CN103969437B - Preparation method of kit for early-to-mid rapid diagnosis of ovarian cancer - Google Patents
Preparation method of kit for early-to-mid rapid diagnosis of ovarian cancer Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention discloses a preparation method of a kit for early-to-mid rapid diagnosis of an ovarian cancer. The method comprises the following steps: 1. preparation of antigens: required antigens are prepared by adopting genes of galectin-3, transthyretin, carcinoma antigen-125 and human epididymis protein 4; 2, preparation of capture antibodies: the antigens are used for immunization of a mammal so as to obtain corresponding monoclonal antibodies; 3, preparation of detection antibodies: the antigens are used for immunization of the mammal so as to obtain corresponding polyclonal antibodies; 4, coating of the capture antibodies; 5, sealing and sample adding. The method has the benefits as follows: the ovarian cancer can be detected in the most comprehensive manner, the benign and malignant of a pelvic lump before menopause or after menopause can be better judged, differential diagnosis can be better performed on gynecology benign ovary lump and cyst as well as the ovarian cancer, the method has the characteristics of high sensitivity and high accuracy, and the sensitivity and accuracy can reach over 96 percent.
Description
Technical field：
The present invention relates to the test kit field of cancer diagnosis detection, specially one kind are quickly examined for ovarian cancer early metaphase
The preparation method of disconnected test kit.
Background technology：
Malignant tumor of ovary is one of common malignant tumor of female sex organ, refer to be grown in it is pernicious swollen on ovary
Tumor, wherein 90%～95% is the cancer of ovarian primary.When medical, 60%～70% is late period, and late case curative effect is not
It is good.Therefore, although the sickness rate of ovarian cancer is less than the 3rd that cervical cancer and carcinoma of endometrium occupy gynecologic malignant tumor, but dead
Rate but exceedes cervical cancer and carcinoma of endometrium sum, is in gynecological cancer first place, is the maximum illness for seriously threatening WomanHealth.
As the fetal development of ovary, anatomic tissue and endocrine function are more complicated, early symptom is not true to type, preoperative discriminating
The organization type of ovarian tumor and good pernicious extremely difficult, so the ovarian cancer no matter really a great problem in diagnosis and treatment.
Up to the present, domestic and international clinical data is counted, its five year survival rate only 25% or so.But if it find that obtain early, 90%
Patient can survive；Relapse rate in ovarian cancer 5 years may be up to 80%, and recurrence time is concentrated mainly on the 3 for the treatment of phase
In year.In women in life, one just being had per five women and pelvic lump occurs, needs are checked pernicious swollen to exclude
The probability of tumor.At present, two kinds of detection meanss of the diagnosis Main Basiss of ovarian cancer.One kind is Transvaginal Ultrasound inspection (TUV).This
Plant imaging method to can be used to check the genitals of women, including uterus, ovary, cervix uteri and vagina.Although its application is more general
Time, but this method can not accurately detect that the lump is benign or pernicious.Additionally, the method also need to it is experienced
Clinical technician testing result is understood.Another kind of common detection methods are to detect tumor markerses CA125, this method
It is considered " goldstandard " of ovarian cancer diagnosis.But, the specificity and sensitivity of CA125 is relatively low, false negative or vacation easily occurs
It is positive.About 50% ovarian cancer I phase patient does not have the elevated phenomenon of CA125 levels, that is to say, that have the ovarian cancer of half
Patient may be failed to pinpoint a disease in diagnosis.Some benign ovarian diseases also result in the rising of CA125 levels, cause false positive.Therefore, examine in clinic
A kind of more preferable diagnostic tool is needed badly with diagnosis of ovarian cancer as early as possible on disconnected.
Tumor markerses (tumor markers, TM) are referred in tumor generation and breeding, by tumor cell itself
Synthesis, release, or the class material that the signal tumor for being reacted to tumor cell and produced by body exists and grows.These materials
The level for not existing in adult normal or occurring in cancer patient is significantly higher than normal person.Current tumor-marker quality testing
Survey technology is considered as the unique channel of the asymptomatic micro- stove tumor of early discovery, this detection technique can prior to X-ray, ultrasound,
The physical examination such as CT, MRI or PET-CT find tumor.Can be used for the examination of high-risk group's malignant tumor, diagnosing tumor and discriminating
Diagnosis, assesses the effect for the treatment of, prediction or monitoring tumor recurrence or transfer.At present, the ovarian cancer diagnosis test kit that hospital occurs
It is the common tumor marker of detection, susceptiveness and accuracy are all low, even if joint inspection also only 70% or so, it is difficult to meet early stage
The requirement of quick diagnosis.
Due to also not coming out for the rapidly and efficiently diagnostic kit of ovarian cancer on market, ovarian cancer morning is badly influenced
Phase finds and treats, has often been in cancer of late stage, misses best occasion for the treatment, greatly reduce when ovarian cancer patients are made a definite diagnosis
The survival rate of patient.If ovarian cancer can be greatly enhanced the survival rate of patient by early diagnosiss.
The content of the invention：
The purpose of the present invention is the deficiency for existing with above-mentioned ovarian cancer diagnosis, there is provided a kind of sensitivity is high, accuracy
The strong preparation method for ovarian cancer early metaphase quick diagnosis reagent kit.
In order to realize the object of the invention, the present invention is adopted the technical scheme that：Try for ovarian cancer early metaphase quick diagnosis
Agent box, including capture antibody, the capture antibody include CBP-35, transthyretin (TTR) and cancer antigen-
Monoclonal antibody obtained in 125 (CA-125) difference.
The capture antibody also includes monoclonal antibody obtained in people's epididymal proteins 4 (HE4).
The test kit also includes detection antibody, and the detection antibody includes CBP-35, turns thyroxine egg
(TTR), cancer antigen -125 (CA-125) and people's epididymal proteins 4 (HE4) distinguish obtained polyclonal antibody in vain.
The capture antibody is by CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
Carrier for expression of eukaryon is cloned into people's epididymal proteins 4 (HE4), and realizes the expression of albumen in mammalian cell, after purification
Corresponding antigen is obtained, the corresponding monoclonal antibody that the antigen immune mammal is obtained.
The step of preparation method of the capture antibody includes is as follows：
(1) preparation of antigen：CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
With people's epididymal proteins 4 (HE4) gene cloning to carrier for expression of eukaryon, and the expression of albumen is realized in mammalian cell, it is pure
Antigen is obtained after change, and this antigen also can be used as standard substance；
(2) capture the preparation of antibody：By above-mentioned antigen immune mammal, obtain corresponding monoclonal antibody and resist for capture
Body.
The detection antibody is by CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
Carrier for expression of eukaryon is cloned into people's epididymal proteins 4 (HE4), and realizes the expression of albumen in mammalian cell, after purification
Corresponding antigen is obtained, the corresponding polyclonal antibody that the antigen immune mammal is obtained.
The step of preparation method of the detection antibody includes is as follows：
(1) preparation of antigen：CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
With people's epididymal proteins 4 (HE4) gene cloning to carrier for expression of eukaryon, and the expression of albumen is realized in mammalian cell, it is pure
Antigen is obtained after change, and this antigen also can be used as standard substance；
(2) preparation of detection antibody：By above-mentioned antigen immune mammal, obtain corresponding polyclonal antibody and resist for detection
Body.
The capture antibody is coated in the hole of microtitration plate in advance, can be simplified step, be improved detection efficiency.
For the preparation method of ovarian cancer early metaphase quick diagnosis reagent kit, which comprises the following steps：
(1) preparation of antigen：By CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
With people's epididymal proteins 4 (HE4) gene cloning to carrier for expression of eukaryon, and the expression of albumen is realized in mammalian cell, it is pure
Antigen needed for obtaining after change, wherein, the antigen also can be used as standard substance；
(2) capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the Dan Ke
Capture antibody of the grand antibody as this test kit；
(3) preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, described many grams
Detection antibody of the grand antibody as this test kit；
(4) capture antibody coating：
1.. the capture antibody that concentration is 1 μ g/ml is coated with into microtitration with carbonate/bicarbonate buffer (pH9.6)
The hole of plate；2.. capping microtitration plate the overnight incubation at 4 DEG C；3.. discard coating buffer (carbonate/bicarbonate buffer
The capture antibody of dilution), and microtitration plate is washed with cleaning mixture twice, add 200 μ l PBST (phosphoric acid every time in micropore
Salt Tween buffer), the gently whipping microtitration plate above the tank removes cleaning mixture, pats microtitration plate on napkin,
Remove remaining drop, dry be put in it is standby in 4 DEG C of environment.
(5) close and be loaded：Add 200 μ l Block buffers (1.2%BSA/PBS) per hole, it is remaining in closing coating hole
Protein binding site.
The inventive method can be improved for the strong spy of the sensitivity high accuracy of ovarian cancer early metaphase quick diagnosis reagent kit
Point, its sensitivity and accuracy reach more than 96%.
For the detection method of ovarian cancer early metaphase quick diagnosis reagent kit, which comprises the following steps：
(1) preparation of antigen：By CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
With people's epididymal proteins 4 (HE4) gene cloning to carrier for expression of eukaryon, and the expression of albumen is realized in mammalian cell, it is pure
Antigen needed for obtaining after change；
(2) capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the Dan Ke
Capture antibody of the grand antibody as this test kit；
(3) preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, described many grams
Detection antibody of the grand antibody as this test kit；
(4) capture antibody coating：
1.. the capture antibody that concentration is 1 μ g/ml is coated with into microtitration with carbonate/bicarbonate buffer (pH9.6)
The hole of plate；
2.. capping microtitration plate the overnight incubation at 4 DEG C；
3.. coating buffer (the capture antibody of carbonate/bicarbonate buffer dilution) is discarded, and washs micro with cleaning mixture
Titer plate adds 200 μ l PBST (phosphate Tween buffer) twice, every time in micropore, and gently whipping is micro- above the tank
Amount titer plate, removes cleaning mixture, pats microtitration plate on napkin, removes remaining drop, dry be put in it is standby in 4 DEG C of environment
With.
(5) close
1.. add 200 μ l Block buffers (1.2%BSA/PBS) in each hole of microtitration plate, in closing coating hole
Remaining protein binding site；
2.. capping microtitration plate is simultaneously incubated at least 1 hour at 37 DEG C, it is preferred that overnight incubation；
(6) it is loaded
1.. the sample of 100 μ l is added to into each hole, is incubated 60 minutes at 37 DEG C；Accurate quantitative result is obtained,
Generally way is to compare the signal of unknown sample and standard curve.Each ELISA Plate must bioassay standard product (double measure or
Triplicate) and blank sample, to guarantee accuracy；
2.. discarding sample, and washing microtitration plate three times, (phosphate is told 200 μ l PBST to be added in micropore every time
Warm buffer)；
3.. the detection antibody that 100 μ l concentration are 0.5 μ g/ml is added to into each hole of microtitration plate；
4.. capping microtitration plate is simultaneously incubated 1 hour at 37 DEG C；
5.. microtitration plate four times is washed with PBST；
6.. 100 μ l labellings two of addition resist；
7.. capping microtitration plate is simultaneously incubated 1 hour at 37 DEG C.
8.. microtitration plate four times is washed with PBST.
(7) detect
1.. by TMB (3,3 ', 5,5 '-tetramethylsBenzidine) solution is added to each hole, is incubated 15-30 minutes, addition etc.
The terminate liquid of volume, then reads optical density at 450nm.
2.. the data obtained by serial dilutions draw standard curve, and concentration is marked in X-axis (logarithmic scale), and extinction
Scale is in Y-axis (lineal scale).Sample concentration is drawn by interpolation on this standard curve.
The present invention filters out 4 tumor markers from numerous tumor markers -- CBP-35
(Galectin-3), transthyretin (TTR), people's epididymal proteins 4 (HE4) and cancer antigen -125 (CA-125) composition ovary
Cancer quick diagnosis reagent kit.Wherein, transthyretin (TTR) is otherwise known as prealbumin (PA), is one kind by liver, arteries and veins
The albumen that network clump and eye are produced, it is relevant with transhipment thyroxine and the A metabolism of Wei Sheng systems.The blood of the patient sustained damage in liver
In clear, the content of TTR is substantially reduced.In hepatocarcinoma, TTR genetic transcriptions are obstructed, and the existing defects on gene structure.And TTR pair
The growth of tumor cell has obvious inhibitory action, therefore TTR genes are a kind of antioncogenes.Produce due to extracting TTR in blood plasma
Low, complex steps are measured, so we can produce TTR in a large number with the method for genetic engineering molecular cloning researches and develops this test kit.Gala
Sugared galectin-3 (Galectin-3) is a kind of galactose-binding protein, is uniquely have embedded structure in Galectin families
Member.CBP-35 (Galectin-3) is distributed widely in tumor tissues, and the expression of hL-31 is with its tumor
Invasion and attack and shift it is closely related.Galectin-3 can be combined with the CD98 of cell surface glycosylation promotes integrin in cell table
Face clustering, strengthens the affinity of integrin and its part indirectly；Also can be directly in conjunction with Integrin alphα1 β 1 and CD11b/18, just
Property or negativity adjust the activity of integrin, affect the combination of integrin and extracellular ligand.Galectin-3 can also increase whole
Expression of the element in cell surface is closed, and promotes collagen in the secretion of cell compartment.As CBP-35 has to polysaccharide
Affinity, it can also be sticked with substrate directly in conjunction with glycosylated cells epimatrix composition, mediated cell.Our research
In showing serum of ovarian cancer patients, Galectin-3 levels are significantly more than healthy population.People's epididymal proteins 4 (HE4) are a kind of new
Tumor markerses.Under normal physiological conditions, HE4 has very low-level expression in respiratory tract, reproductive system and ovary tissue,
But express in many ovarian cancer tissues and patients serum's camber.Cancer antigen -125 (CA-125 or Cancer antigen 125), also by
Referred to as Mucin1 6, it, by the protein of MUC16 coded by said gene, is that go out from ovarian epithelial carcinoma Detection of antigen can coverlet gram to be
A kind of glycoprotein that grand antibody OC125 is combined.CA-125 to the latent effect of the early detection of ovarian cancer be it is controversial, not yet
It is widely used in and screens in asymptomatic women.The use of the subject matter of CA-125 this biomarker is that it lacks sensitivity, it is special
It is not which lacks specificity when ovarian cancer is detected, especially in the early stage of pre-menopausal women.These limit it is meant that
CA-125 tests ovarian cancer often gives people wrong report, allows patient anxiety, so as to do further unnecessary screening (sometimes including handss
Art).Additionally, these restrictions detect false negative it is meant that the women of many early ovarian cancers will receive from CA-125, not
Obtain the state of an illness of oneself further to treat.What food and medicine Surveillance Authority of the U.S. (FDA) ratified can be used for the ovarian cancer of clinic
Blood serum designated object includes CA125 and HE4, both the overall sensitivity to ovarian cancer is used in combination and specificity is respectively 76%
With 95%, but the early diagnosiss for ovarian cancer lack enough Sensitivity and Specificities.
Compared with prior art, beneficial effects of the present invention are as follows：Ovarian cancer is most comprehensively detected, preferably can be judged
Premenopausal or post menopausal pelvic lump it is good pernicious, preferably gynecological's benign ovarian lump and cyst are differentiated with ovarian cancer
Diagnosis, with sensitivity high accuracy it is strong the characteristics of, its sensitivity and accuracy reach more than 96%.
Description of the drawings
Fig. 1 is schematic diagram of the present invention for ovarian cancer early metaphase quick diagnosis reagent kit；
Fig. 2 is that the present invention is detected in blood before and after treatment of ovarian cancer for ovarian cancer early metaphase quick diagnosis reagent kit
The change comparison diagram of Galectin-3；
Fig. 3 is that the present invention is detected in blood before and after treatment of ovarian cancer for ovarian cancer early metaphase quick diagnosis reagent kit
The change comparison diagram of TTR；
Fig. 4 is that the present invention is detected in blood before and after treatment of ovarian cancer for ovarian cancer early metaphase quick diagnosis reagent kit
The change comparison diagram of HE4；
Fig. 5 is that the present invention is detected in blood before and after treatment of ovarian cancer for ovarian cancer early metaphase quick diagnosis reagent kit
The change comparison diagram of CA125.
Specific embodiment
The present invention is carried out for ovarian cancer early metaphase quick diagnosis reagent kit in detail below in conjunction with the drawings and specific embodiments
Thin description explanation.
For ovarian cancer early metaphase quick diagnosis reagent kit, including capture antibody, antibody Block buffer, standard is captured
Product, traget antibody, cleaning mixture and nitrite ion etc..The traget antibody is anti-from HRP labellings two.The capture antibody includes gala
Monoclonal antibody obtained in sugared galectin-3, transthyretin (TTR) and cancer antigen -125 (CA-125) difference；By right
The common quick detection of CBP-35, transthyretin (TTR) and cancer antigen -125 (CA-125), can be effective
Raising Detection accuracy and sensitivity, effectively overcome only according to not enough caused by detection cancer antigen -125 (CA-125).
The capture antibody also includes and monoclonal antibody obtained in people's epididymal proteins 4 (HE4), by detecting that galactose coagulates
Collection element -3, transthyretin (TTR), cancer antigen -125 (CA-125) and people's epididymal proteins 4 (HE4) so as to sensitivity and
Accuracy reaches more than 96%, and this is that available reagent box is incomparable, can fast and accurately diagnosis of ovarian cancer, greatly
Improve the survival rate of patient in ground.
The capture antibody is by CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
Carrier for expression of eukaryon is cloned into people's epididymal proteins 4 (HE4), and realizes the expression of albumen in mammalian cell, after purification
Corresponding antigen is obtained, the corresponding monoclonal antibody that the antigen immune mammal is obtained.
The step of preparation method of the capture antibody includes is as follows：
(1) preparation of antigen：By the method for molecular cloning CBP-35, transthyretin (TTR),
Cancer antigen -125 (CA-125) and people's epididymal proteins 4 (HE4) gene cloning to carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, antigen is obtained after purification, this antigen also can be used as standard substance；The preparation of the antigen can also be used
Prepared by existing conventional method, here is no longer burdensome.
(2) capture the preparation of antibody：By above-mentioned antigen immune mammal, obtain corresponding monoclonal antibody and resist for capture
Body.The mammal is mice and rabbit, preferably mice.The preparation of the capture antibody can also use existing conventional method
Prepare, here is no longer burdensome.
The detection antibody includes CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
With polyclonal antibody obtained in people's epididymal proteins 4 (HE4) difference.
The detection antibody is by CBP-35, transthyretin (TTR), cancer antigen -125 (CA-125)
Carrier for expression of eukaryon is cloned into people's epididymal proteins 4 (HE4), and realizes the expression of albumen in mammalian cell, after purification
Corresponding antigen is obtained, the corresponding polyclonal antibody that the antigen immune mammal is obtained.The preparation of the detection antibody
The step of method includes is as follows：
(1) preparation of antigen：By the method for molecular cloning CBP-35, transthyretin (TTR),
Cancer antigen -125 (CA-125) and people's epididymal proteins 4 (HE4) gene cloning to carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, antigen is obtained after purification, this antigen also can be used as standard substance；
(2) preparation of detection antibody：By above-mentioned antigen immune mammal, obtain corresponding polyclonal antibody and resist for detection
Body.The mammal is mice and rabbit, preferably rabbit.
Wherein, in the hole of the microtitration plate that the capture antibody can be coated in PVC materials in advance, step can be simplified
Suddenly, improve detection efficiency.
For the preparation method of ovarian cancer early metaphase quick diagnosis reagent kit, which comprises the following steps：
(1) preparation of antigen：By the method for molecular cloning by CBP-35, transthyretin (TTR),
Cancer antigen -125 (CA-125) and people's epididymal proteins 4 (HE4) gene cloning to carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, the antigen needed for obtaining after purification, wherein, the antigen also can be used as standard substance；
(2) capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the Dan Ke
Capture antibody of the grand antibody as this test kit；
(3) preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, described many grams
Detection antibody of the grand antibody as this test kit；
(4) capture antibody coating：
1.. the capture antibody that concentration is 1 μ g/ml is coated in into micro drop with carbonate/bicarbonate buffer (pH9.6)
In the hole of fixed board；
2.. microtitration plate is covered with bond plastic product and the overnight incubation at 4 DEG C；
3.. coating buffer (the capture antibody of carbonate/bicarbonate buffer dilution) is discarded, and washs micro with cleaning mixture
Titer plate adds 200 μ l PBST (phosphate Tween buffer) twice, every time in micropore, and gently whipping is micro- above the tank
Amount titer plate, removes cleaning mixture, pats microtitration plate on napkin, removes remaining drop, dry be put in it is standby in 4 DEG C of environment
With；The cleaning mixture is that PBS (phosphate buffer) is middle adds a certain amount of Tween20, the mass percent of the Tween20
Concentration is 0.05%；
(5) close
1.. it (is containing 1.2%BSA (bovine serum albumin) to add 200 μ l Block buffers in every hole of microtitration plate
PBS (phosphate buffer)), for closing remaining protein binding site in coating hole；
2.. microtitration plate is covered with bond plastic product and be incubated at least 1 hour at 37 DEG C, it is preferred that be incubated
Night.
For the detection method of ovarian cancer early metaphase quick diagnosis reagent kit, which comprises the following steps：
(1) preparation of antigen：By the method for molecular cloning by CBP-35, transthyretin (TTR),
Cancer antigen -125 (CA-125) and people's epididymal proteins 4 (HE4) gene cloning to carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, the antigen needed for obtaining after purification, wherein, the antigen also can be used as standard substance；
(2) capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the Dan Ke
Capture antibody of the grand antibody as this test kit；
(3) preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, described many grams
Detection antibody of the grand antibody as this test kit；
(4) capture antibody coating：
1.. the capture antibody that concentration is 1 μ g/ml is coated with into microtitration with carbonate/bicarbonate buffer (pH9.6)
The hole of plate；
2.. microtitration plate is covered with bond plastic product and the overnight incubation at 4 DEG C；
3.. coating buffer (the capture antibody of carbonate/bicarbonate buffer dilution) is discarded, and washs micro with cleaning mixture
Titer plate adds 200 μ l PBST twice, every time in micropore, and (phosphate Tween buffer can be containing 0.05% tween 20
PH value be 7.4 phosphate buffer), the gently whipping microtitration plate above the tank removes cleaning mixture, light on napkin
Clap microtitration plate, remove remaining drop, dry be put in it is standby in 4 DEG C of environment；The cleaning mixture is PBS (phosphate-buffered
Liquid) in add a certain amount of Tween20, the mass percent concentration of the Tween20 is 0.05%；
(5) close
1.. add 200 μ l Block buffers (1.2%BSA/PBS) in each hole of microtitration plate, in closing coating hole
Remaining protein binding site；
2.. microtitration plate is covered with bond plastic product and be incubated at least 1 hour at 37 DEG C, it is preferred that at 4 DEG C
Overnight incubation；
(6) it is loaded
1.. the sample that 100 μ l suitably dilute (20 times of dilution) is added to into each hole, is incubated 60 minutes at 37 DEG C；Will
Obtain accurate quantitative result, it is common practice that compare the signal of unknown sample and standard curve.Each ELISA Plate must be surveyed
Determine standard substance (double measure or triplicate) and blank sample, to guarantee accuracy；
2.. discarding sample, and washing microtitration plate three times, (phosphate is told 200 μ l PBST to be added in micropore every time
Warm buffer)；
3.. the detection antibody that 100 μ l concentration are 0.5 μ g/ml is added to into each hole of microtitration plate；
4.. microtitration plate is covered with bond plastic product and be incubated 1 hour at 37 DEG C；
5.. microtitration plate four times is washed with PBST；
6.. 100 μ l labellings two of addition resist, and which is just diluting 10000 times (1 in PBS using front：10000).The mark
It can be HRP labelling goat-anti rabbits that note two is anti-.
7.. microtitration plate is covered with bond plastic product and be incubated 1 hour at 37 DEG C.
8.. microtitration plate four times is washed with PBST.
(7) detect
1.. TMB (3,3 ', 5,5 '-tetramethyl benzidine) solution is added to into each hole, 15-30 minutes, addition etc. is incubated
Terminate liquid (the 2M H of volume2SO4), then optical density is read at 450nm.
2.. the data obtained by serial dilutions draw standard curve, and concentration is marked in X-axis (logarithmic scale), and extinction
Scale is in Y-axis (lineal scale).Sample concentration is drawn by interpolation on this standard curve.
The present invention includes CBP-35, transthyretin for ovarian cancer early metaphase quick diagnosis reagent kit
(TTR), in cancer antigen -125 (CA-125) and people's epididymal proteins 4 (HE4) four indices two raised as in diagnosis morning simultaneously
The standard of phase ovarian cancer, Cleaning Principle are as shown in Figure 1；CBP-35, transthyretin (TTR), cancer antigen-
Two marks for declining as treatment of ovarian cancer recruitment evaluation simultaneously in 125 (CA-125) and people's epididymal proteins 4 (HE4) four indices
It is accurate.Can be used for entering action clinically by the height of 3 tumor marker levels in detection blood to the therapeutic effect of ovarian cancer
State is assessed.Can be additionally used for the application of relapse and metastasis clinically to ovarian cancer and Index for diagnosis；Can also be used for pulmonary carcinoma morning
Mid-term quick diagnosis.
The present inventor always worked on the research related to tumor of galactose agglutinin and its inhibitor in recent years, achieved perhaps
There is advanced international standard scientific achievement more.CBP-35 (Galectin-3) is becoming new study hotspot, generation
The many laboratorys in boundary all take up the research of this respect.It will be apparent that research of the present inventor in this field is walked in the world
Prostatitis.Meanwhile, inventor is also studied with the dependency of ovarian cancer to TTR, our research indicate that Galectin-3 pair
The Sensitivity and Specificity of ovarian cancer is respectively 78.2% and 82.6%；TTR is respectively to the Sensitivity and Specificity of ovarian cancer
65.5% and 85.2%, see the table below：
In order to accurately be diagnosed to patient as early as possible, we have invented a kind of most comprehensive ovary on this basis
Cancer detection method, using Galectin-3, TTR, HE4 and CA125 joint-detection, this joint inspection method can also preferably judge menopause
Front or post menopausal pelvic lump it is good pernicious, discriminating is carried out with ovarian cancer to gynecological's benign ovarian lump and cyst preferably and is examined
Disconnected, concrete Detection results see the table below：
It can be seen that, the characteristics of this test kit has sensitivity high accuracy strong, its sensitivity and accuracy reach 96% with
On.
Test effect explanation
The clinical therapeutic effect to ovarian cancer carries out dynamic evaluation
Dynamic evaluation is carried out for determining that can this test kit be used for the therapeutic effect to ovarian cancer, we have collected 483 parts
Serum before and after ovarian cancer patients treatment.The testing result of 483 patients referring to 2-5, as a result show ovarian cancer patients treatment before
Galectin-3 in serum afterwards, there were significant differences for the level of TTR, HE4 and CA125.Galectin- in serum after effectively treatment
3, TTR, HE4 can be greatly reduced with the level of CA125, point out this test kit be used for entering Mobile state to the therapeutic effect of ovarian cancer
Assessment.
The preferred embodiment of the present invention described in detail above, it will be appreciated that the ordinary skill of this area is without the need for wound
The property made work just can make many modifications and variations with design of the invention.Therefore, all technical staff in the art
Pass through logical analyses, reasoning in prior art basis or according to the limited available technology of experiment according to present inventive concept
Scheme, should be among the protection domain determined by the claims.
Claims (1)
1. be used for ovarian cancer early metaphase quick diagnosis reagent kit preparation method, it is characterised in that. including the step of it is as follows：
(1) preparation of antigen：By CBP-35, transthyretin, 4 gene of cancer antigen -125 and people's epididymal proteins
Carrier for expression of eukaryon is cloned into, and realizes the expression of albumen in mammalian cell, the antigen needed for obtaining after purification；
(2) capture the preparation of antibody：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody；
(3) preparation of detection antibody：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody；
(4) capture antibody coating：
1.. the capture antibody that concentration is 1 μ g/ml is coated with the hole of microtitration plate with carbonate/bicarbonate buffer；②.
Cover microtitration plate and the overnight incubation at 4 DEG C；3.. coating buffer is discarded, and washs microtitration plate with cleaning mixture twice, often
It is secondary to add 200 μ l PBST in micropore, remove cleaning mixture, remove remaining drop, dry be put in it is standby in 4 DEG C of environment；
(5) close and be loaded：Add 200 μ l Block buffers per hole, remaining protein binding site in closing coating hole is used
Bond plastic product covers microtitration plate and is incubated at 37 DEG C at least 1 hour,
The step of preparation method of the detection antibody includes is as follows：
(1) preparation of antigen：By the method for molecular cloning CBP-35, transthyretin, cancer antigen-
125 and 4 gene cloning of people's epididymal proteins to carrier for expression of eukaryon, and realize the expression of albumen in mammalian cell, purification
After obtain antigen；
(2) preparation of detection antibody：By above-mentioned antigen immune mammal, corresponding polyclonal antibody is obtained for detection antibody.
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| CN107085110A (en) * | 2017-05-31 | 2017-08-22 | 江西乐成生物医疗有限公司 | One kind is used for thyroid cancer external diagnosis reagent case and its detection method |
| CN108181464A (en) * | 2017-12-29 | 2018-06-19 | 广州优迪生物科技股份有限公司 | A kind of preparation method of TTR time resolutions immunofluorescent reagent box |
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