CN106771202A - CA215 detection kits and preparation method thereof and application method - Google Patents
CA215 detection kits and preparation method thereof and application method Download PDFInfo
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Abstract
The present invention discloses a kind of CA215 detection kits and preparation method thereof and application method.The CA215 detection kits include the anti-coated micro reaction plate of CA215 antibody, enzyme conjugates, chemical luminous substrate A, chemical luminous substrate B and calibration object, wherein, the enzyme conjugates is the anti-CA215 antibody of enzyme mark, the marker enzyme of the enzyme conjugates is horseradish peroxidase, the chemical luminous substrate A contains luminol, the chemical luminous substrate B contains hydrogen peroxide, the calibration object is prepared using CA215 sterlings, and its sign concentration is respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/ml, 50AU/ml, 100AU/ml.The CA215 detection kits that the present invention is provided have the advantages that accuracy is high, specific high, precision is good, sensitivity is high and good stability, are 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 kits and its system
Standby, application method and application.
Background technology
Since nearly half a century, the morbidity and mortality of tumour are raised year by year, as the focus in medical field focus.20
Century 70, what the U.S. proposed reflects the anxiety that society remains high to tumor mortality rate to " tumour is marched ", meanwhile,
In past decades, tumor-marker technically has breakthrough with definition, and everything has all promoted what tumor-marker was studied
It is fast-developing.
CA215 is tumor associated antigen, and the antigen is that the monoclonal to being extracted from the ovarian cancer cell of culture in 1987 resists
What body RP215 had found.RP215 is proved to (general with positioned at the heavy chain immunoglobulin mainly by expression in most of cancer cells
Referred to as CA215) the related epitope of carbohydrate reacts.
Because CA215 is that height is associated with cancer cell or cancerous tissue, and can not typically be found in health adult tissue,
Except in hyperplastic epithelium cell or tissue, such as skin, esophagus, cervix and several immunologically privileged siteses, including nerve, eye
Eyeball and testis.
CA215 has potential using value as a general cancer biomarker, and can be with other known cancer
The clinic of the parallel immunodiagnosis in many different types of cancers of the mankind for comparing to record CA215 of biomarker should
With.
It was found that cancer patient and normal human serum CA215 levels are significant difference (P<0.001).Due to clinical stages,
In the case of oophoroma, compared to its positive rate scope of normal individual from 58-86%.For cervical cancer patient, in cancer evening
The stage positive rate of phase is up to 66-94% respectively.
Cervical carcinoma and the result of oophoroma these clinical researches are also shown that average serum CA215 levels, and 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 is determined after seven days, and change of serum C A215 levels are remarkably decreased.Based on these researchs, it can be deduced that conclusion thinks, cancer patient's
Surgery excision or chemotherapy between cervix and oophoroma cause statistically to be substantially reduced in CA215 levels.As a result
Show, CA215 is produced in cancer patient's most probable from tumor locus, and the tumor load can be by the change of serum C A215 of cancer patient
Level fully reflects and detects.Therefore, meter of the customary detection of the change of serum C A215 levels of cancer patient to monitoring cancer development
The state for the treatment of or operative treatment is beneficial after drawing.
Used as a general carcinoma marker, CA215 is than carcinomebryonic antigen (carcino-embryonic antigen, CEA) or β 2
Microglobulin more preferably, has display positive rate higher in most of cancers.For most of human cancer CA215 sun
Property recall rate is all higher, including oophoroma.However, in the case of cervix cancer, using CA215 combine detections than individually use
CA125 has recall rate higher, and recall rate is promoted to 81% from 13%.However, for the oophoroma group of CA215 and CA125
Conjunction also has more preferable recall rate, and verification and measurement ratio is promoted to 82% from 59%.For lung cancer, CA215 and CYFRA21-1 combine detections
With preferable positive rate.For liver cancer, the group of CEA or alpha-fetoprotein (alpha-fetoprotein, AFP) and CA215
Conjunction seems have positive rate higher.In a word, other biomarker for cancer can be improved with the combination of CA215 and faced routinely
Positive rate in bed diagnosis cancer.
Report carries out EIA enzyme immunoassay to last century the mid-1970s Arakawe with luminous signal first, using luminous change
Response analysis ultra micro quantity of material is learned, particularly for checking ultramicron active material in clinical immunoassays.At present, this technology
Clinical medical conventional detection means are transitioned into from the rare technology in laboratory.Chemiluminescence immune assay
(Chemiluminescence Immunoassay, CLIA) is that chemiluminescence or bioluminescence system are mutually tied with immune response
Close, a kind of novel markings immunoassay for detecting trace antigen or antibody.Its Cleaning Principle and radio-immunity
(Radioimmunoassay, RIA) and enzyme are immune, and ((enzyme immunoassay, EIA) is similar, is a difference in that with luminous
Material is directly measured as substrate by the luminous intensity of its own.
Two parts, i.e. immune response system and chemiluminescence analysis system are included in chemiluminescence immune assay.Exempt from
Epidemic disease reaction system, the same Enzyme-multiplied immune technique of its general principle (enzyme linked immunosorbent assay, ELISA).
The principle of chemiluminescence analysis system is enzyme effect in immune response in luminous substrate.Luminous substrate in the presence of enzyme,
Substrate chemically reacts and discharges substantial amounts of energy, produces the intermediate of excitation state.This excitation state intermediate, when its time
During to stable ground state, can simultaneously launch photon.It is measurable quantum yield of luminscence, the light quantity using luminous signal measuring instrument
Sub- yield is directly proportional to the amount of the test substance in sample.It is possible thereby to set up standard curve and calculate test substance in sample
Content.
Chemiluminescence immune assay had both had the high sensitivity of radio-immunity, and easy to operate, fast with enzyme linked immunological
Fast the characteristics of, it is easy to normalizing operation.And do not use harmful reagent, reagent to be kept for the phase long in test, it is applied to biology, doctor
Research and clinical trial diagnostic work are learned, as one of most promising method in on-radiation immunoassay.
The content of the invention
The present invention provides a kind of CA215 detection kits, can with the concentration of quantitative determination human cancer mark CA215,
There is accuracy high, specific height, precision is good, sensitivity is high and stability is good.
A kind of CA215 detection kits, it includes the anti-coated micro reaction plate of CA215 antibody, enzyme conjugates, chemistry hair
Light substrate A, chemical luminous substrate B and calibration object；
Wherein,
The enzyme conjugates is the anti-CA215 antibody of enzyme mark, and the marker enzyme of the enzyme conjugates is horseradish peroxidase
Enzyme；
The chemical luminous substrate A contains luminol, and the chemical luminous substrate B contains hydrogen peroxide；
The calibration object using CA215 sterlings prepare, its sign concentration be respectively 0AU/ml, 5AU/ml, 10AU/ml,
20AU/ml、50AU/ml、100AU/ml；
The anti-coated micro reaction plate of CA215 antibody is prepared in accordance with the following methods：
Coating：After anti-CA215 antibody is using coating buffer dilution, micro reaction plate is taken, the anti-CA215 antibody that will be diluted is added
To in the micropore of the micro reaction plate, the addition of each micropore is l00 μ l, and it is small that (18~25 DEG C) of room temperature places 12~24
When；
Closing：Coating buffer is discarded, is patted dry on blotting paper, add the μ l/ holes of confining liquid 250, room temperature (18~25 DEG C) is put
Put 6~24 hours；
Stabilization：Confining liquid is discarded, the μ l/ holes of stabilizing solution 250 are added, (18~25 DEG C) of room temperature is placed 4~24 hours；
Envelope：Stabilizing solution is discarded, is patted dry on blotting paper, vacuumize 16~24 hours in vacuum drying chamber at room temperature, so
Carry out vacuum sealing bag immediately afterwards, it is labelled after 2~8 DEG C of preservations.
Preferably, the micro reaction plate material is opaque polystyrene or polyethylene.
Preferably, the compound method of the coating buffer is as follows：Take the Na of lmol/L2HPO477.4ml's and lmol/L
Na2HPO422.6ml is mixed, plus deionized water is settled to 1000ml, then is diluted 10 times and used.
Preferably, the confining liquid is to contain the BSA, 1% casein and 0.05% preservative that mass fraction is 3%
Phosphate buffer, the preservative is Proclin-300, and the pH of the phosphate buffer is 7.4.
Preferably, the stabilizing solution is slow containing the sucrose and the phosphate of 0.05% preservative that mass fraction is 4%
Fliud flushing, the preservative is Proclin-300, and the pH of the phosphate buffer is 7.4.
Preferably, the calibration object is prepared according to following method：Phosphate buffer with pH 7.4 is dilute by CA215 sterlings
Various concentrations point is interpreted into, and calibrates the calibration object for obtaining various sign concentration, the phosphate buffer is including mass fraction
The preservative of 3% BSA, 0.5% Creamophpr EL and 0.05%, wherein preservative are Proclin-300.
Preferably, the CA215 detection kits also include concentrated cleaning solution, and the concentrated cleaning solution is pH7.0~8.0
Phosphate buffer, the phosphate buffer includes sodium chloride and 2.5% Tween-20 that mass fraction is 17%.
Present invention simultaneously provides a kind of preparation method of CA215 detection kits, the CA215 detection kits include anti-
The coated micro reaction plate of CA215 antibody, enzyme conjugates, chemical luminous substrate A, chemical luminous substrate B, calibration object and concentration
Cleaning solution, its preparation method comprises the following steps：
1) the anti-coated micro reaction plate of CA215 antibody is prepared：
Coating：After anti-CA215 antibody is using coating buffer dilution, micro reaction plate is taken, the anti-CA215 antibody that will be diluted is added
To in the micropore of the micro reaction plate, the addition of each micropore is l00 μ l, and it is small that (18~25 DEG C) of room temperature places 12~24
When；
Closing：Coating buffer is discarded, is patted dry on blotting paper, add the μ l/ holes of confining liquid 250, room temperature (18~25 DEG C) is put
Put 6~24 hours；
Stabilization：Confining liquid is discarded, the μ l/ holes of stabilizing solution 250 are added, (18-25 DEG C) of room temperature is placed 4~24 hours；
Envelope：Stabilizing solution is discarded, is patted dry on blotting paper, vacuumize 16~24 hours in vacuum drying chamber at room temperature, so
Carry out vacuum sealing bag immediately afterwards, it is labelled after 2~8 DEG C of preservations；
2) enzyme conjugates is prepared：
Anti- CA215 antibody is crosslinked with the horseradish peroxidase for marking using periodates oxidizing process, by gradient
Dilution determines the concentration of the enzyme conjugates, with comprising sodium chloride, 3% BSA, 0.05% that mass fraction is 0.85%
EDTA and 0.05% preservative the phosphate buffers of pH 7.4 preserve enzyme conjugates, wherein preservative be Proclin-
300；
3) chemical luminous substrate A and chemical luminous substrate B is dispensed；
Chemical luminous substrate A and chemical luminous substrate the B packing that will directly purchase, wherein, the chemical luminous substrate A contains
There is luminol, the chemical luminous substrate B contains hydrogen peroxide；
4) calibration object series is prepared：
CA215 sterlings are diluted using the phosphate buffers of pH 7.4, calibration object is obtained, the phosphate buffer includes matter
Amount fraction is 3% BSA, the preservative of 0.5% Creamophpr EL and 0.05%, and wherein preservative is Proclin-
300, the sign concentration of calibration object series be respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/ml, 50AU/ml and
100AU/ml；
5) concentrated cleaning solution is prepared：
28g disodium hydrogen phosphates are weighed, 170g sodium chloride measures 25ml Tween-20s, 1000ml is settled to water, adjusted
PH7.0~8.0；
6) by the anti-coated micro reaction plate of CA215 antibody, enzyme conjugates, chemical luminous substrate A, chemical luminous substrate B,
Calibration object and concentrated cleaning solution are assembled into the CA215 detection kits.
Preferably, the confining liquid is to contain the BSA, 1% casein and 0.05% preservative that mass fraction is 3%
Phosphate buffer, the preservative is Proclin-300, and the pH of the phosphate buffer is 7.4.
Present invention simultaneously provides the application method of CA215 detection kits mentioned above, comprise the following steps：
CA215 detection kits are taken out, the CA215 detection kits are balanced to room temperature, and by the detection reagent
Reagent in box takes out standby；
Dilution concentrated cleaning solution：It is standby after the concentrated cleaning solution is diluted into 50 times with fresh purified water；
Prepare luminous substrate：Chemical luminous substrate A and chemical luminous substrate B is pressed 1:1 volume ratio is configured to after mixing
Luminous substrate；
Serum sample to be measured is taken out to balance to room temperature；
Take out the anti-coated micro reaction plate of CA215 antibody, toward each micropore of micro reaction plate in be separately added into 100 μ l
Calibration object and serum sample to be measured, are well mixed, and put 37 DEG C of insulating boxs and are incubated 60 minutes；
Washing：The liquid in the micropore of the micro reaction plate is discarded, rinses described using the concentrated cleaning solution after dilution
Micro reaction plate, then pats dry；
100 μ l enzyme conjugates are added in toward each micropore of the micro reaction plate, is fully mixed, put 37 DEG C of insulating boxs and incubate
Educate 60 minutes；
Washing：The liquid in the micropore of the micro reaction plate is discarded, rinses described using the concentrated cleaning solution after dilution
Micro reaction plate, then pats dry；
100 μ l luminous substrates are added in each hole, inspection is added immediately into chemical illumination immunity analysis instrument after fully mixing
Survey；
Luminous value is directly chemically read on luminescence immunoassay instrument or according in defaulting in chemical illumination immunity analysis instrument
CA215 detection dosage-reaction normal curve obtain the concentration value of test serum sample and derive.
CA215 detection kits provided compared to prior art, the present invention and preparation method thereof and application method, have
Following beneficial effect：
First, CA215 can be used for the potential risk of diagnosing human cancer as a general biomarker for cancer, and the present invention is carried
The CA215 detection kits of confession can go out the content of patients serum CA215 with quantitative determination, can also combine with other cancer markers
The monitoring of auxiliary diagnosis and related diseases people's therapeutic effect for cancer, such as with AFP, CEA, CA125, CA19-9, CA15-3 or
Cyfra21-1 joints are detected, compared to individually detection single creature mark, it can be observed that cancer detection rate higher,
Clinically it is with a wide range of applications.
2nd, the CA215 detection kits that the present invention is provided have high specific, high sensitivity and good precision, and
And easy to operate, quick, good stability and testing result are accurate.
3rd, closing and stabilization are separately carried out in the step of anti-CA215 antibody coating micro reaction plate that the present invention is provided, and are increased
The strong stability of coating plate.
4th, the present invention provide anti-CA215 antibody coating micro reaction plate the step of in, coating, closing and stabilization when
Between scope it is wider than the conventional time, bring the great convenience, and coating process to be carried out under room temperature environment to experimental implementation person, it is and normal
37 degree different with 4 degree, reduce the use of instrument.
5th, the closing formula of liquid that the present invention is provided, sealing effect more preferably, reduces non-specific binding.
6th, provided by the present invention for prepare the phosphate buffer of calibration object including BSA that mass fraction is 3%,
The preservative of 0.5% Creamophpr EL and 0.05%, makes the calibration object stability of preparation better than conventional buffer solution.
Brief description of the drawings
Technical scheme in order to illustrate more clearly the embodiments of the present invention, below will be to that will make 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, other can also be obtained according to these accompanying drawings
Accompanying drawing, wherein：
Fig. 1 detects dosage-reaction normal curve for the CA215 detection kits CA215 that the present invention is provided；
The preparation method flow chart of steps of the CA215 detection kits that Fig. 2 is provided for the present invention；
The application method flow chart of steps of the CA215 detection kits that Fig. 3 is provided for the present invention.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.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.
Know-why of the invention is as follows：
The present invention detects the concentration value of general carcinoma marker CA215 using chemiluminescence immunoassay technology, there is provided CA215
Detection kit is coated with micro reaction plate with CA215 monoclonal antibodies, and CA215 calibration objects or to be measured are added in micro reaction plate
Serum sample, coated CA215 monoclonal antibodies are combined with CA215 antigen molecules on micro reaction plate, add horseradish peroxide
The CA215 monoclonal antibodies of compound enzyme (horseradish peroxidase, HRP) mark are reacted, on micro reaction plate
The CA215 monoclonal antibodies of coated CA215 monoclonal antibodies and horseradish peroxidase-labeled respectively with CA215 antigen molecules
Different epitopes combine, formed " sandwich " structure.The CA215 monoclonal antibodies of the horseradish peroxidase-labeled being not bound with
It is removed in washing step.Luminous substrate is added, luminous value (RLU), the intensity and sample of the light of transmitting are read on light-emitting appearance
CA215 concentration in product is directly proportional, and detects that dosage-reaction normal curve can quantitatively obtain the concentration of CA215 by CA215
Value, CA215 detection dosage-reaction normal curves are as shown in Figure 1.
It is used for coated anti-CA215 antibody in embodiment and for marking the anti-CA215 antibody of horseradish peroxidase equal
By Gregory Lee, Ph.D. is granted.The CA215 sterlings prepared in calibration object are purchased from chemux companies, chemical luminous substrate A,
Chemical luminous substrate B is purchased from Beijing Key-Biotechnology Co., Ltd, and direct packaging is that can be used.
Embodiment one
CA215 detection kits
The CA215 detection kits include the anti-coated micro reaction plate of CA215 antibody, enzyme conjugates, chemiluminescence
Substrate A, chemical luminous substrate B, calibration object and concentrated cleaning solution, coated anti-CA215 antibody is wherein in micro reaction plate
CA215 monoclonal antibodies.
The micro reaction plate material is opaque polystyrene or polyethylene.
The enzyme conjugates is the anti-CA215 antibody of enzyme mark, and the marker enzyme of the enzyme conjugates is horseradish peroxidase
Enzyme, the anti-CA215 antibody and the horseradish peroxidase are obtained using the crosslinking of periodates oxidizing process, wherein, it is described anti-
CA215 antibody is CA215 monoclonal antibodies.
The luminous substrate A contains luminol, and the luminous substrate B contains hydrogen peroxide, and it is business procurement, and packing is
Can.
The calibration object is prepared using CA215 sterlings, in the present embodiment, 6 kinds of calibration objects of sign concentration has been prepared altogether,
Its sign concentration is respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/ml, 50AU/ml, 100AU/ml.
The concentrated cleaning solution for pH7.0~8.0 phosphate buffer, including the sodium chloride that mass fraction is 17% and
2.5% Tween-20.
Fig. 2 is referred to, is the preparation method flow chart of steps of the CA215 detection kits that the present invention is provided.
Embodiment two
The preparation method of CA215 detection kits
The preparation method of the CA215 detection kits comprises the following steps：
1) the anti-coated micro reaction plate of CA215 antibody is prepared：
Coating：After anti-CA215 antibody is using coating buffer dilution, micro reaction plate is taken, the anti-CA215 antibody that will be diluted is added
To in the micropore of the micro reaction plate, the addition of each micropore is l00 μ l, and it is small that (18~25 DEG C) of room temperature places 12~24
When, wherein the compound method of the coating buffer is as follows：Take lmol/L Na2HPO477.4ml and lmol/L NaH2PO4 22.6ml
Mix, add deionized water to be settled to l000ml, 10 times need to be again diluted when using；
Closing：Coating buffer is discarded, is patted dry on blotting paper, add the μ l/ holes of confining liquid 250, room temperature (18~25 DEG C) is put
Put 6~24 hours, wherein, the confining liquid is to contain the BSA, 1% casein and 0.05% anti-corrosion that mass fraction is 3%
The phosphate buffer of agent, the preservative is Proclin-300, and the pH of the phosphate buffer is 7.4；
Stabilization：Confining liquid is discarded, the μ l/ holes of stabilizing solution 250 are added, (18~25 DEG C) of room temperature is placed 4~24 hours, wherein
The stabilizing solution is to contain the sucrose and the phosphate buffer of 0.05% preservative that mass fraction is 4%, the preservative
It is Proclin-300, the pH of the phosphate buffer is 7.4；Closed using the confining liquid, effect is good, reduced non-
Specific binding.
Envelope：Stabilizing solution is discarded, is patted dry on blotting paper, taken out in vacuum drying chamber 16~24 hours at room temperature, entered immediately
Row vacuum sealing bag, it is labelled after 2~8 DEG C of preservations；
Need to check for gas leakage, such as gas leakage after vacuum sealing bag, need envelope labelled preservation again again.
The method for coating will be closed and stabilization is separately carried out, and increased the stability of coating plate, while being coated with, closing and steady
Fixed time range is more long than the conventional time, is brought great convenience to experimental implementation person, and coating process is under room temperature environment
Carry out, it is different from conventional 37 degree and 4 degree, reduce the use of instrument.
2) enzyme conjugates is prepared：
Anti- CA215 antibody is crosslinked with the horseradish peroxidase for marking using periodates oxidizing process, by gradient
Dilution determines the concentration of the enzyme conjugates, with comprising sodium chloride, 3% BSA, 0.05% that mass fraction is 0.85%
EDTA and 0.05% preservative the phosphate buffers of pH 7.4 preserve enzyme conjugates, wherein preservative be Proclin-
300。
3) packing chemical luminous substrate A, chemical luminous substrate B：
Chemical luminous substrate A and chemical luminous substrate B is directly purchased, is then dispensed, wherein, the chemical luminous substrate A
Containing luminol, the chemical luminous substrate B contains hydrogen peroxide；
4) calibration object is prepared：
CA215 sterlings are diluted using the phosphate buffers of pH 7.4, CA215 sterlings are diluted to various concentrations point, and it is fixed
Mark obtains calibration object, the phosphate buffer including BSA, 0.5% Creamophpr EL that mass fraction is 3% and
0.05% preservative, wherein preservative are Proclin-300, and the sign concentration of the calibration object is respectively 0AU/ml, 5AU/
Ml, 10AU/ml, 20AU/ml, 50AU/ml and 100AU/ml.
Calibration product are prepared using the phosphate buffer dilution CA215 sterlings including 0.5% Creamophpr EL, relatively
In conventional buffer solution, stability is more preferable.
5) concentrated cleaning solution is prepared：
The concentrated cleaning solution is the phosphate buffer of pH7.0~8.0, comprising sodium chloride that mass fraction is 17% and
2.5% Tween-20, its compound method is as follows：28g disodium hydrogen phosphates are weighed, 170g sodium chloride measures 25ml Tween-20s, uses water
1000ml is settled to, pH7.0~8.0 are adjusted.
6) by the anti-coated micro reaction plate of CA215 antibody, enzyme conjugates, chemical luminous substrate A, chemical luminous substrate B,
Calibration object and concentrated cleaning solution are assembled into the CA215 detection kits.
Fig. 3 is referred to, is the application method flow chart of steps of the CA215 detection kits that the present invention is provided.
Embodiment three
The application method of CA215 detection kits
The application method of CA215 detection kits, comprises the following steps：
S1, taking-up CA215 detection kits, the CA215 detection kits are balanced to room temperature, and the detection is tried
Reagent in agent box takes out standby：
Specifically, balanced 30 minutes under the CA215 detection kits being put into room temperature (18~25 DEG C)；
S2, dilution concentrated cleaning solution：It is standby after the concentrated cleaning solution is diluted into 50 times with fresh purified water；
S3, preparation luminous substrate：Chemical luminous substrate A and chemical luminous substrate B is pressed 1:1 volume ratio is prepared after mixing
Into luminous substrate, matching while using；
S4, taking-up serum sample to be measured are balanced to room temperature：
Specifically, serum sample to be measured is put into (18~25 DEG C) of room temperature to balance 30 minutes.
S5, take out the anti-coated micro reaction plate of CA215 antibody, toward each micropore of micro reaction plate in be separately added into
100 μ l calibration objects and serum sample to be measured, are well mixed, and put 37 DEG C of insulating boxs and are incubated 60 minutes；
Specifically micro reaction plate is positioned on micropore frame, be separately added into micropore concentration for 0AU/ml, 5AU/ml,
Six calibration objects of 10AU/ml, 20AU/ml, 50AU/ml and 100AU/ml and serum sample to be measured；
S6, washing：The liquid in the micropore of the micro reaction plate is discarded, institute is rinsed using the concentrated cleaning solution after dilution
Micro reaction plate is stated, is then patted dry：
Washing methods is as follows：The micropore of micro reaction plate is filled it up with the concentrated cleaning solution after dilution, 5 seconds are stood, dried, weight
Patted dry after multiple 5 times；Or washed with board-washing machine washing：The concentrated cleaning solution after 300 μ l dilutions is added per hole, is repeated 4 times, clapped after washing
It is dry；
S7, toward the enzyme conjugates that 100 μ l/ holes are separately added into the micropore of the micro reaction plate, fully mix, put
37 DEG C of insulating boxs are incubated 60 minutes；
S8, washing：The liquid in the micropore of the micro reaction plate is discarded, institute is rinsed using the concentrated cleaning solution after dilution
Micro reaction plate is stated, is then patted dry；
Washing methods is with step S6；
100 μ l luminous substrates are added in S9, past each hole, is added immediately to chemical illumination immunity analysis instrument after fully mixing
In detected；
S10, acquisition testing result：Luminous value is directly chemically read on luminescence immunoassay instrument or according to defaulting in chemistry
CA215 detections dosage-reaction normal curve in luminescence immunoassay instrument obtains the concentration value of test serum sample and derives.
Example IV
The analytical performance evaluation of CA215 detection kits
CA215 standard items are detected first with chemical illumination immunity analysis instrument, draws 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 kits
Carry out the evaluation of accuracy, specificity, precision, LDL and stability.
1) accuracy
The methodology identification of CA215 immue quantitative detection reagent boxes
Calibrating knot is carried out to the CA215 detection kits of embodiment 1 according to manufacture conventional in the art and identification code
Fruit is as follows：
Known content sample is detected with the kit prepared in the embodiment of the present invention 1, the rate of recovery (rate of recovery is calculated
=yield/addition × 100%), it is as a result as shown in the table：
| Addition AU/ml | 22 | 25 | 26 | 31 | 40 | 53 |
| Yield AU/ml | 21.92 | 23.58 | 24.95 | 32.58 | 40.36 | 51.95 |
| Rate of recovery % | 99.64 | 94.32 | 95.96 | 105.10 | 100.90 | 98.02 |
2) specificity
Known content analog is detected with the CA215 detection kits in the embodiment of the present invention 1, as a result such as following table
It is shown：
3) precision
The luminous value of detection quality-control product 1 and quality-control product 2, quality-control product 1 and quality-control product 2 do 10 parallel hole replications respectively
Its luminous value, (Coefficient of Variance, coefficient of dispersion are the hundred of standard deviation and toaverage ratio to calculate CV values
Divide ratio), it is as a result as shown in the table：
| Title | CV% |
| Quality-control product 1 | 4.17 |
| Quality-control product 2 | 5.6 |
4) LDL
Replication sign concentration is zero calibration object (or Sample dilution) 10 times, calculates the average of luminous valueWith standard deviation (SD), incite somebody to actionReacting dose substitute into CA215 detection dose-response curve, calculate phase
Concentration value, as LDL are answered, it is as a result as shown in the table：
5) stability
Each group splits 37 DEG C of test accuracy, precision, LDL and curve correlation coefficients after 7 days in kit,
Result is as follows：
Result above is collected and is drawn：
| Detection project | Test stone | Assay |
| Accuracy | The rate of recovery is 85%~115% | Meet standard |
| Specificity | This kit measurement result should be not more than 1AU/ml | Meet standard |
| Precision CV (%) | Less than 15% (n=10) | Meet standard |
| LDL | Less than 1AU/ml | Meet standard |
| Stability | Each group splits 37 DEG C at least 7 days in kit | Meet standard |
Be can see from above-mentioned summary sheet and illustrate that the CA215 detection kits property indices are qualified, and had
Accuracy is high, specific high, precision is good, sensitivity advantage high and good stability.
Embodiment five：
The determination of the content and critical value of the CA215 of normal person
The CA215 detection kits randomly selected in 120 Healthy Human Serum embodiments 1 are measured, and collect all
Measured value, its critical value (reference of 95% digit is limited) is determined with method of percentiles, final to determine that critical value is 6.3AU/ml.
Embodiment six：
60 serum of ovarian cancer patients are randomly selected, is surveyed with the CA215 quantification kits of the preparation in embodiment 1
It is fixed, collect all measured values, mean concentration is 51.016AU/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
It is fixed, collect all measured values, mean concentration is 70.143AU/ml, is significantly higher than Healthy Human Serum measured value.
CA215 detection kits that the present invention is provided and preparation method thereof and application method, have the advantages that：
First, CA215 can be used for the potential risk of diagnosing human cancer as a general biomarker for cancer, and the present invention is carried
The CA215 detection kits of confession can go out the content of patients serum CA215 with quantitative determination, can also combine with other cancer markers
The monitoring of auxiliary diagnosis and related diseases people's therapeutic effect for cancer, such as with AFP, CEA, CA125, CA19-9, CA15-3 or
Cyfra21-1 joints are detected, compared to individually detection single creature mark, it can be observed that cancer detection rate higher,
Clinically it is with a wide range of applications.
2nd, the CA215 detection kits that the present invention is provided have high specific, high sensitivity and good precision, and
And easy to operate, quick, good stability and testing result are accurate.
3rd, closing and stabilization are separately carried out in the step of anti-CA215 antibody coating micro reaction plate that the present invention is provided, and are increased
The strong stability of coating plate.
4th, the present invention provide anti-CA215 antibody coating micro reaction plate the step of in, coating, closing and stabilization when
Between scope it is wider than the conventional time, greatly facilitate to experimental implementation person, and coating process carries out under room temperature environment, it is and conventional
37 degree different with 4 degree, reduce the use of instrument.
5th, the closing formula of liquid that the present invention is provided, sealing effect more preferably, reduces non-specific binding.
6th, provided by the present invention for prepare the phosphate buffer of calibration object including BSA that mass fraction is 3%,
The preservative of 0.5% Creamophpr EL and 0.05%, makes the calibration object stability of preparation better than conventional buffer solution.
The preferred embodiments of the present invention are the foregoing is only, is not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.It is 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 kits, it is characterised in that it includes that the anti-coated micro reaction plate of CA215 antibody, enzyme are combined
Thing, chemical luminous substrate A, chemical luminous substrate B and calibration object；
Wherein,
The enzyme conjugates is the anti-CA215 antibody of enzyme mark, and the marker enzyme of the enzyme conjugates is horseradish peroxidase；
The chemical luminous substrate A contains luminol, and the chemical luminous substrate B contains hydrogen peroxide；
The calibration object is prepared using CA215 sterlings, and its sign concentration is respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/
ml、50AU/ml、100AU/ml；
The anti-coated micro reaction plate of CA215 antibody is prepared in accordance with the following methods：
Coating：After anti-CA215 antibody is using coating buffer dilution, micro reaction plate is taken, the anti-CA215 antibody for diluting is added to institute
State in the micropore of micro reaction plate, the addition of each micropore is l00 μ l, and room temperature is placed 12~24 hours；
Closing：Coating buffer is discarded, is patted dry on blotting paper, add the μ l/ holes of confining liquid 250, room temperature is placed 6~24 hours；
Stabilization：Confining liquid is discarded, the μ l/ holes of stabilizing solution 250 are added, room temperature is placed 4~24 hours；
Envelope：Stabilizing solution is discarded, is patted dry on blotting paper, vacuumize 16~24 hours, Ran Houli in vacuum drying chamber at room temperature
Vacuum sealing bag is carried out, it is labelled after 2~8 DEG C of preservations.
2. CA215 detection kits according to claim 1, it is characterised in that the micro reaction plate material is impermeable
Bright polystyrene or polyethylene.
3. CA215 detection kits according to claim 1, it is characterised in that the compound method of the coating buffer is as follows：
Take the Na of lmol/L2HPO4The Na of 77.4ml and lmol/L2HPO422.6ml is mixed, plus deionized water is settled to 1000ml, then
10 times of dilution is used.
4. CA215 detection kits according to claim 1, it is characterised in that the confining liquid is to contain mass fraction
It is the phosphate buffer of 3% BSA, 1% casein and 0.05% preservative, the preservative is Proclin-300,
The pH value of the phosphate buffer is 7.4.
5. CA215 detection kits according to claim 1, it is characterised in that the stabilizing solution is to contain mass fraction
It is 4% sucrose and the phosphate buffer of 0.05% preservative, the preservative is Proclin-300, the phosphate
The pH of buffer solution is 7.4.
6. CA215 detection kits according to claim 1, it is characterised in that the calibration object is according to following method system
It is standby：CA215 sterlings are diluted to various concentrations point with the phosphate buffer of pH 7.4, and calibration obtains various sign concentration
Calibration object, the phosphate buffer includes BSA, 0.5% Creamophpr EL and 0.05% that mass fraction is 3%
Preservative, wherein preservative are Proclin-300.
7. CA215 detection kits according to claim 1, it is characterised in that the CA215 detection kits also include
Concentrated cleaning solution, the concentrated cleaning solution is the phosphate buffer of pH7.0~8.0, and the phosphate buffer includes quality
Fraction is 17% sodium chloride and 2.5% Tween-20.
8. a kind of preparation method of CA215 detection kits, it is characterised in that the CA215 detection kits include anti-CA215
The coated micro reaction plate of antibody, enzyme conjugates, chemical luminous substrate A, chemical luminous substrate B, calibration object and thickening and washing
Liquid, its preparation method comprises the following steps：
1) the anti-coated micro reaction plate of CA215 antibody is prepared：
Coating：After anti-CA215 antibody is using coating buffer dilution, micro reaction plate is taken, the anti-CA215 antibody for diluting is added to institute
State in the micropore of micro reaction plate, the addition of each micropore is l00 μ l, and room temperature is placed 12-24 hours；
Closing：Coating buffer is discarded, is patted dry on blotting paper, add the μ l/ holes of confining liquid 250, room temperature is placed 6~24 hours；
Stabilization：Confining liquid is discarded, the μ l/ holes of stabilizing solution 250 are added, room temperature is placed 4~24 hours；
Envelope：Stabilizing solution is discarded, is patted dry on blotting paper, vacuumize 16~24 hours, Ran Houli in vacuum drying chamber at room temperature
Vacuum sealing bag is carried out, it is labelled after 2~8 DEG C of preservations；
2) enzyme conjugates is prepared：
Anti- CA215 antibody is crosslinked with the horseradish peroxidase for marking using periodates oxidizing process, by gradient dilution
The concentration of the enzyme conjugates is determined, with including sodium chloride, 3% BSA, 0.05% that mass fraction is 0.85%
The phosphate buffers of pH 7.4 of the preservative of EDTA and 0.05% preserve enzyme conjugates, and wherein preservative is Proclin-300；
3) chemical luminous substrate A and chemical luminous substrate B is dispensed；
Chemical luminous substrate A and chemical luminous substrate the B packing that will directly purchase, wherein, the chemical luminous substrate A contains Shandong
Minot, the chemical luminous substrate B contains hydrogen peroxide；
4) calibration object series is prepared：
CA215 sterlings are diluted using the phosphate buffers of pH 7.4, the calibration object of various sign concentration is obtained, the phosphate delays
Fliud flushing includes BSA, the preservative of 0.5% Creamophpr EL and 0.05% that mass fraction is 3%, and wherein preservative is
Proclin-300, the sign concentration of the calibration object series is respectively 0AU/ml, 5AU/ml, 10AU/ml, 20AU/ml, 50AU/
Ml and 100AU/ml；
5) concentrated cleaning solution is prepared：
28g disodium hydrogen phosphates and 170g sodium chloride are weighed, 25ml Tween-20s are measured, 1000ml is settled to water, adjust pH7.0
~8.0；
6) by the anti-coated micro reaction plate of CA215 antibody, enzyme conjugates, chemical luminous substrate A, chemical luminous substrate B, calibration
Product and concentrated cleaning solution are assembled into the CA215 detection kits.
9. CA215 according to claim 8 examines the preparation method of testing cassete, it is characterised in that the confining liquid be containing
Mass fraction is the phosphate buffer of 3% BSA, 1% casein and 0.05% preservative, and the preservative is
Proclin-300, the pH of the phosphate buffer is 7.4.
10. the application method of CA215 detection kits according to any one of claim 1 to 7, it is characterised in that bag
Include following steps：
CA215 detection kits are taken out, the CA215 detection kits are balanced to room temperature, and by the detection kit
Reagent take out it is standby；
Dilution concentrated cleaning solution：It is standby after the concentrated cleaning solution is diluted into 50 times with fresh purified water；
Prepare luminous substrate：Chemical luminous substrate A and chemical luminous substrate B is pressed 1:1 volume ratio is configured to light after mixing
Substrate；
Serum sample to be measured is taken out to balance to room temperature；
Take out the anti-coated micro reaction plate of CA215 antibody, toward each micropore of micro reaction plate in be separately added into 100 μ l calibration
Product and serum sample to be measured, are well mixed, and put 37 DEG C of insulating boxs and are incubated 60 minutes；
Washing：The liquid in the micropore of the micro reaction plate is discarded, the micropore is rinsed using the concentrated cleaning solution after dilution
Reaction plate, then pats dry；
100 μ l enzyme conjugates are added in toward each micropore of the micro reaction plate, is fully mixed, put 37 DEG C of insulating boxs and be incubated 60
Minute；
Washing：The liquid in the micropore of the micro reaction plate is discarded, the micropore is rinsed using the concentrated cleaning solution after dilution
Reaction plate, then pats dry；
100 μ l luminous substrates are added in each hole, detection is added immediately into chemical illumination immunity analysis instrument after fully mixing；
Luminous value is directly chemically read on luminescence immunoassay instrument or according in defaulting in chemical illumination immunity analysis instrument
CA215 detections dosage-reaction normal curve obtains the concentration value of test serum sample and derives.
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| CN201611116166.5A CN106771202A (en) | 2016-12-07 | 2016-12-07 | CA215 detection kits and preparation method thereof and application method |
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Cited By (1)
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| CN108680740A (en) * | 2018-05-21 | 2018-10-19 | 苏州佑君环境科技有限公司 | A kind of dilution horseradish peroxidase mark antigen solution and preparation method thereof |
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