CN102507951B - Enzyme-linked immuno sorbent assay (ELISA) kit for performing joint detection on tumor marker - Google Patents
Enzyme-linked immuno sorbent assay (ELISA) kit for performing joint detection on tumor marker Download PDFInfo
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- CN102507951B CN102507951B CN201110380835.0A CN201110380835A CN102507951B CN 102507951 B CN102507951 B CN 102507951B CN 201110380835 A CN201110380835 A CN 201110380835A CN 102507951 B CN102507951 B CN 102507951B
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Abstract
The invention discloses an enzyme-linked immuno sorbent assay (ELISA) kit for performing joint detection on a tumor marker. The ELISA kit consists of a perforated plate coated with neuron specific enolase (NSE), a cytokeratin fragment 21-1 (CYFRA21-1) and a Dkkopf 1 (DKK1) monoclonal antibody, horseradish peroxidase-labeled NSE, horseradish peroxidase-labeled CYFRA21-1, horseradish peroxidase-labeled DKK1, a chromogenic substrate TMB and a substrate stop solution. The ELISA kit has the advantages of simplicity, convenience, sensitivity, stability, high repeatability and the like and has practical value.
Description
Technical field
The present invention relates to detection kit field, be specifically related to a kind of enzyme-linked immunologic detecting kit of joint-detection tumor markers.
Background technology
Lung cancer is one of malignant tumour that in world wide, the most common lethal number is maximum, and its incidence of disease growth rate is also in first of each malignant tumour, due to the concealment of lung cancer onset.Still lack at present examination effectively and method of early diagnosis, patient occurs that symptom is mostly to be late period, prognosis is poor, must within 5 years, survive and be no more than 15%, there is symptom to be less than 10%, but in the patients with lung cancer of diagnosis, the prognosis of operative treatment has clear improvement medium and advanced lung cancer in early days, and its survival rate can reach 70%.Traditional diagnostic method is as methods such as rabat, bronchoscope, phlegm cytology checkings clinically.But lack enough specificitys and sensitivity.Therefore, be badly in need of clinically a kind of can be for the method for lung cancer early diagnosis.At present, for the more simple and efficient method of lung cancer early diagnosis, be by enzyme linked immunological kit, and for the kit of pulmonary cancer diagnosis, be both at home and abroad all to detect for a certain mark of lung cancer, but lung cancer does not have a species specific marker for lung cancer up till now, be all some relevant labels, simultaneously due to lung cancer complicated classification, the expression of different pathological mark in period is different, just for a certain mark, diagnose, can bring greatly Misdiagnosis.
Currently used enzyme linked immunological kit joint-detection is that the ELISA Plate of several marks is operated simultaneously, has reached the object of joint-detection, and this kind of operation can bring very large personal error.
Summary of the invention
The object of the invention is to according to problems such as the personal error existing in existing detection kit are large, provide a kind of and can in an ELISA Plate, to two or three mark, carry out the kit quantitatively detecting simultaneously simultaneously, can eliminate personal error, have advantage easy and simple to handle, with low cost concurrently.
Another object of the present invention is to provide the preparation method of above-mentioned detection kit.
A further object of the invention is to provide the application of above-mentioned detection kit.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
A kind of enzyme-linked immunologic detecting kit of joint-detection tumor markers, by being coated with NSE (NSE), CYFRA21-1 (CYFRA21-1) and Dkkopf 1(DKK1) porous plate of monoclonal antibody, horseradish peroxidase target NSE, CYFRA21-1 and DKK1, chromogenic substrate TMB and substrate stop buffer form; The every hole of described porous plate is coated with NSE monoclonal antibody 1 ~ 4ug/mL; Every hole is coated with CYFRA21-1 monoclonal antibody 1 ~ 2ug/mL; Every hole is coated with DKK1 monoclonal antibody 1 ~ 2ug/mL.NSE, CYFRA21-1, tri-kinds of detection property antibody of DKK1 add in ELISA Plate hole in 4:4:2 ratio, and cumulative volume is 100 uL.
As a kind of preferred version, in above-mentioned porous plate, the coating buffer in every hole is 100uL.
As a kind of preferred version, the working concentration extension rate of described horseradish peroxidase target NSE, CYFRA21-1 and DKK1 is 1:500 ~ 1:2500.
As a kind of preferred version, enzyme conjugates protection liquid is obtained by the phosphate buffer dilution of 0.01mol/L, pH7.4, in described phosphate buffer containing 0.1% bovine serum albumin(BSA) and 0.1% thimerosal.
The preparation method of the enzyme-linked immunologic detecting kit of joint-detection tumor markers of the present invention comprises the steps: that preparation is coated with the porous plate of NSE, CYFRA21-1, DKK1 monoclonal antibody; With pH 8 ~ 10 carbonate buffer solutions, by NSE, CYFRA21-1, the dilution of DKK1 monoclonal antibody, then above-mentioned NSE, CYFRA21-1, DKK1 monoclonal antibody dilution are joined in hole; 37 ℃ of coated 1h, join each hole of ELISA Plate by 5% bovine serum albumin solution, 37 ℃ of sealing 1 h; After sealing, with phosphate buffer, wash version and pat dry, be stored in 4 ℃.
The enzyme-linked immunologic detecting kit of joint-detection tumor markers of the present invention can be applied in the detection of tumor related marker thing.
Compared with prior art, the present invention has following beneficial effect:
Kit of the present invention can detect two or three tumor associated antigen simultaneously, the specificity of detection and highly sensitive, and production cost is low.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of ELISA Plate coated antibody in detection kit of the present invention;
Fig. 2 is the stability analysis figure of detection kit of the present invention.
Embodiment
Below in conjunction with embodiment, further explain the present invention, but embodiment does not limit in any form to the present invention.
The preparation process of embodiment 1 kit
(1) with carbonate buffer solution, dilute tri-kinds of detection property antibody of NSE, CYFRA21-1, DKK1 and add in ELISA Plate hole in 4:4:2 ratio, cumulative volume is 100 uL, is placed in 37 ℃, wrapper sheet 2h.
(2) pat dry liquid in plate, add cleansing solution to wash plate, every hole 200uL, each 30s, washs 5 times.
(3) with 5% bovine serum albumin solution sealase target, every hole 200uL, 37 ℃ of sealing 1 h; After sealing, with phosphate buffer, wash version and pat dry, be stored in 4 ℃.
(4) pat dry liquid in plate, add cleansing solution to wash plate, every hole 200uL, each 30s, washs 5 times.
(5) with Fresco Bag, vacuumize, 4 ℃ of placements are standby.
embodiment 2 double antibodies sandwich method enzyme linked immunological kits detect NSE, CK19, tri-kinds of antigens of DKK1
(1) in reacting hole, add respectively different serum to be checked, every example serum to be checked is established three parallel multiple holes, 5 times of serum dilutions to be checked, and every hole adds 100uL, is placed in 37 ℃, 1h.
(2) pat dry liquid in plate, add cleansing solution to wash plate, every hole 200uL, each 30s, washs 5 times.
(3) each hole adds the enzyme labelled antibody after dilution, every hole 100uL, 37 ℃, incubation 1h.
(4) pat dry liquid in plate, add cleansing solution to wash plate, every hole 200uL, each 30s, washs 5 times.
(5) each hole adds nitrite ion TMB, every hole 100uL, and 37 ℃ of reaction 10 min, add stop buffer TMB, 450nm reading (Fig. 1).
embodiment 3 double antibodies sandwich ELISA method repeatability are analyzed
Choose high, medium and low 3 parts of different positive serum samples, use with a collection of kit and detect, every duplicate samples is parallel survey 8 holes respectively, carry out kit and criticize interior repeatability detection.
Table 1
Choose high, medium and low 3 parts of different positive serum samples, with the double antibodies sandwich method diagnostic kit of 6 parts of different batches, detect, the parallel survey of every duplicate samples 3 holes, carry out kit tippet repeatability and detect.
Table 2
embodiment 3 kit stability analyses
Kit is deposited in 37 ℃ of environment and preserved, regularly detect, till knowing the maximal value drop by half of its quality index, it has been generally acknowledged that at 37 ℃ and deposit and within one day, be equivalent to 4-10 ℃ and deposit 1.5 months.As 37 ℃ of this kits of table analysis at least keep 7 days, be equivalent to preserve more than 10 months (Fig. 2) at 4 ℃.
Table 3
| First day light absorption value | Second day light absorption value | The 3rd day light absorption value | The 4th day light absorption value | The 5th day light absorption value | The 6th day light absorption value | The 7th day light absorption value |
| 1.243 | 1.064 | 1.057 | 1.077 | 0.79 | 1.056 | 0.607 |
| 1.256 | 1.122 | 1.076 | 1.084 | 0.813 | 1.054 | 0.82 |
| 1.295 | 1.101 | 1.131 | 1.067 | 0.821 | 1.07 | 0.753 |
| 1.319 | 1.1 | 1.27 | 1.054 | 0.766 | 0.882 | 0.639 |
| 1.21 | 1.091 | 1.311 | 1.089 | 0.813 | 0.914 | 0.685 |
determining of embodiment 5 kit gray areas
(1) take out 4 ℃ of standby kits, the patients serum of 48 routine normal persons and the 4 normal diseases of routine lung is added to respectively in each hole, every example serum to be checked is established three parallel multiple holes, 5 times of serum dilutions to be checked, and every hole adds 100uL, is placed in 37 ℃, 1h.
(2) pat dry liquid in plate, add cleansing solution to wash plate, every hole 200uL, each 30s, washs 5 times.
(3) each hole adds the enzyme labelled antibody after dilution, every hole 100uL, 37 ℃, incubation 1h.
(4) pat dry liquid in plate, add cleansing solution to wash plate, every hole 200uL, each 30s, washs 5 times.
(5) each hole adds nitrite ion TMB, every hole 100uL, and 37 ℃ of reaction 10 min, add stop buffer TMB, 450nm reading.
Get at random 88 normal human serums as follows by multispecific antibody pulmonary cancer diagnosis kit measurement result:
88 OD value averages (X)=0.208 that normal human serum is measured, standard deviation (SD)=0.0589.So the cut off value (critical value) of its OD value for average (X)+3SD=0.208+0.1767=0.3847 gray area OD value scope be exactly between 0 to 0.3847, meet gray area patient and will check for suspicious.(the OD value that table 4:88 name normal human serum records).
The OD value that table 4:88 name normal human serum records
| 0.267 | 0.153 | 0.13 | 0.136 | 0.182 | 0.278 | 0.018 | 0.23 |
| 0.217 | 0.127 | 0.123 | 0.116 | 0.28 | 0.288 | 0.217 | 0.273 |
| 0.086 | 0.193 | 0.326 | 0.103 | 0.338 | 0.166 | 0.256 | 0.292 |
| 0.258 | 0.123 | 0.135 | 0.138 | 0.239 | 0.207 | 0.21 | 0.281 |
| 0.233 | 0.386 | 0.136 | 0.117 | 0.233 | 0.211 | 0.091 | 0.17 |
| 0.278 | 0.133 | 0.167 | 0.137 | 0.253 | 0.223 | 0.172 | 0.282 |
| 0.303 | 0.103 | 0.136 | 0.33 | 0.216 | 0.293 | 0.221 | 0.28 |
| 0.207 | 0.098 | 0.152 | 0.132 | 0.29 | 0.226 | 0.291 | 0.128 |
| 0.166 | 0.269 | 0.235 | 0.22 | 0.183 | 0.236 | 0.229 | 0.213 |
| 0.202 | 0.203 | 0.163 | 0.131 | 0.201 | 0.295 | 0.112 | 0.213 |
| 0.213 | 0.266 | 0.223 | 0.357 | 0.225 | 0.168 | 0.235 | 0.371 |
Claims (5)
1. an enzyme-linked immunologic detecting kit for joint-detection tumor markers, is characterized in that by the porous plate that is coated with NSE, CYFRA21-1 and DKK1 monoclonal antibody, horseradish peroxidase target antibody, and chromogenic substrate TMB and substrate stop buffer form; The every hole of described porous plate is coated with NSE monoclonal antibody 1 ~ 4ug/mL; Every hole is coated with CYFRA21-1 monoclonal antibody 1 ~ 2ug/mL; Every hole is coated with DKK1 monoclonal antibody 1 ~ 2ug/mL, and NSE, CYFRA21-1, tri-kinds of detection property antibody of DKK1 add in ELISA Plate hole in 4:4:2 ratio, and cumulative volume is 100 uL.
2. the enzyme-linked immunologic detecting kit of joint-detection tumor markers according to claim 1, is characterized in that in described porous plate, and the coating buffer in every hole is 100uL.
3. the enzyme-linked immunologic detecting kit of joint-detection tumor markers according to claim 1, is further characterized in that the working concentration extension rate of described horseradish peroxidase target antibody is 1:500 ~ 1:2500.
4. the enzyme-linked immunologic detecting kit of joint-detection tumor markers according to claim 1; be further characterized in that enzyme conjugates protection liquid is obtained by the phosphate buffer dilution of 0.01mol/L, pH7.4, in described phosphate buffer containing 0.1% bovine serum albumin(BSA) and 0.1% thimerosal.
5. the preparation method of the enzyme-linked immunologic detecting kit of joint-detection tumor markers described in any claim in claim 1 ~ 4, is characterized in that comprising the steps: that preparation is coated with the porous plate of NSE, CYFRA21-1, DKK1 monoclonal antibody; With pH 8 ~ 10 carbonate buffer solutions, by NSE, CYFRA21-1, the dilution of DKK1 monoclonal antibody, then above-mentioned NSE, CYFRA21-1, DKK1 monoclonal antibody dilution are joined in hole; 37 ℃ of coated 1h, join each hole of ELISA Plate by 5% bovine serum albumin solution, 37 ℃ of sealing 1 h; After sealing, with phosphate buffer, wash plate and pat dry, be stored in 4 ℃.
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| CN105588941A (en) * | 2015-02-28 | 2016-05-18 | 苏州飞康生物医药有限公司 | Enzyme-linked immunosorbent assay kit for detecting concentration of tumor marker DKK1 |
| CN105203773A (en) * | 2015-09-28 | 2015-12-30 | 成都博奥新景医学科技有限公司 | Quantitative detection kit for human Dickkopf-1 protein (DKK-1) |
| CN109085355A (en) * | 2017-06-13 | 2018-12-25 | 中国医学科学院肿瘤医院 | Serum protein markers combine the application in screening lung cancer and diagnosis and treatment |
| CN109959796B (en) * | 2017-12-26 | 2022-05-10 | 复旦大学附属华山医院 | Human serum DKK1 enzyme-linked aptamer adsorption detection kit |
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Address after: 510006 Guangdong City, Guangzhou province outside the University of East Ring Road, No. 280 Patentee after: Guangdong Pharmaceutical University Address before: 510006 Guangdong City, Guangzhou province outside the University of East Ring Road, No. 280 Patentee before: Guangdong Pharmaceutical University |