CN108802388A - A kind of probe reagent box and its application method for the label detection of colon cancer high throughput - Google Patents

Abstract

The invention discloses a probe kit for high-throughput marker detection of colon cancer, comprising biotin-labeled ND-1 monoclonal antibody, horseradish catalase-labeled antibody, and streptavidin-coated enzyme Target body; the biotin-labeled ND-1 monoclonal antibody is prepared by labeling the monoclonal antibody with biotin aminoacetyl hydrazide through the sugar chain; the horseradish catalase-labeled antibody is prepared by using glutaraldehyde two-step method Labeled monoclonal antibodies were prepared. The present invention also provides a method for using the above-mentioned probe kit. By adopting square matrix titration to complete high-throughput colon cancer diagnosis, compared with the prior art, the sensitivity and accuracy of detection and diagnosis are higher, and the operation of detection and diagnosis The method is simpler and faster, and the requirements for the professional quality of the operators are relatively low.

Description

A probe kit for high-throughput marker detection of colon cancer and its application method

technical field

The invention belongs to the field of colon cancer diagnosis, and in particular relates to a probe kit for high-throughput marker detection of colon cancer and a use method thereof.

Background technique

In the existing technology, for example, a non-invasive high-throughput methylation colon cancer diagnosis, research and treatment method with patent number 201710550895.X is based on the application of next-generation sequencing technology based on gene methylation, through cfRNA and methyl The establishment of next-generation sequencing libraries and the enrichment of target sites can be realized by the combination of chemical profiles, which can detect tumors in specific organs or parts of the human body at an early stage.

Although this method has high diagnostic sensitivity and accuracy, it is complex to operate and requires high professional skills for instruments, equipment, and operators, making it difficult to apply to general enterprises or scientific research institutions.

Contents of the invention

In view of the above deficiencies in the prior art, the present invention provides a probe kit for high-throughput marker detection of colon cancer, which is specifically realized through the following technologies.

A probe kit for high-throughput marker detection of colon cancer, including biotin-labeled ND-1 monoclonal antibody, horseradish catalase-labeled antibody, and streptavidin-coated microplate body;

The biotin-labeled ND-1 monoclonal antibody is prepared by labeling the monoclonal antibody with biotin aminoacetyl hydrazide through the sugar chain; the horseradish catalase-labeled antibody is prepared by using the glutaraldehyde two-step method to label the monoclonal antibody Prepared.

Preferably, the monoclonal antibody is inoculated into the peritoneal cavity of a mouse by expanding the cell line that secretes the monoclonal antibody stably, so that it proliferates in the mouse in the form of ascites tumor, thereby obtaining a large amount of ascites containing the monoclonal antibody; Then it is prepared by vacuum freeze-drying.

Preferably, when the biotinylated ND-1 monoclonal antibody is prepared, the molecular ratio of biotinaminoacetylhydrazide to monoclonal antibody is 200:1.

Preferably, the preparation method of the glutaraldehyde-labeled monoclonal antibody of the horseradish catalase-labeled antibody is:

Weigh 25 mg of horseradish catalase and dissolve it in 1.25% glutaraldehyde solution, let it stand at room temperature overnight, then elute it with normal saline through a Sephadex G-25 chromatography column, and collect the brown effluent;

Dilute 12.5 mg of monoclonal antibody to 5 ml with normal saline, add dropwise to the brown effluent with stirring, then add 0.25 ml of carbonic acid buffer solution with a concentration of 1M and a pH value of 9.5, and continue stirring for 3 hours to obtain solution I;

Add 0.25ml of lysine with a concentration of 0.2M to the solution I, mix it at room temperature and let it stand for 2 hours, then add 10mL of saturated ammonium sulfate solution dropwise while stirring, and let it stand at 4°C for 1 hour to obtain solution II;

The obtained solution II was centrifuged at 3000 rpm for 0.5 h, the precipitate was washed twice with half-saturated ammonium sulfate, and then dissolved in a phosphate buffer solution with a concentration of 0.15M and a pH value of 7.4 to obtain a solution III;

Put the obtained solution III into a dialysis bag, dialyze with phosphate buffer buffer, remove ammonium ions, and then centrifuge at 10,000 rpm for 30 min to remove the precipitate, and take the supernatant and freeze it to obtain the horseradish catalase-labeled antibody.

Preferably, the preparation method of the elisa plate body coated with streptavidin is as follows: dissolve streptavidin in 0.02M carbonic acid solution with a pH value of 9.5, and the concentration is 10 mg/L; then add 96 wells 100 μL per well of the polystyrene ELISA plate, overnight at 4°C; then wash 3 times with 0.01M Tween-20 phosphate buffer with a pH value of 7.4, soaking for 3 minutes each time; finally add 100 μL serum albumin and stand at 4°C Overnight; repeat the above method 3 times and then dry it.

The present invention also provides a method for using a probe kit for high-throughput marker detection of colon cancer, comprising the following steps:

S1. Dilute biotin-labeled ND-1 monoclonal antibody and horseradish catalase-labeled antibody with 50 mM 2-[N-morpholine]-ethanesulfonic acid buffer, and then mix to obtain mixed solution I for later use;

S2. Add 50 μL of the sample to be tested to each well of the streptavidin-coated ELISA plate body, then add 50 μL of mixed solution I, incubate at 37° C. for 2 h, and then wash with 0.02 M Tris-Tween;

S3. Add 2,2-azino-bis[3-ethylbenzothiazolinesulfonic acid-6]ammonium salt substrate solution, develop color at 37°C for 30min, and then terminate the reaction with 2M sulfuric acid;

S4. Measure the absorbance value at 405nm, use the culture supernatant of CCL187 cells as a positive control, and use the serum of a healthy person as a negative control to finally diagnose colon cancer.

Compared with the prior art, the present invention is beneficial in that: using the probe kit provided by the present invention, the sensitivity and accuracy of detection and diagnosis are higher, and the operation method of detection and diagnosis is simpler and faster, and the operator's labor is not affected. Professional quality requirements are relatively low.

Detailed ways

The technical solution of the present invention will be clearly and completely described below, obviously, the described embodiments are only some embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

This example provides a probe kit for high-throughput marker detection of colon cancer.

A probe kit for high-throughput marker detection of colon cancer, including biotin-labeled ND-1 monoclonal antibody, horseradish catalase-labeled antibody, and streptavidin-coated microplate body;

The biotin-labeled ND-1 monoclonal antibody is prepared by labeling the monoclonal antibody with biotinaminoacetylhydrazide through sugar chains, and the molecular ratio of biotinaminoacetylhydrazide to monoclonal antibody is 200:1.

The horseradish catalase-labeled antibody is prepared by using the glutaraldehyde two-step method to label the monoclonal antibody, and the preparation method is as follows:

Weigh 25 mg of horseradish catalase and dissolve it in 1.25% glutaraldehyde solution, let it stand at room temperature overnight, then elute it with normal saline through a Sephadex G-25 chromatography column, and collect the brown effluent;

Dilute 12.5 mg of monoclonal antibody to 5 ml with normal saline, add dropwise to the brown effluent with stirring, then add 0.25 ml of carbonic acid buffer solution with a concentration of 1M and a pH value of 9.5, and continue stirring for 3 hours to obtain solution I;

Add 0.25ml of lysine with a concentration of 0.2M to the solution I, mix it at room temperature and let it stand for 2 hours, then add 10mL of saturated ammonium sulfate solution dropwise while stirring, and let it stand at 4°C for 1 hour to obtain solution II;

The obtained solution II was centrifuged at 3000 rpm for 0.5 h, the precipitate was washed twice with half-saturated ammonium sulfate, and then dissolved in a phosphate buffer solution with a concentration of 0.15M and a pH value of 7.4 to obtain a solution III;

Put the obtained solution III into a dialysis bag, dialyze with phosphate buffer buffer, remove ammonium ions, and then centrifuge at 10,000 rpm for 30 min to remove the precipitate, and take the supernatant and freeze it to obtain the horseradish catalase-labeled antibody.

The monoclonal antibody is to inoculate the cell line stably secreting the monoclonal antibody in the peritoneal cavity of the mouse through expanded culture, so that it proliferates in the mouse in the form of ascites tumor, thereby obtaining a large amount of ascites containing the monoclonal antibody; Prepared by freeze-drying.

The preparation method of the elisa plate body coated with streptavidin is: the streptavidin is dissolved in the 0.02M carbonic acid solution of pH value 9.5, and concentration is 10mg/L; Then add 96 hole polystyrene 100 μL per well of the ELISA plate, overnight at 4°C; then wash 3 times with 0.01M Tween-20 phosphate buffer with a pH value of 7.4, soaking for 3 minutes each time; finally add 100 μL serum albumin and let stand overnight at 4°C; Repeat the above method 3 times and then dry it.

Application example: Sensitivity and effectiveness analysis of the qualitative diagnosis of colon cancer using the probe kit for high-throughput marker detection of colon cancer in Example 1

1. Clinical materials

60 cases of colorectal cancer were confirmed by pathological diagnosis, including 25 cases of well-differentiated, 30 cases of moderately differentiated, and 5 cases of poorly differentiated; Dukes stage: 10 cases of stage I, 22 cases of stage II, 13 cases of stage III, and 15 cases of stage IV. 40 cases of other various diseases.

The method for the qualitative diagnosis of colon cancer by the probe kit of embodiment 1 is:

2. Statistical processing

Calculate the sample mean and standard deviation. Sensitivity, specificity and validity of detection. ￡ test for the sample mean, u test for the overall rate, and ^x2 test for independence.

3. The activity of each reagent in the kit

The ascites titer obtained was 1:800. When the purified concentration is 1mg/ml, the antibody titer is about 1:10000. When the concentration is 1mg/ml, the titer of biotin-labeled antibody is 1:1600, and the titer of enzyme-labeled antibody is 1:7000. The molecular ratio of enzyme to antibody was 3.2; the working concentrations of detection antibody and capture antibody were 0.6mg/L and 1.25mg/L, respectively.

4. Perform detection and diagnosis according to the use method of the following kits

S1. Dilute biotin-labeled ND-1 monoclonal antibody and horseradish catalase-labeled antibody with 50 mM 2-[N-morpholine]-ethanesulfonic acid buffer, and then mix to obtain mixed solution I for later use;

S2. Add 50 μL of the sample to be tested to each well of the streptavidin-coated ELISA plate body, then add 50 μL of mixed solution I, incubate at 37° C. for 2 h, and then wash with 0.02 M Tris-Tween;

S3. Add 2,2-azino-bis[3-ethylbenzothiazolinesulfonic acid-6]ammonium salt substrate solution, develop color at 37°C for 30min, and then terminate the reaction with 2M sulfuric acid;

S4. Measure the absorbance value at 405nm, use the culture supernatant of CCL187 cells as a positive control, and use the serum of a healthy person as a negative control to finally diagnose colon cancer.

5. Sensitivity and effectiveness analysis of colorectal cancer patients with different degrees of differentiation and different Dukes stages The analysis results of sensitivity and effectiveness are shown in Table 1 below.

Table 1 Diagnosis of cell membrane surface antigens in colorectal cancer patients with different Dukes stages and differentiation degrees

It can be seen from the above table that the sensitivity of colon cancer-associated cell membrane surface antigens in the diagnosis of well-differentiated and moderately differentiated colorectal cancers is significantly higher than that of poorly differentiated colorectal cancers, and the difference is significant. The sensitivity and effectiveness of this antigen for the diagnosis of colorectal cancer patients with different Dukes stages did not change with the changes of Dukes stages.

Claims (6)

1. a kind of probe reagent box for the label detection of colon cancer high throughput, which is characterized in that including biotin labeling ND-1 Monoclonal antibody, horseradish peroxidase labelled antibody and the coated ELISA Plate body of Streptavidin；

The biotin labeling ND-1 monoclonal antibodies are to be prepared labeling of monoclonal antibody by sugar chain using biotin aminoacethydrazide 's；The horseradish peroxidase labelled antibody is prepared using glutaraldehyde two step method labeled monoclonal antibody.

2. a kind of probe reagent box for the label detection of colon cancer high throughput according to claim 1, which is characterized in that The monoclonal antibody is to be inoculated in the cell strain of stably excreting monoclonal antibody in mouse peritoneal by expanding culture, makes it It is proliferated in mouse in the form of ascites tumor, to obtain the largely ascites containing monoclonal antibody；Then through vacuum freeze drying system It is standby to form.

3. a kind of probe reagent box for the label detection of colon cancer high throughput according to claim 1, which is characterized in that In the preparation, the molecular proportion of biotin aminoacethydrazide and monoclonal antibody is 200 to the biotin labeling ND-1 monoclonal antibodies：1.

4. a kind of probe reagent box for the label detection of colon cancer high throughput according to claim 1, which is characterized in that The preparation method of the glutaraldehyde two step method labeled monoclonal antibody of the horseradish peroxidase labelled antibody is：

It weighs horseradish peroxidase 25mg to be dissolved in 1.25% glutaraldehyde solution, be stayed overnight in being stored at room temperature, then pass through SephadexG-25 chromatographic columns are eluted with physiological saline, collect brown efflux；

It takes 12.5mg monoclonal antibodies normal saline dilution to 5ml, is added dropwise under stirring in the brown efflux, then add Enter a concentration of 1M of 0.25ml, the carbonic acid buffer of pH value 9.5 continues stirring 3h and obtains solution I；

Add the lysine of a concentration of 0.2M of 0.25ml in the solution I, mixing stands 2h at room temperature, then adds dropwise while stirring Enter 10mL saturated ammonium sulfate solution, standing 1h at 4 DEG C obtains solution II；

Acquired solution II is centrifuged into 0.5h at 3,000 rpm, taking precipitate is washed secondary with semi-saturation ammonium sulfate, is re-dissolved in a concentration of 0.15M, in the phosphate buffer of pH value 7.4 solution III；

Acquired solution III is fitted into bag filter, is dialysed with phosphate buffer buffer solution, 10000rpm is centrifuged after removing ammonium ion 30min removal precipitations, take supernatant freezen protective, as horseradish peroxidase labelled antibody.

5. a kind of probe reagent box for the label detection of colon cancer high throughput according to claim 1, which is characterized in that The preparation method of the coated ELISA Plate body of Streptavidin is：The 0.02M carbonic acid that Streptavidin is dissolved in pH value 9.5 is molten In liquid, a concentration of 10mg/L；Then 96 hole polystyrene ELISA Plates are added per 100 μ L of hole, 4 DEG C overnight；PH value 7.4 is used again 0.01M Tween-20 phosphate buffers are washed 3 times, impregnate 3min every time；It is eventually adding at 100 4 DEG C of μ L seralbumins and stood Night；It is dried after being repeated 3 times according to the method described above to obtain the final product.

6. the application method described in any one of Claims 1 to 4 for the probe reagent box of colon cancer high throughput label detection, It is characterized in that, includes the following steps：

S1, with 2- [N- morpholines]-ethanesulfonic acid buffer of 50mM respectively by biotin labeling ND-1 monoclonal antibodies, horseradish hydrogen peroxide Enzymic-labelled antibody dilute, remix mixed liquor I is spare；

S2,50 μ L samples to be tested are added per hole in the coated ELISA Plate body of the Streptavidin, then add 50 μ L mixed liquors I, 2h is incubated at 37 DEG C, is then washed with the Tris- tweens of 0.02M；

S3 plus 2,2- azine groups-two [3- ethylbenzthiazoline sulfonates -6] ammonium salt substrate solution, develop the color 30min at 37 DEG C, then It is terminated and is reacted with 2M sulfuric acid；

S4,405nm absorbance values are surveyed, is negative right with Healthy Human Serum using CCL187 cell culture supernatants as positive control According to last diagnostic colon cancer.