CN106405111A - Preparation method of enzyme linked immunosorbent assay kit for detection of breast cancer tumor marker CA153 based on elastase fluorescent substrate - Google Patents

Preparation method of enzyme linked immunosorbent assay kit for detection of breast cancer tumor marker CA153 based on elastase fluorescent substrate Download PDF

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CN106405111A
CN106405111A CN201610814364.2A CN201610814364A CN106405111A CN 106405111 A CN106405111 A CN 106405111A CN 201610814364 A CN201610814364 A CN 201610814364A CN 106405111 A CN106405111 A CN 106405111A
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ala
buffer solution
elastoser
elastase
breast cancer
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常津
武玉东
宫晓群
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Tianjin University
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Abstract

The invention relates to a preparation method of an enzyme linked immunosorbent assay kit for detection of a breast cancer tumor marker CA153 based on an elastase fluorescent substrate; two technologies of enzyme linked immunosorbent assay and fluorescence detection are combined, and a detection probe is prepared by an elastase and a tumor marker corresponding antibody Ab2. The probe, the tumor marker and another tumor marker antibody Ab1 embedded in the enzyme linked immunosorbent assay ELISA kit form a sandwich structure through the action between the antibodies and the antigens, finally, the fluorescence is produced by the action of the enzyme and the fluorescent substrate, the CA153 breast cancer related tumor marker is detected in a qualitative and quantitative manner, and a method for detection of the protein marker is established. The whole preparation process is simple and suitable for industrial production; according to the fluorescence characteristics of the substrate, and the action with the enzyme, the protein marker can be detected in a qualitative and quantitative manner, and the specificity is good; and in the whole detection process, the new method for detection of the protein marker is established.

Description

Detect the enzyme of breast cancer tumour mark CA153 based on elastoser fluorogenic substrate Linked immunologic reagent box preparation method
Technical field
The present invention relates to disease detection technical field, more particularly to a kind of based on the inspection of elastoser fluorogenic substrate Survey the enzyme linked immunological kit preparation method of breast cancer tumour mark CA153.
Background technology
Breast cancer has become the threat able-bodied kinds of tumor of women at present.Global breast cancer incidence is from 20th century 70 Age Mo starts in rising trend always.8 women of the U.S. just have 1 people in life and suffer from breast cancer.According to National Cancer Center and The pathogenesis of breast carcinoma data in 2009 that prevention and control of diseases office of the Ministry of Public Health announces for 2012 show：National tumour registration area mammary gland Cancer morbidity occupies the 1st of female malignant, and female mammary gland cancer morbidity (rough and careless) whole nation adds up to 42.55/10 ten thousand, city City is 51.91/10 ten thousand, and rural area is 23.12/10 ten thousand.Breast cancer has become the great public health problem of current social.From 20 generation The whole world Death Rate of Breast Cancer nineties of recording presents downward trend；Trace it to its cause, one is the development of breast cancer screening, makes morning The ratio of phase case increases；Two is the development of comprehensive therapy of breast cancer, improves curative effect.CA15-3 is the most important of breast cancer Specific marker.The CA15-3 of the patient with breast cancer of 30%-50% is significantly raised, and the change of its content is close with therapeutic effect Correlation, is patient with breast cancer's diagnosis and the optimal parameter of monitoring postoperative recurrence, observation curative effect.CA15-3 dynamic measurement contributes to II The early detection of recurrence after phase and III primary breast cancer Case treatment；When CA15-3 is more than 100U/ml it is believed that there being metastatic Pathology.
Immunofluorescence technique (Immunofluorescence technique), also known as fluorescent antibody technics, is labelled immune Develop earliest one kind in technology.It is setting up on the basis of immunology, biochemistry and microscopy Technology.Just there are some scholars to attempt to be combined antibody molecule with some probe materials very early on, entered using antigen-antibody reaction Row tissue or the positioning of intracellular antigen material.Because antigen-antibody reaction has the specificity of height, so working as antigen-antibody When reacting, only it is to be understood that one of factor is it is possible to find another factor.Immunofluorescence technique is exactly will not shadow Ring the active fluorochrome mark of antigen-antibody on antibody (or antigen), after its corresponding antigen (or antibody) combines, A kind of specificity fluorescent reaction is assumed under fluorescence microscope.
Elastoser (Elastase) fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110 is that a kind of bisamide spreads out Biological rhodamine 110 (Rhodamine110) molecular probe, be one sensitive and selectively mensure protease molten Substrate in liquid or in living cells.These fluorogenic substrates contain a kind of amino acid or peptide is covalently attached to each Rhodamine110 Amino, after enzymatic lysis, non-fluorescence bisamide substrate is first converted into hypofluorescence monoamides, is then converted to hyperfluorescence Rhodamine110, fluorescence can increase further.Because Rhodamine110 has larger extinction coefficient, so the letter of experiment Make an uproar higher than very.Additionally, for compared to common fluorescein, the fluorescence of Rhodamine110 can keep stable between pH3-9. Fluoroscopic examination at present has been widely used in the aspects such as spike, imaging and mark because having excellent optical property, and The advantages of ELISA is then because of its sensitivity height, high specificity is in special consequence in detection field.So intending herein Develop the enzyme linked immunological kit detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate, to breast cancer Tumor markers CA153 is detected.
Content of the invention
In view of the unique optical property of critical role in lesion detection for the protein marker, fluoroscopic examination and enzyme connection are exempted from The advantage of epidemic disease detection technique.We will be in conjunction with enzyme linked immunosorbent detection and two kinds of technology of fluoroscopic examination, using elastoser and swollen Tumor markers corresponding antibodies Ab2Prepared detection probe.Probe, tumor markers and be embedded in enzyme linked immunological ELISA kit Another kind of tumor markers antibody A b1By the effect between antibody antigen, form a kind of similar sandwich structure, finally by The effect of enzyme and fluorogenic substrate produces fluorescence, and CA153 this breast cancer related neoplasms mark is carried out with the detection of qualitative, quantitative, Set up the new method of protein markers analyte detection, high degree improves detection sensitivity.
The enzyme linked immunological detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate of present invention preparation Kit, is characterized in that from elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110 selective degradation produces Raw fluorescence carries out quantitative determination to breast cancer tumour mark CA153, and its structural formula is as follows：
Technical scheme is as follows：
A kind of enzyme linked immunological kit detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate； Its step is as follows：
1) by the new sodium metaperiodate NalO preparing4Solution is added in elastoser Elastase solution, keeps away under room temperature After light stirring, processed with acetate buffer dialysed overnight, make system pH reach 9.0～9.3 by adding carbonate buffer solution, Add CA153 labelled antibody Ab immediately2, under room temperature, after lucifuge stirring, add a small amount of sodium borohydride NaBH4PBS is used after fully reacting Buffer solution dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by CA153 coated antibody Ab1Add 96 hole elisa Plates, after standing overnight process, rinse 96 holes with lavation buffer solution ELISA Plate, rinses 96 hole elisa Plates with lavation buffer solution after adding BSA Seal treatment again, adds standard CA153 antigen afterwards Or patient's sample slowly rocks incubation, rinse 96 hole elisa Plates with lavation buffer solution, add above-mentioned enzyme to join detection probe Elastase-Ab1, rinse 96 hole elisa Plates with lavation buffer solution, be eventually adding trypsase fluorogenic substrate (CBZ-Ala-Ala- Ala-Ala)2- R110, reads 96 hole elisa Plates fluorescence intensity levels with fluorescence microplate reader.
Described acetate buffer is the buffer solution of 0.01M pH4.4；Carbonate buffer solution is the buffering of 0.2M pH9.5 Liquid；PBS is the buffer solution of 0.15M pH7.4.
Described elastoser：Sodium metaperiodate mass ratio is 1:0.8～1.2；Elastoser：CA153 labelled antibody matter Amount ratio is 1:1～5；Elastoser：Sodium borohydride mass ratio is 15:1.
Described CA153 coated antibody Ab1Concentration is 10～50 μ g/mL.
Described lavation buffer solution is the PBS of the 0.01M pH7.4 containing 0.05～0.5%Tween-20.
Described Cleaning Principle is according to trypsase fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110 degraded produces Fluorescence intensity level and CA153 standard antigen concentration between relation Criterion curve, qualitative and quantitative detection oophoroma tumor Mark CA125.
By sodium metaperiodate NalO4CA153 is remembered antibody A b by effect2Connect with elastoser Elastase, obtain enzyme connection Detection probe Elastase-Ab2.CA153 coated antibody Ab1 is added 96 hole elisa Plates, is overnight washed with lavation buffer solution afterwards for 4 DEG C Wash 96 hole elisa Plates, wash 96 hole elisa Plates with lavation buffer solution after adding 37 DEG C of Seal treatment of BSA, add in 96 hole elisa Plates Wash 96 hole elisa Plates with lavation buffer solution after entering reaction at 37 DEG C of measuring samples, add above-mentioned Elastase-Ab2Detection probe Wash 96 hole elisa Plates with lavation buffer solution after reacting at 37 DEG C, add elastoser fluorogenic substrate (CBZ-Ala-Ala- Ala-Ala)2- R110, finally uses fluorescence microplate reader reading.If detection sample is with the presence of CA153 antigen A g, probe Elastase-Ab2, the CA153 antigen A g and CA153 coated antibody Ab being embedded in 96 hole elisa Plates1Between antibody antigen Effect, form a kind of similar sandwich structure Elastase-Ab2-Ag-Ab1, now elastoser fluorogenic substrate (CBZ- Ala-Ala-Ala-Ala)2- R110 degrades in the presence of enzyme and produces fluorescence；If detection sample does not have CA153, antigen A g deposits In then detection probe Elastase-Ab of 96 hole elisa Plates2By washed buffer solution for cleaning, now elastoser fluorescence bottom Thing (CBZ-Ala-Ala-Ala-Ala)2- R110 is non-degradable, does not have fluorescence to occur.Variable concentrations are added on 96 hole elisa Plates Standard CA153 antigen, Criterion curve, qualitative and quantitative detection breast cancer tumour mark CA153.
The enzyme linked immunological detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate of present invention preparation Kit advantage is：
1. adopt elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110 is this to have excellent optics Rhodamine 110 (R110) molecular probe of the bisamide derivatives of property, because Rhodamine110 has larger delustring system Number, so the signal to noise ratio of experiment is very high.Additionally, for compared to common fluorescein, the fluorescence of Rhodamine110 is in pH3-9 Between can keep stable.
2. using this traditional sensing techniques with sensitivity height, high specificity of enzyme linked immunosorbent detection ELISA, Be conducive to improving detection efficiency.
3. adopt sodium metaperiodate NalO4CA153 is remembered antibody A b by effect2Connect with elastoser Elastase and obtain enzyme Connection detection probe Elastase-Ab2, the fluorescence intensity that sandwich structure degraded fluorogenic substrate produces, qualitative and quantitative detection breast cancer Tumor markers CA153, significantly reduces detection fluorescence background.
As shown in figure 1, elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2After-R110 enzymatic lysis, non-glimmering Light bisamide substrate takes the lead in being converted into the monoamides of a pair of hypofluorescence, is then converted to the Rhodamine110 of hyperfluorescence, fluorescence Increase further, realize super sensitivity detection.As shown in Fig. 2 it is strong according to addition variable concentrations standard CA153 antigen gained fluorescence Degree, Criterion curve understands that the elastoser fluorogenic substrate of present invention preparation detects breast cancer tumour mark CA153's Enzyme linked immunological kit detection sensitivity is higher, reaches 1*10-10U/mL；Fig. 3 according to add not synantigen it is known that system of the present invention Standby elastoser fluorogenic substrate detects the enzyme linked immunological kit specificity of breast cancer tumour mark CA153 very well.
Brief description
The enzyme connection detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate of Fig. 1 present invention preparation Immune reagent kit degradation of substrates schematic diagram.
The enzyme connection detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate of Fig. 2 present invention preparation Immune reagent kit calibration curve and ultraviolet picture.
The enzyme connection detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate of Fig. 3 present invention preparation The special linearity curve of immune reagent kit and ultraviolet picture.
Specific embodiment
In following case study on implementation, the invention will be further elaborated, but the invention is not restricted to this.
Case study on implementation 1：
1) by the new 0.2mL sodium metaperiodate NalO preparing4Solution (21.39mg/mL) is added to 1mL elastoser In Elastase solution (5mg/mL), after lucifuge stirring 20min under room temperature, with 1mM PH4.4 acetate buffer dialysed overnight Process, make system pH reach 9.0～9.3 by adding 20 μ L 0.2M PH9.5 carbonate buffer solutions, add 10mg immediately CA153 labelled antibody Ab2, under room temperature, after lucifuge stirring 2h, add 0.1mL sodium borohydride NaBH4(4mg/mL) use after fully reacting 0.15M PH7.4PBS buffer solution dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by 100 μ L CA153 coated antibody Ab1(24 μ g/mL) adds 96 hole elisa Plates, and 4 DEG C stand overnight use after process Lavation buffer solution rinses 96 hole elisa Plates, adds 37 DEG C of closing 1h of 300 μ L BSA (10mg/mL) to use washing buffer again after processing Liquid rinses 96 hole elisa Plates, adds 100 μ L CA153 standard antigens slowly to rock incubation afterwards, rinses 96 holes with lavation buffer solution ELISA Plate, adds 100 μ L above-mentioned enzyme connection detection probe Elastase-Ab2Dilution, rinses 96 hole enzyme marks with lavation buffer solution Plate, is eventually adding 100 μ L elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110, is read with fluorescence microplate reader Take 96 hole elisa Plates fluorescence intensity levels.
Case study on implementation 2：
2) by the new 0.2mL sodium metaperiodate NalO preparing4Solution (20mg/mL) is added to 1mL elastoser In Elastase solution (5mg/mL), after lucifuge stirring 20min under room temperature, with 1mM PH4.4 acetate buffer dialysed overnight Process, make system pH reach 9.0～9.3 by adding 20 μ L 0.2M PH9.5 carbonate buffer solutions, add 10mg immediately CA153 labelled antibody Ab2, under room temperature, after lucifuge stirring 2h, add 0.1mL sodium borohydride NaBH4(4mg/mL) use after fully reacting 0.15M PH7.4PBS buffer solution dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by 100 μ L CA153 coated antibody Ab1(24 μ g/mL) adds 96 hole elisa Plates, and 4 DEG C stand overnight use after process Lavation buffer solution rinses 96 hole elisa Plates, adds 37 DEG C of closing 1h of 300 μ L BSA (10mg/mL) to use washing buffer again after processing Liquid rinses 96 hole elisa Plates, adds 100 μ L CA153 standard antigens slowly to rock incubation afterwards, rinses 96 holes with lavation buffer solution ELISA Plate, adds 100 μ L above-mentioned enzyme connection detection probe Elastase-Ab2Dilution, rinses 96 hole enzyme marks with lavation buffer solution Plate, is eventually adding 100 μ L elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110, is read with fluorescence microplate reader Take 96 hole elisa Plates fluorescence intensity levels.
Case study on implementation 3：
3) by the new 0.2mL sodium metaperiodate NalO preparing4Solution (30mg/mL) is added to 1mL elastoser In Elastase solution (5mg/mL), after lucifuge stirring 20min under room temperature, with 1mM PH4.4 acetate buffer dialysed overnight Process, make system pH reach 9.0～9.3 by adding 20 μ L 0.2M PH9.5 carbonate buffer solutions, add 10mg immediately CA153 labelled antibody Ab2, under room temperature, after lucifuge stirring 2h, add 0.1mL sodium borohydride NaBH4(4mg/mL) use after fully reacting 0.15M PH7.4PBS buffer solution dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by 100 μ L CA153 coated antibody Ab1(24 μ g/mL) adds 96 hole elisa Plates, and 4 DEG C stand overnight use after process Lavation buffer solution rinses 96 hole elisa Plates, adds 37 DEG C of closing 1h of 300 μ L BSA (10mg/mL) to use washing buffer again after processing Liquid rinses 96 hole elisa Plates, adds 100 μ L CA153 standard antigens slowly to rock incubation afterwards, rinses 96 holes with lavation buffer solution ELISA Plate, adds 100 μ L above-mentioned enzyme connection detection probe Elastase-Ab2Dilution, rinses 96 hole enzyme marks with lavation buffer solution Plate, is eventually adding 100 μ L elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110, is read with fluorescence microplate reader Take 96 hole elisa Plates fluorescence intensity levels.
Case study on implementation 4：
4) by the new 0.2mL sodium metaperiodate NalO preparing4Solution (21.39mg/mL) is added to 1mL elastoser In Elastase solution (5mg/mL), after lucifuge stirring 20min under room temperature, with 1mM PH4.4 acetate buffer dialysed overnight Process, make system pH reach 9.0～9.3 by adding 20 μ L 0.2M PH9.5 carbonate buffer solutions, add 5mg immediately CA153 labelled antibody Ab2, under room temperature, after lucifuge stirring 2h, add 0.1mL sodium borohydride NaBH4(4mg/mL) use after fully reacting 0.15M PH7.4PBS buffer solution dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by 100 μ L CA153 coated antibody Ab1(10 μ g/mL) adds 96 hole elisa Plates, and 4 DEG C stand overnight use after process Lavation buffer solution rinses 96 hole elisa Plates, adds 37 DEG C of closing 1h of 300 μ L BSA (10mg/mL) to use washing buffer again after processing Liquid rinses 96 hole elisa Plates, adds 100 μ L CA153 standard antigens slowly to rock incubation afterwards, rinses 96 holes with lavation buffer solution ELISA Plate, adds 100 μ L above-mentioned enzyme connection detection probe Elastase-Ab2Dilution, rinses 96 hole enzyme marks with lavation buffer solution Plate, is eventually adding 100 μ L elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110, is read with fluorescence microplate reader Take 96 hole elisa Plates fluorescence intensity levels.
Case study on implementation 5：
5) by the new 0.2mL sodium metaperiodate NalO preparing4Solution (21.39mg/mL) is added to 1mL elastoser In Elastase solution (5mg/mL), after lucifuge stirring 20min under room temperature, with 1mM PH4.4 acetate buffer dialysed overnight Process, make system pH reach 9.0～9.3 by adding 20 μ L 0.2M PH9.5 carbonate buffer solutions, add 25mg immediately CA153 labelled antibody Ab2, under room temperature, after lucifuge stirring 2h, add 0.1mL sodium borohydride NaBH4(4mg/mL) use after fully reacting 0.15M PH7.4PBS buffer solution dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by 100 μ L CA153 coated antibody Ab1(50 μ g/mL) adds 96 hole elisa Plates, and 4 DEG C stand overnight use after process Lavation buffer solution rinses 96 hole elisa Plates, adds 37 DEG C of closing 1h of 300 μ L BSA (10mg/mL) to use washing buffer again after processing Liquid rinses 96 hole elisa Plates, adds 100 μ L CA153 standard antigens slowly to rock incubation afterwards, rinses 96 holes with lavation buffer solution ELISA Plate, adds 100 μ L above-mentioned enzyme connection detection probe Elastase-Ab2Dilution, rinses 96 hole enzyme marks with lavation buffer solution Plate, is eventually adding 100 μ L elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110, is read with fluorescence microplate reader Take 96 hole elisa Plates fluorescence intensity levels.
Case study on implementation 6：
1) by the new 0.2mL sodium metaperiodate NalO preparing4Solution (20mg/mL) is added to 1mL elastoser In Elastase solution (5mg/mL), after lucifuge stirring 20min under room temperature, with 1mM PH4.4 acetate buffer dialysed overnight Process, make system pH reach 9.0～9.3 by adding 20 μ L 0.2M PH9.5 carbonate buffer solutions, add 10mg immediately CA153 labelled antibody Ab2, under room temperature, after lucifuge stirring 2h, add 0.1mL sodium borohydride NaBH4(4mg/mL) use after fully reacting 0.15M PH7.4PBS buffer solution dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by 100 μ L CA153 coated antibody Ab1(24 μ g/mL) adds 96 hole elisa Plates, and 4 DEG C stand overnight use after process Lavation buffer solution rinses 96 hole elisa Plates, adds 37 DEG C of closing 1h of 300 μ L BSA (10mg/mL) to use washing buffer again after processing Liquid rinses 96 hole elisa Plates, afterwards every hole add the concentration of 100 μ L CA153 standard antigens be respectively 100U/mL, 10U/mL, 1U/mL, 0.1U/mL, 0.01U/mL, 0.001U/mL, 0.0001U/mL, 0U/mL, slowly rock incubation, are rushed with lavation buffer solution Wash 96 hole elisa Plates, add 100 μ L above-mentioned enzyme connection detection probe Elastase-Ab2Dilution, rinses 96 holes with lavation buffer solution ELISA Plate, is eventually adding 100 μ L elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110, uses luciferase mark Instrument reads 96 hole elisa Plates fluorescence intensity levels.Criterion curve.
Case study on implementation 7：
1) by the new 0.2mL sodium metaperiodate NalO preparing4Solution (20mg/mL) is added to 1mL elastoser In Elastase solution (5mg/mL), after lucifuge stirring 20min under room temperature, with 1mM PH4.4 acetate buffer dialysed overnight Process, make system pH reach 9.0～9.3 by adding 20 μ L 0.2M PH9.5 carbonate buffer solutions, add 10mg immediately CA153 labelled antibody Ab2, under room temperature, after lucifuge stirring 2h, add 0.1mL sodium borohydride NaBH4(4mg/mL) use after fully reacting 0.15M PH7.4PBS buffer solution dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by 100 μ L CA153 coated antibody Ab1(24 μ g/mL) adds 96 hole elisa Plates, and 4 DEG C stand overnight use after process Lavation buffer solution rinses 96 hole elisa Plates, adds 37 DEG C of closing 1h of 300 μ L BSA (10mg/mL) to use washing buffer again after processing Liquid rinses 96 hole elisa Plates, and every hole is separately added into 100 μ L HCG, BSA, PBS, CEA, CA125, AFP, PSA, CA153 and resists afterwards Former, slowly rock incubation, rinse 96 hole elisa Plates with lavation buffer solution, add 100 μ L above-mentioned enzyme connection detection probe Elastase- Ab2Dilution, rinses 96 hole elisa Plates with lavation buffer solution, is eventually adding 100 μ L elastoser fluorogenic substrate (CBZ-Ala- Ala-Ala-Ala)2- R110, reads 96 hole elisa Plates fluorescence intensity levels with fluorescence microplate reader.Set up non-specific adsorption curve.

Claims (7)

1. a kind of enzyme linked immunological kit preparation detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate Method, is characterized in that from elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110 selective degradation produces Fluorescence quantitative determination is carried out to breast cancer tumour mark CA153, its structural formula is as follows：
2. the enzyme linked immunological examination detecting breast cancer tumour mark CA153 based on elastoser fluorogenic substrate of claim 1 Agent box preparation method is it is characterised in that step is as follows：
1) by the new sodium metaperiodate NalO preparing4Solution is added in elastoser Elastase solution, lucifuge stirring under room temperature Afterwards, with acetate buffer dialysed overnight process, by add carbonate buffer solution make system pH reach 9.0～9.3, immediately plus Enter CA153 labelled antibody Ab2, under room temperature, after lucifuge stirring, add a small amount of sodium borohydride NaBH4PBS is used after fully reacting Dialysed overnight, obtains enzyme connection detection probe Elastase-Ab2；
2) by CA153 coated antibody Ab1Add 96 hole elisa Plates, after standing overnight process, rinse 96 hole enzyme marks with lavation buffer solution Plate, rinses 96 hole elisa Plates with lavation buffer solution after adding BSA Seal treatment again, adds standard CA153 antigen or disease afterwards People's sample slowly rocks incubation, rinses 96 hole elisa Plates with lavation buffer solution, adds above-mentioned enzyme to join detection probe Elastase- Ab1, rinse 96 hole elisa Plates with lavation buffer solution, be eventually adding trypsase fluorogenic substrate (CBZ-Ala-Ala-Ala-Ala)2- R110, reads 96 hole elisa Plates fluorescence intensity levels with fluorescence microplate reader.
3. method as claimed in claim 2, is characterized in that acetate buffer is the buffer solution of 0.01M pH4.4；Carbonate Buffer solution is the buffer solution of 0.2M pH9.5；PBS is the buffer solution of 0.15M pH7.4.
4. method as claimed in claim 2, is characterized in that elastoser：Sodium metaperiodate mass ratio is 1:0.8～1.2；Bullet Property protease：CA153 labelled antibody mass ratio is 1:1～5；Elastoser：Sodium borohydride mass ratio is 15:1.
5. method as claimed in claim 2, is characterized in that CA153 coated antibody Ab1Concentration is 10～50 μ g/mL.
6. method as claimed in claim 2, is characterized in that lavation buffer solution is containing 0.05～0.5%Tween-20 The PBS of 0.01M pH7.4.
7. method as claimed in claim 2, is characterized in that according to elastoser fluorogenic substrate (CBZ-Ala-Ala-Ala- Ala)2Relation Criterion curve between the fluorescence intensity level of-R110 degraded generation and CA153 standard antigen concentration, qualitative Quantitative determination oophoroma tumor marker CA125.
CN201610814364.2A 2016-09-10 2016-09-10 Preparation method of enzyme linked immunosorbent assay kit for detection of breast cancer tumor marker CA153 based on elastase fluorescent substrate Pending CN106405111A (en)

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