CN103983791A - Human CA125 protein quantum dot labeled double-sandwiched immunoassay detection kit - Google Patents

Human CA125 protein quantum dot labeled double-sandwiched immunoassay detection kit Download PDF

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CN103983791A
CN103983791A CN201410232892.8A CN201410232892A CN103983791A CN 103983791 A CN103983791 A CN 103983791A CN 201410232892 A CN201410232892 A CN 201410232892A CN 103983791 A CN103983791 A CN 103983791A
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concentration
quantum dot
mouse
solution
people
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夏曦
郭忠正
卢运萍
罗红志
杨绘
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SHENZHEN CHANGHONG TECHNOLOGY Co Ltd
SHENZHEN BOOMINGSHING MEDICAL DEVICE Co Ltd
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SHENZHEN CHANGHONG TECHNOLOGY Co Ltd
SHENZHEN BOOMINGSHING MEDICAL DEVICE Co Ltd
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    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2800/00—Detection or diagnosis of diseases
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Abstract

The invention provides a human CA125 protein quantum dot labeled double-sandwiched immunoassay detection kit. The human CA125 protein quantum dot labeled double-sandwiched immunoassay detection kit comprises the following components: CA125 protein standards with different fixed concentrations, a positive quality control product, a negative quality control product, 0.01M phosphate buffered solution with pH value of 7.2, polyclonal antibody diluent, a mouse anti-human CA125 monoclonal antibody coated ELISA plate, solution of CdTe quantum dot specifically combined with CA125 protein and mouse anti-human CA125 monoclonal antibody couplet, and 25*plate washing liquid. According to the kit, the polyclonal antibody used as a detection antibody, is labeled by the CdTe quantum dot, and the quantum dot labeling method has the characteristics of wide excitation spectrum, weak autofluorescence, stable fluorescence, long half-life period and the like, and therefore, the resolution ratio, the sensitivity and the specificity during detection can be greatly improved.

Description

The double fastener heart immunoassay detection kit that a kind of people CA125 albumen is quantum dot-labeled
Technical field
The invention belongs to immune diagnostic technique field, particularly the immue quantitative detection reagent box of people CA125 albumen.
Background technology
CA125 is that a kind of relative molecular mass is the glycoprotein of 200kD, is generally distributed in the mesothelial tissue cell surfaces such as pleura, pericardium, peritonaeum, endometrium, genital tract and amnion.In the time that these positions malignant change occur or are subject to inflammatory stimulus, in serum, the level of CA125 will significantly raise.CA125 monitors for oophoroma curative effect and check as the outstanding feature of oophoroma.The CA125 concentration of most of ovarian cancer patients can present a kind of continuation rising trend, can be used as the mark of patient disease progress, prognosis and inspection chemotherapeutic efficacy.In the time of malignant tumor of digestive tract (as cancer of pancreas, liver cancer, cancer of the stomach, intestinal cancer) and some benign hepatic diseases, adenocarcinoma of lung, pelvic inflammatory pathology, endometriosis, level of serum CA 125 also can raise.In the puncture fluid of ovary elium under malignant change and pernicious cystoepithelioma, CA125 level can obviously raise.Therefore CA125 can not be served as the detection index that oophoroma is single.To early ovarian cancer, this detection often checks the accuracy as improved diagnosis together with the pelvioscopy of Transvaginal Ultrasound and per rectum vagina with other.In addition, jointly detect or carry out continuous detecting and also can reduce the false positive results of diagnosis with other tumor markerses.This detection is also very valuable to the result for the treatment of of prediction ovarian cancer patients, shows that chemotherapy is effective if chemotherapeutic period CA125 reduces, if the rear lasting masculin for the treatment of often indicates the recurrence of tumour.At present mainly contain the method for exempting from of putting, ELISA method, chemoluminescence method etc. for measuring the method for CEA clinically.
Past is subject to methodological restriction to put CA125 (CA125) the mensuration kit of exempting from as representative, its sensitivity and antijamming capability wretched insufficiency, have very large drawback, substantially withdraw from the market, applying at present more is enzyme linked immunosorbent detection technology and chemiluminescence.Wherein, enzyme immunoassay (EIA) sensitivity is low, and influence factor is more, easily causes false negative and false positive results; And quantum dot (QDs) has continuous and wide exciting light spectrum, any light source that its fluorescence can be less than its quantum confinement peak by wavelength excites, and fluorescence spectra position can regulate and control by changing quantum dot physical size.So only just can excite the quantum dot of multiple different colours fluorescence to carry out multiplex fluorescence detection with a kind of excitation source of wavelength.Resolution and sensitivity are effectively improved.Therefore, quantum dot immune analytic approach is far superior to enzyme immunoassay (EIA).
Also there is not at present the method that adopts CA125 content in quantum dot immune analyzing and testing human serum, do not have relative kit yet.
Summary of the invention
The object of the invention is to develop a kind of kit that adopts the method for quantum dot immune analysis to detect CA125 content in human serum, its detection speed is fast and highly sensitive.
The invention provides the quantum dot-labeled double fastener heart immunoassay detection kit of a kind of people CA125 albumen, comprise following component:
Each fixed concentration is respectively the CA125 protein standard substance of 0U/ml, 10U/ml, 50U/ml, 250U/ml, 500U/ml, 1000U/ml;
Positive quality control product;
Negative quality-control product;
The phosphate buffer of the 0.01M of pH7.2;
Polyclonal antibody dilution;
Be coated with the ELISA Plate of mouse-anti people CA125 monoclonal antibody;
The couplet solution of the CdTe quantum dot that can be combined with CA125 protein-specific and mouse-anti people CA125 polyclonal antibody;
25 × wash plate liquid.
The preparation method of the CA125 protein standard substance of wherein said each fixed concentration is preferably: cultivate in a large number Proliferation of Human Ovarian Cell Ovcar-3, get cell culture fluid, after dialysis is concentrated, use molecular sieve to isolate the CA125 albumen of purifying, the PBS damping fluid that to re-use pH7.2, concentration be 0.01M is diluted to 0U/ml, 10U/ml, 50U/ml, 250U/ml, 500U/ml, 1000U/ml.
The preparation method of the CA125 albumen of described purifying is preferably as follows:
Step 1: use containing 10%FBS, 3.5g/L Hepes1.9g/L NaHCO 3dMEM complete culture solution cultivate Proliferation of Human Ovarian Cell Ovcar-3,37 DEG C, 5%CO 2cultivate 72 hours;
Step 2: collecting cell nutrient solution, use the bag filter dialysis that the pH7.2 of 20 times of cell culture fluid volumes, PBS damping fluid that concentration is 0.01M are 5kD with molecular cut off, after dialysis, concentrate with containing 20 times of PBS damping fluids that 30% PEG-6000 and pH7.2, concentration are 0.01M again;
Step 3: the cell culture fluid after step 2 is concentrated flows through the molecular sieve of molecular cut off as 50-400kD taking the speed of 2ml/min, is used quick protein purification instrument to collect the albumen at the peak of 200kD left and right, is CA125 albumen.
Step 4: the CA125 protein 20 of collecting is doubly concentrated, and the same step 2 of method, obtains the CA125 antigen after purifying.
Described negative quality-control product is sample diluting liquid, the phosphate buffer that described sample diluting liquid is is 0.01M containing 1%BSA, 0.05% Tween-20 and pH7.2, concentration; Described positive quality control product is that the CA125 antigen sample diluting liquid after purifying is diluted to the solution that concentration is 800U/ml; Described 25 × PBS damping fluid that to wash plate liquid and be containing 0.5%Tween-20 and pH7.2, concentration be 0.01M.
The phosphate buffer that it is 0.01M that described polyclonal antibody dilution is preferably containing 0.5% bovine serum albumin(BSA), 0.1% Tween-20 and pH7.2, concentration.
The preparation method of the described ELISA Plate that is coated with mouse-anti people CA125 monoclonal antibody is preferably as follows:
It is 2 μ g/ml that the mouse-anti people CA125 phosphate buffer that for monoclonal antibody, pH7.2, concentration are 0.01M is adjusted to concentration, the every hole of ELISA Plate adds 100 μ l, and 4 DEG C of overnight incubation, discard coating buffer, then add 300 μ l/ holes containing the PBST solution washing of 0.1%Tween-20 three times, pat dry; Every hole adds the phosphate buffer that 200 μ l are 0.01M containing 0.1%Tween-20,5% bovine serum albumin(BSA) and pH7.2, concentration again, be confining liquid, 4 DEG C of sealings are spent the night, and discard confining liquid, then add 300 μ l/ holes containing the PBST solution washing of 0.1%Tween-20 three times, pat dry.
The preparation method of described mouse-anti people CA125 monoclonal antibody is preferably as follows:
1) select 8～10 weeks BALB/c female mices, before inoculation hybridoma 1～2 week, first to mouse peritoneal injection 0.5ml incomplete Freund's adjuvant;
2) collect well-grown hybridoma, centrifuge washing 1 time, is resuspended in the NaHCO containing 3.5g/L Hepes1.9g/L 3the DMEM nutrient solution of serum-free in, adjusting cell density is 1～2 × 10 6/ ml, obtains cell suspension;
3) every mouse peritoneal injection 0.5ml cell suspension, inoculating cell 7～12 days, mouse web portion obviously expands, and while touch with hand, skin has tension, the skin of abdomen of can sterilizing, connect syringe needle No. 8 with 5ml syringe, thrust abdominal cavity, unload syringe, raise mouse head, ascites is splashed in centrifuge tube; 2～3 days, interval, after ascites regeneration is gathered, gets with method again, and a mouse generally can extract 2～3 times, and the ascites of collecting, through the centrifugal 15min of 3000rpm, is discarded to upper strata grease, cell component and other sediments, draws faint yellow ascites; Adopt the thick purifying of saturated ammonium sulphate method and affinity column method purifying hybridoma ascites, obtain the mouse-anti people CA125 monoclonal antibody after purifying that concentration is 1.2mg/ml.
The preparation method of the couplet solution of the described CdTe quantum dot that can be combined with CA125 protein-specific and mouse-anti people CA125 polyclonal antibody is preferably as follows: the CdTeQDs600ul that gets 0.1mM, adding 18ul concentration is that after the ethylene dichloride of 1mg/ml and the methyl alcohol of 800ul mix, lucifuge is shaken 30min with 800rpm, add again the beta-mercaptoethanol of 8ul to stop being the QDs after activation, get the mouse-anti people CA125 polyclonal antibody pH7.2 after the purifying of 0.75mg, concentration is that the phosphate buffer of 0.01M is diluted to 4 μ g/ml, add in the QDs10ul after activation and mix with it, lucifuge 800rpm concussion 2 hours, after finishing, reaction add again mercaptoethanol 20 μ l to stablize quantum dot, then the bag filter that is 12kD with molecular cut off dialysis, after dialysis under 16300 × g condition centrifugal 3min, remove supernatant, be precipitated, the coupling that is purifying has the mouse-anti people CA125 polyclonal antibody of quantum dot, precipitation is resuspended in to pH7.2, concentration is in the phosphate buffer of 0.01M, obtaining concentration is the CdTe quantum dot of 5mg/ml and the couplet solution of mouse-anti people CA125 polyclonal antibody.
The preparation method of described CdTeQDs is preferably as follows:
By 2.5 × 10 -4the CdCl of mol 22.5H 2o is dissolved in the ultrapure water of 25ml, adds 3 × 10 -4the two hydration trisodium citrates, 0.5 × 10 of glutathione GSH, the 0.1g of mol -4the Na of mol 2teO 3with 2.4 × 10 -4the NaBH of mol 4under condition with magnetic stirring apparatus with 200rpm, stir 30 minutes, NaOH with 0.01M in whipping process regulates PH to 10.5, after completing, stirring puts into the microwave device that power is made as the band backflow of 600W, in the time that becoming light green, solution colour starts to reflux, back flow reaction 4h, along with the difference of return time forms the quantum dot taking glutathione as stabilizing agent of a series of different-grain diameter 1-10nm, reacted solution is cooled to after room temperature and is precipitated with two volumes absolute ethyl alcohol centrifugal 5min under the condition of 4000rpm, remove excessive Cd in supernatant 2+, TeO 3 2-deng impurity, similarity condition repeats 3 times, after ethanol volatilizees completely, in the pH7.2 that precipitation is resuspended in, the phosphate buffer that concentration is 0.01M, obtains the CdTeQDs of 0.1mM;
The preparation method of the mouse-anti people CA125 polyclonal antibody after described purifying is preferably as follows:
1) animal immune
CA125 antigen 1 0000U after above-mentioned purifying is dissolved in the phosphate buffer solution that 1ml pH7.2, concentration are 0.01M and obtains antigenic solution, in 1ml incomplete Freund's adjuvant, add the Much's bacillus of deactivation to make Freund's complete adjuvant, and add above-mentioned 1ml antigenic solution, concuss makes it the fully emulsified antigen emulsion that obtains, extract this antigen emulsion with 3ml syringe, rabbit is carried out to injections of antigens emulsion, and carry out several times booster immunization;
2) collect antiserum
7～10 days collection blood after booster immunization injection, a large amount of blood of collecting after generation antibody to be determined, blood is placed in to 37 DEG C of constant temperature ovens to be placed 30 minutes, again 4 DEG C of hold over night, medication shovel is dialled blood clot fall from tube wall, by blood transfer to plastic centrifuge tube, 4 DEG C, centrifugal 10 minutes of 10,000g, collects supernatant and is antiserum;
3) antibody purification
In antiserum, adding solid sodium azide to concentration is 0.05%, 4 DEG C, 15, centrifugal 5 minutes of 000g, shift out supernatant and remove by filter unnecessary fat through the filtrator of 0.45 μ m again, dilute with the volume ratio of 1:5 removing by filter the antiserum pH7.4 of unnecessary fat and TBS buffer solution that concentration is 0.1M, filter with the filtrator of 0.45 μ m again, with the speed of 0.5ml/min by the antiserum-TBS buffer solution after filtering to GE1ml Protein A Sepharose prepacked column, continuous upper prop 2 times also retains loading efflux, the TBS buffer solution that is 0.1M by pH7.4 and concentration cleans pillar to A λ 280nmglycocoll-the HC1 that is 0.1M by pH4.5, concentration again after <0.008 is with the speed wash-out of 0.5ml/min, and eluent goes to dialysed overnight in the PBS damping fluid that pH7.2 and concentration are 0.01M immediately, collects as stated above eluent to A λ 280nm<0.008, obtains the anti-human CA125 polyclonal antibody of rabbit after purifying.
The present invention also provides the detection method of the quantum dot-labeled double fastener heart immunoassay detection kit of described people CA125 albumen, comprises the steps:
Step 1: get the ELISA Plate that is coated with mouse-anti people CA125 monoclonal antibody, room temperature is placed 30min;
Step 2: add in the lath hole of ELISA Plate in step 1 with 100 μ l/ hole testing samples, make negative control, join respectively in the lath hole of ELISA Plate in step 1 with the CA125 protein standard substance 100 μ l that positive quality control product 100 μ l make positive control, each fixed concentration with negative quality-control product 100 μ l simultaneously, 37 DEG C of reactions after 1 hour with 25 × wash the solution flushing of plate liquid after 25 times of dilutions 3 times, each 3min, pats dry;
Step 3: the CdTe quantum dot that can be combined with CA125 protein-specific with polyclonal antibody dilution and the couplet solution 1:10000 of mouse-anti people CA125 polyclonal antibody dilution, every hole adds 100 μ L, 37 DEG C of reactions after 1 hour with 25 × wash the solution flushing of plate liquid after 25 times of dilutions 3 times, pat dry, then add the phosphate buffer of the 0.01M of the pH7.2 of 100 μ L;
Step 4: the ELISA Plate of handling well is measured to fluorescence intensity level via fluorescence microplate reader, by fluorescence intensity level and the corresponding concentration Criterion curve of CA125 protein standard substance, obtain the detection line that r value is greater than 0.990, negative quality-control product and positive quality control product numerical value all in the range of linearity, calculation sample content.
In kit of the present invention, carry out mark as the polyclonal antibody that detects antibody with CdTe quantum dot, because the quantum dot-labeled method adopting has exciting light spectrum width, a little less than autofluorescence, the features such as the stable and long half time of fluorescence, can improve the resolution, sensitivity and the specificity that detect greatly while therefore detection.
Brief description of the drawings
Fig. 1 is the proteins gel electrophoresis picture of the CA125 antigen after purifying.
Fig. 2 is the OD450nm value of measuring the anti-human CA125 polyclonal antibody of rabbit after purifying by full-automatic microplate reader, and taking dilutability as horizontal ordinate, the typical curve that OD value is drawn for ordinate.
Fig. 3 is the OD450nm value of measuring the mouse-anti people CA125 monoclonal antiserum after purifying by full-automatic microplate reader, and taking dilutability as horizontal ordinate, the typical curve that OD value is drawn for ordinate.
Fig. 4 is the fluorescence intensity level of using the ELISA Plate that fluorescence microplate reader Check processing is good.Taking the CA125 antigen after the purifying of variable concentrations as horizontal ordinate, taking corresponding glimmering light intensity value as ordinate, curve plotting.
Embodiment
Following instance is used for illustrating the present invention, but is not used for limiting the scope of the invention.
The Purification of embodiment 1.CA125 albumen
A large amount of Proliferation of Human Ovarian Cell Ovcar-3 that cultivate, get cell culture fluid, use molecular sieve separation of C A125 albumen, and concrete steps are as follows:
Step 1: use DMEM complete culture solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS 3) cultivation Proliferation of Human Ovarian Cell Ovcar-3 (Proliferation of Human Ovarian Cell Ovcar-3 is purchased from U.S. ATCC), 37 DEG C, 5%CO 2cultivate 72 hours.
Step 2: collecting cell nutrient solution, use the PBS damping fluid (pH7.2 of 20 times of cell culture fluid volumes, concentration is 0.01M) dialysis (bag filter molecular cut off 5kD), after dialysis, 20 times of the PBS damping fluids (pH7.2, concentration is 0.01M) with the PEG-6000 that contains 30% (v/v) are concentrated again.
Step 3: use quick protein purification instrument to collect destination protein (CA125), the cell culture fluid after step 2 is concentrated flows through molecular sieve (GE Sephadex with the speed of 2ml/min tMg-20050-400kD), use quick protein purification instrument to collect the albumen at the peak of 200kD left and right, be CA125 albumen.
Step 4: by concentrated 20 times of the protein liquids (being the CA125 albumen of step 3) of collecting, the same step 2 of method, obtains the CA125 antigen after purifying.
Detect the purity of the CA125 antigen after purifying by proteins gel electrophoresis, as shown in Figure 1, measure its concentration is 20000U/ml to result, for follow-up test.
The anti-human CA125 polyclonal antibody preparation of embodiment 2. rabbit and qualification
1. animal immune
(the large ear of Japan is white to prepare two adult rabbits, 4-6 month, body weight 2Kg, male), be that CA125 antigen (20000U/ml) after the purifying obtaining in 0.5ml embodiment 1 dissolves in 1ml phosphate buffer solution (pH7.2, concentration is 0.01M) and obtains antigenic solution by 10000U.At 1ml incomplete Freund's adjuvant (paraffin oil: amnion fat=2: add the Much's bacillus (final concentration is 5mg/mL) of deactivation to make Freund's complete adjuvant 1), and add above-mentioned 1ml antigenic solution, concuss makes it the fully emulsified antigen emulsion that obtains, extract this antigen emulsion with 3ml syringe, connect 25G syringe needle, the bubble in Inside Syringe.From cage, taking out rabbit is placed on smooth, at 4 different positions: two are in back, two are in thigh place carries out hypodermic injection, the skin of comforting the rabbit hair of injection place and exposing with ethanol disinfection, pinch out skin, the angle inserting needle that syringe needle is spent with relative skin 15, depth of needle is 1～2cm, carefully do not thrust in muscle, at 4 different parts each injection approximately 500 μ l antigen emulsions respectively, after injection finishes, pin after placing several seconds, is extracted gently in injection place again, and sterilize in injection place with ethanol, every 2～3 weeks repeat aforesaid operations injections of antigens emulsion at above-mentioned 4 positions, carry out altogether 5 booster immunizations and within 7～10 days, collect blood according to following steps 2 after booster immunization injection.Front to the blood of collection and the injection blood of collecting is compared, check and whether have antibody to produce.After generation antibody to be determined, can collect in a large number blood, but every rabbit collection blood can not be more than 40ml to prevent shock.
2. collect antiserum
The method of collecting blood is: rabbit is put on fixed mount gently, dimethylbenzene is applied to the upper middle part of ear's blood vessel, with blade tilt 45 ° of otch that cut out 0.23～0.3cm at this place blood can be flowed out freely, with sterilization after pipe collect the blood oozing, dab incision if there is solidifying available warm water before finishing, continue again to collect, collect the gauze after available sterilization after appropriate blood and dab affected part, after 10～20 seconds definite blood flows in flicking affected part stop, can finishing.Blood is placed in to 37 DEG C of constant temperature ovens and places 30 minutes, then 4 DEG C of hold over night.Medication shovel is dialled blood clot fall from tube wall, and by blood transfer, to plastic centrifuge tube, 4 DEG C, centrifugal 10 minutes of 10,000g, collects supernatant and be antiserum (can preserve the several years at-20 DEG C).
3. antibody purification
In antiserum, adding solid sodium azide to concentration is 0.05% (m/v), and 4 DEG C, centrifugal 5 minutes of 15,000g, shifts out supernatant and pass through filter again (0.45 μ m) removes by filter unnecessary fat.
The above-mentioned antiserum TBS buffer solution (pH7.4, concentration is 0.1M) that removes by filter unnecessary fat is diluted with the volume ratio of 1:5, then (0.45 μ m) filters to use filtrator.With the speed of 0.5ml/min by the antiserum-TBS buffer solution after filtering to post (GE1ml Protein A Sepharose prepacked column), be the combination that ensures antiserum and filler, need continuous upper prop 2 times also retains loading efflux.With TBS buffer solution (pH7.4,0.1M) clean pillar to A λ 280nmafter <0.008, use again glycocoll-HC1 (pH4.5, concentration is 0.1M) with the speed wash-out of 0.5ml/min, eluent goes to dialysed overnight in PBS damping fluid (pH7.2,0.01M) immediately, collects as stated above eluent to A λ 280nm<0.008, obtains the anti-human CA125 polyclonal antibody of rabbit after purifying, and recycling spectrophotometric determination its concentration is 2.8mg/ml, after packing 2～8 DEG C of preservations.
4. the qualification of the anti-human CA125 polyclonal antibody of rabbit
The sensitivity of the anti-human CA125 polyclonal antibody of 4.1 indirect ELISA Preliminary detection rabbit
1) coated
With PBS damping fluid (pH7.20.01M), the CA125 antigen after purifying (concentration of embodiment 1 is 20000U/ml) is diluted to protein concentration and is respectively 20000U/ml, 10000U/ml, 5000U/ml, 2500U/ml, 1500U/ml, 1000U/ml, 500U/ml, 250U/ml, 100U/ml, 10U/ml, the protein sample 100 μ l that get dilution add in the reacting hole of each polystyrene board.Next day, discards solution in hole, uses containing the PBS damping fluid (pH7.2,0.01M) of 0.1%Tween-20 and washes 3 times, each 2min.
2) application of sample
Anti-human rabbit after step 3 purifying CA125 polyclonal antibody is pressed to 1:500,1:1000,1:2000,1:5000,1:10000 doubly dilutes, the liquid 100 μ l that get different dilute concentrations are added in above-mentioned coated reacting hole, putting 37 DEG C hatches 2 hours, with the PBS damping fluid (pH7.2 containing 0.1%Tween-20, concentration is 0.01M) wash each 2min 3 times.
3) add enzyme labelled antibody
In each reacting hole, add the goat anti-rabbit igg of HRP mark of 1:80000 dilution (purchased from Abnova company, 111-035-003,2ml dress) 100 μ l, hatch 1 hour for 37 DEG C, wash 3 times through the PBS damping fluid (pH7.2, concentration is 0.01M) containing 0.1%Tween-20, each 2min, then pats dry.
4) add substrate solution colour developing
In each reacting hole, add TMB nitrite ion (purchased from green skies department, P0209,100ml dress) 100 μ l, colour developing 10min.
5) cessation reaction
In each reacting hole, add 2M H 2sO 450 μ l cessation reactions.
Result is judged: on ELISA detector, survey OD value in 450nm place.Taking albumen dilutability as horizontal ordinate, taking 450nmOD value as ordinate, figure runs a curve.
Result demonstration, it is 0U～1000U that the anti-human CA125 polyclonal antibody of the rabbit 1:10000 after purifying dilutes between the detection zone that has good linear relationship.
4.2 the western blotting of the anti-human CA125 polyclonal antibody of rabbit qualification
1) electrophoresis
Preparation 12%SDS-PAGE gel, the CA125 antigen after the purifying that embodiment 1 is obtained carries out point sample by the amount of 500U, 250U, 150U, 100U, 50U, 25U, 10U respectively, uses 80V constant voltage electrophoresis;
2) transferring film
Adopting half-dried electricity to turn method forwards the albumen in PAGE gel on NC film to;
3) sealing
NC film is put in the PBS damping fluid (pH7.2, concentration is 0.01M) that contains 5%BSA, and 100rpm seals 1h;
4) hatch primary antibodie
Primary antibodie adds the anti-human CA125 polyclonal antibody of the rabbit after step 3 purifying doubly diluting by 1:500,1:1000,1:2000,1:5000,1:10000, hatch 1h for 37 DEG C, wash three times through the PBS damping fluid (pH7.2, concentration is 0.01M) containing 0.1%Tween-20;
5) hatching two resists
The goat anti-rabbit igg (purchased from Abnova company, 111-035-003,2ml dress) of the two anti-HRP marks that are 1:10000 dilution, hatches 1h for 37 DEG C, washes three times through the PBS damping fluid (pH7.2, concentration is 0.01M) containing 0.1%Tween-20;
6) colour developing
Add nitrite ion (DAB6.0mg; 0.01M PBS10.0ml), lucifuge color development at room temperature is to occurring dark band,
7) stop
Finally put into distilled water cessation reaction;
Result demonstration, the anti-human CA125 polyclonal antibody of rabbit after the purifying that 1:2000 doubly dilutes can well be identified the CA125 antigen after 10U purifying.
4.3 the titration of the anti-human CA125 polyclonal antibody of rabbit after purifying
Adopt indirect elisa method, its concrete steps are as follows:
1) wrapper sheet
Utilize phosphate buffer that pH7.2, concentration are 0.01M by the CA125 antigen diluent after purifying in embodiment 1 to optimum concentration 50U/100ul, in each ELISA Plate, add 100ul, put into wet box, 4 DEG C of coated spending the night;
2) sealing
Discard coating buffer next day, use the PBST (pH7.2 containing 0.1% (v/v) Tween-20,0.01M) the every reacting hole of detersive enzyme target 4 times, 300ul/ time, each 5min, dries, add confining liquid (the PBS pH of buffer 7.2 containing 0.1% (v/v) Tween-20,5% (m/v) bovine serum albumin(BSA), 0.01M) 300 μ l/ hole sealings, put into wet box, and 4 DEG C are spent the night;
3) enzyme mark primary antibodie
Discard confining liquid, the PBS doubling dilution (1:(2 that the same washing is 0.01M by pH7.2, concentration n× 100), the integer of n>=0) the anti-human CA125 polyclonal antibody of rabbit (step 3) 100 μ l/ holes after purifying, set up blank, the positive and negative control, 37 DEG C of wet box incubation 1.5h simultaneously;
4) ELIAS secondary antibody
Discard serum, the same washing, then add HRP-goat anti-rabbit igg (purchased from Abnova company, 111-035-003,2ml dress) the 100 μ l that are diluted to 1:80000 with PBS (pH7.20.01M) in every hole, 37 DEG C of incubations 1 hour.
5) add substrate solution colour developing
Discard ELIAS secondary antibody, the same washing, TMB develops the color (with step 4.1), 37 DEG C of lucifuge colour developing 10min.
6) cessation reaction
Every hole adds the 2M H of 50 μ l 2sO 4stop buffer cessation reaction.
Result is measured: measure OD450nm value by full-automatic microplate reader, the results are shown in accompanying drawing 2, horizontal ordinate is dilution log value, and ordinate is OD value.
Result shows, the tiring as 1:25600 of the anti-human CA125 polyclonal antibody of rabbit after purifying.
Embodiment 3. preparations of mouse-anti people CA125 monoclonal antibody and qualification
1. the preparation of mouse-anti people CA125 monoclonal antibody
1.1 animal immune
A. the CA125 antigen (20000U/ml) after purifying in embodiment 1 is mixed with isopyknic Freund's complete adjuvant (with embodiment 2), it is fully emulsified that concuss makes it, make Water-In-Oil CA125 antigen emulsion, through the subcutaneous multi-point injection in back of SPF level pure lines BALB/c, the female mice in 6～8 week age, every 100 μ l.Every mouse immune dosage is CA125 antigen emulsion 1000U.
B. after two weeks, mix (with embodiment 2) with isopyknic incomplete Freund's adjuvant with the CA125 antigen (20000U/ml) after purifying in embodiment 1, fully emulsified, through the subcutaneous multi-point injection in back of the SPF of step a level pure lines BALB/c, the female mice in 6～8 week age, every 100 μ l.Every mouse immune dosage is CA125 antigen emulsion 1000U.
C. respectively at booster immunization first, (be that step is carried out booster immunization for the 2nd, 4,6 weeks after b), its each booster immunization method of operating, injections of antigens emulsion and injected dose are all with step b.
Detect after each booster immunization the tiring of serum antibody of three days with indirect elisa method (with embodiment 2 steps 4.3), still can identify CA125 antigen in the time that the serum antibody titer detecting reaches 1:25600 time, the antibody that has produced anti-CA 125 albumen in Mice Body is described.
First 3 days mouse of Fusion of Cells (reach 1:25600 and still can identify CA125 antigen because be actually to tire after the 4th booster immunization, therefore this time is after the 4th booster immunization, but do not get rid of the time of second and third booster immunization yet) tail vein injection or lumbar injection 100U (using the antigen emulsion of step b) booster immunization again, for step 1.3 Fusion of Cells is used.
The recovery of 1.2SP2/0 cell and cultivation
From liquid nitrogen container, take out and contain SP2/0 cell cryopreservation tube (taking from Wuhan University's cell bank), drop into immediately in 37 DEG C of water-baths, after thawing, in the centrifugal 5～10min of 1000rpm, abandon supernatant; Preparation is containing DMEM nutrient solution (3.5g/L Hepes, the 1.9g/L NaHCO of 10% calf serum 3) cultivation recovery cell.By cell (1 × 10 6individual) to be inoculated in the both sides, female mice back in normal BALB/c, 6～8 week age subcutaneous.Treat that knurl grows to 3～5cm left and right, carry out the aseptic knurl of plucking, with serum-free DMEM nutrient solution (3.5g/L Hepes1.9g/L NaHCO 3) after washing 3 times, be cut into diameter 2mm left and right fritter with little scissors, add and added in advance 2～3ml serum-free DMEM nutrient solution (3.5g/L Hepes1.9g/L NaHCO 3) 200 order copper sieves in, grind, squeeze out single tumour cell with piston, put DMEM complete culture solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS 3) middle cellar culture, make cell maintain exponential phase.Merge first 1 day for SP2/0 cell changes nutrient solution one time, after cell counter counting, with DMEM complete culture solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS 3) regulate cell density be 1～5 × 10 5individual/ml, merges and got approximately 1～5 × 10 the same day 7in the aseptic centrifuge tube of individual SP2/0 cell harvesting to 1 50ml, the centrifugal supernatant of abandoning, adds 5ml serum-free DMEM nutrient solution (3.5g/L Hepes1.9g/L NaHCO 3), mix, obtain SP2/0 cell solution, cell count is 1 × 10 7individual/ml, for subsequent use.
1.3 separate fusion mouse boosting cell
Get the mouse (first 3 days tail vein injections of Fusion of Cells or lumbar injection are crossed the antigen emulsion of 100U step b) of booster immunization in 1.1, pluck eyeball blood sampling and put to death, 75% alcohol-pickled 5～10 minutes, be fixed on cake wax; With after 75% alcohol disinfecting skin, cut off skin of abdomen, expose peritonaeum also with 75% alcohol wipe sterilization; Draw serum-free DMEM nutrient solution 5ml with glass syringe and inject mouse peritoneal, with syringe suction (attention can not puncture the digestive organs of mouse) repeatedly in abdominal cavity; Extract abdomen intracavity liquid out with this syringe, inject in 50ml centrifuge tube; Change tweezers, mention peritonaeum, change scissors, expose abdominal cavity, the aseptic spleen of winning, carefully cuts off periphery fat and manadesma fast, with serum-free DMEM nutrient solution (3.5g/L Hepes1.9g/L NaHCO 3) wash 1～2 time, then spleen is put into the plate of containing 200 order copper sieves, and break coating, grind, push splenocyte with piston and cross net, draw serum-free DMEM nutrient solution (3.5g/L Hepes1.9g/L NaHCO 3) 5ml piping and druming copper sieve, the splenocyte of collecting after net is put into the aseptic centrifuge tube of 50ml; The centrifugal 5min of speed by aseptic centrifuge tube with l000rpm; Abandon supernatant, add 5ml serum-free DMEM nutrient solution (3.5g/L Hepes1.9g/LNaHCO 3) re-suspended cell becomes splenocyte solution, cell count is 1 × 10 5individual/ml, stand-by; With DMEM nutrient solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS 3) macrophage in the abdominal cavity of resuspended precipitation, obtain macrophage solution (2 × 10 4individual/ml), add 96 well culture plates, 100 μ l/ holes, are then placed in 37 DEG C, 5%CO 2in incubator, cultivate, for subsequent use.
1.4 the selectivity of Fusion of Cells and hybridoma is cultivated
Above-mentioned 1.2 SP2/0 cell solution is mixed to 20ml altogether with 1.3 splenocyte solution by the volume ratio of 1:5, put into the aseptic centrifuge tube of 50ml, with the centrifugal 5min of speed of l000rpm; Abandon supernatant, flick the pipe end, make precipitation loosening, slowly drip the PBS solution (pH7.2 containing PEG45% (v/v) of 37 DEG C of pre-temperature along centrifugal tube wall, 0.01M) lml slowly rotates centrifuge tube to mix cell simultaneously, in 1min, PBS solution is added; Put in 37 DEG C of water-baths after 1min, then in 5min, slowly add the serum-free DMEM nutrient solutions of 37 DEG C of pre-temperature (containing 3.5g/L Hepes1.9g/L NaHCO 3) 8ml, stir and make cell become the suspension of homogeneous gently simultaneously; Put in 37 DEG C of water-baths after 5min, with the centrifugal 5min of speed of l000rpm, abandon supernatant; Add the HAT nutrient solution of 37 DEG C of pre-temperature [with DMEM nutrient solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS 3) be basis, add 2% (v/v) HAT solution (purchased from Sigam company) and be made into], gently outstanding cell, cell count adds up to 2 × 10 6individual/ml.By 1 × 10 5individual splenocyte/hole is added drop-wise in the 96 porocyte culture plates containing feeder cells, is placed in 37 DEG C, 5%CO 2in incubator, cultivate.Within after merging 5～10 days, can use HAT nutrient solution (the same) half amount to change liquid according to clonal growth situation; Merge and cultivate 2 weeks rear interchangeable HT nutrient solutions [with DMEM complete culture solution (the 3.5g/L Hepes1.9g/L NaHCO of 10%FBS 3) be basis, add 1% (v/v) HAT solution (purchased from Sigam company) and be made into], change liquid and cultivate interchangeable DMEM nutrient solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS after 3 weeks 3); Continuing to cultivate 3～5 days is that visible little clone occurs, hybrid cell is larger, rounded and transparent, and other cell light transmissions are poor and dead gradually; Cultivate 8～12 days, clonal growth is to 1/3～1/2 of hole floorage again, and now desirable culture supernatant, carries out antibody test; Once the clone cell of the predetermined antibody of secretion detected, should be in time by positive colony transferred species to 24 well culture plates, more further proceed to culture flask and expand and cultivate, frozen part clone cell carries out cloning cultivation simultaneously.
1.5 the screening of the hybridoma of secretion monoclonal antibody specific
After Fusion of Cells, once the clone who grows suitable size, should select in time the hybrid cell clone of the predetermined antibody of sensitive, quick, reliable immunological method screening secretion.This experiment adopts the indirect elisa method of recombined human ES antigen to detect, and wherein primary antibodie is Hybridoma Cell Culture supernatant, and two resist for HRP-mountain sheep anti-mouse igg (dilutability is 1:80,000).Concrete operation step detects with 4.3 mouse serum titers in embodiment 2.In the time that the culture supernatant antibody detecting reaches 1:25600, still can identify the CA125 antigen after purifying, get the single hole monoclonal cell strain of wherein tiring the highest and be labeled as A1.
1.6 subclonings are cultivated
Cloning is cultivated for the hybridoma cell strain that obtains secretion monospecific antibody most important.Generally need to carry out 3～4 subclonings and cultivate, to ensure to secrete the stability of sex clone growth.Adopt limiting dilution assay, concrete steps are as follows: at DMEM complete culture solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS 3) in add the hybridoma in A1 hole (step 1.5), making cell density is 4 × 10 2the cell suspension of individual/μ l, inoculation 96 well culture plates, every hole 50 μ l, 37 DEG C, 5%CO 2overnight incubation in incubator; Clone in inferior daily micropipettor piping and druming Hybridoma Cell Culture plate mesopore, is suspended in DMEM complete culture solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS 3) in; Sampling, with blood counting chamber counting, is used respectively DMEM complete culture solution (the 3.5g/L Hepes1.9g/L NaHCO containing 10%FBS 3) adjust cell density to 20/ml, 5/ml; Respectively by the hybridoma suspension inoculation of two kinds of density in containing in the culture plate of feeder cells, every hole 50 μ l, make every hole respectively containing 2,1,0.5 cells; Be placed in 37 DEG C, 5%CO 2in incubator, cultivate, a Zhou Houjia adds DMEM complete culture solution (the 3.5g/L Hepes1.9g/LNaHCO of 100 μ l containing 10%FBS 3), cultivate 12～15 days, to there being clone's culture supernatant to carry out antibody test (method with in embodiment 2 4.3); To the cloning cultivation (condition of culture is the same) again of positive monoclonal cell, until clone's secreting specificity antibody of 100%; Positive colony is further expanded to cultivation, frozen simultaneously.
A large amount of preparations and the purifying of 1.7 mouse-anti people CA125 monoclonal antibodies
In cell cultivation process, hybridoma can produce and secrete monoclonal antibody, approximately 10～100 μ g/ml.In order to obtain a large amount of high-titer antibodies, conventionally hybridoma is implanted in BALB/c mouse body, to prepare and collect the ascites containing monoclonal antibody specific, method is as follows:
1) select 8～10 weeks BALB/c female mices, before inoculation hybridoma 1～2 week, first to mouse peritoneal injection 0.5ml incomplete Freund's adjuvant (formula is with embodiment 2);
2) collect well-grown hybridoma, centrifuge washing 1 time, is resuspended in DMEM nutrient solution (the 3.5g/L Hepes1.9g/L NaHCO of serum-free 3) in, adjusting cell density is 1～2 × 10 6/ ml, obtains cell suspension;
3) every mouse peritoneal injection 0.5ml cell suspension; The health status of close observation mouse and ascites sign, inoculating cell 7～12 days, visible mouse web portion obviously expands, and while touch with hand, skin has tension, the skin of abdomen of can sterilizing, connect syringe needle No. 8 with 5ml syringe, thrust abdominal cavity, unload syringe, raise mouse head, ascites is splashed in centrifuge tube; 2～3 days, interval, after ascites regeneration is gathered, gets with method again, and a mouse generally can extract (extracting 1ml) 2～3 times at every turn.The ascites of collecting, through the centrifugal 15min of 3000rpm, is discarded to upper strata grease, cell component and other sediments, draw faint yellow ascites; Adopt the thick purifying of saturated ammonium sulphate method and affinity column method (GE1ml Protein A Sepharose prepacked column) purifying hybridoma ascites, obtain the mouse-anti people CA125 monoclonal antibody after purifying, its concentration is 1.2mg/ml.
2. the qualification of the mouse-anti people CA125 monoclonal antibody after purifying
Mouse-anti people CA125 monoclonal antibody sensitivity after 2.1 indirect ELISA Preliminary detection purifying
CA125 antigen after the purifying of embodiment 1 is pressed respectively to 2000U, 1000U, 500U, 250U, 150U, 100U, 50U, 25U, 10U, the amount of 1U is coated with (method is with 4.1 steps in embodiment 2), primary antibodie adds by 1:500, 1:1000, 1:2000, mouse-anti people CA125 monoclonal antibody (gained in step 1.7) after the purifying that 1:4000 doubly dilutes, the goat anti-mouse IgG of the two anti-HRP marks that are 1:80000 dilution is (purchased from Abnova company, 111-035-003, 2ml dress), hatch 1h for 37 DEG C, through the PBS damping fluid (pH7.2 containing 0.1%Tween-20, concentration is 0.01M0.1%Tween-20) pat dry after washing three times, add TMB nitrite ion (purchased from green skies company) 100 μ l, colour developing 10min, add 2M H 2sO 450 μ l cessation reactions, 450nm surveys OD value.Concrete grammar is with step 4.1 in embodiment 2.Taking albumen dilutability as horizontal ordinate, taking 450nmOD value as ordinate, figure runs a curve.
Result shows, the mouse-anti people CA125 monoclonal antibody dilutability after purifying exists 0U～1000U between the detection zone of good linear relationship while being 1:4000.
2.2 the western blotting of mouse-anti people CA125 monoclonal antibody qualification
CA125 antigen after embodiment 1 purifying is carried out to SDS-PAGE glue point sample by the amount of 500U, 250U, 150U, 100U, 50U, 25U, 10U respectively, primary antibodie adds the mouse-anti people CA125 monoclonal antibody (collecting gained in step 1.7) after the purifying doubly diluting by 1:500,1:1000, the goat anti-mouse IgG of the two anti-HRP marks that are 1:10000 dilution is (purchased from Abnova company, 111-035-003,2ml dress), concrete grammar is with step 4.2 in embodiment 2.
As a result, the mouse-anti people CA125 monoclonal antibody after the purifying that dilutability is 1:1000 can well be identified the CA125 antigen after 10U purifying.
The titration of anti-CA 125 monoclonal antibody after 2.3 purifying
Adopt indirect elisa method, coated with the CA125 antigen 50U/100 μ l after the purifying of embodiment 1,4 DEG C are spent the night; After washing, 5% skimmed milk power sealing is spent the night; Using mouse-anti people CA125 monoclonal antibody (the embodiment 3 steps 1.7) serial dilution after purifying as primary antibodie, 37 DEG C of incubation 2h; The goat anti-mouse IgG 1:80 of HRP-mark after washing, 000 dilution is anti-as two, 37 DEG C of incubation 1h; After washing, add substrate colour developing and stop in time, surveying OD450 value, other actual conditions is with 4.3 of embodiment 2.
The results are shown in accompanying drawing 3, the tiring as 1:512 of the mouse-anti people CA125 monoclonal antibody after purifying.
2.4 the immunoglobulin class of mouse-anti people CA125 monoclonal antibody and hypotype qualification
With reference to Sigma company antibody subtype detection kit instructions, adopt Antigen-Mediated ELISA method to measure Ig classification and the hypotype of monoclonal antibody, the mouse-anti people CA125 monoclonal antibody (embodiment 3 steps 1.7) after the gained purifying of mensuration is alpha hypotype.
Embodiment 4 prepares the couplet solution of CdTe quantum dot and mouse-anti people CA125 polyclonal antibody
Synthesizing of 4.1CdTe quantum dot
By CdCl 22.5H 2o (2.5 × 10 -4mol) be dissolved in the ultrapure water of 25ml, add glutathione GSH (3 × 10 -4mol), two hydration trisodium citrates (0.1g), Na 2teO 3(0.5 × 10 -4and NaBH mol) 4(2.4 × 10 -4mol), under condition with magnetic stirring apparatus with 200rpm, stir 30 minutes, in whipping process, regulate PH to 10.5 with NaOH (0.01M), it is all under room temperature environment that institute responds, after completing, stirring puts into the microwave device (power is made as 600W) that band refluxes, in the time that becoming light green, solution colour starts to reflux, back flow reaction 4h, along with the difference of return time forms the quantum dot taking glutathione as stabilizing agent of a series of different-grain diameters (1-10nm).Reacted solution is cooled to after room temperature and is precipitated with two volumes absolute ethyl alcohol centrifugal 5min under the condition of 4000rpm.Remove excessive Cd in supernatant 2+, TeO 3 2-deng impurity, similarity condition repeats 3 times, after ethanol volatilizees completely, in the PBS damping fluid (pH7.4,0.01M) that precipitation is resuspended in, obtains CdTeQDs (CdTe quantum dot), is the CdTeQDs (0.1mM) after activation.
4.2CdTe the coupling of quantum dot and mouse-anti people CA125 polyclonal antibody and purifying (serve as a mark two anti-)
Get the CdTeQDs600ul of above-mentioned concentration, add the EDC (ethylene dichloride) of 18ul (1mg/ml) to mix rear lucifuge concussion 30min (800rpm) with the methyl alcohol of 800ul, add again the beta-mercaptoethanol of 8ul to stop being the QDs after activation, get mouse-anti people CA125 polyclonal antibody (embodiment 2) PBS (pH7.2 after the purifying of 0.75mg, concentration is 0.01M) be diluted to 4 μ g/ml, add in the QDs10ul after activation and mix with it, 2 hours (800rpm) of lucifuge concussion, after finishing, reaction add again mercaptoethanol 20 μ l to stablize quantum dot, then dialysis (bag filter molecular cut off 12kD).After dialysis under 16300 × g condition centrifugal 3min, remove supernatant, be precipitated, the coupling that is purifying has the mouse-anti people CA125 polyclonal antibody of quantum dot, precipitation is resuspended in PBS (pH7.20.01M), the couplet solution that obtains CdTe quantum dot and mouse-anti people CA125 polyclonal antibody, concentration is 5mg/ml, 4 DEG C of preservations.
Through indirect elisa method (method is with embodiment 2 steps 4.3), primary antibodie is mouse-anti people CA125 monoclonal antibody, mark two resists the couplet solution for CdTe quantum dot and mouse-anti people CA125 polyclonal antibody, detects and finds that mark two resists that under the diluting condition of 1:5000, to detect effect best.
Embodiment 5
The present embodiment provides people CA125 albumen quantum dot-labeled double fastener heart immunoassay detection kit and detection method thereof.
The double fastener heart immunoassay detection kit that described people CA125 albumen is quantum dot-labeled, comprising:
Be coated with the ELISA Plate of mouse-anti people CA125 monoclonal antibody;
Each fixed concentration (0U/ml, 10U/ml, 50U/ml, 250U/ml, 500U/ml, 1000U/ml)
CA125 protein standard substance;
Positive quality control product;
Negative quality-control product (sample diluting liquid);
The phosphate buffer (PBS) of the 0.01M of pH7.2;
Polyclonal antibody dilution;
The couplet solution of the CdTe quantum dot that can be combined with CA125 protein-specific and mouse-anti people CA125 polyclonal antibody;
25 × wash plate liquid.
Wherein:
1) preparation of various reagent solutions:
A. the CA125 antigen (embodiment 1 that the CA125 protein standard substance of each fixed concentration is purifying, 20000U/ml) use PBS damping fluid (pH7.2,0.01M) to be diluted to 0U/ml, 10U/ml, 50U/ml, 250U/ml, 500U/ml, 1000U/ml;
B. positive quality control product is that the CA125 antigen after purifying (embodiment 1,20000U/ml) sample diluting liquid is below diluted to the solution that concentration is 800U/ml;
C. negative quality-control product is the sample diluting liquid that does not contain CA125 antigen; Sample diluting liquid is the phosphate buffer (pH7.2,0.01M) containing 1%BSA, 0.05% Tween-20;
Preparation method: take 1g bovine serum albumin(BSA) (BSA) and be dissolved in 100ml phosphate buffer (pH7.2,0.01M), then add 0.05ml Tween-20, mix.
The phosphate buffer (PBS) of the 0.01M of D.pH7.2;
Preparation method: take potassium dihydrogen phosphate (KH 2pO 4) 0.2g, sodium hydrogen phosphate (Na 2hPO 412H 2o) 2.9g, sodium chloride (NaCl) 8.0g, potassium chloride (KCl) 0.2g, is dissolved in 9500ml water, regulates pH value to 7.2 with 1M HCL, is settled to 1000ml with distilled water.
Preparation method: take 5g bovine serum albumin(BSA) and be dissolved in 100ml phosphate buffer (pH7.2,0.01M);
E. polyclonal antibody dilution is the phosphate buffer (pH7.2,0.01M) containing 0.5% bovine serum albumin(BSA), 0.1% Tween-20;
Preparation method: take 0.5g bovine serum albumin(BSA) and be dissolved in 100ml phosphate buffer (pH7.2,0.01M), then add 0.1ml Tween-20, mix.
F. be coated with the ELISA Plate of mouse-anti people CA125 monoclonal antibody.
By phosphate buffer (pH7.2 for mouse-anti people CA125 monoclonal antibody (embodiment 3 steps 1.7), 0.01M) adjusting concentration is 2 μ g/ml, the every hole of ELISA Plate adds 100 μ l antibody diluents, 4 DEG C of overnight incubation, discard coating buffer, then add 300 μ l/ hole PBST solution [PBS (pH7.2,0.01M), 0.1% (v/v) Tween-20] wash three times, pat dry; Every hole adds confining liquid (the PBS pH of buffer 7.2 of 200 μ l containing 0.1% (v/v) Tween-20,5% (m/v) bovine serum albumin(BSA) again, 0.01M), 4 DEG C of sealings are spent the night, discard confining liquid, then add the PBST (pH7.2 of 300 μ l/ holes containing 0.1% (v/v) Tween-20,0.01M) washing three times, pats dry.
The couplet solution of the CdTe quantum dot that G. can be combined with CA125 protein-specific and mouse-anti people CA125 polyclonal antibody (from embodiment 4 4.2).
H.25 × wash plate liquid and be the PBS damping fluid (PH7.2,0.01M) containing 0.5%Tween-20;
Preparation method: take potassium dihydrogen phosphate (KH 2pO 4) 5g, sodium hydrogen phosphate (Na 2hPO 412H 2o) 72.5g, sodium chloride (NaCl) 200g, potassium chloride (KCl) 5g, is dissolved in 950ml water, with 1M HCL adjusting ph value to 7.2, then add 125ml Tween-20, be finally settled to 1000ml with distilled water.
2) sample preparation: tested serum is made to the dilution of proper proportion (as 1:4) of sample diluting liquid.
3) the detection method step of the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen is as follows:
A. get the ELISA Plate that is coated with mouse-anti people CA125 monoclonal antibody, room temperature is placed 30min.
B. application of sample: in 100 μ l/ hole testing samples (step 2)) add in the lath hole of ELISA Plate in steps A, establish the CA125 protein standard substance 100 μ l of negative control (negative quality-control product) 100 μ l, positive control (positive quality control product) 100 μ l, each fixed concentration simultaneously, 37 DEG C of reactions after 1 hour with 1 × wash plate liquid (by 25 × wash plate liquid tri-distilled water to be diluted to 1 ×) flushing 3 times, each 3min, pats dry.
C. add couplet solution: the CdTe quantum dot that can be combined with CA125 protein-specific with polyclonal antibody dilution and the couplet solution 1:10000 of mouse-anti people CA125 polyclonal antibody dilution, every hole adds 100 μ L, 37 DEG C of reactions after 1 hour with 1 × wash plate liquid (by 25 × wash plate liquid tri-distilled water to be diluted to 1 ×) flushing 3 times, pat dry, then add the phosphate buffer (PBS) of the 0.01M of the pH7.2 of 100 μ L.
D. detect: the ELISA Plate of handling well is measured to fluorescence intensity level via fluorescence microplate reader.By fluorescence intensity level and the corresponding concentration Criterion curve of CA125 protein standard substance, obtain detection line (r value is greater than 0.990), linear relationship is fine, can be used for sample size and calculates; Negative quality-control product and positive quality control product numerical value all, in the range of linearity, illustrate that this system can be used for sample detection.
4) kit performance index examination
(1) between the detection zone of kit
1) get the ELISA Plate (F ELISA Plate in above-described embodiment 5) that is coated with good mouse-anti people CA125 monoclonal antibody and sealing, room temperature is placed half an hour.
2) add CA125 protein standard substance (the CA125 antigen after the purifying of embodiment 1) to do the dilution (standard items of the each fixed concentration in embodiment 5) after the dilution of proper proportion, each sample does 3 repeating holes, 100ul/ hole, 37 DEG C of reactions were washed plate 3 times with washing plate liquid after 1 hour, and ELISA Plate is patted dry.
3) with PBS (pH7.2,0.01M) after the dilution anti-human CA125 polyclonal antibody of rabbit (embodiment 2) 1:10000 dilution, every hole adds 100ul, 37 DEG C of reactions after 1 hour with 25 × wash plate liquid to wash plate 3 times, after patting dry, add 100ul PBS (pH7.2,0.01M).
By step 3) ELISA Plate handled well measures fluorescence intensity level via fluorescence microplate reader.Taking the protein standard substance of the CA125 of variable concentrations as horizontal ordinate, taking corresponding glimmering light intensity value as ordinate, curve plotting.
As a result, as shown in Figure 4, it is 0U-1000U that this kit has between the detection zone of good linear relationship, if detected serum exceedes 1000U, suggestion is with detecting after sample diluting liquid dilution again.
(2) kit sensitivity (detection limit)
Be used as sample with zero normative reference product (A point) (being sample diluting liquid), through indirect elisa method, be method with the detection method of the quantum dot-labeled double fastener heart immunoassay detection kit of (in embodiment 5 3) people CA125 albumen), use fluorescence microplate reader 488nm wavelength measurement 20 times, calculate its fluorescent value average and standard deviation.Add that taking the average of A point measured value concentration that the fluorescent value substitution typical curve equation of the standard deviation gained of 2 times calculates is as its detection limit.
Result demonstration, detection limit is <3U.
(3) stability of kit
Three batches of reagent of self-control kit are positioned over respectively to 4 DEG C of half a year and 1 year, place after 7 days for 37 DEG C, pass through respectively the detection method of the quantum dot-labeled double fastener heart immunoassay detection kit of indirect elisa method (in embodiment 5 3) people CA125 albumen), use 488nm wavelength measurement fluorescence intensity level, linear relationship and zero standard product fluorescence intensity level relatively and between the front normative reference product each point fluorescence intensity level of placement, measuring stability.Result demonstration, this kit has good stability.
The evaluation of the anti-human CA125Elisa detection kit of embodiment 6
For detecting the effect of double antibodies sandwich enzyme linked immunological (DAS-ELISA) detection kit, the present embodiment uses the kit of embodiment 5 to 594 parts of normal human serum samples (standard of normal human serum sample: serum sample physical examination result is all without liver, brain, kidney, disease of digestive tract, without blood transfusion and major operation history, women is not in the gestational period and lactation in half a year) in people CA125 protein content measure.The wherein male sex 348 people (18～78 years old age), women 246 people (21～75 years old age), detection minimum is 0U/ml, mxm. is 39.40U/ml, mean concentration (X) is 9.63U/ml, and standard deviation (SD) is 6.26U/ml.Wherein, the concentration value of most of samples (88.94%) is below 10U/ml, and concentration has 57 parts at the sample of 10.0～36.0U/ml, in the time that concentration value is below 36U/ml, has comprised 98.38% sample.Comprehensive existing research and clinical existing reference value, CA125 serum normal reference value when this kit is detected is set as 0～36.0U/ml, can set according to the difference of each department sample the reference range of oneself when each hospital is used.

Claims (10)

1. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen, is characterized in that, comprises following component:
Each fixed concentration is respectively the CA125 protein standard substance of 0U/ml, 10U/ml, 50U/ml, 250U/ml, 500U/ml, 1000U/ml;
Positive quality control product;
Negative quality-control product;
The phosphate buffer of the 0.01M of pH7.2;
Polyclonal antibody dilution;
Be coated with the ELISA Plate of mouse-anti people CA125 monoclonal antibody;
The couplet solution of the CdTe quantum dot that can be combined with CA125 protein-specific and mouse-anti people CA125 polyclonal antibody;
25 × wash plate liquid.
2. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen according to claim 1, it is characterized in that, the preparation method of the CA125 protein standard substance of wherein said each fixed concentration is: cultivate in a large number Proliferation of Human Ovarian Cell Ovcar-3, get cell culture fluid, after dialysis is concentrated, use molecular sieve to isolate the CA125 albumen of purifying, the PBS damping fluid that to re-use pH7.2, concentration be 0.01M is diluted to 0U/ml, 10U/ml, 50U/ml, 250U/ml, 500U/ml, 1000U/ml.
3. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen according to claim 2, is characterized in that, the preparation method of the CA125 albumen of described purifying is as follows:
Step 1: use containing 10%FBS, 3.5g/L Hepes1.9g/L NaHCO 3dMEM complete culture solution cultivate Proliferation of Human Ovarian Cell Ovcar-3,37 DEG C, 5%CO 2cultivate 72 hours;
Step 2: collecting cell nutrient solution, use the bag filter dialysis that the pH7.2 of 20 times of cell culture fluid volumes, PBS damping fluid that concentration is 0.01M are 5kD with molecular cut off, after dialysis, concentrate with containing 20 times of PBS damping fluids that 30% PEG-6000 and pH7.2, concentration are 0.01M again;
Step 3: the cell culture fluid after step 2 is concentrated flows through the molecular sieve of molecular cut off as 50-400kD taking the speed of 2ml/min, is used quick protein purification instrument to collect the albumen at the peak of 200kD left and right, is CA125 albumen.
Step 4: the CA125 protein 20 of collecting is doubly concentrated, and the same step 2 of method, obtains the CA125 antigen after purifying.
4. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen according to claim 3, it is characterized in that, described negative quality-control product is sample diluting liquid, the phosphate buffer that described sample diluting liquid is is 0.01M containing 1%BSA, 0.05% Tween-20 and pH7.2, concentration; Described positive quality control product is that the CA125 antigen sample diluting liquid after purifying is diluted to the solution that concentration is 800U/ml; Described 25 × PBS damping fluid that to wash plate liquid and be containing 0.5%Tween-20 and pH7.2, concentration be 0.01M.
5. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen according to claim 4, it is characterized in that the phosphate buffer that described polyclonal antibody dilution is is 0.01M containing 0.5% bovine serum albumin(BSA), 0.1% Tween-20 and pH7.2, concentration.
6. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen according to claim 5, is characterized in that,
The preparation method of the described ELISA Plate that is coated with mouse-anti people CA125 monoclonal antibody is as follows:
It is 2 μ g/ml that the mouse-anti people CA125 phosphate buffer that for monoclonal antibody, pH7.2, concentration are 0.01M is adjusted to concentration, the every hole of ELISA Plate adds 100 μ l, and 4 DEG C of overnight incubation, discard coating buffer, then add 300 μ l/ holes containing the PBST solution washing of 0.1%Tween-20 three times, pat dry; Every hole adds the phosphate buffer that 200 μ l are 0.01M containing 0.1%Tween-20,5% bovine serum albumin(BSA) and pH7.2, concentration again, be confining liquid, 4 DEG C of sealings are spent the night, and discard confining liquid, then add 300 μ l/ holes containing the PBST solution washing of 0.1%Tween-20 three times, pat dry.
7. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen according to claim 6, is characterized in that, the preparation method of described mouse-anti people CA125 monoclonal antibody is as follows:
1) select 8～10 weeks BALB/c female mices, before inoculation hybridoma 1～2 week, first to mouse peritoneal injection 0.5ml incomplete Freund's adjuvant;
2) collect well-grown hybridoma, centrifuge washing 1 time, is resuspended in the NaHCO containing 3.5g/L Hepes1.9g/L 3the DMEM nutrient solution of serum-free in, adjusting cell density is 1～2 × 10 6/ ml, obtains cell suspension;
3) every mouse peritoneal injection 0.5ml cell suspension, inoculating cell 7～12 days, mouse web portion obviously expands, and while touch with hand, skin has tension, the skin of abdomen of can sterilizing, connect syringe needle No. 8 with 5ml syringe, thrust abdominal cavity, unload syringe, raise mouse head, ascites is splashed in centrifuge tube; 2～3 days, interval, after ascites regeneration is gathered, gets with method again, and a mouse generally can extract 2～3 times, and the ascites of collecting, through the centrifugal 15min of 3000rpm, is discarded to upper strata grease, cell component and other sediments, draws faint yellow ascites; Adopt the thick purifying of saturated ammonium sulphate method and affinity column method purifying hybridoma ascites, obtain the mouse-anti people CA125 monoclonal antibody after purifying that concentration is 1.2mg/ml.
8. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen according to claim 7, it is characterized in that, the preparation method of the couplet solution of the described CdTe quantum dot that can be combined with CA125 protein-specific and mouse-anti people CA125 polyclonal antibody is as follows: the CdTeQDs600ul that gets 0.1mM, adding 18ul concentration is that after the ethylene dichloride of 1mg/ml and the methyl alcohol of 800ul mix, lucifuge is shaken 30min with 800rpm, add again the beta-mercaptoethanol of 8ul to stop being the QDs after activation, get the mouse-anti people CA125 polyclonal antibody pH7.2 after the purifying of 0.75mg, concentration is that the phosphate buffer of 0.01M is diluted to 4 μ g/ml, add in the QDs10ul after activation and mix with it, lucifuge 800rpm concussion 2 hours, after finishing, reaction add again mercaptoethanol 20 μ l to stablize quantum dot, then the bag filter that is 12kD with molecular cut off dialysis, after dialysis under 16300 × g condition centrifugal 3min, remove supernatant, be precipitated, the coupling that is purifying has the mouse-anti people CA125 polyclonal antibody of quantum dot, precipitation is resuspended in to pH7.2, concentration is in the phosphate buffer of 0.01M, obtaining concentration is the CdTe quantum dot of 5mg/ml and the couplet solution of mouse-anti people CA125 polyclonal antibody.
9. the quantum dot-labeled double fastener heart immunoassay detection kit of people CA125 albumen according to claim 8, is characterized in that,
The preparation method of described CdTeQDs is as follows:
By 2.5 × 10 -4the CdCl of mol 22.5H 2o is dissolved in the ultrapure water of 25ml, adds 3 × 10 -4the two hydration trisodium citrates, 0.5 × 10 of glutathione GSH, the 0.1g of mol -4the Na of mol 2teO 3with 2.4 × 10 -4the NaBH of mol 4under condition with magnetic stirring apparatus with 200rpm, stir 30 minutes, NaOH with 0.01M in whipping process regulates PH to 10.5, after completing, stirring puts into the microwave device that power is made as the band backflow of 600W, in the time that becoming light green, solution colour starts to reflux, back flow reaction 4h, along with the difference of return time forms the quantum dot taking glutathione as stabilizing agent of a series of different-grain diameter 1-10nm, reacted solution is cooled to after room temperature and is precipitated with two volumes absolute ethyl alcohol centrifugal 5min under the condition of 4000rpm, remove excessive Cd in supernatant 2+, TeO 3 2-deng impurity, similarity condition repeats 3 times, after ethanol volatilizees completely, in the pH7.2 that precipitation is resuspended in, the phosphate buffer that concentration is 0.01M, obtains the CdTeQDs of 0.1mM;
The preparation method of the mouse-anti people CA125 polyclonal antibody after described purifying is as follows:
1) animal immune
CA125 antigen 1 0000U after above-mentioned purifying is dissolved in the phosphate buffer solution that 1ml pH7.2, concentration are 0.01M and obtains antigenic solution, in 1ml incomplete Freund's adjuvant, add the Much's bacillus of deactivation to make Freund's complete adjuvant, and add above-mentioned 1ml antigenic solution, concuss makes it the fully emulsified antigen emulsion that obtains, extract this antigen emulsion with 3ml syringe, rabbit is carried out to injections of antigens emulsion, and carry out several times booster immunization;
2) collect antiserum
7～10 days collection blood after booster immunization injection, a large amount of blood of collecting after generation antibody to be determined, blood is placed in to 37 DEG C of constant temperature ovens to be placed 30 minutes, again 4 DEG C of hold over night, medication shovel is dialled blood clot fall from tube wall, by blood transfer to plastic centrifuge tube, 4 DEG C, centrifugal 10 minutes of 10,000g, collects supernatant and is antiserum;
3) antibody purification
In antiserum, adding solid sodium azide to concentration is 0.05%, 4 DEG C, 15, centrifugal 5 minutes of 000g, shift out supernatant and remove by filter unnecessary fat through the filtrator of 0.45 μ m again, dilute with the volume ratio of 1:5 removing by filter the antiserum pH7.4 of unnecessary fat and TBS buffer solution that concentration is 0.1M, filter with the filtrator of 0.45 μ m again, with the speed of 0.5ml/min by the antiserum-TBS buffer solution after filtering to GE1ml Protein A Sepharose prepacked column, continuous upper prop 2 times also retains loading efflux, the TBS buffer solution that is 0.1M by pH7.4 and concentration cleans pillar to A λ 280nmglycocoll-the HC1 that is 0.1M by pH4.5, concentration again after <0.008 is with the speed wash-out of 0.5ml/min, and eluent goes to dialysed overnight in the PBS damping fluid that pH7.2 and concentration are 0.01M immediately, collects as stated above eluent to A λ 280nm<0.008, obtains the anti-human CA125 polyclonal antibody of rabbit after purifying.
10. according to the detection method of the quantum dot-labeled double fastener heart immunoassay detection kit of the people CA125 albumen described in claim 1-9 any one, it is characterized in that, comprise the steps:
Step 1: get the ELISA Plate that is coated with mouse-anti people CA125 monoclonal antibody, room temperature is placed 30min;
Step 2: add in the lath hole of ELISA Plate in step 1 with 100 μ l/ hole testing samples, make negative control, join respectively in the lath hole of ELISA Plate in step 1 with the CA125 protein standard substance 100 μ l that positive quality control product 100 μ l make positive control, each fixed concentration with negative quality-control product 100 μ l simultaneously, 37 DEG C of reactions after 1 hour with 25 × wash the solution flushing of plate liquid after 25 times of dilutions 3 times, each 3min, pats dry;
Step 3: the CdTe quantum dot that can be combined with CA125 protein-specific with polyclonal antibody dilution and the couplet solution 1:10000 of mouse-anti people CA125 polyclonal antibody dilution, every hole adds 100 μ L, 37 DEG C of reactions after 1 hour with 25 × wash the solution flushing of plate liquid after 25 times of dilutions 3 times, pat dry, then add the phosphate buffer of the 0.01M of the pH7.2 of 100 μ L;
Step 4: the ELISA Plate of handling well is measured to fluorescence intensity level via fluorescence microplate reader, by fluorescence intensity level and the corresponding concentration Criterion curve of CA125 protein standard substance, obtain the detection line that r value is greater than 0.990, negative quality-control product and positive quality control product numerical value all in the range of linearity, calculation sample content.
CN201410232892.8A 2014-05-29 2014-05-29 Human CA125 protein quantum dot labeled double-sandwiched immunoassay detection kit Pending CN103983791A (en)

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