CN1232636C - Method for detecting human tumor antigen P 185 Her-2 and its application in diagnosing tumor - Google Patents
Method for detecting human tumor antigen P 185 Her-2 and its application in diagnosing tumor Download PDFInfo
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Abstract
The present invention relates to a method for detecting double antibody sandwich ELISA of soluble p185<HER-2> antigens, which is characterized in that purified p185<HER-2> monoclonal antibodies prepared by adopting a surface epitope embedding method are used as coated antibodies to be combined on solid supporters, samples containing soluble p185<HER-2>, purified p185<HER-2> standard antigens with known concentration and the coated antibodies are incubated respectively, and another kind of purified p185<HER-2> monoclonal antibodies which are marked by horseradish peroxidase and are perpared by adopting a surface epitope embedding method is used as detecting antibodies. The purified p185<HER-2> standard antigens are prepared by that affinity chromatography is carried out to samples containing p185<HER-2> antigens by using anti p185<HER-2> extracellular region monoclonal antibodies prepared by adopting a surface epitope embedding method, and affinity balls of Sepharose 4B-ProteinG covalent cross-linking. The detecting method has the advantages of strong specificity of the detection of soluble p185<HER-2> antigens, and high purity of standard soluble antigens. The detecting method can be used for the diagnosis of tumor sera.
Description
Technical field:
The present invention relates to the preparation of tumour antigen, the immunology detection technology of solubility tumor marker and the application in diagnosing tumor thereof.
Background technology:
P185
HER-2Be a kind of important tumor cell surface transmembrane glycoprotein, surplus mammary gland, ovary, lung, stomach, liver etc. ten, all show the p185 of overexpression in kind of the cancer by oncogene neu/erbB-2/HER-2 coding
HER-2Albumen.According to U.S.'s " science " magazine (Science, 1989,244:707-712) report has the mammary cancer more than 30% and the cell of ovarian cancer patient tumor tissues p185 to occur approximately
HER-2High expression level, such patient's malignancy of tumor degree height, poor prognosis." Britain's cancer magazine " (Br J Cancer, 1994,70,739-742) report detects p185
HER-2Differential diagnosis, judging prognosis to kinds of tumors are all significant down to treatment.Especially to mammary cancer, p185
HER-2By internationally recognized be an important clinical index." Chinese IMMUNOLOGY KEY WORDS INDEX (2000,16; 539-541) report is by the p185 of cell surface epi-position entrapping method development
HER-2Monoclonal antibody can be specifically and high expression level p185
HER-2The combination of cytolemma outskirt, to high expression level p 185
HER-2Tumor tissues has very strong film polarization and the dyeing of low endochylema, is particularly suitable for p185 in the clinical case tissue
HER-2Carrying out immunohistochemical method detects.Though the immunohistochemical methods method is at present comparatively conventional detection method, this method must could be used under the prerequisite of surgical resection sufferer tissue, and is not easy to detect gross sample and to the long-term follow of postoperative patient, and patient's perioperatively p185
HER-2Expression level to prognosis with instruct treatment plan to have important effect.Serum p185
HER-2Detection technique can dynamic observe and simple and easy to do patient, is more suitable for clinical needs.U.S.'s " journal of biological chemistry " (JBiol Chem, 1990; 266:1716-20.) report, use antibody at genetically modified high expression level p185
HER-2The cell strain culture supernatant in can detect solubility p185
HER-2It is the p105 level; And point out p185
HER-2Similar with other cell-membrane receptor molecules, its extracellular region can come off and enters serum from cell surface when overexpression.Dutch subsequently " breast cancer research and treatment " magazine (Breast cancer Res treat 1993; 24:97-102) and U.S.'s " cancer " magazine (Oncology1997; 54:475-481) etc. priority has been reported and has been utilized anti-p185
HER-2Antibody also adopts enzyme linked immunological absorption (ELISA) technology to carry out serum p185
HER-2Detect." U.S.'s clinical pathology magazine " (Am J ClinPathol, 1999:112 (suppl.1): S53-S67) summarized 16 relevant serum p185 in laboratory in the world
HER-2Result of study, point out: serum p185 antigen levels rising and tumor recurrence or transfer have very strong dependency, and are indicating the resistance to chemotherapy, radiotherapy and hormonotherapy, so serum p185
HER-2Antigen can be used as an important tumor marker for clinical use.
Existing preparation p185
HER-2Antigenic method mainly contains: the A method: as U.S.'s " clinical analysis magazine " (Journal ofClinical Analysis, 12:298-303,1998) report with Superose 12HR chromatography method from containing p185
HER-2Purifying p185 in the proteic cell pyrolysis liquid
HER-2Albumen, this method are to carry out separation and purification according to proteic molecular weight, do not have specificity, so p185
HER-2Antigen is lost easily, and purification efficiency is low; The B method: U.S.'s " cancer research " (Cancer Research, 51,2593-2598, May 15,1991) report with lectin affinity post from containing p185
HER-2Affinity chromatography p185 in the proteic cell pyrolysis liquid
HER-2Albumen, the affinity column that this method is used comprises p185 to all glycoprotein
HER-2Albumen all has adsorption, so specificity is low, and the antigenic purity of gained is low; The C method, as U.S.'s " cancer research " (CancerResearch, 51,2593-2598, May15,1991) report with activatory Sepharose 4B affinity column from containing p185
HER-2Affinity chromatography p185 in the cells and supernatant of extracellular region protein
HER-2Albumen, the affinity column that uses in this method needs antibody on the first mark, but the site of antibody conjugated antigen on localized diversity on the affine pearl of Sepharose 4B makes antibody is not exposed to outside the affine pearl fully, again because p185
HER-2Molecular weight bigger, therefore caused certain sterically hindered, p185
HER-2Antigen can not be fully with affinity column on antibodies, chromatography efficient is low; The D method, as U.S.'s " immunological method magazine " (Journal of Immunological Methods, 132,1990,73-80) Bao Dao elder generation is to containing p185
HER-2The cells and supernatant of extracellular region protein is carried out affinity chromatography, carry out ion exchange chromatography again, increased ion exchange chromatography in this method, ion exchange chromatography is to carry out purifying according to the difference of albumen iso-electric point, there is not specificity, therefore not only step is loaded down with trivial details, and has increased the chance of antigen losses.
The existing human serum p185 that detects
HER-2Method mainly contain: the A method, as the homogeneous phase magnetic particle method of Holland's " breast cancer research and treatment " (Breastcancer research and treatment, 43:87-95,1997) magazines report.This detection method with the magnetic particle of antibody labeling as solid phase carrier, operational requirement is higher, detect antibody and need acridiniumeaster on the mark, during detection acridinium easter is activated, the signal that produces is a fluorescent signal, need with special fluorescence detector, expense is higher, still is not easy to promote.The B method, as U.S.'s " tumor research " (cancer research 51,2593-2598, May 15,1991) Bao Dao radioimmunoassay: this detection method need will detect isotropic substance on the antibody labeling, the signal that produces is a gamma-rays, needs to detect with the γ calculating instrument, needs special protection during operation.The C method, as U.S.'s " clinical chemistry " (Clinical Chemistry 45, No2,1999,292-295) Bao Dao lectin-enzyme-linked immunoassay method (lectin-ELISA method), this method replaces antibody as detection reagent with lectin, specificity is low.The D method, as the tri-antibody sandwich enzyme-linked immune detection method (tri-antibody sandwich ELISA method) of U.S.'s " anticancer research " (Anticancerresearch 18:2891-2894,1998) report, this method steps is comparatively loaded down with trivial details, not only needs to prepare anti-p185
HER-2Monoclonal antibody, also to prepare anti-p185
HER-2How anti-, many anti-specificitys are strong inadequately, the stability between batch is not easy to keep.The E method, (Journal of Clinical Oncology, Vol 10, N09 (September), 1992,1436-1443) Bao Dao double-antibody sandwich enzyme-linked immunoassay method (double-antibody sandwich elisa method) as U.S.'s " clinical cancer magazine ".This method adopts the ELISA enzyme plate as solid-phase matrix, and is simple to operate; The signal of reaction is the reacted colour-change of enzyme substrates, and human body does not need to contact radio isotope, safety; Adopt the double antibody sandwich method high specificity, the stability of the monoclonal antibody that immortality cell produces is easy to keep.But existing E method is because the standard antigen of its use and A, B, C, D method are the same, normally adopt lectin affinity column affinity chromatography method, ion exchange layer analysis method, Superose 12HR chromatography method or Sepharose 4B affinity chromatography method to obtain, specificity is strong inadequately, and antigenic purity is low.
Summary of the invention:
The objective of the invention is: a kind of standard solubility p185 is provided
HER-2Antigenic preparation method, solubility p185
HER-2Antigenic double-antibody sandwich elisa detection method.
Standard solubility p185 of the present invention
HER-2Antigenic preparation method comprises preparing with antibody linked affine pearl, preparation and contains p185
HER-2Antigenic sample, will contain p185
HER-2Antigenic sample mixes with the affinity pearl, mixture is adorned post and wash-out antigen; It is characterized in that: described and antibody linked affine pearl is the anti-p185 that adopts surperficial epi-position entrapping method preparation
HER-2The affine pearl of the monoclonal antibody of extracellular region and Sepharose 4B-ProtcinG covalent cross-linking.
Solubility p185 of the present invention
HER-2Antigenic double-antibody sandwich elisa detection method comprises a kind of p185
HER-2Monoclonal antibody is incorporated on the solid support as coated antibody, will contain solubility p185 respectively
HER-2Sample and the p185 of the concentration known of purifying
HER-2Standard antigen and the antibody incubation that wraps quilt, p185 wherein
HER-2The coated antibody of antigen is optionally caught, and uses the p185 of another kind of enzyme labelling again
HER-2Monoclonal antibody makes to detect antibody, enzyme-added then substrate, and the enzyme immunity detection method of employing color reaction comes the photoabsorption of test sample; P185 with the purifying of concentration known
HER-2Standard antigen compares, and with the absorbance value of color reaction the concentration of standard antigen is mapped, and formulates standard straight-line; The absorbance value of testing sample is compared p185 in the calculation sample with the absorbance value of standard antigen
HER-2Concentration; It is characterized in that: (1) described p185 that is used in the double-antibody sandwich elisa detection method
HER-2Monoclonal antibody is purified and p185 horseradish peroxidase-labeled, that adopt surperficial epi-position entrapping method preparation
HER-2Monoclonal antibody; (2) p185 of described purifying
HER-2Standard antigen is the anti-p185 that adopts surperficial epi-position entrapping method preparation
HER-2The affine pearl of the monoclonal antibody of extracellular region and Sepharose4B-ProteinG covalent cross-linking is to containing p185
HER-2Antigenic sample carries out that the method for affinity chromatography obtains.
Solubility p185 of the present invention
HER-2The purposes of antigenic double-antibody sandwich elisa detection method in the tumour serum diagnosis, comprising will definitely dissolubility p185 with result's acceptance of the bid of double-antibody sandwich elisa color reaction
HER-2Antigenic absorbance value is to standard solubility p185
HER-2Antigenic concentration known mapping obtains a standard straight-line; The value that the average light absorption value of normal human serum reaction is added twice standard variance standard straight-line therewith relatively finds this to be worth pairing p185
HER-2Antigen concentration is made as threshold value; The absorbance value and the standard straight-line of human serum reaction to be measured are compared, calculate p185 in the serum
HER-2Antigenic concentration then is diagnosed as p185 greater than threshold value
HER-2The tumour patient of high expression level; It is characterized in that: the p185 of described purifying
HER-2Standard antigen is to adopt the affine pearl of the monoclonal antibody of surperficial epi-position entrapping method preparation and Sepharose 4B-ProteinG covalent cross-linking to containing p185
HER-2Antigenic sample carries out that the method for affinity chromatography obtains.
The present invention compared with prior art has the following advantages and specificity:
(1) solubility p185 of the present invention
HER-2The preparation method of standard antigen compared with prior art has the following advantages: with respect to the A method: present method has p185
HER-2Antigen and chromatography column bonded high specificity, the advantage that purification efficiency is high; With respect to the B method, present method also has p185
HER-2Antigen and chromatography column bonded high specificity, the advantage that the antigen purification degree is high; With respect to the C method, present method has and has overcome the advantage sterically hindered, that purification efficiency is high; With respect to the D method, present method has the advantage that step is simple, purification efficiency is high.Owing to utilize antibody to carry out the affinity chromatography method, be to carry out purifying, rather than carry out purifying whether to carry out purifying according to glycosylation according to the difference of molecular weight according to antigen-antibody bonded specificity, neither carry out purifying according to the difference of iso-electric point, so specificity improves greatly; Owing to adopt affine pearl of Sepharose 4B-ProteinG rather than Sepharose 4B or Sepharose4B-protein A as matrix, when antibody and its covalent cross-linking, Fc γ 1 fragment of antibody is by the proteinG specificity combination on the Sepharose 4B, on the antibody in conjunction with p185
HER-2Antigenic Fab fragment makes p185 outside the affine pearl of Sepharose 4B-ProteinG
HER-2Antigen and affine pearl bonded are sterically hindered to be reduced greatly, and purification efficiency is improved.
(2) solubility p185 of the present invention
HER-2Antigenic double-antibody sandwich elisa detection method is to adopt the monoclonal antibody of cell surface epi-position entrapping method preparation to detect solubility p185
HER-2Antigenic double-antibodies sandwich ELISA is further to improve on the basis of former E method, therefore has all advantages of former E method.And because standard antigen preparation method's improvement, with respect to former E method, the inventive method has purification of soluble standard p185
HER-2Antigenic efficient height, high specificity, the advantage that antigenic purity is high.Used antibody is the anti-p185 that adopts surperficial epi-position entrapping method preparation in the method
HER-2The monoclonal antibody of extracellular region, these monoclonal antibodies only are used in immunohistochemical methods at present and detect on the pathological section, and the tissue sample of excision when this detection need provide operation does not see that the ELISA that uses it for soluble tumour antigen detects and purifying p185
HER-2Antigenic report.We further discover, with horseradish peroxidase on the mark behind the antibody purification, and the character of antibody before and after the mark are carried out the detection of immunoblotting, and the result shows before and after the monoclonal antibody mark of these a few strain purifying and p185
HER-2The combination of extracellular region specificity, and stability is fine, and the affinity that we are different according to antibody is selected the array mode and the antibody working concentration of two kinds of the suitableeest antibody, is applied to solubility p185 first
HER-2Antigenic ELISA detection method, this detection do not need to perform an operation, and have good detection specificity and sensitivity.
(3) adopt solubility p185 of the present invention according to us
HER-2The double-antibody sandwich elisa detection method 88 routine tumour patients and 137 routine normal peoples' serum is carried out the result of repeated detection, prove detection method repeatability of the present invention, good stability.This detection method detects p185
HER-2Sensitivity reach 10 to 30ng/ml.x
2The result who analyzes shows that this method can obviously be distinguished tumour patient and normal population (P＜0.025), has practicality, can be applicable to clinical diagnosis.
Embodiment:
Embodiment 1, preparation contain solubility p185
HER-2Antigenic cell pyrolysis liquid and cells and supernatant:
(1) preparation contains solubility p185
HER-2Antigenic cell pyrolysis liquid:
Collect respectively the NIH/3T3 cell (inoblast of a kind of mouse) grow to 80% above full scale and T6-17 cell (transfection people's the NIH/3T3 cell of HER-2 gene, this cell surface high expression level p185
HER-2), PBS with cold 0.01M, PH7.2 washes once, the cold lysis buffer that adding is made up of 50mM Tris-HCl PH7.5,1%Trion X-100,150mM NaCl and 8mMPMSF, ice bath vibration 30 minutes, collect lysate, 4 ℃ are centrifugal, 12000rpm 10 minutes, and the supernatant of T6-17 cell is the cell pyrolysis liquid that contains the p185 crude antigen.The NIH/3T3 cell pyrolysis liquid is as negative control.
(2) preparation contains solubility p185
HER-2Antigenic cells and supernatant:
Use serum-free culture during the T6-17 passage instead, when to be covered with, collect the supernatant of cultivating, the culture supernatant of NIH/3T3 cell is as negative control.
Below the cell pyrolysis liquid that uses among each embodiment all by this method preparation, do not indicate in addition.
Embodiment 2, detection solubility p185
HER-2The foundation of antigenic double-antibody sandwich elisa method condition:
(1) cultivates the anti-p185 of energy specific secretion that obtains with cell surface epi-position entrapping method
HER-2The hybridoma of the immortality of extracellular region monoclonal antibody such as A18 and A21, amplification back plants in the abdominal cavity of Balb/c mouse in 6 to 8 weeks, treat long ascites after, take out ascites, be rough monoclonal antibody A18 and A21.
Monoclonal antibody A18 and A21 in the sad-ammonium sulfate two-step precipitation method purifying ascites of (2) employing routine
(3) the monoclonal antibody A18 that adopts the sodium periodate method that improves to prepare the purifying of peroxidase labelling is A18-HRP: claim 5mg HRP to be dissolved in the distilled water, add the 0.1M sodium periodate NaIO of the new preparation of 0.2ml
4, the room temperature lucifuge stirred 20 minutes; In 4 ℃ of NaAC solution dialysed overnight to 1mM, PH4.4; In 4 ℃ with antibody to be marked to 0.01M NaHCO
3The damping fluid dialysed overnight; In the good HRP solution of dialysis, add 20ul0.2M NaHCO
3Solution is transferred to pH value between PH9.0 and the PH9.5, adds the 1ml antibody-solutions of the good 10mg/ml of dialysis immediately, and the room temperature lucifuge stirred two hours; The boron sodium cyanide NaBH of the 4mg/ml that adding 0.1ml newly joins
4Solution stirred 2 hours in 4 ℃; In 4 ℃ to 0.01M PH7.2PBS dialysed overnight.The antibody that the mol ratio of mark and mark rate all meet the traget antibody service requirements is the good detection antibody A 1 g-HRP solution of mark, wherein adds 60% sterile glycerol of equivalent ,-20 ℃ of preservations.
(4) determine the working concentration of coated antibody A21:
With antibody purified A21 serial dilution in 0.05MNaHCO
3, PH is that 9.5 bag is cushioned liquid, presses the 100ul/ hole and adds the ELISA enzyme plate, spent the night in 4 ℃ of bags; Add the NIH/3T3 cell pyrolysis liquid in contrast of serial dilution respectively and contain solubility p185 after the encapsulant sealing with general Te Jin company
HER-2Antigenic T6-17 cell pyrolysis liquid; The washing back adds the A18-HRP of fixed concentration; Add and face the colour developing of phenylenediamine (OPD) substrate solution, the 492nm photometry absorbs (OD) value.With detected p185
HER-22 times of 10ug/ml of the A21 concentration when the antigen amount reaches capacity substantially are as the concentration of coated antibody A21.
(5) definite working concentration that detects antibody A 1 8-HRP:
Step is substantially with (4), and just the concentration of T6-17 cell pyrolysis liquid and NIH/3T3 cell pyrolysis liquid changes fixed concentration into, and A18-HRP concentration changes serial dilution into.With absorbance value ODT6-17 and the ODNIH/3T3 respectively antagonist concentration mapping of cell pyrolysis liquid, obtain two smooth curves at the 490nm place; With ODT6-17 during greater than the ODNIH/3T3 of twice the A18-HRP concentration of correspondence as the positive.Antibody concentration in the positive scope is the working concentration of antibody.Usually select the working concentration of 5ug/ml as A18-HRP.
Embodiment 3, adopt anti-p185
HER-2Monoclonal antibody and the crosslinked affine pearl purifying p185 of Sepharose 4B-Protein G
HER-2Antigenic method
The concrete steps of this method are:
(1) preparation and anti-p185
HER-2The crosslinked affine pearl of Sepharose 4B-Protein G of monoclonal antibody:
With antibody purified solution with mix through the abundant swollen Sepharose of damping fluid 4B-Protein G glue particle, 4 ℃ of rotations are incubated overnight; With the 0.08M of 10 times of volumes, the sodium tetraborate damping fluid centrifuge washing twice of PH9.0, each rotating speed is 3000g, the time is 2 minutes; Abandon supernatant, affine pearl be resuspended in the sodium tetraborate damping fluid of 10 times of volumes, add solid bifunctional reagent DMP, to final concentration be 20mM, room temperature was slightly vibrated 30 minutes; With the 0.2M of 10 times of volumes, the thanomin damping fluid centrifuge washing of PH8.0 once; Resuspended affine pearl is in the above-mentioned thanomin damping fluid of 10 times of volumes, and room temperature was slightly vibrated 2 hours; With 10 times of volumes consist of 50mM Tris-HCl PH7.5,150mM NaCl, 1%Triton X-100, the Binding Buffer washing of 0.025%NaN3 is once.
By the SDS-PAGE electrophoresis the affine pearl of equal-volume before and after adding DMP is detected, concentrated glue is gum concentration 5%, and separation gel is a gum concentration 10%.With affine pearl mix with sample-loading buffer boil after, sample on the centrifuging and taking supernatant; Adding in the swimming lane before the DMP has the antibody band, and adding in the swimming lane behind the DMP does not have the antibody band, and crosslinked success is described.The affine pearl of crosslinked good antibody is stored among the Binding Buffer standby at 4 ℃.
(2) will contain solubility p185
HER-2Antigenic liquid (cell pyrolysis liquid or cell culture supernatant) mixes with the affine pearl of antibody, dress post, washing column and wash-out and collection antigen.
To contain solubility p185
HER-2Antigenic cell pyrolysis liquid or cell culture supernatant mix with the affine pearl of crosslinked good antibody, and 4 ℃ of rotations are incubated overnight; Mixed solution is adorned post; Make liquid slowly by chromatography column, collect all and pass through liquid; With the last again post of same flow velocity, fraction collection is equipped with by liquid surveys (flow velocity in following each step can suitably be accelerated) with it; With the Binding Buffer washing chromatography column of 20 times of column volumes, fraction collection washings; With the 10mM phosphate of 20 times of column volumes, the pre-elutriant washing chromatography column of PH6.8, the pre-elutriant of fraction collection is equipped with to be surveyed; With the 100mM Glysine of 5 times of column volumes, the elutriant wash-out antigen of PH 2.5 contains the 1.5MTris-HCl of 0.1 times of volume in the fraction collection elutriant, every collection tube, and the neutralization buffer of PH8.8 is so that the elutriant pH value returns to neutrality rapidly.
(3) with sandwich ELISA method of the present invention and conventional immunoblotting to the solubility p185 in the sample of each step fraction collection
HER-2Antigen carries out check and analysis:
A. adopt sandwich ELISA method of the present invention to measure in the chromatography process solubility p185 in the various collection liquid
HER-2Antigen distributes:
The anti-p185 of purifying
HER-2NaHCO3 solution to the final concentration that monoclonal antibody A21 is diluted in 0.05M, PH9.6 is 10ug/ml, and the bag quilt is in the ELISA enzyme plate, and every hole 100ul is incubated overnight in 4 ℃; It is inferior to give a baby a bath on the third day after its birth with the TPBS washings of the 0.01M that contains 0.05% tween, PH7.2, each two minutes; Every hole adds encapsulant of 350ul general Te Jin company, room temperature sealing 40 minutes; The same washing; Every hole adds each collection sample that 100ul is diluted in confining liquid, and 4 ℃ are incubated overnight; The same washing; Every hole adds the A18-HRP that 100ul 5ug/ml is diluted in confining liquid, in 37 ℃ of incubations 1.5 hours of preserving moisture; The same washing; Every hole adds 100ul 1mg/ml OPD substrate solution, room temperature lucifuge reaction about 10 minutes; Every hole adds 100ul 1M H2SO4, termination reaction; On enzyme mark photoelectric color comparator, survey the absorbance value OD490 of 490nm.Color reaction result shows, cell pyrolysis liquid, washings and pre-elutriant pass through in the liquid antigenic content seldom, the antigen of acquisition concentrates in the elutriant.Explanation most of soluble antigen when last sample all is attracted on the antibody affinity column, by all being the foreign protein that is not adsorbed in the liquid; By the washing of specificity elutriant, the antigen that is adsorbed by antibodies specific is eluted, and the p185 in the elutriant also is described
HER-2Antigen has obtained purifying.
B. adopt in the western blot determination chromatography process solubility p185 in the various collection liquid
HER-2Antigenic content and immunologic opsonin:
To carry out conventional SDS-PAGE electrophoresis by liquid, concentrated glue is gum concentration 5% earlier, and separation gel is a gum concentration 7%; Electrotransfer is to the Hybond-ECL film; Ponceau dyeing; The washing back is with sealing the damping fluid closing membrane; The anti-p185 that adds 2ug/ml
HER-2Monoclonal antibody, 37 ℃ of incubations 2 hours; Washing adds the sheep anti-mouse igg of the horseradish peroxidase souvenir of 1:2000 dilution, 37 ℃ of incubations 1.5 hours; Washing, the fluorogenic substrate colour developing is with the exposure of X-ray sheet; The result that result and a measure coincide, that is: the sandwich ELISA method is accredited as the male elution samples in the swimming lane of ponceau dyeing and immunoblotting, and a band is respectively arranged near 185KD and 105KD, is respectively p185
HER-2Antigen and extracellular region thereof.And the sandwich ELISA method is accredited as no immunoblotting band in the negative elution samples swimming lane.Illustrate that the p185 antigen in the elutriant is purified the p185 of purifying
HER-2Antigen has by anti-p185
HER-2The specificity of monoclonal antibody identification, the antigen concentration of purifying is more than 1ug/ml.
Embodiment 4, detect solubility p185 in cell pyrolysis liquid or the cells and supernatant with sandwich ELISA method of the present invention
HER-2Antigen:
With embodiment 3 (3) a.Just sample is respectively cell pyrolysis liquid and cells and supernatant, with the solubility p185 of purifying
HER-2Antigen in contrast.The result of color reaction shows the solubility p185 of purifying
HER-2Between antigenic concentration and the OD value good linear dependence is arranged; The OD value of T6-17 cell pyrolysis liquid and T6-17 cells and supernatant is more than the twice of OD value of NIH/3T3 cell pyrolysis liquid and cells and supernatant respectively.Illustrate that this method can detect the p185 of the solubility in cell pyrolysis liquid and the cells and supernatant
HER-2Antigen.
Embodiment 5, the present invention are used for human serum solubility p185
HER-2Test experience:
(1) human serum sample's detection test:
Utilize double-antibodies sandwich ELISA of the present invention to detect 137 routine normal peoples, the solubility p185 in 65 routine mammary cancer patients and 33 other cancer patient serum of example
HER-2Level.Each experiment solubility p185 of standard
HER-2Antigen in contrast.Concrete steps are seen embodiment 3.Solubility p185 with concentration known among the result of color reaction
HER-2The absorbance value of standard antigen is to standard solubility p185
HER-2Antigenic concentration mapping obtains a standard straight-line; The average light absorption value of all normal human serums reactions that detect in the experiment adds the value standard straight-line comparison therewith of twice standard variance, finds this to be worth pairing p185
HER-2Antigen concentration is made as threshold value, is 30ng/ml; The absorbance value of everyone sero-reaction and standard straight-line are relatively calculated p185 in the serum
HER-2Antigenic concentration then is diagnosed as p185 greater than 30ng/ml
HER-2The tumour patient of high expression level.
(2) statistical analysis of serum sample detected result:
After using double-antibodies sandwich ELISA of the present invention above-mentioned tumour patient serum and normal human serum being detected, the measurement result side of card (x2) checked.The result shows the P value less than 0.025, illustrates that present method can obviously distinguish patient and control group, and detected result has statistical significance, has reached the clinical practice requirement.
Claims (2)
1, a kind of standard solubility p185
HER-2Antigenic preparation method comprises preparing with antibody linked affine pearl, preparation and contains p185
HER-2Antigenic sample, will contain p185
HER-2Antigenic sample mixes with the affinity pearl, mixture is adorned post and wash-out antigen; It is characterized in that: described and antibody linked affine pearl is the anti-p185 that adopts surperficial epi-position entrapping method preparation
HER-2The affine pearl of the monoclonal antibody of extracellular region and Sepharose 4B-ProteinG covalent cross-linking.
2, a kind of solubility p185
HER-2Antigenic double-antibody sandwich elisa detection method comprises a kind of p185
HER-2Monoclonal antibody is incorporated on the solid support as coated antibody, will contain solubility p185 respectively
HER-2Sample and the p185 of the concentration known of purifying
HER-2Standard antigen and the antibody incubation that wraps quilt, p185 wherein
HER-2The coated antibody of antigen is optionally caught, and uses the p185 of another kind of enzyme labelling again
HER-2Monoclonal antibody makes to detect antibody, enzyme-added then substrate, and the enzyme immunity detection method of employing color reaction comes the photoabsorption of test sample; P185 with the purifying of concentration known
HER-2Standard antigen compares, and with the absorbance value of color reaction the concentration of standard antigen is mapped, and formulates standard straight-line; The absorbance value of testing sample is compared p185 in the calculation sample with the absorbance value of standard antigen
HER-2Concentration; It is characterized in that: (1) described p185 that is used in the double-antibody sandwich elisa detection method
HER-2Monoclonal antibody is purified and p185 horseradish peroxidase-labeled, that adopt surperficial epi-position entrapping method preparation
HER-2Monoclonal antibody; (2) p185 of described purifying
HER-2Standard antigen is the anti-p185 that adopts surperficial epi-position entrapping method preparation
HER-2The affine pearl of the monoclonal antibody of extracellular region and Sepharose 4B-ProteinG covalent cross-linking is to containing p185
HER-2Antigenic sample carries out that the method for affinity chromatography obtains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01113787 CN1232636C (en) | 2001-07-13 | 2001-07-13 | Method for detecting human tumor antigen P 185 Her-2 and its application in diagnosing tumor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01113787 CN1232636C (en) | 2001-07-13 | 2001-07-13 | Method for detecting human tumor antigen P 185 Her-2 and its application in diagnosing tumor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1397803A CN1397803A (en) | 2003-02-19 |
| CN1232636C true CN1232636C (en) | 2005-12-21 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 01113787 Expired - Fee Related CN1232636C (en) | 2001-07-13 | 2001-07-13 | Method for detecting human tumor antigen P 185 Her-2 and its application in diagnosing tumor |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043058A (en) * | 2010-11-23 | 2011-05-04 | 中生北控生物科技股份有限公司 | Detection kit of acetyl amantadine for predicting tumors |
| CN103698536B (en) * | 2013-12-20 | 2016-03-16 | 安徽安科生物工程（集团）股份有限公司 | Oncoprotein P185 detection kit |
| CN105158474A (en) * | 2015-09-18 | 2015-12-16 | 安徽省立医院 | Reagent kit for detecting serum HER2 and application |
| CN106501529A (en) * | 2016-11-17 | 2017-03-15 | 南京健安医疗科技有限公司 | A kind of chemiluminescence enzyme immunoassay kit of detection proto-oncogene HER2 and application |
| CN107022030B (en) * | 2017-03-23 | 2020-08-28 | 广东谷盈生物科技产业研究院有限公司 | Monoclonal antibody for detecting alpha-fetoprotein, kit and application |
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2001
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