CN105510585A - Double-antibody sandwich ELISA kit for human beta3 acetylglucosaminyltransferase 8 (beta3GnT8) - Google Patents
Double-antibody sandwich ELISA kit for human beta3 acetylglucosaminyltransferase 8 (beta3GnT8) Download PDFInfo
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Abstract
The invention discloses a double-antibody sandwich ELISA kit for human beta3GnT8. The kit comprises a cleaning solution, a sample diluent, a coloring solution, a stopping solution, an antibody-coated ELISA plate, a detection antibody, an enzyme-labeled antibody and a standard control protein. Thus, the anti-human beta3GnT8 double-antibody sandwich ELISA kit is prepared; and a double-antibody sandwich ELISA assay and the kit are employed to quantitatively detect the content of the beta3GnT8 protein in the serum of a patient, especially a patient with colorectal carcinoma. An important reference basis is provided for early diagnosis of colorectal carcinoma by detecting the level of the beta3GnT8 protein in the serum of the patient with colorectal carcinoma and comparing the level of the beta3GnT8 protein in the serum of the patient with the level of the beta3GnT8 protein in the serum of a normal person.
Description
Technical field
The present invention relates to virus detection techniques field, particularly people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit.
Background technology
β 3 acetylglucosamine based transferase 8/ β 1,3 galactosyltransferase 7 genes (AY277592EC2.4.1-) be by the good teach problem group of University Of Suzhou Wu scholar by electronic cloning method in the world first clone obtain and first called after β 1, the new gene of β 3 glycosyl transferase extended familys of 3 galactosyltransferases, carried out the research of cloning and expressing to our laboratory of this gene, Japanese scholars carries out RNTO β 3 acetylglucosamine based transferase (β 3GnT8) after enzyme activity determination to it afterwards.This room is carried out further checking research to its catalysis and is namely carried out β 1 respectively in the recent period, the enzymatic reaction of 3 galactosyltransferases and β 3-N-acetylglucosaminyl transferase reaction system, high performance liquid chromatography (HPLC) is utilized to carry out result detection, show that β 3GalT7 has more weak β 1 simultaneously, the activity of 3 galactosyltransferases and higher β 3-N-acetylglucosaminyl transferase, therefore preliminary RNTO β 3 acetylglucosamine based transferase (β 3GnT8)/β 1, 3 galactosyltransferases (β 3GalT7), be abbreviated as β 3GnT8 or β 3GalT7.
β 3GnT8 plays an important role in the synthesis of tumor marker antigen poly lactose amine.Abroad in Recent Years and our research show, β 3GnT8 exists overexpression in various human tumor tissues.Many polylactosamine structure that it catalyzes and synthesizes are a kind of distinctive glycan containing N-acetyl lactosamine (LacNAc) repetitive sequence, it is added on O-glycan, on N-glycan and glycolipid. many polylactosamine structure are often carried some important carbohydrate structures by modification, such as Lewis related antigen, HNK-1 antigen etc., play important physiological and pathological function.In metastases aspect, poly lactose amine and dependency structure thereof occupy key player in cell-cell interaction, effect between cell and extracellular matrix, immune response and cancer cell migration ability.There are some researches show, the many polylactosamine structure in triantennary, four antenna N-glycan β 1,6 branches participate in numerous malignancy of tumor phenotypes, affect cell proliferation and transfer ability.
We successfully have developed rabbit anti-human β 3GnT8 polyclonal antibody in 2010, research shows that this antibody has in immunoassay and preferably applies, and there is comparatively high specific, and these work also just solve one of most critical issue in the development of this kit.The method of application double-antibody sandwich elisa (enzyme-linked immunosorbent assay) kit, to the detection of β 3GnT8, has simple to operate, quick, sensitive, feature accurately.And the application glycosyl transferase examination of ELISA method to tumor patient has relevant report, the ELISA kit such as like ceramide for the examination of tumor patient or patients with viral infections's serum, all show good using value and prospect.The method using double-antibody sandwich elisa to detect β 3GnT8 is first carried out examination to tumor patients such as colon cancers by the present invention; By the examination of this kit to clinical tumor sample, detect the content of serum beta 3GnT8 with this kit.By to the content of β 3GnT8 in dissimilar cancer patient's serum and normal human serum comparision contents, an important reference frame can be provided by these differences to tumour early detection, and these work will have greatly application prospect and Development volue.
Summary of the invention
The technical matters that the present invention mainly solves is to provide that a kind of high specificity, degree of accuracy are high, easy and simple to handle, anti-human β 3GnT8 double-antibody sandwich elisa kit fast.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: provide people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit, comprising: the ELISA Plate of cleansing solution, sample diluting liquid, nitrite ion, stop buffer, coated antibody, detection antibody, enzyme labelled antibody, standard control albumen.
Preferably, the ELISA Plate of described coated antibody is for coated antibody with mouse-anti people β 3 acetylglucosamine based transferase 8 monoclonal antibody, diluted by coated antibody with coating buffer, every hole adds the coated antibody after dilution, and is washed by rear cleansing solution at the bag that spends the night; In ELISA Plate, add confining liquid again, wait question response, last cleansing solution washing.
Preferably, described detection antibody is rabbit anti-human β 3 acetylglucosamine based transferase 8 polyclonal antibody.
Preferably, described enzyme labelled antibody is the goat anti-rabbit antibodies of horseradish peroxidase-labeled.
Preferably, described standard control albumen: comprise positive control β 3GnT8 albumen and negative control coli somatic albumen.
Preferably, the concentration after the coated liquid dilution of described coated antibody is 5 μ g/mL, and the every hole of the coated antibody after dilution addition is 100 μ L, and the temperature of the bag quilt that spends the night is 4 DEG C; The bag that spends the night is 3 ~ 4 times by rear cleansing solution washing times; The amount adding confining liquid in ELISA Plate is every hole 100 μ L, and the temperature of reaction adding confining liquid in ELISA Plate is 37 DEG C, and the reaction time is 1 ~ 2 hour, and it is 3 ~ 4 times that reaction terminates rear cleansing solution washing times.
Preferably, the preparation method of described coated antibody is:
The first, β 3 acetylglucosamine based transferase 8 albumen is mixed with Freund's complete adjuvant, carries out subcutaneous inoculation by BALB/c mouse;
The second, after first respectively at BALB/c mouse, be replaced by incomplete Freund's adjuvant and second time and third time immunity are carried out to BALB/c mouse;
3rd, in BALB/c mouse third time immunity, lumbar injection booster immunization is carried out to BALB/c mouse;
4th, conveniently hybridoma preparation method prepares the fusion hybridoma cell strain of spleen bone-marrow-derived lymphocyte and SP2/0 cell, carry out monoclonal cell sorting cultivation, obtained positive hybridoma cell strain through bag by the ELISA Screening and Identification of β 3 acetylglucosamine based transferase 8 albumen;
5th, prepare mouse-anti people β 3 acetylglucosamine based transferase 8 monoclonal antibody by BALB/c mouse ascites tumor, ascites obtains the coated antibody being used for double-antibody sandwich elisa kit after separation and purification.
Preferably, in the described first step, the blending ratio of β 3 acetylglucosamine based transferase 8 albumen and Freund's complete adjuvant is 1:1; Every BALB/c mouse immunity total amount 0.2mL carries out subcutaneous inoculation; The concrete time of carrying out second time and third time immunity to BALB/c mouse in described second step is rear BALB/c mouse initial immunity 3 weeks and after 5 weeks; The concrete time of carrying out lumbar injection booster immunization to BALB/c mouse in the 3rd step is BALB/c mouse third time immunity 2 weeks afterwards and first 1 day of Fusion of Cells.
In such scheme, described coating buffer compound method is: 1.59gNa2CO3,2.93gNaHCO3, and deionized water dissolving is also settled to 1L.
The invention has the beneficial effects as follows: a kind of people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit is provided, utilize anti-human β 3GnT8 monoclonal antibody and anti-human β 3GnT8 polyclonal antibody, prepare anti-human β 3GnT8 double-antibody sandwich elisa kit; It adopts anti-human β 3GnT8 monoclonal antibody and anti-human β 3GnT8 polyclonal antibody as the coated antibody in this kit and detects antibody, strong eliminating nonspecific interference, and can realize detecting the different epi-positions of antigen protein, make result have more accuracy.And detection of the present invention is simple to operate, quick, testing cost is low, be applicable to the detection of extensive sample, is easy to popularize.It adopts DASELISA immuno absorbence ELISA method, detects the content of serum beta 3GnT8 protein level, especially colorectal cancer patients with this kit.By the height of colorectal cancer patients serumβ 3GnT8 protein level, and contrast with normal human serum level, provide an important reference frame by the early diagnosis that is colon cancer of these differential expressions.
Embodiment
Below preferred embodiment of the present invention is described in detail, can be easier to make advantages and features of the invention be readily appreciated by one skilled in the art, thus more explicit defining is made to protection scope of the present invention.
One, the preparation of mouse-anti people β 3GnT8 monoclonal antibody
1. the immune mouse of antigen sensibilization bone-marrow-derived lymphocyte obtains
Adding after albumen sample 0.01MPBS (pH7.2) 10 × dilution of β 3GnT8 prokaryotic expression purifying after isopyknic Freund's complete adjuvant fully mixes is injected in female SPF level Balb/c mouse peritoneal in 55 week ages by 30ug/ albumen dosage, incomplete Freund's adjuvant peritoneal immunity is added respectively once by the above-mentioned albumen dosage that waits after two weeks, undertaken four every one week by 30ug/ albumen dosage (without adjuvant) again after three immunity to exempt from, four exempt from latter 5 ~ 7 days, and orbital vein blood sampling is got serum and made test antibodies; To fix standard antigen with described β 3GnT8 purifying protein (1mg/ml), do indirect ELISA, tire to show to obtain more than 1 ︰ 1000 and can produce the immune mouse of more efficient valency for prokaryotic expression product specific antibody, existing antigen sensibilization bone-marrow-derived lymphocyte clone in this mouse spleen is described simultaneously.
2. the acquisition of fused cell clone and the screening of secretory antibody clone strain
A. fused cell growth clone cultivates
Asepticly get an above-mentioned qualified mice spleen, be placed in sterile petri dish, 10%FBS+RPMI-1640 is cut into small pieces after washing 2-3 time, use sterile glass slice lapping, extrude splenocyte, 30-40ml10%FBS+RPMI1640 dispels re-suspended cell, cross 200 order nylon membranes carry out cell sieve after (unicellular be easy to Fusion of Cells) wash 2-3 time with RPMI-1640 nutrient culture media again and count;
In the ratio of splenocyte and myeloma cell line SP2/0 cell 10:1, the SP2/0 cell being in exponential phase is added in Spleen cell suspensions centrifuge tube, with RPMI-1640 nutrient culture media centrifuge washing 2-3 time, PEG Fusion of Cells agent Fusion of Cells method carries out splenocyte and SP2/0 merges routinely; After merging, centrifugal segregation merges liquid, be resuspended in 120ml20%FBS+RPMI-1640 nutrient solution, and by 100ul/ hole plant in 12 pieces in advance length have Balb/c mouse peritoneal huge bite or 96 orifice plates in thymocyte feeder layer hole in, plant 12 piece of 96 orifice plate, every hole adds 100ul containing 2 × HAT, carry out 2/3 and change liquid with containing 1 × HAT20%FBSPLUSRPMI-1640 nutrient solution and continue cultivation after 37 DEG C of 5%CO2 cultivate one week, after 2 weeks, turn out multiple HAT resistance clone to be screened.
B. secretory antibody clone screening and subclone
Respectively get clone hole supernatant and be divided into two parts (50ul/ parts) as antibody to be checked, detect the previous day β 3GnT8 Prokaryotic expression, purification albumen (1mg/ml) 0.1MNa2CO3/NaHCO3 (pH9.6) is done 1:15 dilution and is coated in 96 hole ELISA Plate by 100ul/ hole, spent the night in 4 DEG C of bags, 2 times are washed with 0.01MPBS (pH7.2), add 200ul/ hole 5%CBS/0.01MPBS (pH7.2) in 37 DEG C of wet floating 2h, 1 time is washed with 0.01MPBS (pH7.2), every hole adds a antibody to be checked, if SP2/0 cells and supernatant control wells, 37 DEG C of wet floating 1h, 0.02%Tween20/0.01MPBS (pH7.2) washs 4 times (4 × 10min), 1 ︰ 300affinitypurifiedHRPconjugatedgoatanti-mouseIgG (H+L) (affine pure horseradish peroxidase-labeled goat anti-mouse IgG (heavy chain and light chain) two resists) (5%CBS/0.01MPBS (pH7.2) dilution) two respectively adding 50ul resists, the same wet floating 1h, after washing 4 times, add the OPD/H2O2 nitrite ion lucifuge colour developing of 50ul/ hole, ELISA microplate reader measures OD490 value, the 2 times of interpretations of control wells OD490 value are greater than for positive hole with test hole OD490 value.
Preparing to detect increases in Tissue Culture Flask by 293T cell the previous day, cultivates in 37 DEG C of 5%CO2; Collect 293T cell, often organize 1 × 105 cell, by cell 0.01MPBS(pH7.2) wash 1 time, often pipe adds 1ml rupture of membranes agent re-suspended cell, and centrifugal 5 minutes of 1000rpm, abandons supernatant, PBS cleaning twice; Every hole adds a antibody to be checked (clone's porocyte supernatant) and establishes SP2/0 cells and supernatant control wells, hatches 0.5h in 4 DEG C; 0.01MPBS(pH7.2) wash 2 times (2 × 5min), every hole adds the 1 ︰ 200affinitypurifiedFITCconjugatedgoatanti-mouseIgG(H+L of 100ul) (affine pure FITC marks goat anti-mouse IgG (heavy chain and light chain) two and resists) (3%CBS/0.01MPBS(pH7.2) dilution) two anti-lucifuges hatch 0.5h, after washing 2 times, carry out the analysis of flow cytometry loading, by the positive hole of control wells interpretation, filter out two positive colony.
Above-mentioned two positive clone is carried out at least 2 and take turns unicellular subclone (method is cultivated with fused cell growth clone), above-mentioned two screening (the same method) is carried out again until after obtaining 100% pair of positive colony when cell grows to 30% cell monolayer, get and wherein grow the most vigorous strain clone and called after 5F3, this clone is carried out expansion to cultivate, and freezing and thawing.
3. induce ascites in target hybridoma body to produce
Select SPF level BALB/c mouse in 5 week age (average weight 21g) 10, before inoculating cell, 1-2 week presses 0.5ml/ lumbar injection whiteruss sensitization in advance.Inoculation hybridoma the previous day, above-mentioned hybridoma is gone down to posterity by 1 ︰ 8 and treats that cell is in exponential phase and blows and beats suspension cell gently, the centrifugal 5min of 1000r/min, abandon nutrient solution, after centrifugal 2 times of serum free medium washing, and with this cultivation keynote cell concentration to 5X106/ml, be only injected in above-mentioned pretreated BALB/c mouse abdominal cavity by 0.5ml/, meticulously raise after injection.Inject and get ascites in 5ml centrifuge tube in latter 8 days, the centrifugal 5min of 1000r/min is to remove the cell in ascites, get supernatant 4 DEG C of centrifugal 15min of 12000r/min, the supernatant that takes a morsel also detects its protein content and purity with ultraviolet spectrophotometer and SDS-PAGE, measure it with indirect elisa method to tire, and packing-80 DEG C is frozen for subsequent use simultaneously.
4. monoclonal antibody immunity globulin antibody hypotype and specificity analyses
A. get ascites, utilize subclass to measure test paper qualification monoclonal antibody immunity tropomyosin isoform classification analysis.
B. the multiple sample western-blot of ascites monoclonal antibody tests
Get escherichia coli prokaryotic expression immunity antigen respectively and L929 turns β 3GnT8 gene overexpression cell extraction albumen as target protein, 12%SDS-PAGE electrophoresis in ice-water bath is splined on by 50ug/line, after electrophoresis, by wet transfer method in 110mA ice-water bath by the protein delivery in glue on NC film, after 0.01MPBS (pH7.2) submergence balance 30min, close with 5% skimmed milk power 4 DEG C and spend the night, after adding above-mentioned primary antibodie incubated at room 90min to be measured, after washing 4 times with 0.02%Tween20/0.01MPBS, 1 ︰ 300affinitypurifiedHRPconjugatedgoatanti-mouseIgM (H+L) (3% skimmed milk power/0.01MPBS (pH7.2) dilution) incubated at room 60min after washing 4 times, lucifuge colour developing in DAB/H2O2 nitrite ion, take pictures after abundant washing.Result shows, and ascites antibody all has good behaviour in the WesternBlot experiment of protokaryon albumen and eukaryotic protein, and this antibody not only can carry out the qualitative analysis of β 3GnT8 albumen, also can carry out quantitatively or semi-quantitative analysis simultaneously.
Two, the preparation of enzyme labelled antibody
Get 30mL polyvalent antibody and be placed in beaker, add 30mL0.01MpH7.2PBS mixing, then dropwise add 80mL saturated ammonium sulfate solution, limit edged stirs, and room temperature leaves standstill 30min, the centrifugal 30min of 3000rpm, removes supernatant.In precipitation, add 80mLPBS dissolution precipitation, then add saturated ammonium sulfate solution and reach 33% saturation degree, limit edged stirs, and room temperature leaves standstill 30Min, the centrifugal 30min of 3000rpm, removes supernatant; So 2-3 time repeatedly.Again sediment is dissolved in 12mL water, to 4 DEG C of dialysis in 0.07MpH6.3PBS solution, until outer liquid and Nessler's reagent or 1% BaCl solution reaction be negative.Dialyse complete, the centrifugal 5min of 3000rpm, removes the IgG solution that supernatant is extraction, for enzyme labelled antibody.
Three, ELISA Plate reaction homogeneity detects
Stochastic choice 3 pieces of ELISA Plate, IgG(M according to 1 μ g/ml rabbit), 100 μ L/ holes add, 4 DEG C are spent the night, discard and wash, repeated washing 3 times, ELISA Plate is patted dry, add cow's serum confining liquid (10%) 200 μ L/ hole carry out 37 DEG C close 1 hour, wash away confining liquid (the same), then 100 μ L/ holes add the goat anti-rabbit antibody (1:500) of horseradish peroxidase-labeled, hatch 1h for 37 DEG C, washing; Then add 100 μ L/ hole nitrite ion lucifuge 15min, add 50 μ L/ hole stop buffers, microplate reader surveys OD value, calculates the coefficient of variation between each hole, detects ELISA Plate homogeneity.
Four, the anti-the best bag of Sheet is determined by concentration and how anti-best effort concentration
Dilute by the ascites monoclonal antibody of coating buffer by purifying, ratio is followed successively by: 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:10000,1:12800,1:25600; Wrap respectively and spent the night (4 DEG C) by plate.With the PBS dilution containing 1% hyclone, the rabbit of purifying resisting is diluted according to 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:10000 morely, carry out application of sample according to square formation.With β 3GnT8 cytolytic proteins do positive, L929-MOCK does negative control, carries out sandwich ELISA, often group establishes 3 multiple holes.According to yin and yang attribute ratio P/N > 2.1, the OD mean value of relatively more each group, determines that the suitableeest monoclonal antibody bag is by concentration and suitable much the most anti-concentration.
Five, optimize confining liquid and off-period
With the suitableeest antibody concentration 4 DEG C of coated elisa plates that spend the night, after washing, close with the confining liquid that 1%BSA, 5%BSA, 1% horse serum, 5% horse serum, 5% skimmed milk power 5 kinds are different respectively, 200 μ l/ holes, close 2 hours for 37 DEG C, carry out double crush syndrome, respectively organize the OD value of yin and yang attribute sample, to determine the sealing effect of different confining liquid.
With the suitableeest antibody concentration 4 DEG C of coated elisa plates that spend the night, after washing, 200 μ l/ holes add best confining liquid 37 DEG C and close, and select 0.5,1.0,1.5,2,3 hour off-period, carry out ELISA respectively, survey OD value.Respectively organize the OD value of yin and yang attribute sample, to determine best off-period.
Six, antibody diluent optimization
With the suitableeest antibody concentration and bag by condition coated elisa plate, yin and yang attribute sample is added after closing, many anti-and ELIAS secondary antibody three each dilution proportion liquid 1:100,1:500 and 1:3000 dilutions respectively, l group 1%BSAPBST, the 2nd group with 5%, 3rd group with 5% horse serum PBST, react after 2 hours at 37 DEG C, through washing, add substrate and to develop the color 15 minutes cessation reactions, microplate reader measures OD450 value, to determine optimum antibody dilution.
Seven, detection limit is determined
With the antibody bag of 5pg/ml by good ELISA Plate, 8 dilutabilitys clinical serum being carried out 1:10,1:50,1:100,1:500,1:1000,1:2000,1:5000,1:10000 carry out ELISA mensuration, set up yin and yang attribute to contrast simultaneously.
Eight, replica test qualification
Repeatability checking in plate: be placed in 37 DEG C of 1h by plate with antibody bag, then 4 DEG C are spent the night, washing rear enclosed, the sample that detection 6 parts is different in same plate: 3 parts of positive reference substances and 3 parts of negative samples, every increment originally repeats 3 holes, carry out ELISA detection according to the top condition optimized, calculate the coefficient of variation CV% of same this OD450 of increment value, be used for evaluating the repeatability detecting sample in plate.
Repeatability checking between plate: wrap by good ELISA Plate under the same conditions with 6 pieces, different time points duplicate detection 6 increment product: 3 parts of positive reference substances and 3 parts of negative samples, every increment originally repeats 3 holes, ELISA detection is carried out according to the top condition optimized, calculate the coefficient of variation CV% of same this OD450 of increment value, be used for evaluating the repeatability detecting sample in plate.With CV% lower than 10%, and there was no significant difference is critical criterion.
Also relate in the present invention by the use of double-antibody sandwich elisa kit in people β 3 acetylglucosamine based transferase 8 albumen vitro detection, the steps include:
1) measuring samples is added: the ELISA Plate of taking out coated antibody, adds the measuring samples (as serum) in 100 μ L/ holes, set up positive control and negative control hole simultaneously, hatch 1h-2h in 37 DEG C, then cleansing solution washing 3-4 time;
2) add and detect antibody: take out the detection antibody in kit, dilute according to the volume ratio of 1:100 with sample diluting liquid and mix, add by 100 μ L/ holes, hatch 0.5h in 37 DEG C, then cleansing solution washs 3-4 time;
3) enzyme labelled antibody is added: take out the enzyme labelled antibody in kit, carry out diluting and mixing according to the ratio of 1:500 with cleansing solution, 100 μ L/ holes add, and hatch 1h in 37 DEG C, then cleansing solution washing 3-4 time;
4) develop the color: add nitrite ion, add by 100 μ L/ holes, develop the color 10-15min under room temperature, adds stop buffer color development stopping by 100 μ L/ holes.
5) optical density value is measured: detect by microplate reader.
In the present invention, cleansing solution, sample diluting liquid, nitrite ion, stop buffer belong to prior art, use the technician of this kit to prepare voluntarily as required, the preferred following condition of the present invention:
Cleansing solution: NaCl8g, KH2PO40.27g, Na2HPO41.42g, KCl0.2g, 800mL distilled water adds 0.5mLTween-20 after dissolving, and fully mixes, and adjustment PH to 7.2, is settled to 1000mL.
Sample diluting liquid: NaCl8g, KH2PO40.27g, Na2HPO41.42g, KCl0.2g, 800mL distilled water adds 10g standard bovine albumin BSA after dissolving, and fully dissolves mixing, and adjustment PH to 7.2, is settled to 1000mL.
Substrate solution A:3,3 ', 5 ', 5-tetramethyl biphenyl diamines 0.2g, absolute ethyl alcohol 100mL, adds distilled water and is settled to 1000mL.
Substrate solution B:Na2HPO414.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea solution 6.4mL, adds distilled water 800mL, and regulate PH to 5.2, supplementary distilled water is settled to 1000mL.
Stop buffer: 2MH2SO4 solution.
The present invention utilizes anti-human β 3GnT8 monoclonal antibody and anti-human β 3GnT8 polyclonal antibody, prepares anti-human β 3GnT8 double-antibody sandwich elisa kit; Adopt DASELISA immuno absorbence ELISA method, detect the content of serum beta 3GnT8 protein level with this kit, especially colorectal cancer patients.By the height of colorectal cancer patients serumβ 3GnT8 protein level, and contrast with normal human serum level, provide an important reference frame by the early diagnosis that is colon cancer of these differential expressions.
Above-described embodiment is only for illustrating technical conceive of the present invention and feature; its object is to person skilled in the art can be understood content of the present invention and be implemented; can not limit the scope of the invention with this; all equivalences done according to Spirit Essence of the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (8)
1. a people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit, is characterized in that: comprising: the ELISA Plate of cleansing solution, sample diluting liquid, nitrite ion, stop buffer, coated antibody, detection antibody, enzyme labelled antibody, standard control albumen.
2. people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit according to claim 1, it is characterized in that: the ELISA Plate of described coated antibody is for coated antibody with mouse-anti people β 3 acetylglucosamine based transferase 8 monoclonal antibody, with coating buffer, coated antibody is diluted, every hole adds the coated antibody after dilution, and is washed by rear cleansing solution at the bag that spends the night; In ELISA Plate, add confining liquid again, wait question response, last cleansing solution washing.
3. people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit according to claim 2, is characterized in that: described detection antibody is rabbit anti-human β 3 acetylglucosamine based transferase 8 polyclonal antibody.
4. people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit according to claim 3, is characterized in that: described enzyme labelled antibody is the goat anti-rabbit antibodies of horseradish peroxidase-labeled.
5. people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit according to claim 4, is characterized in that: described standard control albumen: comprise positive control β 3GnT8 albumen and negative control coli somatic albumen.
6. the people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit according to claim 2 to 4 any one, it is characterized in that: the concentration after the coated liquid dilution of described coated antibody is 5 μ g/mL, coated antibody after dilution every hole addition is 100 μ L, and the temperature of the bag quilt that spends the night is 4 DEG C; The bag that spends the night is 3 ~ 4 times by rear cleansing solution washing times; The amount adding confining liquid in ELISA Plate is every hole 100 μ L, and the temperature of reaction adding confining liquid in ELISA Plate is 37 DEG C, and the reaction time is 1 ~ 2 hour, and it is 3 ~ 4 times that reaction terminates rear cleansing solution washing times.
7. the people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit according to claim 6 any one, is characterized in that: the preparation method of described coated antibody is:
The first, β 3 acetylglucosamine based transferase 8 albumen is mixed with Freund's complete adjuvant, carries out subcutaneous inoculation by BALB/c mouse;
The second, after first respectively at BALB/c mouse, be replaced by incomplete Freund's adjuvant and second time and third time immunity are carried out to BALB/c mouse;
3rd, in BALB/c mouse third time immunity, lumbar injection booster immunization is carried out to BALB/c mouse;
4th, conveniently hybridoma preparation method prepares the fusion hybridoma cell strain of spleen bone-marrow-derived lymphocyte and SP2/0 cell, carry out monoclonal cell sorting cultivation, obtained positive hybridoma cell strain through bag by the ELISA Screening and Identification of β 3 acetylglucosamine based transferase 8 albumen;
5th, prepare mouse-anti people β 3 acetylglucosamine based transferase 8 monoclonal antibody by BALB/c mouse ascites tumor, ascites obtains the coated antibody being used for double-antibody sandwich elisa kit after separation and purification.
8. the people β 3 acetylglucosamine based transferase 8 double-antibody sandwich elisa kit according to claim 7 any one, is characterized in that: in the described first step, the blending ratio of β 3 acetylglucosamine based transferase 8 albumen and Freund's complete adjuvant is 1:1; Every BALB/c mouse immunity total amount 0.2mL carries out subcutaneous inoculation; The concrete time of carrying out second time and third time immunity to BALB/c mouse in described second step is rear BALB/c mouse initial immunity 3 weeks and after 5 weeks; The concrete time of carrying out lumbar injection booster immunization to BALB/c mouse in the 3rd step is BALB/c mouse third time immunity 2 weeks afterwards and first 1 day of Fusion of Cells.
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