | ????5×10 3 |
| ??D5 | ?19.103 | ????5×10 2 | ??E5 | ?18.205 | ????5×10 2 |
| ??D6 | ?20.062 | ????5×10 1 | ??E6 | ?18.311 | ????5×10 1 |
| ??D7 | ?20.487 | ????5×10 0 | ??E7 | ?20.006 | ????5×10 0 |
| ??D8 | ?20.318 | Blank | ??E8 | ?20.279 | Blank |
Wherein, the MG7-Ag standard items are to represent with the lysate behind the tumour cell counting.
According to typical curve, as shown in table 2 in conjunction with the antigenic content that its Ct value obtains test serum:
Table 2 cancer is made a definite diagnosis MG7-Ag real-time fluorescence PCR testing result in the group and the serum of healthy population group
| The product to be tested sequence number | The Ct value | ??MG7-Ag | The product to be tested sequence number | The Ct value | ??MG-7Ag | Clinical diagnosis |
| ??A1 | ??17.166 | ??653.856 | ??B1 | ??16.340 | ??1520.41 | The orifice of the stomach well-differentiated adenocarcinoma, the little curved metastatic carcinoma of seeing |
| ??A2 | ??15.791 | ??2697.87 | ??B2 | ??15.248 | ??4665.91 | Poorly differentiated adenocarcinoma in the orifice of the stomach, the part carcinoma muco-cellulare |
| The product to be tested sequence number | The Ct value | ??MG7-Ag | The product to be tested sequence number | The Ct value | ??MG-7Ag | Clinical diagnosis |
| ??A3 | ??14.716 | ??8070.28 | ??B3 | ??15.394 | ??4027.03 | Poorly differentiated adenocarcinoma, artery lymph node 3/5 lesser curvature 2/5 has transfer |
| ??A4 | ??14.562 | ??9335.67 | ??B4 | ??15.173 | ??5001 | Greater curvature senior middle school differentiation gland cancer |
| ??A5 | ??15.652 | ??3075.44 | ??B5 | ??15.011 | ??5901.54 | The little curved lymph node of postoperative cardiac carcinoma is seen transfer |
| ??A6 | ??14.698 | ??8129.36 | ??B6 | ??14.601 | ??8969.4 | The orifice of the stomach poorly differentiated adenocarcinoma, the lesser curvature side lymph node is seen metastatic carcinoma |
| ??A7 | ??13.893 | ??18481.2 | ??B7 | ??13.646 | ??23786.9 | Poorly differentiated adenocarcinoma in the cardielcosis type |
| ??A8 | ??13.122 | ??40606.9 | ??B8 | ??14.024 | ??16176.3 | Remnant gastric cancer T4N1M0 |
| ??F1 | ??18.326 | ??200.093 | ??G1 | ??19.951 | ??38.0854 | The healthy population group |
| ??F2 | ??19.055 | ??95.1216 | ??G2 | ??18.989 | ??101.681 | The healthy population group |
| ??F3 | ??15.974 | ??2209.24 | ??G3 | ??17.475 | ??477.205 | The healthy population group |
| ??F4 | ??16.013 | ??2123.43 | ??G4 | ??15.266 | ??4549.38 | The healthy population group |
| ??F5 | ??19.814 | ??43.8007 | ??G5 | ??17.337 | ??549.325 | The healthy population group |
| ??F6 | ??15.843 | ??2524.54 | ??G6 | ??15.199 | ??4870.46 | The healthy population group |
| The product to be tested sequence number | The Ct value | ??MG7-Ag | The product to be tested sequence number | The Ct value | ??MG-7Ag | Clinical diagnosis |
| ??F7 | ??16.801 | ??949.235 | ??G7 | ??17.332 | ??552.434 | The healthy population group |
| ??F8 | ??18.071 | ??259.601 | ??G8 | ??18.884 | ??113.228 | The healthy population group |
Wherein, the result of MG7-Ag be expressed as with test serum in the suitable cancer of the stomach MKN45 cell number of MG7-Ag content that comprises;
A, B group is made a definite diagnosis group for cancer, and F, G group is the healthy population group, can clearly distinguish cancer with the MG7-Ag content that detects gained and make a definite diagnosis group and healthy population, and the Mg7-Ag content in cancer patient's serum exceeds more than 10 times than normal population mean value; The present invention is to have susceptibility and specific as the detection by quantitative of micro-antigen MG7-Ag;
And the MG7-Ag testing result of comparison A, B group, it can also be seen that detection method of the present invention obtain the result of MG7 in the serum traced into cancer of the stomach by stages, the variation of the relevant relevant antigenic content of classification: MG7Ag content is very high in cancer patient's serum that low differentiation companion shifts, well-differentiated carcinoma, do not see that MG7Ag content is low slightly among the patients serum of transfer, but exceed normal yet; Part precancerous lesion patient detected value is higher needs follow-up observation; Show that this method detects MG7Ag and contains the desired accuracy of measurer clinical diagnosis, can be used for the diagnosis of cancer of the stomach and people at highest risk's examination, and testing result process applicant's repeated detection shows to have stability and repeatability.
Claims (5)
1. the quantitative detecting method based on MG7-Ag in the serum of double antibody identification is characterized in that, may further comprise the steps:
1) bag quilt: be cushioned liquid with monoclonal antibody MGb2 concentration dilution to 10 μ g/ml with bag, join then in the realtime-PCR reaction tube, each reaction tube adds 50 μ l, hatches behind the 1h for 37 ℃ and washs with PBS-T liquid;
2) sealing: each reaction tube adds 5mg/ml BSA as confining liquid, hatches behind the 1h with the washing of PBS-T liquid for 37 ℃;
3) antigen-antibody reaction: after sealing is finished, add standard items or the test sample of 50 μ l in the corresponding reaction tube, 4 ℃ are spent the night, and discard liquid, then with the washing of PBS-T liquid;
Described standard items are the MG7-Ag standard items of gradient concentration dilution; Described test sample is a test serum;
4) biotinylated MG7 antibodies: after antigen-antibody reaction was finished, each reaction tube added the biotinylated MG7 antibody of 50 μ l, hatched behind the 1h with the washing of PBS-T liquid for 37 ℃;
5) streptavidin combination: after biotinylated MG7 antibodies was finished, each reaction tube added the streptavidin of 50 μ l, hatched 30min for 37 ℃, the washing of PBS-T liquid;
6) biotinylated DNA reporter molecules combination: streptavidin is in conjunction with after finishing, and each reaction tube adds the biotinylation DNA of 50 μ l, hatches behind the 30min with the washing of PBS-T liquid for 37 ℃;
7) real-time fluorescence quantitative PCR reacts: each reaction tube gained biotinylation DNA is that template is carried out the real-time fluorescence quantitative PCR reaction respectively after finishing with washing;
The PCR reaction conditions is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 45s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s; Extended 10 minutes in 72 ℃ after 35～40 loop ends;
Elongating temperature with pcr amplification biotinylation DNA locates to read the fluoroscopic examination amount for 72 ℃, obtains the pcr amplification curve of every hole correspondence;
8) drawing standard curve: according to one group of pcr amplification curve of standard QC correspondence, set the fluoroscopic examination threshold value, obtain one group of period of the corresponding standard QC at this fluoroscopic examination threshold value place; Standard items concentration according to each standard QC reaches the corresponding relation of the period of fluoroscopic examination threshold value with it, sets up typical curve;
9) determine the period of test sample: one group of pcr amplification curve according to test sample pipe correspondence obtains the period that every hole test sample reaches fluoroscopic examination threshold value place;
10) determine the MG7-Ag content of test sample:, determine its MG7-Ag content according to the period of every hole test sample according to typical curve.
2. a kind of quantitative detecting method based on MG7-Ag in the serum of double antibody identification as claimed in claim 1 is characterized in that described biotinylation DNA is biotin labeled non-specific sequence DNA.
3. a kind of quantitative detecting method based on MG7-Ag in the serum of double antibody identification as claimed in claim 1 is characterized in that the fluoroscopic examination threshold value that sets is in the index stage of pcr amplification curve.
4. a kind of quantitative detecting method based on MG7-Ag in the serum of double antibody identification as claimed in claim 1 is characterized in that described standard items are the MG7-Ag of purifying.
5. a kind of quantitative detecting method based on MG7-Ag in the serum of double antibody identification as claimed in claim 1 is characterized in that described standard items are the suspending liquid of the stomach cancer cell of surface expression MG7-Ag.
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| CN109666718A (en) * | 2018-12-17 | 2019-04-23 | 扬州大学 | A kind of remaining PCR pipe formula detection method of antibody-mediated trace imidacloprid pesticide |
| CN110687293A (en) * | 2018-07-06 | 2020-01-14 | 中国人民解放军第四军医大学 | Kit for detecting gastric cancer antigen MG7-Ag and application thereof |
| CN115651966A (en) * | 2022-12-13 | 2023-01-31 | 北京天浚元生生物科技有限公司 | Method for detecting trace biomarkers through double-antibody mediated PCR |
| CN116640830A (en) * | 2023-05-04 | 2023-08-25 | 河北国高生物科技有限公司 | immuno-PCR working solution and preparation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103383396A (en) * | 2012-05-03 | 2013-11-06 | 苏州博泰安生物科技有限公司 | Artificial DNA replication regulation and control switch and its preparation method and use in antibody detection |
| CN103383396B (en) * | 2012-05-03 | 2015-08-12 | 苏州博泰安生物科技有限公司 | Artificial dna copies regulating switch and preparation method thereof and the application detecting antibody |
| CN110687293A (en) * | 2018-07-06 | 2020-01-14 | 中国人民解放军第四军医大学 | Kit for detecting gastric cancer antigen MG7-Ag and application thereof |
| CN109666718A (en) * | 2018-12-17 | 2019-04-23 | 扬州大学 | A kind of remaining PCR pipe formula detection method of antibody-mediated trace imidacloprid pesticide |
| CN115651966A (en) * | 2022-12-13 | 2023-01-31 | 北京天浚元生生物科技有限公司 | Method for detecting trace biomarkers through double-antibody mediated PCR |
| CN116640830A (en) * | 2023-05-04 | 2023-08-25 | 河北国高生物科技有限公司 | immuno-PCR working solution and preparation method and application thereof |
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