CN106501515A - CA215 detection kit and preparation method thereof and using method - Google Patents
CA215 detection kit and preparation method thereof and using method Download PDFInfo
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- CN106501515A CN106501515A CN201611116897.XA CN201611116897A CN106501515A CN 106501515 A CN106501515 A CN 106501515A CN 201611116897 A CN201611116897 A CN 201611116897A CN 106501515 A CN106501515 A CN 106501515A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The present invention discloses a kind of CA215 detection kit and preparation method thereof and using method.The CA215 detection kit includes Magneto separate agent, the CA215 antibody of tracer-labelling, Chemoluminescent substrate and calibration object；The Magneto separate agent is to be coated the magnetic particle solution of CA215 antibody；The Chemoluminescent substrate be diethanolamine buffer, the diethanolamine buffer include AMPPD；The calibration object is prepared using CA215 sterlings, and which indicates concentration and is respectively 0A μ/ml, 5A μ/ml, 10A μ/ml, 20A μ/ml, 50A μ/ml, 100A μ/ml.The CA215 detection kit that the present invention is provided has the advantages that sensitivity height, range of linearity width, specificity are high and stability is good, is clinically with a wide range of applications.
Description
Technical field
The present invention relates to belong to the external clinical detection technique field of cancer, more particularly to CA215 detection kit and its system
Preparation Method and using method.
Background technology
Since nearly half a century, the M ＆ M of tumor is raised year by year, becomes the focus in medical circle focus.20
Century 70, what the U.S. proposed reflect society to the high anxiety of tumor mortality rate to " tumor is marched ", meanwhile,
In past decades, tumor-marker technically has breakthrough with definition, and everything has all promoted tumor-marker research
Fast-developing.
CA215 is tumor associated antigen, and the antigen is the monoclonal anti extracted by the ovarian cancer cell from culture in 1987
Body RP215 has found.RP215 is proved to and is located at the main heavy chain immunoglobulin by expression in most of cancerous cell (typically
Referred to as CA215) the related epi-position of carbohydrate reacts.
As CA215 is that height is associated with cancerous cell or cancerous tissue, and typically can not find in health adult tissue,
Except in hyperplastic epithelium cell or tissue, such as skin, esophagus, cervix uteri and several immunologically privileged siteses, including nerve, eye
Eyeball and testis.
CA215 as a general cancer biomarker with potential using value, and can be with other known cancer
The clinic for comparing the immunologic diagnosises in many different types of cancers of the mankind to record CA215 parallel of disease biomarker should
With.
It was found that cancer patient and normal human serum CA215 levels are significant difference (P<0.001).Due to clinical stagess,
In the case of ovarian cancer, normal individual its positive rate scope is compared from 58-86%.For cervical cancer patient, in cancer evening
The stage positive rate of phase is up to 66-94% respectively.
The result of cervical cancer and ovarian cancer these clinical researches it is also shown that average serum CA215 levels the stage is simultaneously in the preoperative
At a relatively high level is maintained within before one week surgical operation or chemotherapy or radiotherapy.Conversely, when operation technique or chemotherapy or
Radiotherapy was determined after seven days, and change of serum C A215 level is remarkably decreased.Based on these researchs, it can be deduced that conclusion thinks, cancer patient's
Excision or chemotherapy between cervix uteri and ovarian cancer causes statistically to substantially reduce in CA215 levels.As a result
Show, CA215 most probables in cancer patient are produced from tumor locus, and the tumor load can be by the serum of cancer patient
CA215 levels fully reflect and detect.Therefore, the customary detection of the change of serum C A215 level of cancer patient is sent out to monitoring cancer
After the plan of exhibition, the state for the treatment of or operative treatment is beneficial.
Used as a general carcinoma marker, CA215 is than carcinoembryonic antigen (carcino-embryonic antigen, CEA) or β 2
Microglobulin more preferably, in most of cancers has higher display positive rate.For most of human cancer CA215 sun
Property recall rate is all higher, including ovarian cancer.However, in the case of cervical cancer, using CA215 combine detections than individually use
CA125 has higher recall rate, recall rate to be promoted to 81% from 13%.However, for the ovarian cancer group of CA215 and CA125
Closing also has more preferable recall rate, verification and measurement ratio to be promoted to 82% from 59%.For pulmonary carcinoma, CA215 and CYFRA21-1 combine detections
There is preferable positive rate.Group for hepatocarcinoma, CEA or alpha-fetoprotein (alpha-fetoprotein, AFP) and CA215
Close and seem have higher positive rate.In a word, other biomarker for cancer and CA215 are combined to improve and are being faced conventional
Positive rate in bed diagnosis cancer.
Last century the mid-1970s Arakawe reported first carries out EIA enzyme immunoassay with luminous signal, using luminous change
Response analysises ultra micro quantity of material is learned, is particularly used in clinical immunoassays, checking ultramicron active substance.At present, this technology
Clinical medical conventional sense means are transitioned into from the rare technology of laboratory.Chemiluminescence immune assay
(Chemiluminescence Immunoassay, CLIA) is mutually to tie chemiluminescence or bioluminescence system with immunoreation
Close, for detecting a kind of novel markings immunoassay of trace antigen or antibody.Its Cleaning Principle and radioimmunity
((enzyme immunoassay, EIA) is similar, and different heres are with luminous for the immunity of (Radioimmunoassay, RIA) and enzyme
Material is used as substrate, and is directly measured by the luminous intensity of its own.
Include two parts, i.e. immune response system and chemiluminescence analysis system in chemiluminescence immune assay.Exempt from
Epidemic disease response system, the same Enzyme-multiplied immune technique of its ultimate principle (enzyme linked immunosorbent assay, ELISA).
The principle of chemiluminescence analysis system is the enzyme effect in immunoreation in luminous substrate.Luminous substrate in the presence of enzyme,
There is chemical reaction and discharge substantial amounts of energy in substrate, produce the intermediate of excited state.This excited state intermediate, when its time
During to stable ground state, photon can be launched simultaneously.It is measurable quantum yield of luminscence using luminous signal measuring instrument, the light quantity
Sub- yield is directly proportional to the amount of the test substance in sample.It is possible thereby to Criterion curve calculate test substance in sample
Content.
Chemiluminescence immune assay had both had the high sensitivity of radioimmunity, and easy to operate, fast with enzyme linked immunological
Fast the characteristics of, it is easy to normalizing operation.And in testing, do not use harmful reagent, reagent to be kept for the phase long, be applied to biology, doctor
Research and clinical experiment diagnostic work is learned, becomes one of most promising method in on-radiation immunoassay.
Content of the invention
The present invention provides a kind of CA215 detection kit, can with the concentration of detection by quantitative human cancer mark CA215,
Have sensitivity height, range of linearity width, non-specific low and stability good.
The CA215 detection kit that the present invention is provided includes that Magneto separate agent, the CA215 antibody of tracer-labelling, chemistry are sent out
Light substrate solution and calibration object；
Wherein,
The Magneto separate agent is to be coated the magnetic particle solution of CA215 antibody；
The Chemoluminescent substrate is diethanolamine buffer, and (2- spirals are golden comprising 3- for the diethanolamine buffer
Firm alkane) -4- methoxyl group -4- (3- phosphorus oxygen acyls)-phenyl -1,2- dioxane disodium salts；
The calibration object using CA215 sterlings prepare, its indicate concentration be respectively 0AU/ml, 5AU/ml, 10AU/ml,
20AU/ml、50AU/ml、100AU/ml.
Preferably, the Magneto separate agent is prepared in accordance with the following methods：
(1) magnetic particle is activated：Take the carboxyl magnetic particle after fully mixing to be put in centrifuge tube, centrifuge tube is positioned over magnetic point
On frame, after carboxyl magnetic particle is adsorbed separating completely, supernatant is removed, MES buffer solution carboxyls magnetic particle is added extremely
Less once；Add EDC solution and NHS solution to activate carboxyl magnetic particle；
(2) coated magnetic particle：With the carboxyl magnetic particle after the activation of MES buffer solutions at least one times, centrifuge tube is placed in
On Magneto separate frame, separate and remove supernatant, add the CA215 antibody of 2.5mg/ml to mix, at room temperature reaction 2～4 hours, instead
After should terminating, then centrifuge tube is placed on Magneto separate frame, removes supernatant, washed with ethanolamine solutions at least one times, Ran Houzai
Ethanolamine solutions are added in centrifuge tube, room temperature reaction 2～4 hours, after reaction terminates, removes supernatant and preserves, wherein, carboxyl
Magnetic particle is 1 with the volume ratio of CA215 antibody：1；
(3) coated magnetic particle prepared by step (2) is diluted to by 0.1～1mg/ml using bead suspension liquid.
Preferably, the carboxyl magnetic particle is Fe3O4/Fe2O3Magnetic nano-particle and the polymerization of high-molecular organic material
Thing, its mean diameter is 0.5～5 μm, preferably 1～3 μm, and the carboxyl magnetic particle surface is with carboxyl, amino and hydroxyl
One or more group.
Preferably, above-mentioned bead suspension is prepared in accordance with the following methods：Weigh Tris- alkali 1.21g, sodium chloride 4.38g and
Proclin300 0.2g add the purified water of 200ml, with hydrochloric acid or sodium hydroxide adjust pH value 8.5 after standing 10min after mixing
± 0.5, it is subsequently adding 2g trehaloses, 10gBSA, 1g gelatin, the zinc chloride of magnesium chloride 0.5ml, 1mol/L of 1mol/L
The T-20 of the calcium chloride 0.1ml and 0.5mL of 0.05ml, 1mol/L, with purified water constant volume to 500mL, is filtrated to get magnetic bead suspension
Liquid.
Preferably, the tracer be horseradish peroxidase, alkali phosphatase, diamantane (obsolete), luminol, different luminol and
At least one in its derivant, acridinium ester, preferably alkali phosphatase.
Preferably, in the CA215 antibody of the tracer-labelling, CA215 antibody concentration is 1～300ng/ml.
Present invention simultaneously provides a kind of preparation method of CA215 detection kit, the CA215 detection kit includes magnetic
Separating medium, the CA215 antibody of tracer-labelling, Chemoluminescent substrate, calibration object and cleaning mixture, its preparation method include as
Lower step：
(1) Magneto separate reagent is prepared
Activation magnetic particle：Carboxyl magnetic particle is taken in centrifuge tube and is fully mixed, centrifuge tube is positioned on Magneto separate frame,
After carboxyl magnetic particle is adsorbed separating completely, supernatant is removed, MES buffer solution carboxyls magnetic particle is added at least one times；
Add EDC solution and NHS solution to activate carboxyl magnetic particle；
Coated magnetic particle：With the carboxyl magnetic particle after the activation of MES buffer solutions at least one times, centrifuge tube is placed in magnetic point
On frame, separate and remove supernatant, add the CA215 antibody of 2.5mg/ml to mix, react 2～4 hours at room temperature, reaction knot
Shu Hou, then centrifuge tube is placed on Magneto separate frame, supernatant is removed, is washed with ethanolamine solutions at least one times, is then added
In centrifuge tube, room temperature reaction 2～4 hours, after reaction terminates, removes supernatant and preserves ethanolamine solutions, and wherein carboxyl magnetic is micro-
Grain is 1 with the volume ratio of CA215 antibody：1；
The coated magnetic particle for preparing is diluted to by the work that 0.1～1mg/ml obtains Magneto separate agent using bead suspension
Liquid；
(2) the CA215 antibody of tracer-labelling is prepared：
Activated alkaline phosphatase：The alkali phosphatase for taking 20mg/ml is placed in the bottom of reaction tube, adds excess
4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group butanimide ester sodium salts of 40mmol/L, concussion are mixed, room
10～30min of standing and reacting under the conditions of temperature, is subsequently adding in the glycine solution of 1mol/L and unnecessary 4- (N- maleimides
Methyl) hexamethylene -1- carboxylic acid sulfonic group butanimide ester sodium salts, with Zeba desalination column purifications after reaction 10min, activated
Alkali phosphatase；
Activation CA215 antibody：The CA215 monoclonal antibodies for taking 5mg/ml are placed in the bottom of reaction tube, add excess
The TCEP solution of 0.5mol/L, concussion are mixed, and standing and reacting 30-60min under room temperature condition is subsequently adding the glycine of 1mol/L
With unnecessary TCEP solution in solution, react 10 minutes, horse back Zeba desalination column purifications after terminating obtain the CA215 for activating
Antibody；
By the alkali phosphatase for having activated and the CA215 antibody in mass ratio 1 for having activated:1 mixing, under the conditions of 2-8 DEG C
Reaction 12h, reaction obtain the good CA215 antibody-enzyme conjugates of purification with the method for sieve chromatography after terminating；
CA215 antibody-enzyme conjugates good for purification are diluted to 1～300ng/ml using Working dilutions, spike is obtained
The working solution of the CA215 antibody of substance markers；
(3) Chemoluminescent substrate is prepared：
3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-phenyl -1,2- dioxane two is weighed respectively
Sodium salt 0.2g, bis--bis- (benzoxazoles -2-) thiophene 0.1g of fluorescent whitening agent 2.5-, bovine serum albumin 5g, BDMQ surfactant
1g and proclin300 10g, are subsequently adding MgSO4 0.1ml, 0.8mmol/LZnCl of 0.4mmol/L20.1ml and
The diethanolamine buffer 500ml of 0.1mol/L, mixes 15 minutes, is subsequently adding the diethanolamine buffer constant volume of 0.1mol/L
1000ml is arrived, mixing filtration is stand-by, keeps in dark place under the conditions of being placed on 2-8 DEG C；
(4) calibration object series is prepared：
CA215 sterlings are diluted using 7.4 phosphate buffers of pH, calibration object is obtained, the phosphate buffer includes matter
Amount fraction is the preservative of 3% BSA and 0.05%, and wherein preservative is Proclin-300, the sign of the calibration object series
Concentration is respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/ml, 50AU/ml and 100AU/ml；
(5) cleaning mixture is prepared：
Take trishydroxymethylaminomethane 2.24g, 8.76g sodium chloride and 0.5ml tween 20s add water and are settled to 1000ml, mix
Even filtration；
(6) by Magneto separate agent, the CA215 antibody of tracer-labelling, Chemoluminescent substrate, calibration object and cleaning mixture group
Dress up the CA215 detection kit.
Preferably, described the step of prepare Magneto separate agent in, the bead suspension is prepared in accordance with the following methods：Weigh
Tris- alkali 1.21g, sodium chloride 4.38g and Proclin300 0.2g add the purified water of 200ml, after standing 10min after mixing
PH value 8.5 ± 0.5 is adjusted with hydrochloric acid or sodium hydroxide, 2g trehaloses, 10gBSA, 1g gelatin, the chlorination of 1mol/L is subsequently adding
The T-20 of the calcium chloride 0.1ml and 0.5mL of zinc chloride 0.05ml, 1mol/L of magnesium 0.5ml, 1mol/L, is arrived with purified water constant volume
500mL, mixes and is filtrated to get bead suspension liquid.
Preferably, described prepare tracer-labelling CA215 antibody the step of in, the Working dilutions each component content
As follows:The bovine serum albumin of 1%-5% volumes, the Mus serum of 1-5% volumes, the new-born calf serum of 1-10% volumes, 1-5%
The horse serum of volume, the porcine blood serum of 1-5% volumes, the sodium chloride of 8g/L, the potassium chloride of 0.2g/L, 2.9g/L disodium hydrogen phosphates,
0.2g/L potassium dihydrogen phosphates, 10-30g/L trehaloses, 0.1～1g/L 4- formylphenyl boronic acids, the glycerol of 0.1-5% volumes,
The ethylene glycol of 0.1-3% volumes, 0.1-20g/L xanthan gum, the tween 20 of 0.01-1% volumes, 0.1-1g/L ethylenediamine tetrems
Acid, the gentamycin of Proclin-300,0.1-0.5% volume of 1% volume.
The present invention also provides a kind of using method using CA215 detection kit mentioned above, comprises the steps：
CA215 detection kit is taken out, the CA215 detection kit is balanced to room temperature, and the CA215 is detected
Reagent in test kit takes out standby；
Take out serum sample to be measured to balance to room temperature；
Quantity according to calibration object and serum sample to be measured prepares reaction tube, and numbers, and is added respectively in test tube
The serum sample and calibration object of 50 μ l, then adds the CA215 antibody of 100 μ l tracer-labellings in every test tube, then divides
The Magneto separate agent of 25 μ l is not added, is fully vibrated at 37 DEG C, be subsequently placed on Magneto separate frame and separate；
Supernatant is poured out, is cleaned with cleaning mixture；
100 μ l of chemical luminous substrate are added in each reaction tube, is put in chemiluminescence measuring instrument after fully mixing
Row detection；
Obtain testing result：The chemiluminescence intensity RLU values of each reaction tube are directly read, the concentration value with calibration object is as horizontal stroke
Coordinate, the RLU values of calibration object are that vertical coordinate draws standard curve, according to the concentration that standard curve calculates serum sample to be measured
Value, the measuring instrument that directly chemically lights read or derive.
CA215 detection kit provided compared to prior art, the present invention and preparation method thereof and using method, have
Following beneficial effect：
First, the CA215 detection kit that the present invention is provided, is a kind of new tumor markerses, other Cancer Biology marks
Thing and CA215 combine the positive rate that can be improved in routine clinical diagnosis cancer.And the reaction pattern of this reagent
It is sandwich assay, specificity is good, sensitivity is high, and without HOOK effects, the range of linearity is wide；
2nd, the present invention is incorporated into carboxyl magnetic particle in the middle of immunoassay technology, makes the system of reagent reacting homogeneous, can pole
The earth improves the efficiency that antigen-antibody is combined, and shortens the response time；
3rd, the labelling of alkali phosphatase is different from general glutaraldehyde method and Over-voltage protection, difunctional crosslinking used
Method, makes test kit possess higher sensitivity and linear measurement range, and non-specific low, good stability.
4th, as a result of the new reinforcing agent technology of AMPPD/ of company's substrate solution, the sensitivity of luminous substrate liquid is high, luminous hold
The continuous time is long, greatly improves the sensitivity of reagent.
Description of the drawings
For the technical scheme being illustrated more clearly that in the embodiment of the present invention, below will be to making needed for embodiment description
Accompanying drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for
For those of ordinary skill in the art, on the premise of not paying creative work, can be obtaining other according to these accompanying drawings
Accompanying drawing, wherein：
CA215 detection kit CA215 detection dosage-reaction normal curve that Fig. 1 is provided for the present invention；
The preparation method flow chart of steps of the CA215 detection kit that Fig. 2 is provided for the present invention；
The using method flow chart of steps of the CA215 detection kit that Fig. 3 is provided for the present invention.
Specific embodiment
Accompanying drawing in below in conjunction with the embodiment of the present invention, to the embodiment of the present invention in technical scheme carry out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiment.It is based on
Embodiment in the present invention, it is all other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
The know-why of the present invention is as follows：
The present invention detects the concentration value of general carcinoma marker CA215 using chemiluminescence immunoassay technology, in reaction tube
CA215 calibration objects or test serum sample, the CA215 monoclonal antibodies for adding tracer-labelling is added to be reacted, then plus
Enter the magnetic particle solution of coated CA215 monoclonal antibodies, coated CA215 monoclonal antibodies and alkali phosphatase enzyme mark
CA215 monoclonal antibodies are combined with the different epitopes of CA215 antigen molecules respectively, form " sandwich " structure.It is not bound with
The CA215 monoclonal antibodies of alkali phosphatase enzyme mark are removed in washing step.Chemoluminescent substrate is added, in light-emitting appearance
Upper reading luminous value (RLU), the intensity of the light of transmitting is directly proportional to the CA215 concentration in sample, by CA215 detection dosage-
Reaction normal curve can quantitatively obtain the concentration value of CA215, and CA215 detection dosage-reaction normal curves are as shown in Figure 1.
In embodiment, CA215 antibody and CA215 sterlings are taught by the Qegory Lee of Univ British Columbia Canada
Award and gift, magnetic particle is purchased from Japanese JSR companies.
Embodiment one
CA215 detection kit
The CA215 detection kit include Magneto separate agent, the CA215 antibody of tracer-labelling, Chemoluminescent substrate,
Calibration object and cleaning mixture.
The Magneto separate agent is to be coated the magnetic particle solution of CA215 antibody, the coating of CA215 antibody in the Magneto separate agent
Concentration is 0.1～1mg/mL.Wherein, magnetic particle is Fe3O4/Fe2O3Magnetic nano-particle and the polymerization of high-molecular organic material
Thing, i.e. Fe3O4/Fe2O3 are kernel, and its microsphere surface is with one or more group in carboxyl, amino and hydroxyl.The magnetic
Microgranule mean diameter is 0.5～5 μm, preferably 1～3 μm.
The tracer for labelling CA215 antibody in the CA215 antibody of the tracer-labelling can be Radix Cochleariae officinalises peroxide
At least one in compound enzyme, alkali phosphatase, diamantane (obsolete), luminol, different luminol and its derivant, acridinium ester, preferably alkali
Acid phosphatase.In the CA215 antibody of the tracer-labelling, CA215 antibody concentration is 1～300ng/ml.
The Chemoluminescent substrate is diethanolamine buffer, and every liter of diethanolamine buffer includes 3- (2- spirals
Diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyls)-phenyl -1,2- dioxane disodium salt (AMPPD) 0.2g, fluorescent whitening agent
2.5- bis--bis- (benzoxazoles -2-) thiophene (EBF) 0.1g, bovine serum albumin (BSA) 5g, BDMQ surfactant 1g and
The MgSO4 0.1ml and 0.8mmol/LZnCl of proclin300 10g, 0.4mmol/L20.1ml, remaining are 0.1mol/L's
Diethanolamine.
The calibration object is prepared using CA215 sterlings, in the present embodiment, has prepared the calibration object of 6 kinds of sign concentration altogether,
Which indicates concentration and is respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/ml, 50AU/ml, 100AU/ml.
The cleaning mixture is the neutral buffered liquid of pH7.0～8.0, takes trishydroxymethylaminomethane 2.24g, 8.76g chlorination
Sodium and 0.5ml tween 20s add water and are settled to 1000ml, mix and filter.
Fig. 2 is referred to, the preparation method flow chart of steps of the CA215 detection kit provided for the present invention.
Embodiment two
The preparation method of CA215 detection kit
The preparation method of the CA215 detection kit comprises the steps：
1) Magneto separate agent is prepared：
Activation magnetic particle：After carboxyl magnetic particle being mixed fully, take the carboxyl magnetic particle of 100 μ l in the centrifuge tube of 1.5ml,
Be placed on Magneto separate frame, to be separated after remove supernatant, take 200 μ l100mmol/L MES buffer (pH 5.0) be added to from
In heart pipe, it is placed on Magneto separate frame, rear removal supernatant to be separated, repeated washing 3 times are rapidly added 50 μ of new preparation
The NHS solution of the EDC solution of l50mg/ml and 50 μ l 50mg/ml mixes carboxyl magnetic in the centrifuge tube equipped with carboxyl magnetic particle
Microgranule fully suspends, and reacts 30min, after reaction terminates, with the MES buffer (pH 5.0) of 100 μ l 100mmol/L under room temperature
Washing 3 times；
Coated magnetic particle：Carboxyl magnetic particle good for above-mentioned activation is placed on Magneto separate frame, is separated and is removed supernatant, added
The CA215 antibody of 100 μ l 2.5mg/ml, soft clearly mixing；2h is reacted at room temperature；Centrifuge tube is placed in magnetic after terminating by reaction
On separator frame, supernatant is removed, add the ethanolamine solutions (pH9.0) of 100 μ l 3mol/L to wash 3 times, then add 100 μ
In centrifuge tube, room temperature reaction 2h, after reaction terminates, removes supernatant to the ethanolamine solutions (pH9.0) of l3mol/L, adds suitable
The preservation liquid of amount is preserved, and the preservation liquid is the buffer containing 1%BSA and 0.1mol/LPBS, its pH=7.4；
Coated for above-mentioned CA215 antibody carboxyl magnetic particle is diluted to by 0.1～1mg/ml using bead suspension, i.e., described
The coated CA215 antibody concentration of Magneto separate agent is 0.1～1mg/ml, obtains the working solution of Magneto separate agent.Wherein described magnetic bead hangs
Supernatant liquid is prepared in accordance with the following methods：Weigh Tris- alkali 1.21g, sodium chloride 4.38g and Proclin300 0.2g and add 200ml's
Purified water, with HCl or NaOH adjusts pH value 8.5 ± 0.5 after standing 10min after mixing, be subsequently adding 2g trehaloses, 10gBSA,
1g gelatin, the calcium chloride 0.1ml and 0.5mL of zinc chloride 0.05ml, 1mol/L of magnesium chloride 0.5ml, 1mol/L of 1mol/L
T-20, with purified water constant volume to 500mL, mixes and is filtrated to get bead suspension.
2) the CA215 antibody of tracer-labelling is prepared：
(1) activated alkaline phosphatase：The alkali phosphatase for taking 0.02ml20mg/ml is placed in the bottom of reaction tube, weighs
4- (N- maleimidomehyls) hexamethylene -1- carboxylic acids sulfonic group butanimide ester sodium salt (sulfo-smcc) of 2mg, with pure
Change water dissolution to 40mmol/L (17.5mg/ml), add the 40mmol/L's of 5ul in the reaction tube containing alkali phosphatase
Sulfo-smcc solution, concussion are mixed, and standing and reacting 10-30min under room temperature condition is subsequently adding the glycine solution of 1mol/L
With unnecessary sulfo-smcc in 0.5 μ l, 10min is reacted, horse back Zeba desalination column purifications after terminating obtain the alkalescence for activating
Phosphatase；
(2) CA215 antibody is activated：The CA215 monoclonal antibodies for taking 0.5ml 5mg/ml are placed in the bottom of reaction tube, claim
The TCEP of 6mg is taken, and the TCEP solution of 0.5mol/L is obtained with the PBS dissolvings of pH 7.2, in the reaction tube containing CA215 antibody
50 μ l TCEP solution of middle addition, concussion are mixed, and standing and reacting 30-60min under room temperature condition is subsequently adding the sweet ammonia of 1mol/L
With unnecessary TCEP in 1 μ l of acid solution, react 10 minutes, horse back Zeba desalination column purifications after terminating obtain the CA215 for activating
Antibody；
(3) by the good antibody of above-mentioned activation and the alkali phosphatase in mass ratio 1 for having activated:1 mixing, in 2-8 DEG C of condition
Lower reaction 12h, reaction obtain the good CA215 antibody-enzyme conjugates of purification with the method for sieve chromatography after terminating；
CA215 antibody-enzyme conjugates good for purification are diluted to 1～300ng/ml using Working dilutions, i.e., described are shown
In the CA215 antibody of track substance markers, CA215 antibody concentration is 1～300ng/ml, obtains the CA215 antibody work of tracer-labelling
Liquid.Wherein described Working dilutions each component content is as follows:The bovine serum albumin of 1%-5% volumes, the Mus blood of 1-5% volumes
Clearly, the new-born calf serum of 1-10% volumes, the horse serum of 1-5% volumes, the porcine blood serum of 1-5% volumes, the sodium chloride of 8g/L,
The potassium chloride of 0.2g/L, 2.9g/L disodium hydrogen phosphates, 0.2g/L potassium dihydrogen phosphates, 10-30g/L trehaloses, 0.1～1g/L 4-
Formylphenyl boronic acid, the glycerol of 0.1-5% volumes, the ethylene glycol of 0.1-3% volumes, 0.1-20g/L xanthan gum, 0.01-1%
The tween 20 of volume, 0.1-1g/L ethylenediaminetetraacetic acid, Proclin-300,0.1-0.5% volume of 1% volume celebrating mould greatly
Element.
3) Chemoluminescent substrate is prepared：
3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-phenyl -1,2- dioxane two is weighed respectively
Sodium salt (AMPPD) 0.2g, bis--bis- (benzoxazoles -2-) thiophene (EBF) 0.1g of fluorescent whitening agent 2.5-, bovine serum albumin (BSA)
5g, BDMQ surfactant 1g and proclin300 10g, is subsequently adding MgSO4 0.1ml, 0.8mmol/L of 0.4mmol/L
ZnCl2The diethanolamine buffer 500ml of 0.1ml and 0.1mol/L, mixes 15 minutes, is subsequently adding the diethyl of 0.1mol/L
Hydramine buffer constant volume is mixed and filters stand-by, keep in dark place under the conditions of being placed on 2-8 DEG C to 1000ml；
4) calibration object is prepared：
CA215 sterlings are diluted using 7.4 phosphate buffers of pH, calibration obtains calibration object, the phosphate buffer bag
Include the preservative of the BSA and 0.05% that mass fraction is 3%, wherein preservative is Proclin-300, the sign of the calibration object
Concentration is respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/ml, 50AU/ml and 100AU/ml.
5) cleaning mixture is prepared：
Take trishydroxymethylaminomethane 2.24g, 8.76g sodium chloride and 0.5ml tween 20s add water and are settled to 1000ml, mix
Even filtration.
6) by Magneto separate agent, the CA215 antibody of tracer-labelling, Chemoluminescent substrate, calibration object and cleaning mixture group
Dress up the CA215 detection kit.
Fig. 3 is referred to, the using method flow chart of steps of the CA215 detection kit provided for the present invention.
Embodiment three
The using method of CA215 detection kit
The using method of CA215 detection kit, comprises the steps：
S1, CA215 detection kit is taken out, the CA215 detection kit is balanced to room temperature, and by the CA215
Reagent in detection kit takes out standby：
Specifically, the CA215 detection kit is put and balance 30 minutes under room temperature (18～25 DEG C)；
S2, taking-up serum sample to be measured is balanced to room temperature：
Specifically, serum sample to be measured is put (18～25 DEG C) of room temperature to balance 30 minutes.
S3, prepare test tube according to the quantity of calibration object and serum sample to be measured, and number, add in test tube respectively
The serum sample and calibration object of 50 μ l, then adds the CA215 antibody of 100 μ l tracer-labellings, then distinguishes in every test tube
The Magneto separate agent of 25 μ l is added, is fully vibrated at 37 DEG C, be subsequently placed on Magneto separate frame and separate；
Specifically, test tube quantity be 7, be separately added in each test tube concentration for 0AU/ml, 5AU/ml, 10AU/ml,
Six calibration objects of 20AU/ml, 50AU/ml and 100AU/ml and serum sample to be measured；
S4, supernatant is poured out, cleaned with cleaning mixture；
100 μ l of chemical luminous substrate are added in S5, each test tube, is carried out with chemiluminescence measuring instrument after fully mixing
Detection；
S6, acquisition testing result：The chemiluminescence intensity RLU values for measuring each test tube are directly read, with the concentration of calibration object
It is worth for abscissa, the RLU values of calibration object are that vertical coordinate draws standard curve, calculate serum sample to be measured according to standard curve
Concentration value, directly chemically light measuring instrument read or derive.
Example IV
The analytical performance evaluation of CA215 detection kit
CA215 standard substance are detected first with chemical illumination immunity analysis instrument, draw detection dosage-reaction normal
Curve, shown in Figure 1, the standard curve is built in computer software, then tests known content sample and calibration object etc.,
Read luminous value or the corresponding concentration of luminous value is calculated according to dose-response standard curve, to the CA215 detection kit
Carry out the evaluation of accuracy, specificity, precision, lowest detectable limit and stability.
1) accuracy
The methodology identification of CA215 immue quantitative detection reagent boxes
Calibrating knot is carried out to the CA215 detection kit of embodiment 1 according to manufacture conventional in the art and identification code
Fruit is as follows：
Known content sample is detected with the test kit prepared in the embodiment of the present invention 1, the response rate (response rate is calculated
=yield/addition × 100%), as a result as shown in the table：
| Addition AU/ml | 5 | 10 | 25 | 50 |
| Yield AU/ml | 4.94 | 10.65 | 26.11 | 51.02 |
| Response rate % | 98.80 | 106.50 | 104.44 | 102.04 |
2) specificity
Known content analog is detected with the CA215 detection kit in the embodiment of the present invention 1, as a result such as following table
Shown：
3) elaboration
Detectable concentration is respectively the luminous value of the quality-control product 2 of the quality-control product 1 and 50AU/ml of 5AU/ml, quality-control product 1 and Quality Control
The concentration of product 2 does 10 parallel hole replication its luminous values respectively, calculate CV values (Coefficient of Variance, from
Scattered coefficient, is the percentage ratio of standard deviation and toaverage ratio), as a result as shown in the table：
4) lowest detectable limit
Replication sign concentration is zero calibration object (or Sample dilution) 10 times, calculates the average of luminous valueWith standard deviation (SD), incite somebody to actionResponse magnitude substitute into CA215 detection dose-response curve, calculate phase
Concentration value, as lowest detectable limit is answered, as a result as shown in the table：
5) stability
In test kit, each group splits 37 DEG C of test accuracies after 7 days, lowest detectable limit, precision, curve correlation coefficient, knot
Fruit is as follows：
Result above is collected and is drawn：
| Detection project | Touchstone | Assay |
| Accuracy | The response rate is 95%～110% | Conformance with standard |
| Specificity | This kit measurement result should be not more than 1AU/ml | Conformance with standard |
| Elaboration CV (%) | It is less than 15% (n=10) | Conformance with standard |
| Sensitivity | It is less than 1AU/ml | Conformance with standard |
| Stability | In test kit, each group splits 37 DEG C at least 7 days | Conformance with standard |
Can see that the explanation CA215 detection kit property indices are qualified from above-mentioned summary sheet, and have
Accuracy is high, specificity is high, elaboration is good, sensitivity is high and the advantage of good stability.
Embodiment five：
The determination of the content and marginal value of the CA215 of normal person
The CA215 detection kit that randomly selects in 200 Healthy Human Serum embodiments 1 is measured, and collects all
Measured value, determines its marginal value (the reference limit of 95% digit), reference range≤6.3AU/ml with method of percentiles.
Embodiment six：
60 serum of ovarian cancer patients are randomly selected, is surveyed with the CA215 quantification kits of the preparation in embodiment 1
Fixed, collect all measured values, mean concentration is 20.74AU/ml, is significantly higher than Healthy Human Serum measured value.
Embodiment seven：
60 cervical cancer patient serum are randomly selected, is surveyed with the CA215 quantification kits of the preparation in embodiment 1
Fixed, collect all measured values, mean concentration is 31.08AU/ml, is significantly higher than Healthy Human Serum measured value.
CA215 detection kit that the present invention is provided and preparation method thereof and using method, have the advantages that：
First, the CA215 detection kit that the present invention is provided, is a kind of new tumor markerses, other Cancer Biology marks
Thing and CA215 combine the positive rate that can be improved in routine clinical diagnosis cancer.And the reaction pattern of this reagent
It is sandwich assay, specificity is good, sensitivity is high, and without HOOK effects, the range of linearity is wide；
2nd, the present invention is incorporated into carboxyl magnetic particle in the middle of immunoassay technology, makes the system of reagent reacting homogeneous, can pole
The earth improves the efficiency that antigen-antibody is combined, and shortens the response time；
3rd, the labelling of alkali phosphatase is different from general glutaraldehyde method and Over-voltage protection, difunctional crosslinking used
Method, makes test kit possess higher sensitivity and linear measurement range, and non-specific low, good stability.
4th, as a result of the new reinforcing agent technology of AMPPD/ of company's substrate solution, the sensitivity of luminous substrate liquid is high, luminous hold
The continuous time is long, greatly improves the sensitivity of reagent.
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, for the skill of this area
For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, made any repair
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of CA215 detection kit, it is characterised in which includes：Magneto separate agent, the CA215 antibody of tracer-labelling, change
Learn luminous substrate liquid and calibration object；
Wherein,
The Magneto separate agent is to be coated the magnetic particle solution of CA215 antibody；
The Chemoluminescent substrate be diethanolamine buffer, the diethanolamine buffer include 3- (2- spiral Buddha's warrior attendants
Alkane) -4- methoxyl group -4- (3- phosphorus oxygen acyls)-phenyl -1,2- dioxane disodium salts；
The calibration object is prepared using CA215 sterlings, and which indicates concentration and is respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/
ml、50AU/ml、100AU/ml.
2. CA215 detection kit according to claim 1, it is characterised in that the Magneto separate agent is in accordance with the following methods
Prepare：
(1) magnetic particle is activated：Take the carboxyl magnetic particle after fully mixing to be put in centrifuge tube, centrifuge tube is positioned over Magneto separate frame
On, after carboxyl magnetic particle is adsorbed separating completely, supernatant is removed, MES buffer solution carboxyls magnetic particle at least is added
Secondary；Add EDC solution and NHS solution to activate carboxyl magnetic particle；
(2) coated magnetic particle：With the carboxyl magnetic particle after the activation of MES buffer solutions at least one times, centrifuge tube is placed in magnetic point
On frame, separate and remove supernatant, add the CA215 antibody of 2.5mg/ml to mix, react 2～4 hours at room temperature, reaction knot
Shu Hou, then centrifuge tube is placed on Magneto separate frame, supernatant is removed, is washed with ethanolamine solutions at least one times, is then added
In centrifuge tube, room temperature reaction 2～4 hours, after reaction terminates, removes supernatant and preserves ethanolamine solutions, and wherein, carboxyl magnetic is micro-
Grain is 1 with the volume ratio of CA215 antibody：1；
(3) coated magnetic particle prepared by step (2) is diluted to by 0.1～1mg/ml using bead suspension.
3. CA215 detection kit according to claim 2, it is characterised in that the carboxyl magnetic particle is Fe3O4/Fe2O3
Magnetic nano-particle and the polymer of high-molecular organic material, its mean diameter are 0.5～5 μm, preferably 1～3 μm, and the carboxylic
Base magnetic particle surface is with one or more group in carboxyl, amino and hydroxyl.
4. CA215 detection kit according to claim 2, it is characterised in that the bead suspension is according to lower section
Method is prepared：The purified water that Tris- alkali 1.21g, sodium chloride 4.38g and Proclin300 0.2g add 200ml is weighed, after mixing
PH value 8.5 ± 0.5 is adjusted with hydrochloric acid or sodium hydroxide after standing 10min, be subsequently adding 2g trehaloses, 10gBSA, 1g gelatin,
The T-20 of the calcium chloride 0.1ml and 0.5mL of zinc chloride 0.05ml, 1mol/L of magnesium chloride 0.5ml, 1mol/L of 1mol/L, uses
Purified water constant volume is mixed and is filtrated to get bead suspension to 500mL.
5. CA215 detection kit according to claim 1, it is characterised in that the tracer is horseradish peroxidase
At least one in enzyme, alkali phosphatase, diamantane (obsolete), luminol, different luminol and its derivant, acridinium ester, preferably alkaline phosphorus
Sour enzyme.
6. CA215 detection kit according to claim 1, it is characterised in that the CA215 antibody of the tracer-labelling
Middle CA215 antibody concentration is 1～300ng/ml.
7. a kind of preparation method of CA215 detection kit, it is characterised in that the CA215 detection kit includes Magneto separate
Agent, the CA215 antibody of tracer-labelling, Chemoluminescent substrate, calibration object and cleaning mixture, its preparation method include following step
Suddenly：
(1) Magneto separate agent is prepared
Activation magnetic particle：Carboxyl magnetic particle is taken in centrifuge tube and is fully mixed, centrifuge tube is positioned on Magneto separate frame, carboxylic is treated
After base magnetic particle is adsorbed separating completely, supernatant is removed, MES buffer solution carboxyls magnetic particle is added at least one times；Again plus
Enter EDC solution and NHS solution to activate carboxyl magnetic particle；
Coated magnetic particle：With the carboxyl magnetic particle after the activation of MES buffer solutions at least one times, centrifuge tube is placed in Magneto separate frame
On, separating and remove supernatant, add the CA215 antibody of 2.5mg/ml to mix, react 2～4 hours at room temperature, reaction terminates
Afterwards, then by centrifuge tube it is placed on Magneto separate frame, removes supernatant, washed with ethanolamine solutions at least one times, then add second
In centrifuge tube, room temperature reaction 2～4 hours, after reaction terminates, removes supernatant and preserves, wherein carboxyl magnetic particle alkanolamine solution
Volume ratio with CA215 antibody is 1：1；
The coated magnetic particle for preparing is diluted to by the working solution that 0.1～1mg/ml obtains Magneto separate agent using bead suspension；
(2) the CA215 antibody of tracer-labelling is prepared：
Activated alkaline phosphatase：The alkali phosphatase for taking 20mg/ml is placed in the bottom of reaction tube, adds excessive 40mmol/L
4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group butanimide ester sodium salts, concussion mix, under room temperature condition
10～30min of standing and reacting, is subsequently adding in the glycine solution of 1mol/L and unnecessary 4- (N- maleimidomehyls) ring
Hexane -1- carboxylic acid sulfonic group butanimide ester sodium salts, with Zeba desalination column purifications after reaction 10min, obtain the alkalescence for activating
Phosphatase；
Activation CA215 antibody：The CA215 monoclonal antibodies for taking 5mg/ml are placed in the bottom of reaction tube, add excess
The TCEP solution of 0.5mol/L, concussion are mixed, and standing and reacting 30-60min under room temperature condition is subsequently adding the glycine of 1mol/L
With unnecessary TCEP solution in solution, react 10 minutes, horse back Zeba desalination column purifications after terminating obtain the CA215 for activating
Antibody；
By the alkali phosphatase for having activated and the CA215 antibody in mass ratio 1 for having activated:1 mixing, reacts under the conditions of 2-8 DEG C
12h, reaction obtain the good CA215 antibody-enzyme conjugates of purification with the method for sieve chromatography after terminating；
CA215 antibody-enzyme conjugates good for purification are diluted to 1～300ng/ml using Working dilutions, tracer-labelling is obtained
The working solution of the CA215 antibody of note；
(3) Chemoluminescent substrate is prepared：
3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-phenyl -1,2- dioxane disodium salt is weighed respectively
0.2g, fluorescent whitening agent 2.5- bis--bis- (benzoxazoles -2-) thiophene 0.1g, bovine serum albumin 5g, BDMQ surfactant 1g and
Proclin300 10g, are subsequently adding MgSO4 0.1ml, 0.8mmol/LZnCl of 0.4mmol/L20.1ml and 0.1mol/L
Diethanolamine buffer 500ml, mix 15 minutes, the diethanolamine buffer constant volume for being subsequently adding 0.1mol/L is arrived
1000ml, mixing filtration are stand-by, keep in dark place under the conditions of being placed on 2-8 DEG C；
(4) calibration object series is prepared：
CA215 sterlings are diluted using 7.4 phosphate buffers of pH, calibration object is obtained, the phosphate buffer includes quality point
Preservative of the number for 3% BSA and 0.05%, wherein preservative are Proclin-300, the sign concentration of the calibration object series
Respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/ml, 50AU/ml and 100AU/ml；
(5) cleaning mixture is prepared：
Take trishydroxymethylaminomethane 2.24g, 8.76g sodium chloride and 0.5ml tween 20s add water and are settled to 1000ml, mixed
Filter；
(6) Magneto separate agent, the CA215 antibody of tracer-labelling, Chemoluminescent substrate, calibration object and cleaning mixture are assembled into
The CA215 detection kit.
8. the preparation method of CA215 detection kit according to claim 7, it is characterised in that described prepare Magneto separate
In the step of agent, the bead suspension is prepared in accordance with the following methods：Weigh Tris- alkali 1.21g, sodium chloride 4.38g and
Proclin300 0.2g add the purified water of 200ml, with hydrochloric acid or sodium hydroxide adjust pH value 8.5 after standing 10min after mixing
± 0.5, it is subsequently adding 2g trehaloses, 10gBSA, 1g gelatin, the zinc chloride of magnesium chloride 0.5ml, 1mol/L of 1mol/L
The T-20 of the calcium chloride 0.1ml and 0.5mL of 0.05ml, 1mol/L, with purified water constant volume to 500mL, mixes and is filtrated to get magnetic bead
Suspension.
9. the preparation method of CA215 detection kit according to claim 7, it is characterised in that described prepare tracer
In the step of CA215 antibody of labelling, the Working dilutions each component content is as follows:The bovine serum albumin of 1%～5% volume
In vain, the Mus serum of 1～5% volume, the new-born calf serum of 1～10% volume, the horse serum of 1～5% volume, 1～5% volume
Porcine blood serum, the sodium chloride of 8g/L, the potassium chloride of 0.2g/L, 2.9g/L disodium hydrogen phosphates, 0.2g/L potassium dihydrogen phosphates, 10～30g/
L trehaloses, 0.1～1g/L 4- formylphenyl boronic acids, the glycerol of 0.1～5% volume, the ethylene glycol of 0.1～3% volume,
0.1～20g/L xanthan gum, the tween 20 of 0.01～1% volume, 0.1～1g/L ethylenediaminetetraacetic acid, 1% volume
Proclin-300, the gentamycin of 0.1～0.5% volume.
10. the using method of CA215 detection kit according to any one of claim 1 to 6, it is characterised in that bag
Include following steps：
CA215 detection kit is taken out, the CA215 detection kit is balanced to room temperature, and by CA215 detection kit
Reagent take out standby；
Take out serum sample to be measured to balance to room temperature；
Quantity according to calibration object and serum sample to be measured prepares reaction tube, adds the serum sample of 50 μ l respectively in test tube
And calibration object, in every test tube, then add the CA215 antibody of 100 μ l tracer-labellings, then the magnetic for being separately added into 25 μ l
Separating medium, fully vibrates at 37 DEG C, is subsequently placed on Magneto separate frame and separates；
Supernatant is poured out, is cleaned with cleaning mixture；
100 μ l of chemical luminous substrate are added in each reaction tube, is put in chemiluminescence measuring instrument after fully mixing and is examined
Survey；
Obtain testing result：The chemiluminescence intensity RLU values of each reaction tube are directly read, and with the concentration value of calibration object as horizontal seat
Mark, the RLU values of calibration object are that vertical coordinate draws standard curve, according to the concentration that standard curve calculates serum sample to be measured
Value, the measuring instrument that directly chemically lights read or derive.
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