CN108008132A - A kind of kit of joint-detection oophoroma tumor marker HE4 and CA125 and its preparation method and application - Google Patents
A kind of kit of joint-detection oophoroma tumor marker HE4 and CA125 and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to technical field of immunoassay, and in particular to a kind of kit of joint-detection oophoroma tumor marker HE4 and CA125 and its preparation method and application.Kit of the present invention, horseradish peroxidase is subjected to silanization treatment, horseradish peroxidase has been blocked to become the possibility of phosphate acceptors in alkaline phosphatase cataluminescence substrate luminescence process, the presence for solving the problems, such as horseradish peroxidase can disturb alkaline phosphatase catalytic luminescence, make it possible to first using luminous intensity size caused by alkaline phosphatase on Chemoluminescent substrate detection magnetic particle, again using the size of the absorbance of colour developing caused by horseradish peroxidase on assay chromogenic substrate solution detection magnetic particle, realize to being detected in sample while HE4 and CA125.Kit detection process of the present invention is simple, the reaction time is short, and reagent dosage is few, cost-effective, and high sensitivity, reagent repeatability and stability are good.
Description
Technical field
The present invention relates to technical field of immunoassay, and in particular to a kind of joint-detection oophoroma tumor marker HE4 and
Kit of CA125 and its preparation method and application.
Background technology
Oophoroma is one of malignant tumour of gynaecologic reproductive system, its incidence in gynecological tumor is only second to cervix cancer
The 3rd is occupied with carcinoma of uterine body, but its lethality occupies first in gynecological disease.Ovary is primarily due to ensconce deeply
In bone cavity, oophoroma early symptom very unobvious clinically also lack effective method of early diagnosis, and many patients are examining
Break when oophoroma has been cancer of late stage.The five year survival rate of early stage patient is more than 90 percent, and late period reaches
Less than 30 percent.Therefore, how as early as possible be diagnosed to be prognosis of the oophoroma for patient and existence is extremely critical.It is near several
Year, scholars always search for having high specific and the tumor marker of accuracy, to improve the early diagnostic rate of oophoroma,
Ensure effective prognosis of patient.
Evaluation, monitoring after early detection of the detection of blood serum tumor markers for tumour, the development of the state of an illness, treatment is multiple
Hair and transfer etc. all have certain application value.Big multi-tumor marker is not just for a kind of cancer, and very much
Cancer also there is over more than one tumor markers.Therefore, each sample only detects a kind of tumor markers and cannot
Meet the needs of clinical diagnosis, in same sample, two or more tumor markers joint-detection is current
The direction of development.
Sugar antigens 125 (CA125) are oophoroma preoperative diagnosis observation of curative effect postoperative recurrences and prognosis evaluation generally acknowledged at present
Most valuable observation index, but the synthesis of CA125 is influenced by factors, as endometriosis, pelvic inflammation etc. are equal
It can be seen that raise to some extent, studies have found that the Serum tumor marker CA125 detected value of the I-II phase ovarian cancer patients of 40%-50% is not
Rise, so still having certain limitation using CA125 as the unique index of oophoroma early diagnosis.
The cDNA of people's epididymal proteins 4 (HE4, also known as WFDC2) gene is earliest by Kirchhoff etc. first from the epithelium of epididymis
Distal end is separated, and is a kind of protease inhibitors that may be related with spermioteleosis degree.Have many experiments at present and show HE4
There is very high application value in oophoroma early diagnosis.Drapkin etc. describes HE4 not with immunohistochemical method
With the expression in organization type oophoroma, find HE4 93% ovarian serous carcinoma, 50% clear cell carcinoma of ovary and
High expression in 100% utero-ovarian inner membrance sample cancer, and do not expressed in ovary mucus cancer and normal ovarian tissue.Moore etc.
Research shows, for unique identification thing, the sensitiveness that HE4 is used for diagnosis of ovarian cancer is up to 72.9%, specificity up to 95%, and
The sensitiveness of CA125 is 27%-66%, and specificity is 75.2%.For joint-detection thing, the detection of CA125 joints HE4 is quick
Perception is up to 76.4%, specific 95%, hence it is evident that higher than the susceptibility of individual event detection HE4 or CA125.Huhtinen etc. researches show that
Joint-detection HE4 and CA125 serum-concentration can increase the accuracy rate of diagnosis of oophoroma, and can be from ovarian endometrial cyst
Middle identification of ovarian cancer.Kim etc. can be successfully by can researches show that joint-detection adnexal masses patients serum HE4 and CA125 level
It can develop into the excessive risk group of oophoroma and low-risk group patient distinguishes that (sensitiveness 87.5%, specificity is 93.8%).Therefore,
Most oophoroma HE4 and CA125 levels can raise simultaneously, and the joint-detection of the two is conducive to improve the recall rate of oophoroma, has
Oophoroma caused by effect improves single tumor markers feminine gender is failed to pinpoint a disease in diagnosis, and is effectively reduced false negative result, is considerably increased ovary
The accuracy of cancer diagnosis.
In immunoassay, the separation of Ag-Ab immune complex is often comparatively laborious, time-consuming, and often signal-to-noise ratio is low
The reason for, magnetic particle is introduced as immune response and separated solid phase carrier, is one of current strategy.Magnetic particle
It is mainly used for being connected with antibody, can capture target substance by immune response forms immune complex, in externally-applied magnetic field
Under effect, immune complex is detained to get off, and realizes separation, so as to remove the interference of sample mesostroma, reduces background
Value.Moreover, compared with the immunoassay of microwell plate, the intervention of magnetic particle not only makes the dosage of reaction reagent reduce about
1/2, and shorten the reaction time, it is often more important that background value is reduced, improves sensitivity.
So far, one kind is lacked in the clinical detection of oophoroma once to test together using magnetic particle immunoassay
When occur two kinds of tumor markers concentration detection process is simple, the reaction time is short, reagent dosage is few, have high sensitivity,
The kit of the detection HE4 and CA125 for many good characteristics such as reagent is reproducible and stability is good.
The content of the invention
The defects of in order to overcome the prior art, the object of the present invention is to provide a kind of joint-detection oophoroma tumor marker
The kit of HE4 and CA125, can examine the HE4 in human serum and CA125 while specificity, sensitivity, accuracy
Survey.
The second object of the present invention is to provide a kind of reagent of joint-detection oophoroma tumor marker HE4 and CA125
The preparation method of box.
The third object of the present invention is to provide a kind of reagent of joint-detection oophoroma tumor marker HE4 and CA125
The application of box.
In order to realize the above object the present invention adopts the following technical scheme that：
A kind of kit of joint-detection oophoroma tumor marker HE4 and CA125, including HE4 antibody and CA125 antibody
The magnetic particle of combined mark, enzyme marker, Chemoluminescent substrate, assay chromogenic substrate solution；Wherein enzyme marker is by alkaline phosphatase
The HE4 antibody of mark and the CA125 antibody composition of silanization horseradish peroxidase-labeled.
Optionally, the Chemoluminescent substrate is that the Tris that chemical luminous substrate solution A is 0.2mol/L pH 9.7 delays
Fliud flushing solution, further includes sodium chloride (NaCl), the CTAC (hexadecyltrimethylammonium chloride) of 0.08mmol/L of 2.7mol/L
With AMPPD (3- (the spiral adamantane of 2-) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes of 2.6mmol/L
Disodium salt), do not contain Sodium azide；
The sodium acetate and the lemon acid buffering of 0.03mol/L that assay chromogenic substrate solution is the 0.33mol/L of pH 6.0 ± 0.5 are molten
Liquid, further includes 10% glycerine, 0.06% 30% hydrogen peroxide (H2O2) and with dimethyl sulfoxide (DMSO) (DMSO) dissolve 0.5g/
The tetramethyl benzidine (TMB) of mL, does not contain Sodium azide.
Optionally, mentioned reagent box further includes calibration object and lavation buffer solution.
Optionally, the cleaning solution is the Tris containing polysorbas20 and sodium chloride, the pH value of concentrated cleaning solution for 7.5~
8.5, the content of wherein sodium chloride is 20~100g/L, the content of polysorbas20 is 0.05%~5%.
Optionally, the calibration object is the standard solution containing HE4 and CA125.
Optionally, silanization horseradish peroxidase is prepared by following methods, including horseradish peroxidase is added
Enter and mixture solution is obtained in the n,N-Dimethylformamide solution of tert-butyl chloro-silicane, silanization horseradish is reacted to obtain in stirring
Peroxidase.
Optionally, the concentration of the n,N-Dimethylformamide solution of the tert-butyl chloro-silicane is 3.02mg/ml；
The N,N-dimethylformamide solution of 128 μ L tert-butyl chloro-silicanes is added in horseradish peroxidase per 1mg.
Optionally, the reaction temperature for stirring reaction is 2~8 DEG C, when the reaction time is 1 small.
Optionally, further included in silanization horseradish peroxidase preparation process and carry out desalination and concentration, tool to enzyme first
Body method is：Horseradish peroxidase is taken, addition removes salt buffer, desalination is carried out using molecular sieve chromatography, then to desalination
The horseradish peroxidase collected afterwards carries out centrifugal concentrating to 3~5mg/ml.
Wherein except the preparation method of salt buffer is：Imidazoles 10.2g accurately is weighed, sodium chloride (NaCl) 5.84g, is dissolved in
In 1000g purified waters, pH=8.2~8.5 are adjusted.
Optionally, further include and silanization horseradish peroxidase is purified and concentrated, specific method is：Using molecule
Sieve chromatography isolates and purifies the product after mixture solution stirring reaction, removes unreacted residue, will after purification
Silanization horseradish peroxidase carry out centrifugal concentrating to 3~5mg/ml.
The preparation method of the kit of above-mentioned joint-detection oophoroma tumor marker HE4 and CA125, including following operation
Step：
1) magnetic particle of HE4 antibody and the antibody combined marks of CA125 is prepared：Including containing HE4 antibody and CA125 antibody
The carboxyl magnetic bead after activation is added in solution, after mixing coupling reaction, the magnetic bead after reaction is closed, up to described
The magnetic particle of HE4 antibody and the antibody combined marks of CA125；
2) enzyme marker is prepared：A：Prepare the HE4 antibody of alkali phosphatase enzyme mark：By activate desalination after HE4 antibody with
The alkaline phosphatase mixing coupling reaction after desalination is activated, up to the HE4 antibody of alkali phosphatase enzyme mark；
B：Prepare the CA125 antibody of silanization horseradish peroxidase-labeled：Horseradish peroxidase after desalination is added
Enter to react in tert-butyl chloro-silicane solution and silanization horseradish peroxidase is made, by silanization horseradish peroxidase
Coupling reaction is mixed with activating the CA125 antibody of desalination after activation desalination, up to silanization horseradish peroxidase-labeled
CA125 antibody；
C：By the HE4 antibody of the step A alkali phosphatase enzyme marks prepared and silanization horseradish peroxidase-labeled
CA125 antibody mixes, up to enzyme marker；
3) Chemoluminescent substrate and assay chromogenic substrate solution are prepared；
4) the HE4 antibody of step 1) preparation and the magnetic particle of the antibody combined marks of CA125, the enzyme mark of step 2) preparation are taken
Chemoluminescent substrate and assay chromogenic substrate solution prepared by thing, step 3), is assembled into kit, that is, completes.
The specific preparation method of the magnetic particle of HE4 antibody and the antibody combined marks of CA125 is in step 1)：
1. the cleaning of magnetic bead：Carboxyl magnetic bead is precipitated after cleaning carboxyl magnetic bead with NaOH solution and pure water and removes supernatant；
2. the activation of magnetic bead：Take EDC and Sulfo-NHS to be dissolved separately in MES buffer solutions respectively, prepare Sulfo-
NHS solution and EDC solution, Sulfo-NHS solution and EDC solution are added to the appearance for filling the carboxyl magnetic bead 1. step is cleaned after
In device, mixed 30 minutes under the conditions of 2-8 DEG C；
3. the cleaning of magnetic bead：Carboxyl magnetic bead after cleaning activation with MES buffer solutions；
4. the desalination of antibody：HE4 antibody-solutions and CA125 antibody-solutions are measured respectively, use GEPD10 desalting columns and MES
After buffer solution carries out desalination to it, diluted or concentrated as the volume (unit of carboxyl magnetic bead mass number after 0.04 times of activation：
Milliliter)；
5. the connection of magnetic bead：4. the antibody-solutions in are added into filling 3. in the container of activated carboxyl magnetic bead,
When room temperature mixing coupling reaction 2 is small；
6. the cleaning and closing of magnetic bead：Supernatant is removed after carboxyl magnetic bead precipitation 5. step is coupled after, is added with step 4.
In the isometric Tris-HCl solution of antibody-solutions, room temperature mixes 30 minutes and carboxyl magnetic bead residue activated sites point sealed
Close；
7. the preservation of magnetic bead：Supernatant is removed after carboxyl magnetic bead precipitation 6. step is closed after, is washed using PBST solution
Wash, carboxyl magnetic bead precipitated after the completion of cleaning and removes supernatant, add with step 4. in the isometric magnetic bead of antibody-solutions preserve and buffer
Liquid is preserved.
The specific preparation method of HE4 antibody of step 2) alkaline phosphatase mark is：
1. the desalination and concentration of antibody:Take anti-HE4 monoclonal antibodies using molecular sieve chromatography to antibody carry out desalination and
Concentration, final concentration of 3~5mg/ml；
2. activation and the desalination of antibody：2- iminothiolanes (2-Iminothiolane hydrochloride) are taken to dissolve
In 2-IT dilutions, final concentration of 13.76mg/ml；It is sub- dissolved with 2- that 10 μ l are added in antibody after 1. being handled per 1mg steps
The 2-IT dilutions of amino sulfane, after mixing stirring reaction at room temperature, carry out it using molecular sieve chromatography to remove salt treatment, receive
It is spare after collection；
3. activation and the desalination of enzyme：Take 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimides
Ester sodium salt (Sulfo-SMCC) is dissolved in purified water, final concentration of 4.36mg/ml；Add according in the alkaline phosphatase of every 1mg
Enter the adding proportion of 50 μ l Sulfo-SMCC solution, Sulfo-SMCC solution is added in alkaline phosphatase, mix stir at room temperature
After mixing reaction, it is carried out using molecular sieve chromatography to remove salt treatment, it is spare after collection；
4. the coupling and purifying of antibody and enzyme：2. the HE4 antibody after the activation that will be obtained in and 3. and the alkalescence after activation
Phosphatase, according to mass ratio 1:1 is mixed, be placed at 2~8 DEG C reaction 4 it is small when；Production is coupled to it using molecular sieve chromatography
Thing is isolated and purified, and obtains the HE4 antibody of alkali phosphatase enzyme mark.
The specific preparation method of the CA125 antibody of silanization horseradish peroxidase-labeled in step 2)：
1. the desalination and concentration of antibody：Anti-CA 125 monoclonal antibody is taken to carry out desalination to antibody using molecular sieve chromatography
And concentration, final concentration of 3-5mg/ml.
2. activation and the desalination of antibody：2- iminothiolanes (2-Iminothiolane hydrochloride) are taken to dissolve
In 2-IT dilutions, final concentration of 13.76mg/ml；It is sub- dissolved with 2- that 10 μ l are added in antibody after 1. being handled per 1mg steps
The 2-IT dilutions of amino sulfane, after mixing stirring reaction at room temperature, carry out it using molecular sieve chromatography to remove salt treatment, receive
It is spare after collection；
3. activation and the desalination of enzyme：Take 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimides
Ester sodium salt oxygen (Sulfo-SMCC) is dissolved in purified water, final concentration of 4.36mg/ml；According to the silanization horseradish mistake of every 1mg
50 μ l Sulfo-SMCC solution are added in oxidizing ferment, after mixing stirring reaction at room temperature, it is carried out using molecular sieve chromatography
It is spare after collection except salt treatment；
4. the coupling and purifying of antibody and enzyme：2. the CA125 antibody after the activation that will be obtained in and 3. and the silicon after activation
Alkanisation horseradish peroxidase, according to mass ratio 1:1 is mixed, be placed at 2-8 DEG C reaction 4 it is small when；Utilize sieve chromatography
Method isolates and purifies its coupled product, obtains the CA125 antibody of silanization horseradish peroxidase-labeled.
The preparation method of above-mentioned 2-IT dilutions is：Triethanolamine 14.92g, sodium hydroxide 4.0g accurately are weighed, is dissolved in
In 1000g purified waters, pH=8.5~9.0 are adjusted.
Preferably, by the HE4 antibody of the alkali phosphatase enzyme mark of preparation and silanization horseradish peroxidase mark in step C
The CA125 antibody equal proportions of note are uniformly mixed, be placed in it is spare at 2~8 DEG C, be enzyme marker.
The kit of above-mentioned joint-detection oophoroma tumor marker HE4 and CA125 detect at the same time in human serum HE4 and
Application in terms of CA125 concentration.
Specific detection method in detection human serum in terms of HE4 and CA125 concentration includes following operating procedure：
Ⅰ：Sample to be detected is taken in reaction cup, adds the magnetic particle of HE4 antibody and the antibody combined marks of CA125, then add
Enter enzyme marker, fully mix, and 16min is incubated at 37 DEG C；Cleaning separation magnetic particle；Chemiluminescence bottom is added into magnetic particle
Thing solution A, reacts and measures luminous intensity；100 μ L chromogenic substrate B solutions are added into magnetic particle again, react 5min and in 620mn
Lower measure absorbance；
Ⅱ：Take the calibration object of concentration known to measure luminous intensity and absorbance according to the method for step I, draw luminous intensity with
The standard working curve of HE4 concentration, obtains the correspondence formula of luminous intensity and HE4 concentration；Draw the mark of absorbance and CA125 concentration
Quasi- working curve, obtains the correspondence formula of absorbance and CA125 concentration；Then test serum sample is measured according to the method for step I
This luminous intensity and absorbance, the luminous intensity of serum sample and absorbance are brought into corresponding relational expression respectively, calculate serum
The concentration of HE4 and CA125 in sample.
Kit of the present invention, is captured in sample at the same time using the magnetic particle of HE4 antibody and the antibody combined marks of CA125
HE4 and CA125, then again using the silanization horseradish peroxide of alkaline phosphatase and CA125 the antibody mark marked by HE4 antibody
The enzyme marker of compound enzyme composition is combined with capturing the magnetic particle of HE4 and CA125 in sample, since the present invention is by horseradish peroxidating
Thing enzyme carries out silanization treatment, has blocked horseradish peroxidase to become alkaline phosphatase cataluminescence substrate luminescence process
The possibility of middle phosphate acceptors, the presence for solving the problems, such as horseradish peroxidase can disturb alkaline phosphatase catalytic luminescence, make
Obtaining can be first using luminous intensity size caused by alkaline phosphatase on Chemoluminescent substrate detection magnetic particle, then using aobvious
The size of the absorbance of colour developing caused by horseradish peroxidase on color substrate solution detection magnetic particle, realize to HE4 in sample and
Detected while CA125.
Using kit of the present invention, the content of HE4 and CA125 in human serum very effective can be detected, it is of the invention
Using the advantage of Magneto separate, chemiluminescence and color developing detection method, making that detection process is simple, the reaction time is short, reagent dosage is few,
It is cost-effective, while the detection kit also has high sensitivity, reagent reproducible and many excellent spies such as stability is good
Property, a kind of very effective detection means is provided for clinical diagnosis and research work, whether is provided for doctor's evaluation for oophoroma
One good booster action.
Brief description of the drawings
Fig. 1 is the related of 4 content results of people's epididymal proteins in 1 kit of embodiment and commercial reagent box measure human serum
Property；
Fig. 2 is the correlation that 1 kit of embodiment measures 125 content results of sugar antigens in human serum with commercial reagent box；
Fig. 3 is the standard curve linear fit information using comparative example kit joint test HE4；
Fig. 4 uses the standard curve linear fit information of 1 kit joint test HE4 of embodiment.
Embodiment
Technical scheme is described in detail below by specific embodiment.
Embodiment 1
The present embodiment provides a kind of kit of joint-detection oophoroma tumor marker HE4 and CA125, including calibration
Product, the magnetic particle of HE4 antibody and the antibody combined marks of CA125, enzyme marker, Chemoluminescent substrate, assay chromogenic substrate solution, washing
Liquid；Wherein enzyme marker is by the HE4 antibody of alkali phosphatase enzyme mark and the CA125 antibody of silanization horseradish peroxidase-labeled
Composition；
Chemoluminescent substrate is the Tris buffer solns that chemical luminous substrate solution A is 0.2mol/L pH 9.7, also
The CTAC (hexadecyltrimethylammonium chloride) and 2.6mmol/L of sodium chloride (NaCl), 0.08mmol/L including 2.7mol/L
AMPPD (3- (the spiral adamantane of 2-) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxane disodium salt), no
Contain Sodium azide；
The sodium acetate and the lemon acid buffering of 0.03mol/L that assay chromogenic substrate solution is the 0.33mol/L of pH 6.0 ± 0.5 are molten
Liquid, further includes 10% glycerine, 0.06% 30% hydrogen peroxide (H2O2) and with dimethyl sulfoxide (DMSO) (DMSO) dissolve 0.5g/
The tetramethyl benzidine (TMB) of mL, does not contain Sodium azide；
Calibration object is the standard solution containing HE4 and CA125；Cleaning solution is the Tris containing polysorbas20 and sodium chloride, dense
The pH value of contracting cleaning solution is 7.5-8.5, and the content of wherein sodium chloride is 20-100g/L, the content of polysorbas20 is 0.05%-5%.
The present embodiment kit is prepared by following methods：
1st, the preparation of calibration object
1. the composition of calibration object buffer solution：By the sodium chloride of PB, 0.15mol/L of 0.1mol/L, 1% bovine serum albumin
The polysorbas20 composition of bletilla 0.05%, pH value 7.4,2-8 DEG C of storage are spare；
2. with 1. middle gained calibration object buffer solution, a certain amount of 4 albumen of recombined human epididymal proteins (is purchased from the U.S.
Meridian companies) and sugar antigens 125 (being purchased from Meridian companies of the U.S.) in same calibration object buffer solution, prepare respectively
Into final concentration of 0pmol/L and 0IU/mL, 30pmol/L and 10IU/mL, 100pmol/L and 40IU/mL, 250pmol/L and
The calibration object of 100IU/mL, 750pmol/L and 300IU/mL, 1500pmol/L and 600IU/mL；
2nd, the preparation of the magnetic particle of HE4 antibody and the antibody combined marks of CA125
1) cleaning of magnetic bead：Taking a certain amount of carboxyl magnetic bead solution, container is placed in magnet stand in the container of certain volume, sinks
Supernatant is removed behind shallow lake, respectively using the NaOH solution of 0.01M and the pure water of precooling, carboxyl magnetic bead is precipitated after the completion of cleaning and is gone
Clearly, carboxyl magnetic bead solid is only stayed；
2) activation of magnetic bead：Accurately measure 0.1 times of magnetic bead quality (unit：Milligram) vulcanization nitrogen HOSu NHS
(Sulfo-NHS), the MES buffer solutions of the 25mM pH5.0 of certain volume precooling are added, it is 5mg/ml to make its concentration；Correct amount
The carbodiimide hydrochloride (EDC) of 0.1 times of magnetic bead quality is taken, adds the MES bufferings of the 25mM pH5.0 of certain volume precooling
Solution, it is 5mg/ml to make its concentration；The EDC solution of the Sulfo-NHS solution of preparation and preparation is added to filling carboxyl in 1)
In the container of magnetic bead solid, this carboxyl magnetic bead suspension is mixed 30 minutes under the conditions of 2~8 DEG C, is completed to carboxyl magnetic bead
Activation；
3) cleaning of magnetic bead：The activation completion of carboxyl magnetic bead is placed on magnet stand, removes supernatant after carboxyl magnetic bead precipitation, uses precooling
The MES buffer solutions of 25mM pH5.0 washed, carboxyl magnetic bead is precipitated after the completion of cleaning and removes supernatant, only stays the carboxyl of activation
Magnetic bead solid；
4) desalination of antibody：4 antibody-solutions of people's epididymal proteins and sugar antigens 125 of accurate 0.01 times of magnetic bead quality of measurement are anti-
Liquid solution, carries out desalting processing, after the completion of desalination using the MES buffer solutions of GE PD10 desalting columns and 25mM pH5.0 to it
4 antibody-solutions of people's epididymal proteins and the dilution of 125 antibody-solutions of sugar antigens or concentration are (single for the volume of 0.04 times of magnetic bead mass number
Position：Milliliter)；
5) connection of magnetic bead：4) antibody-solutions in are added to the container that activated carboxyl magnetic bead solid is filled in 3)
In, when room temperature mixing 2 is small, complete the coupling of carboxyl magnetic bead and 125 antibody of 4 antibody of people's epididymal proteins and sugar antigens；
6) cleaning and closing of magnetic bead：After the completion of carboxyl magnetic bead and 125 antibody coupling of 4 antibody of people's epididymal proteins and sugar antigens
Magnet stand is placed in, the Tris-HCl solution of supernatant, addition and 4) isometric 100mM pH7.4, room temperature are gone after carboxyl magnetic bead precipitation
Mix 30 minutes and carboxyl magnetic bead residue activated sites point is closed；
7) preservation of magnetic bead：The closing completion of carboxyl magnetic bead is placed on magnet stand, removes supernatant after carboxyl magnetic bead precipitation, uses
0.1%PBST (0.14M NaCl, 3mM KCl, 10mM Na2HPO4、2mM KH2PO4, 0.8mM Tween-20s, pH=7.2 ±
0.05) washed, carboxyl magnetic bead is precipitated after the completion of cleaning and removes supernatant, added and 4) and 6) isometric magnetic bead preserves buffer solution
(2.6%H2O·NaH2PO4, 14.4%2H2O·Na2HPO4, 0.5%Proclin300,1.4%NaCl, 5%BSA, pH=7.4
± 0.05) preserved.
3rd, the preparation of enzyme marker
1) the HE4 antibody of alkali phosphatase enzyme mark
1. the desalination and concentration of antibody:Anti- HE4 monoclonal antibodies 0.5mg is accurately weighed, is resisted using molecular sieve chromatography
Body carries out desalination and concentration, final concentration of 3~5mg/ml；
2. activation and the desalination of antibody：Accurately weigh 2- iminothiolanes (2-Iminothiolane
Hydrochloride) 2-6mg, being dissolved in 2-IT dilutions, (14.92g triethanolamines, 4.0g sodium hydroxides, are dissolved in 1000g
In purified water, pH=8.5~9.0 are adjusted) in, final concentration of 13.76mg/ml；Added in antibody after 1. being handled per 1mg steps
10 μ L are dissolved with the 2-IT dilutions of 2- iminothiolanes, and stirring reaction 20 minutes is mixed under room temperature (17-23 DEG C), completes HE4
Antibody surface dissociates the activation of primary amino radical, produces free sulfhydryl groups；The HE4 antibody after activation is removed using molecular sieve chromatography
Salt treatment, it is spare after collection；
3. activation and the desalination of enzyme：Accurately weigh 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group ambers
Imide ester sodium salt (Sulfo-SMCC) 2~4mg, is dissolved in purified water, final concentration of 4.36mg/ml；Accurately weigh alkalescence
Phosphatase ALP 0.75mg, the ratio of 50 μ L Sulfo-SMCC solution is added in the alkaline phosphatase according to every 1mg, will be above-mentioned
Sulfo-SMCC solution is added in alkaline phosphatase, and stirring reaction 15 minutes is mixed under room temperature (17~23 DEG C), completes the work of enzyme
Change；The ALP after activation is carried out using molecular sieve chromatography to remove salt treatment, it is spare after collection；
4. the coupling and purifying of antibody and enzyme：2. the HE4 antibody after the activation that will be obtained in and 3. and the ALP after activation by
According to mass ratio 1:1 is mixed, be placed at 2~8 DEG C reaction 4 it is small when；Its coupled product is divided using molecular sieve chromatography
From purifying, the HE4 antibody of alkali phosphatase enzyme mark is obtained.
2) the CA125 antibody of silanization horseradish peroxidase-labeled
1st, the preparation of silanization horseradish peroxidase：
1. desalination and the concentration of enzyme：Horseradish peroxidase (HRP) 0.75mg accurately is weighed, with molecular sieve chromatography to it
Desalination is carried out, determines the collection concentration of HRP at 280nm with ultraviolet-uisible spectrophotometer；Set with centrifugal ultrafiltration pipe and centrifugation
It is standby that concentration is carried out to HRP, it is concentrated into 3~5mg/ml；Use except salt buffer preparation method for：Accurately weigh imidazoles
10.2g, sodium chloride (NaCl) 5.84g, is dissolved in 1000g purified waters, adjusts pH=8.2~8.5；
2. the silanization of enzyme：Tert-butyl chloro-silicane (TBDMSCL) 2-5mg accurately is weighed, with N, N- dimethyl methyls
Acid amides (DMF) is dissolved to 3.02mg/ml；DMF according to the 128 μ L TBDMSCL added in every 1mg horseradish peroxidases is molten
The ratio of liquid, adds the DMF solution of TBDMSCL in the 1. horseradish peroxidase of middle concentration, is placed at 2-8 DEG C and mixes stirring
React 1 it is small when, complete the silanization of HRP；
3. the purifying and concentration of silanization enzyme：The HRP of 2. middle silanization is isolated and purified with molecular sieve chromatography, is removed
Dereaction residue；Silanization HRP ultraviolet-uisible spectrophotometers after purification are determined to collection concentration at 280nm, are used
Centrifugal ultrafiltration pipe and centrifugation apparatus carry out concentration to it, are concentrated into 3~5mg/ml, are placed in preserving at 2~8 DEG C；
2nd, the CA125 antibody of silanization horseradish peroxidase-labeled：
1. the desalination and concentration of antibody:Anti-CA 125 monoclonal antibody 0.5mg accurately is weighed, utilizes molecular sieve chromatography pair
Antibody carries out desalination and concentration, final concentration of 3~5mg/ml；
2. activation and the desalination of antibody：Accurately weigh 2- iminothiolanes (2-Iminothiolane
Hydrochloride) 2~6mg, being dissolved in 2-IT dilutions, (14.92g triethanolamines, 4.0g sodium hydroxides, are dissolved in 1000g
In purified water, pH=8.5~9.0 are adjusted) in, final concentration of 13.76mg/ml；It is dissolved with according to 10 μ L are added in every 1mg antibody
The ratio of the 2-IT dilutions of 2- iminothiolanes, is added to what is 1. prepared by the 2- iminothiolanes for being dissolved in 2-IT dilutions
Stirring reaction 20 minutes is mixed in antibody, under room temperature (17-23 DEG C), CA125 antibody surfaces is completed and dissociates the activation of primary amino radical, production
Raw free sulfhydryl groups；The CA125 antibody after activation is carried out using molecular sieve chromatography to remove salt treatment, it is spare after collection.
3. activation and the desalination of enzyme：Accurately weigh 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group ambers
Imide ester sodium salt (Sulfo-SMCC) 2~4mg, is dissolved in purified water, final concentration of 4.36mg/ml；Accurately weigh silane
The HRP 0.75mg of change, the ratio of 50 μ L Sulfo-SMCC solution is added according to every 1mg silanizations HRP, and Sulfo-SMCC is molten
Liquid is added in silanization HRP, is mixed under room temperature (17-23 DEG C) after stirring reaction 15 minutes, the surface for completing silanization HRP is dissociated
The activation of primary amino radical；The silanization HRP after activation is carried out using molecular sieve chromatography to remove salt treatment, it is spare after collection；
4. the coupling and purifying of antibody and enzyme：2. the CA125 antibody after the activation that will be obtained in and 3. and the silicon after activation
Alkanisation HRP is according to mass ratio 1:1 is mixed, be placed at 2~8 DEG C reaction 4 it is small when；Production is coupled to it using molecular sieve chromatography
Thing is isolated and purified, and obtains the CA125 antibody of silanization horseradish peroxidase-labeled.
3) preparation of enzyme marker：By 1) -4. in obtained alkali phosphatase enzyme mark HE4 antibody and 2) -2- 4. in
The CA125 antibody equal proportions of obtained silanization horseradish peroxidase-labeled are uniformly mixed, be placed in it is spare at 2-8 DEG C, be
Enzyme marker.
4th, Chemoluminescent substrate is prepared
It is accurate to measure Tris 24g, CTAC (hexadecyltrimethylammonium chloride) 25.5mg, AMPPD (3- (spiral Buddha's warrior attendants of 2-
Alkane) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxane disodium salt) 1g, sodium chloride (NaCl) 160g, purifying
Water is dissolved to 1000ml, and it is 9.7 ± 0.05 to mix 30min adjustment pH value, that is, obtains this kit Chemoluminescent substrate；
5th, assay chromogenic substrate solution is prepared
It is accurate to measure tetramethyl benzidine (TMB) 0.15g, it is dissolved in 3mL dimethyl sulfoxide (DMSO)s (DMSO)；By the solution with
The sodium acetate of 27.2g, the citric acid of 3.2g, the 30%H of 0.6mL2O21000ml is dissolved to purified water with the glycerine of 100mL, is mixed
Even 30min adjustment pH value is 6.0 ± 0.5, that is, obtains this kit assay chromogenic substrate solution；
6th, cleaning solution is prepared
Accurate to measure Tris 8.4g, NaCl63g, Tween-20 7g, purified water is settled to 1000ml, mixes 30min tune pH
For 8.0 ± 0.5,0.22 μm of cleaning solution filtered up to 7 times of concentrations.
7th, by above-mentioned calibration object, the magnetic particle of HE4 antibody and the antibody combined marks of CA125, enzyme marker, chemiluminescence bottom
Thing liquid, assay chromogenic substrate solution and cleaning solution are dispensed and are sealed, up to the kit.
Embodiment 2
The kit that the present embodiment provides embodiment 1 is applied to while detects the HE4 in sample and CA125, specific inspection
Survey method is：
Ⅰ：Sample to be detected is taken in reaction cup, adds the magnetic particle of HE4 antibody and the antibody combined marks of CA125, then add
Enter enzyme marker, fully mix, and 16min is incubated at 37 DEG C；Cleaning separation magnetic particle；Chemiluminescence bottom is added into magnetic particle
Thing solution A, reacts and measures luminous intensity；100 μ L chromogenic substrate B solutions are added into magnetic particle again, react 5min and in 620mn
Lower measure absorbance；
Ⅱ：Take the calibration object of concentration known to measure luminous intensity and absorbance according to the method for step I, draw luminous intensity with
The standard working curve of HE4 concentration, obtains the correspondence formula of luminous intensity and HE4 concentration；Draw the mark of absorbance and CA125 concentration
Quasi- working curve, obtains the correspondence formula of absorbance and CA125 concentration；Then test serum sample is measured according to the method for step I
This luminous intensity and absorbance, the luminous intensity of serum sample and absorbance are brought into corresponding relational expression respectively, calculate serum
The concentration of HE4 and CA125 in sample.
Comparative example
The difference of this comparative example kit and 1 kit of embodiment is not carry out silane to horseradish peroxidase
Change, enzyme marker is made of the HE4 antibody of alkali phosphatase enzyme mark and the CA125 antibody of horseradish peroxidase-labeled.
The performance evaluation for the kit that 1 embodiment of the present invention 1 of test example provides
1st, the detection method that the kit provided the embodiment of the present invention 1 is provided according to embodiment 2 is in human serum sample
Oophoroma tumor marker HE4 and CA125 carry out joint-detection, to its linear, accuracy, sensitivity, precision and stabilization
Property is evaluated, as a result shown in following Tables 1 and 2：
1 HE4 inspection results of table
2 CA125 inspection results of table
From the testing result of Tables 1 and 2, joint-detection oophoroma tumor marker HE4 prepared by the present invention and
The kit of CA125, its indices meet relevant standard, the kit good properties of preparation.
2 kit of the present invention of test example and commercial reagent box measured value correlation test
1st, for trying kit
Kit, HE4 projects kit prepared by embodiment 1 are ARCHITECT HE4 Reagent Kit, CA125
Project kit is CanAg CA125 EIA
2nd, test method and result
By using the kit prepared by the embodiment of the present invention 1 with being tried purchased from the chemiluminescence particulate immune detection of Abbott Laboratories
The content of people's epididymal proteins 4 in agent box ARCHITECT HE4 Reagent Kit measure serum samples, by its respective people's epididymis
4 measured value of albumen is compared, and result of the test is shown in Fig. 1；With the kit prepared by the embodiment of the present invention 1 with being purchased from health
The content of sugar antigens 125, respective by its in the enzyme-linked immunologic detecting kit CanAg CA125 EIA measure serum samples of lattice
125 measured value of sugar antigens is compared, and result of the test carries out regression analysis for shown in Fig. 2.It can be seen that from Fig. 1 and Fig. 2
Kit of the present invention and commercial reagent box all have good related in serum on 125 measured value of people's epididymal proteins 4 and sugar antigens
Property, show that kit of the present invention can be realized special, sensitive, exactly and the HE4 in human serum and the content of CA125 are joined
Detection is closed, using the advantage of Magneto separate, chemiluminescence and color developing detection method, makes that detection process is simple, the reaction time is short, reagent
Dosage is few, cost-effective.
Influence of the different enzyme marker of test example 3 to kit performance
1st, for trying kit
The kit prepared by kit, comparative example prepared by embodiment 1
2nd, test method and result
Kit prepared by the embodiment of the present invention 1 is surveyed respectively with comparative example kit according to 2 the method for embodiment
Determine HE4 standard curves, be fitted using software, respective calibration curve information is compared, result of the test Fig. 3, shown in 4；
It can be seen that comparative example kit, which can make HE4 item detections signal value decline and influence its collimation, causes song from Fig. 3 and Fig. 4
Line, which can not be fitted, to be passed through, and 1 kit of the embodiment of the present invention to HE4 projects without significantly interfering with, its curve matching by and signal
Value is relatively stablized.
It is of the invention by marking CA125 antibody again after carrying out silylating reagent to horseradish peroxidase from the result,
Horseradish peroxidase is blocked to become the possibility of phosphate acceptors in alkaline phosphatase cataluminescence substrate solution luminescence process, solution
Determined horseradish peroxidase presence interference alkaline phosphatase shine the problem of, realize the connection to HE4 in human serum and CA125
Close detection.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
The present invention is described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that：It still may be used
To modify to the technical solution described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic；
And these modification or replace, do not make appropriate technical solution essence depart from various embodiments of the present invention technical solution spirit and
Scope.
Claims (10)
- A kind of 1. kit of joint-detection oophoroma tumor marker HE4 and CA125, it is characterised in that including HE4 antibody and The magnetic particle of the antibody combined marks of CA125, enzyme marker, Chemoluminescent substrate, assay chromogenic substrate solution；Wherein enzyme marker is by alkali The HE4 antibody of acid phosphatase mark and the CA125 antibody composition of silanization horseradish peroxidase-labeled.
- 2. the kit of joint-detection oophoroma tumor marker HE4 and CA125 as claimed in claim 1, it is characterised in that The Chemoluminescent substrate be 0.2mol/L pH 9.7 Tris buffer solns, further include 2.7mol/L sodium chloride, The hexadecyltrimethylammonium chloride of 0.08mmol/L and 3- (the spiral adamantane of the 2-) -4- methoxyl group -4- (3- of 2.6mmol/L Phosphorus oxygen acyl)-phenyl -1,2- dioxane disodium salt；Assay chromogenic substrate solution is the sodium acetate of 0.33mol/L and the citric acid solution of 0.03mol/L of pH 6.0 ± 0.5, also Including 10% glycerine, the tetramethyl benzidine of 0.06% 30% hydrogen peroxide and the 0.5g/mL with dmso solution.
- 3. the kit of joint-detection oophoroma tumor marker HE4 and CA125 as claimed in claim 1 or 2, its feature exist In further including calibration object and lavation buffer solution.
- 4. the kit of joint-detection oophoroma tumor marker HE4 and CA125 as claimed in claim 1, it is characterised in that Silanization horseradish peroxidase is prepared by following methods, including horseradish peroxidase is added tert-butyldimethylsilyl chloride Mixture solution is obtained in the n,N-Dimethylformamide solution of silane, silanization horseradish peroxidase is reacted to obtain in stirring.
- 5. the kit of joint-detection oophoroma tumor marker HE4 and CA125 as claimed in claim 4, it is characterised in that The concentration of the N,N-dimethylformamide solution of the tert-butyl chloro-silicane is 3.02mg/ml；Horseradish peroxide per 1mg The N,N-dimethylformamide solution of 128 μ L tert-butyl chloro-silicanes is added in compound enzyme.
- 6. the kit of joint-detection oophoroma tumor marker HE4 and CA125 as claimed in claim 4, it is characterised in that Further included in silanization horseradish peroxidase preparation process and be to enzyme progress desalination and concentration, specific method first：Take horseradish Peroxidase, addition remove salt buffer, carry out desalination using molecular sieve chromatography, then the horseradish peroxide to being collected after desalination Compound enzyme carries out centrifugal concentrating to 3~5mg/ml.
- 7. the kit of joint-detection oophoroma tumor marker HE4 and CA125 as claimed in claim 4, it is characterised in that Further include and silanization horseradish peroxidase is purified and concentrated, specific method is：Using molecular sieve chromatography to mixing Product after the stirring reaction of thing solution is isolated and purified, and unreacted residue is removed, by silanization horseradish mistake after purification Oxide enzyme carries out centrifugal concentrating to 3~5mg/ml.
- A kind of 8. preparation side of the kit of joint-detection oophoroma tumor marker HE4 and CA125 as claimed in claim 1 Method, it is characterised in that including following operating procedure：1) magnetic particle of HE4 antibody and the antibody combined marks of CA125 is prepared：Including the solution containing HE4 antibody and CA125 antibody The middle carboxyl magnetic bead added after activation, after mixing coupling reaction, closes the magnetic bead after reaction, resists up to the HE4 The magnetic particle of body and the antibody combined marks of CA125；2) enzyme marker is prepared：A：Prepare the HE4 antibody of alkali phosphatase enzyme mark：By the HE4 antibody after activation desalination and activation Alkaline phosphatase mixing coupling reaction after desalination, up to the HE4 antibody of alkali phosphatase enzyme mark；B：Prepare the CA125 antibody of silanization horseradish peroxidase-labeled：Horseradish peroxidase after desalination is added into uncle Reacted in butyldimethylchlorosilane solution and silanization horseradish peroxidase is made, silanization horseradish peroxidase is activated Coupling reaction is mixed with activating the CA125 antibody of desalination after desalination, the CA125 up to silanization horseradish peroxidase-labeled resists Body；C：The HE4 antibody of the step A alkali phosphatase enzyme marks prepared and the CA125 of silanization horseradish peroxidase-labeled are resisted Body mixes, up to enzyme marker；3) Chemoluminescent substrate and assay chromogenic substrate solution are prepared；4) take HE4 antibody prepared by step 1) and enzyme marker prepared by the magnetic particles of the antibody combined marks of CA125, step 2), Chemoluminescent substrate and assay chromogenic substrate solution prepared by step 3), is assembled into kit, that is, completes.
- 9. a kind of kit of joint-detection oophoroma tumor marker HE4 and CA125 as claimed in claim 1 is examined at the same time Survey the application in terms of HE4 and CA125 concentration in human serum.
- 10. application as claimed in claim 3, it is characterised in that specific detection method includes following operating procedure：Ⅰ：Take to be checked Test sample sheet adds the magnetic particle of HE4 antibody and the antibody combined marks of CA125, adds enzyme marker in reaction cup, fully mixed It is even, and incubate 16min at 37 DEG C；Cleaning separation magnetic particle；Chemical luminous substrate solution A is added into magnetic particle, reacts and surveys Determine luminous intensity；100 μ L chromogenic substrate B solutions are added into magnetic particle again, 5min is reacted and measures absorbance under 620mn；Ⅱ：Take the calibration object of concentration known to measure luminous intensity and absorbance according to the method for step I, draw luminous intensity and HE4 is dense The standard working curve of degree, obtains the correspondence formula of luminous intensity and HE4 concentration；Draw the standard work of absorbance and CA125 concentration Make curve, obtain the correspondence formula of absorbance and CA125 concentration；Then test serum sample is measured according to the method for step I Luminous intensity and absorbance, the luminous intensity of serum sample and absorbance are brought into corresponding relational expression respectively, calculate serum sample In HE4 and CA125 concentration.
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| CN110221084A (en) * | 2019-01-03 | 2019-09-10 | 河南大学 | A kind of nanometer selenium kit of quick detection HE4 and CA125 |
| CN111693703A (en) * | 2020-07-17 | 2020-09-22 | 北京信诺卫康科技有限公司 | Spondin1 and HE4 combined as early ovarian cancer biomarker and kit |
| CN111707825A (en) * | 2020-07-29 | 2020-09-25 | 四川携光生物技术有限公司 | Kit for combined detection of tumor markers MCT1 and MCT4, and preparation method and application thereof |
| CN112798781A (en) * | 2020-12-30 | 2021-05-14 | 北京联众泰克科技有限公司 | Composition for detecting human epididymis protein 4, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
| CN113311154A (en) * | 2021-05-28 | 2021-08-27 | 宁波瑞源生物科技有限公司 | Coupling method, diluent and application of immunomagnetic particles |
| CN114280301A (en) * | 2022-03-03 | 2022-04-05 | 北京华瑞康源生物科技发展有限公司 | Method and kit for simultaneously detecting two tumor markers |
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| CN110275028A (en) * | 2019-01-03 | 2019-09-24 | 河南大学 | A kind of anti-HE4 antibody kit of colloid gold label and preparation method thereof |
| CN110221084B (en) * | 2019-01-03 | 2022-07-26 | 河南大学 | Nano-selenium kit for rapidly detecting HE4 and CA125 |
| CN110275028B (en) * | 2019-01-03 | 2022-07-26 | 河南大学 | Colloidal gold-labeled anti-HE4 antibody kit and preparation method thereof |
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| CN112798781A (en) * | 2020-12-30 | 2021-05-14 | 北京联众泰克科技有限公司 | Composition for detecting human epididymis protein 4, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
| CN113311154A (en) * | 2021-05-28 | 2021-08-27 | 宁波瑞源生物科技有限公司 | Coupling method, diluent and application of immunomagnetic particles |
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Address after: 102200, Beijing Changping District science and Technology Park, super Road, No. 37 Applicant after: Beijing Huizhong Medical Equipment Co., Ltd Applicant after: Liu Xiangdai Address before: 102200, Beijing Changping District science and Technology Park, super Road, No. 37 Applicant before: Beijing Runnuosi Medical Science & Technology Co., Ltd. Applicant before: Liu Xiangdai |
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