CN102507951A

CN102507951A - Enzyme-linked immuno sorbent assay (ELISA) kit for performing joint detection on tumor marker

Info

Publication number: CN102507951A
Application number: CN2011103808350A
Authority: CN
Inventors: 臧林泉; 刘大路; 潘雪刁
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2011-11-25
Filing date: 2011-11-25
Publication date: 2012-06-20
Anticipated expiration: 2031-11-25
Also published as: CN102507951B

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

A kind of enzyme-linked immunologic detecting kit of joint-detection tumor markers

Technical field

The present invention relates to the 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 the most common deadly number is maximum in the world wide, and its incidence of disease growth rate also is in first of each malignant tumour, because the concealment of lung cancer onset.Still lack examination effectively and method of early diagnosis at present; It is to be mostly late period that symptom appears in the patient, and prognosis is relatively poor, must survive in 5 years in being no more than 15%; The symptom is arranged 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%.Methods such as traditional clinically diagnostic method such as rabat, bronchoscope, phlegm cytology checking.But lack enough specificitys and sensitivity.Therefore, be badly in need of a kind of method that can be directed against the lung cancer early diagnosis clinically.At present, be by enzyme linked immunological kit for the more simple and efficient method of lung cancer early diagnosis, and all be to detect both at home and abroad to a certain mark of lung cancer for the kit of pulmonary cancer diagnosis; But lung cancer does not have a species specific marker for lung cancer up till now; All be some relevant labels, owing to the lung cancer complicated classification, the expression of different pathological mark in period is different simultaneously; Just diagnose, can bring greatly mistaken diagnosis situation to a certain mark.

The enzyme linked immunological kit joint-detection that is adopted at present is to operate the ELISA Plate of several kinds of marks simultaneously, has reached the purpose of joint-detection, and this kind operation can bring very big personal error.

Summary of the invention

The objective of the invention is to according to problems such as the personal error that exists in the existing detection kit are big; A kind of kit that can be simultaneously on an ELISA Plate, two or three mark be carried out simultaneously detection by quantitative is provided; Can eliminate personal error, have advantage easy and simple to handle, with low cost concurrently.

Another purpose 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 through following technical scheme:

A kind of enzyme-linked immunologic detecting kit of joint-detection tumor markers; By the porous plate that is coated with neuron specific enolase thing enzyme (NSE), cytokeratin fragment 21-1 (CYFRA21-1) and Dkkopf 1 (DKK1) monoclonal antibody; Horseradish peroxidase target NSE, CYFRA21-1 and DKK1, chromogenic substrate TMB and substrate stop buffer are formed; The every hole of said porous plate encapsulates NSE monoclonal antibody 1 ~ 4ug/mL; Every hole encapsulates CYFRA21-1 monoclonal antibody 1 ~ 2ug/mL; Every hole encapsulates DKK1 monoclonal antibody 1 ~ 2ug/mL.NSE, CYFRA21-1, three kinds of detection property of DKK1 antibody add in the ELISA Plate hole in the 4:4:2 ratio, and cumulative volume is 100 uL.

As a kind of preferred version, in the above-mentioned porous plate, the coating buffer in every hole is 100uL.

As a kind of preferred version, the working concentration extension rate of said 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, contains 0.1% bovine serum albumin(BSA) and 0.1% thimerosal in the said phosphate buffer.

The preparation method of the enzyme-linked immunologic detecting kit of joint-detection tumor markers of the present invention comprises the steps: to prepare the porous plate that is coated with NSE, CYFRA21-1, DKK1 monoclonal antibody; , then above-mentioned NSE, CYFRA21-1, DKK1 monoclonal antibody dilution are joined in the hole NSE, CYFRA21-1, the dilution of DKK1 monoclonal antibody with pH 8 ~ 10 carbonate buffer solutions; 37 ℃ encapsulate 1h, and 5% bovine serum albumin solution is joined each hole of ELISA Plate, 37 ℃ of sealing 1 h; The sealing back is washed the version bat with phosphate buffer and is done, and it is 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 tumour correlating markings thing.

Compared with prior art, the present invention has following beneficial effect:

Kit according to the invention can detect two or three tumor associated antigen simultaneously, the specificity of detection and highly sensitive, and production cost is low.

Description of drawings

Fig. 1 is the synoptic diagram of ELISA Plate coated antibody in the detection kit of the present invention;

Fig. 2 is the stability analysis figure of detection kit of the present invention.

Embodiment

Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.

The preparation process of embodiment 1 kit

(1) dilute NSE, CYFRA21-1, three kinds of detection property of DKK1 antibody in the 4:4:2 ratio adding ELISA Plate hole with carbonate buffer solution, cumulative volume is 100 uL, places 37 ℃, wrapper sheet 2h.

(2) clap liquid in the dry plate, add cleansing solution and 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; The sealing back is washed the version bat with phosphate buffer and is done, and it is stored in 4 ℃.

(4) clap liquid in the dry plate, add cleansing solution and wash plate, every hole 200uL, each 30s washs 5 times.

(5) vacuumize with Fresco Bag, 4 ℃ of placements are subsequent use.

Embodiment 2 double antibodies sandwich method enzyme linked immunological kits detect NSE, CK19, three kinds of antigens of DKK1

(1) add different serum to be checked in the reacting hole respectively, 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, places 37 ℃, 1h.

(3) each hole adds the enzyme labelled antibody after diluting, every hole 100uL, 37 ℃, incubation 1h.

(5) each hole adds colour developing liquid TMB, every hole 100uL, and 37 ℃ of reaction 10 min add stop buffer TMB, 450nm reading (Fig. 1).

Embodiment 3 double antibodies sandwich ELISA methods repeatability is 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, detect with the double antibodies sandwich method diagnostic kit of 6 parts of different batches, the parallel survey of every duplicate samples 3 holes are carried out kit tippet repeatability and are detected.

Table 2

Embodiment 3 kit stability analyses

Kit deposited in 37 ℃ of environment preserve, regularly detect, know till the maximal value drop by half of its quality index, it has been generally acknowledged that to deposit to be equivalent to 4-10 ℃ in one day and to deposit 1.5 months at 37 ℃.At least kept 7 days like 37 ℃ of this kits of table analysis, be equivalent to and 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

Confirming of embodiment 5 kit gray areas

(1) take out 4 ℃ of subsequent use kits, the patients serum of the normal disease of 48 routine normal persons and 4 routine lungs 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, places 37 ℃, 1h.

(5) each hole adds colour developing liquid TMB, every hole 100uL, and 37 ℃ of reaction 10 min add stop buffer TMB, the 450nm reading.

It is following with multispecific antibody pulmonary cancer diagnosis kit measurement result to get 88 normal human serums at random:

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 then gray area OD value scope be exactly between 0 to 0.3847, meet the 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

1. the enzyme-linked immunologic detecting kit of a joint-detection tumor markers; It is characterized in that by the porous plate that is coated with NSE, CYFRA21-1 and DKK1 monoclonal antibody; Horseradish peroxidase target NSE, CYFRA21-1 and DKK1, chromogenic substrate TMB and substrate stop buffer are formed; The every hole of said porous plate encapsulates NSE monoclonal antibody 1 ~ 4ug/mL; Every hole encapsulates CYFRA21-1 monoclonal antibody 1 ~ 2ug/mL; Every hole encapsulates DKK1 monoclonal antibody 1 ~ 2ug/mL, and NSE, CYFRA21-1, three kinds of detection property of DKK1 antibody add in the ELISA Plate hole in the 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 the said porous plate that the coating buffer in every hole is 100uL.

3. the enzyme-linked immunologic detecting kit of joint-detection tumor markers according to claim 1, its characteristic are that also the working concentration extension rate of said horseradish peroxidase target NSE, CYFRA21-1 and DKK1 is 1:500 ~ 1:2500.

4. the enzyme-linked immunologic detecting kit of joint-detection tumor markers according to claim 1; Its characteristic is that also enzyme conjugates protection liquid is obtained by the phosphate buffer dilution of 0.01mol/L, pH7.4, contains 0.1% bovine serum albumin(BSA) and 0.1% thimerosal in the said phosphate buffer.

5. the preparation method of the enzyme-linked immunologic detecting kit of any said joint-detection tumor markers of claim in the claim 1 ~ 4 is characterized in that comprising the steps: to prepare the porous plate that is coated with NSE, CYFRA21-1, DKK1 monoclonal antibody; , then above-mentioned NSE, CYFRA21-1, DKK1 monoclonal antibody dilution are joined in the hole NSE, CYFRA21-1, the dilution of DKK1 monoclonal antibody with pH 8 ~ 10 carbonate buffer solutions; 37 ℃ encapsulate 1h, and 5% bovine serum albumin solution is joined each hole of ELISA Plate, 37 ℃ of sealing 1 h; The sealing back is washed the version bat with phosphate buffer and is done, and it is stored in 4 ℃.

6. the application of enzyme-linked immunologic detecting kit in the detection of tumour correlating markings thing of any said joint-detection tumor markers of claim in the claim 1 ~ 4.