CN111077312A - Application of group of tumor-associated antigens in preparation of cardiac cancer early screening kit - Google Patents

Abstract

The invention belongs to the fields of molecular biology and oncology, and particularly relates to application of a group of tumor-associated antigens in preparation of a cardiac cancer early screening kit. The combination of tumor-associated antigens CD38, CCND1, RB1, KMT2D, FAT1 and EP300 for preparing the cardiac cancer early screening kit. The ELISA kit for screening the early cardia cancer comprises a solid phase carrier and tumor-associated antigens coated on the solid phase carrier, wherein the tumor-associated antigens are CD38, CCND1, RB1, KMT2D, FAT1 and EP 300. The invention uses the six tumor-associated antigens of CD38, CCND1, RB1, KMT2D, FAT1 and EP300 as a combination for the first time to detect the cardiac cancer, especially the early cardiac cancer, and the detection sensitivity of the kit is 94.74 percent and the specificity is 84.21 percent.

Description

Application of group of tumor-associated antigens in preparation of cardiac cancer early screening kit

Technical Field

The invention belongs to the fields of molecular biology and oncology, and particularly relates to application of a group of tumor-associated antigens in preparation of a cardiac cancer early screening kit.

Background

With the development of society and the change of environment, cardiac cancer has become a malignant tumor that seriously threatens the health and safety of human life. Cardia cancer refers to cancer occurring in the anatomical region of the cardia, and is generally considered to be located within 2cm below the junction of the esophagus and the stomach in China. The city of Linzhou, Henan province is one of the high-incidence regions of esophageal cancer in China, and researches show that cardiac cancer is high in incidence in the high-incidence regions of esophageal cancer. This phenomenon suggests that in the local living environment, strong carcinogenic factors may exist, which act on the mucosa of the upper digestive tract for a long time, and lead to high incidence of both esophageal cancer and cardiac cancer through some similar mechanisms.

Traditionally, "asymptomatic people with high incidence area, over 40 years old, male, smoking, drinking and positive family history" is generally defined as "people with high risk or high risk of cardiac cancer", and is also a main object for early screening of cardiac cancer. At present, endoscopy, mucosal biopsy and histopathology examination, particularly the general survey and follow-up of asymptomatic residents in high incidence areas, still remain the most effective means for finding patients with early cancer and precancerous lesions. However, with the expansion of the screening range, the methods expose some problems, which restrict the further popularization of the screening, such as the detection rate and the present disease rate of the endoscopic screening, the compliance of the examined person, the endoscopic screening technology and the histopathological diagnosis level have close relations. The lack of effective biological indexes and methods for early warning and early diagnosis of high risk groups in the prior art is a main reason for causing the late time of clinic cardiac cancer patients and high mortality.

The occurrence and development of cardia cancer are a multi-factor, multi-stage and slow development process, and a research team in the leaders of the professor Wanglidong of the first subsidiary hospital of Zhengzhou university finds that the cardia cancer is determined as a patient with precancerous lesion through endoscopic biopsy, and the digestive tract mucosa of the population can be further converted into cancer from atypical hyperplasia (precancerous lesion); however, some people can stop when they progress to a certain stage, and do not progress further to malignancy, and even can change from precancerous lesion to normal epithelial tissue, which suggests that cardiac precancerous lesion has the characteristic of bidirectional development. Cardiac cancer has oncogene activation and oncogene inactivation during the onset of cancer, and these molecular biological changes promote the development and progression of cancer cells.

In recent years, it has been discovered that cancer cells synthesize and release a unique set of autocrine antigens, i.e., Tumor Associated Antigens (TAAs), during their development and progression. Some of the TAAs have been used for clinical auxiliary diagnosis, however, cardiac cancer specific associated antigen kits that can be used for clinical diagnosis are still very lacking.

On the basis of a genomic database established by technologies such as whole genome sequencing and whole genome exon sequencing in the early stage, the research team screens out six tumor-associated antigen (TAA) autoantibodies by using an autoantibody chip technology, wherein the six TAAs are CD38, CCND1, RB1, KMT2D, FAT1 and EP300 respectively. The proteins are all related to early-stage cardiac cancer and precancerous lesion, and the detection of the change of the autoantibody by combining a plurality of tumor-associated antigens is favorable for playing a certain early warning role for asymptomatic high-risk population of cardiac cancer. However, the use of multiple autoantibody combination detection methods and corresponding kits for early diagnosis of cardiac cancer is still rare. Therefore, the invention provides an ELISA kit for early diagnosis of cardiac cancer, which can effectively detect cardiac cancer, especially early cardiac cancer.

Disclosure of Invention

In view of the problems and deficiencies of the prior art, the present invention is directed to a novel use of a panel of tumor antigens, and it is another object of the present invention to provide a kit made with the panel of tumor associated antigens.

Based on the above purpose, the technical scheme adopted by the invention is as follows:

the application of the combination of tumor-associated antigens CD38, CCND1, RB1, KMT2D, FAT1 and EP300 in preparing an early cardiac cancer screening kit.

An ELISA kit for screening early cardiac cancer comprises a solid phase carrier and tumor-associated antigens coated on the solid phase carrier, wherein the tumor-associated antigens are CD38, CCND1, RB1, KMT2D, FAT1 and EP 300.

Further, the kit further comprises a sample diluent, a second antibody diluent, positive control serum, negative control serum, a developing solution, a stop solution and a washing solution; both the sample diluent and the second antibody diluent were PBST buffer containing 1% (W/V) BSA; the color development liquid consists of a color development liquid A and a color development liquid B, wherein the color development liquid A is 0.02% (W/V) TMB (3, 3,5, 5' -tetramethylbenzidine), and the color development liquid B is 0.006% (W/V) urea peroxide; the termination solution is concentrated sulfuric acid with the weight percent of 10 percent; the wash was PBST buffered solution containing pH =7.4 and 0.05 v% tween-20.

Further, the second antibody carries a detectable label.

Further, the label is horseradish peroxidase.

Further, the second antibody is RecA protein.

Further, the positive control serum is KMT2D positive control serum, and the negative control serum is KMT2D negative control serum.

Further, the KMT2D positive control serum is cardiac cancer patient serum with positive KMT2D antibodies detected by indirect ELISA and Western blot method; the KMT2D negative control serum is normal human serum with KMT2D antibody expression level detected by indirect ELISA and Western blot method as the average content of antibody in normal human serum.

Further, the solid phase carrier is an enzyme label plate.

Further, the detection object of the ELISA kit is human serum.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention takes the six tumor-related antigens of CD38, CCND1, RB1, KMT2D, FAT1 and EP300 as a combination for the first time, jointly detects the antibody expression levels of the six TAAs in human serum, can effectively detect the cardiac cancer, particularly the early cardiac cancer, has the detection sensitivity of 94.74 percent (namely the ratio of correctly diagnosing the early cardiac cancer by using the 6 tumor-related antigens in the early cardiac cancer patients is 94.74 percent), and has the specificity of 84.21 percent (namely the ratio of the patients without cardiac cancer determined to be 84.21 percent when using the six tumor-related antigens for jointly detecting the non-cardiac cancer patients is higher than the detection rate of the cardiac cancer screened by the existing clinical endoscope, therefore, the ELISA kit has higher sensitivity and specificity, can be used for screening asymptomatic people in the cardiac cancer high incidence area in large scale, and can greatly improve the detection rate of the early cardiac cancer, is beneficial to screening and early discovery of asymptomatic high risk population, thereby greatly reducing the mortality of cardia cancer patients.

(2) The ELISA kit prepared by the invention can synchronously detect the expression levels of six cardia cancer related antibodies in a serum sample, and compared with the method for singly detecting a certain TAA antibody, the six TAA antibodies are jointly detected, so that the detection success rate is high, the technical reproducibility is good, the material consumption is less, the cost is low, the operation is simple, the use is convenient and quick, the detection efficiency and the diagnosis efficiency of clinical cardia cancer are greatly improved, and the kit can be popularized and used in common laboratories.

Drawings

FIG. 1 is a schematic diagram of an indirect enzyme-linked immunosorbent assay;

FIG. 2 is a diagram showing the antigen coating layout of a 48-well ELISA plate in an ELISA kit according to the present invention (wherein the name of the antigen indicates that the well is coated with the antigen, to-be-detected serum is added for detecting the expression level of the corresponding antibody in the to-be-detected serum, "+" indicates a positive control well to which a positive control serum is added, "-" indicates a negative control well to which a negative control serum is added, and "blank" indicates a blank control well to which a sample diluent without serum is added, and the other operations are the same, and the blank control is used for the background value in the reaction experiment);

FIG. 3 is a scattergram showing the relative expression amounts of autoantibodies to tumor-associated antigens in the sera of the cardiac cancer group and the control group;

FIG. 4 is a ROC curve of the detection of early cardiac cancer by the combination of six tumor-associated antigens with autoantibodies.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.

The experimental procedures described in the following examples, unless otherwise specified, are conventional in the art or according to the conditions recommended by the manufacturers; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.

The invention prepares an autoantibody combined detection ELISA kit for screening early cardiac cancer according to the principle of indirect enzyme-linked immunosorbent assay. The principle of indirect enzyme-linked immunosorbent assay is shown in figure 1, and comprises the steps of linking an antigen to a solid phase carrier, combining an antibody to be detected in a sample with the solid phase carrier to form a solid phase antigen-detected antibody complex, combining an enzyme-labeled secondary antibody with an antibody in the solid phase antigen-detected antibody complex to form the solid phase antigen-detected antibody-enzyme-labeled secondary antibody complex, and then measuring the color development degree after adding a substrate (color development agent) to determine the content of the antibody to be detected.

EXAMPLE 1 preparation of the kit

1. Experimental materials and reagents:

(1) six tumor-associated antigen proteins (CD 38, CCND1, RB1, KMT2D, FAT1, EP 300) purchased from Wuhan Eimei technologies, Inc.;

(2) 48-hole enzyme label plate: purchased from Corning corporation;

(3) coating liquid: 50mM carbonate buffer, pH = 9.6;

(4) sealing liquid: PBST buffer containing 2% (W/V) BSA;

(5) sample diluent: PBST buffer containing 1% (W/V) BSA;

(6) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;

(7) enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein (Invitrogen corporation);

(8) washing liquid: PBST buffered solution pH =7.4 and 0.05 v% tween-20.

(9) Positive control serum: KMT2D positive control serum, namely cardia cancer patient serum with positive KMT2D antibody detected by indirect ELISA and Western blot;

(10) negative control serum: KMT2D negative control serum, namely normal human serum with KMT2D antibody expression level equal to the average content of normal human serum antibody by indirect ELISA and Western blot detection;

(11) color developing solution A: 0.02% (W/V) TMB, formulation: dissolving 0.005g of methylbenzidine (TMB) in 25mL of deionized water;

(12) color developing solution B: 0.006% (W/V) of urea peroxide, formulation: 4.665g of citric acid and 4.40g of Na2HPO418.40g of citric acid are fully dissolved in 400mL of deionized water, 3.2mL of 0.75% urea hydrogen peroxide is added, the pH value is adjusted to 5.0-5.5, deionized water is added to the solution until the volume reaches 500mL, and the solution is uniformly mixed and stored at 4 ℃;

(13) stopping liquid: 10 wt% of concentrated sulfuric acid;

(14) an enzyme-labeling instrument: star Fax 2100 (aware. us).

2. Preparing an antigen-coated ELISA plate:

(1) six tumor-associated antigen solutions were prepared:

dissolving the six tumor-associated proteins in the coating solution respectively, and mixing to obtain six antigen solutions. Wherein the concentration of the CD38 solution is 0.125 mu g/mL, the concentration of the CCND1 solution is 0.25 mu g/mL, the concentration of the RB1 solution is 1.0 mu g/mL, the concentration of the FAT1 solution is 1.0 mu g/mL, the concentration of the EP300 solution is 0.75 mu g/mL, and the concentration of the KMT2D solution is 0.35 mu g/mL.

(2) Coating an enzyme label plate:

respectively adding the prepared six tumor-associated antigen solutions into sample application holes of a 48-hole enzyme label plate according to the layout shown in figure 2, wherein the sample application amount is 100 mu L/hole; KMT2D antigen solution is added into the positive control well and the negative control well, coating solution is added into the blank control well, the positive control well and the negative control well are incubated for 1h at 37 ℃, and the coating solution is removed after overnight incubation at 4 ℃.

(3) And (3) sealing:

adding a sealing solution into the sample application holes of the coated 48-hole enzyme label plate, wherein the sample application amount is 300 mu L/hole, incubating for 2 hours at room temperature, and then removing the sealing solution.

(4) And (3) drying and packaging:

and (3) placing the 48-hole ELISA plate subjected to sealing treatment in a 37 ℃ drying box for drying, and then packaging to obtain the antigen-coated 48-hole ELISA plate, and storing at 4 ℃ for later use.

3. The kit comprises the following components:

(1) the 48-hole enzyme label plate coated by the antigen prepared in the step 2;

(2) sample diluent: PBST buffer containing 1% (W/V) BSA;

(3) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;

(4) enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein (Invitrogen corporation);

(5) color development liquid: the color development liquid consists of a color development liquid A and a color development liquid B, wherein the color development liquid A is 0.02% (W/V) TMB, and the color development liquid B is 0.006% (W/V) urea peroxide; when in use, the color development liquid A and the color development liquid B are uniformly mixed in equal volume according to the ratio of 1: 1;

(6) stopping liquid: 10 wt% of concentrated sulfuric acid;

(7) washing liquid: PBST buffer solution pH =7.4 and containing 0.05 v% tween-20;

(8) positive control serum: KMT2D positive control serum;

(9) negative control serum: KMT2D negative control serum;

and the reagents (2) - (9) are packaged respectively and then form a kit with the antigen-coated ELISA plate.

Example 2: method of using kit

1. Incubation of serum samples:

and (3) diluting the serum sample to be detected with a sample diluent according to the ratio of 1: 500, adding the diluted serum sample into a reaction hole of a 48-hole enzyme label plate coated with the antigen, wherein the sample adding amount is 100 mu L/hole, placing the reaction hole in a constant-temperature incubator at 37 ℃ for incubation for 1h, then discarding the liquid in the reaction hole, and washing the reaction hole for 3 times and 3min each time by using a washing solution.

2. Incubation with enzyme-labeled secondary antibody:

horseradish peroxidase-labeled RecA protein was diluted with secondary antibody at 1: 40000, adding the diluted RecA protein labeled by horseradish peroxidase into reaction wells of a 48-well enzyme label plate, adding the protein at a loading rate of 100 mu L/well, incubating in a constant-temperature incubator at 37 ℃ for 50min, discarding the liquid in the wells, and washing with a washing solution for 3 times, 3min each time.

3. Color development and termination reaction:

and (3) uniformly mixing the color development liquid A and the color development liquid B in an equal volume according to a ratio of 1:1, then quickly adding the mixed color development liquid into reaction holes of a 48-hole enzyme label plate, wherein the sample addition amount is 100 mu L/hole, placing the plate at 37 ℃ in a dark place for reaction for 15min, then adding 50 mu L of stop solution into each reaction hole, stopping the reaction, reading the OD450 optical density value by using an enzyme label instrument within 20 min, and zeroing by using a blank control hole.

4. And (4) judging a result:

the Mean of the OD values in the positive control well and the negative control well plus two standard deviations (Mean +2 SD) were used as a cutoff value (Cut off value), and above this value, positive was judged, and below this value, negative was judged.

Example 3: diagnostic value analysis of the kit of the invention

The kit of the embodiment 1 of the invention is used for detecting serum samples of patients with early cardiac cancer and normal persons so as to evaluate and analyze the value of the kit for screening and diagnosing early cardiac cancer.

1. Sample source

Serum samples from an esophageal cancer focus open laboratory of Henan province, a first subsidiary hospital of Zhengzhou university were collected, wherein the serum of 190 parts (control group) of normal persons and 190 parts (early gastric cardia cancer group) of patients with early gastric cardia cancer were collected. 190 normal human sera were from healthy physical population in the laboratory cooperative hospital physical center without any evidence of tumor association. Of 190 normal persons, 108 men and 92 women had a mean age of 58.7 ± 9.2 years and an age range of 35-82 years. 190 sera of patients with early stage cardiac cancer were obtained from histopathologically confirmed patients with early stage (stage 0 + stage I) cardiac cancer who had not received radiotherapy or chemotherapy. Of 190 patients with cardiac cancer, 108 men and 92 women had a mean age of 58.5 ± 6.3 years, with the age range of 48-81 years.

2. Serum preparation

5mL of fasting venous blood is extracted into a centrifuge tube, kept stand for 30 minutes at room temperature, centrifuged (2000 rpm), and the upper serum is sucked and subpackaged, wherein each tube is 100 mu L, and stored in a refrigerator at minus 80 ℃.

3. Experimental methods

The relative expression levels of the six TAA autoantibodies in the sera of 190 normal persons (control group) and 190 cardia cancer patients (cardia cancer group) were determined using the kit prepared in example 1 and the kit used in example 2.

The results of using prism software to plot scattergrams of the relative expression amounts of six tumor-associated antigen autoantibodies in the early cardiac cancer group and the control group, etc. (see fig. 3) were statistically significant. The diagnostic value of autoantibodies for detecting cardiac cancer was then evaluated using the evaluation method of the screening test (see table 1 and fig. 4 for results).

4. Analysis of results

Table 1 shows the combined detection results of different tumor-associated antigen and autoantibody combinations, and it can be seen from table 1 that as the number of antigen combinations increases, the sensitivity of early cardiac cancer diagnosis increases with the number of antigen combinations; when six tumor-associated antigens are combined, the sensitivity is as high as 94.74%, and the specificity can still reach 84.21%, which indicates that the six tumor-associated antigens are ideal screening method and means for early stage cardiac cancer.

FIG. 3 shows the relative expression levels of six tumor-associated antigen autoantibodies in the serum of 190 cardiac cancer groups and 190 control groups, and it can be seen that the average expression levels of these six tumor-associated antigen autoantibodies in the early cardiac cancer group were relatively high, and the average relative expression levels were fluctuated around 0.6. The six tumor-associated antigen autoantibodies are expressed at a relatively low average level in the control group, and the average relative expression amount is about 0.1. As shown in FIG. 3, the mean serum levels of the six TAAs in the patients with early cardiac cancer were significantly higher than those in the control group, suggesting that the autoantibodies of the six TAAs can be used for screening early cardiac cancer.

FIG. 4 is a ROC curve of autoantibody detection of six tumor associated antigens for early stage cardiac cancer, wherein six points marked with numbers in FIG. 4 represent six different antigen combinations, respectively, and the numbers indicate the sensitivity of the corresponding combinations. 0.20 shows the detection sensitivity of autoantibodies against tumor associated antigens of CD38 antigen for 1 index for early stage cardiac cancer; 0.36 shows the detection sensitivity of autoantibody combinations of 2 indices CD38+ CCND1 tumor associated antigens for early stage cardiac cancer; 0.45 represents the detection sensitivity of the autoantibody combination of 3 indices CD38+ CCND1+ RB1 tumor-associated antigens to early cardiac cancer; 0.56 indicates the detection sensitivity of 4 indices of autoantibody combination of CD38+ CCND1+ RB1+ KMT2D tumor-associated antigen for early stage cardia cancer; 0.79 indicates the detection sensitivity of 5 kinds of autoantibody combinations indicating CD38+ CCND1+ RB1+ KMT2D + FAT1 tumor-associated antigens for early cardiac cancer; 0.95 shows the detection sensitivity of the combination of six tumor-associated antigens for autoantibodies against early stage cardiac cancer; these six points indicate that the area of the Roc curve formed is larger as the number of relevant antigens in the combination is larger.

As can be seen from FIG. 4, with the increase of the number of antigen combinations, the area under the ROC curve is increased significantly, which indicates that the six tumor-associated antigen autoantibody combined detection ELISA kits have higher judgment accuracy and diagnosis value for early cardiac cancer than any one, two, three, four or even five tumor-associated antigen autoantibody combined detection ELISA kits, and further confirms that the kit of the present invention can be used as an ideal screening method and means for early cardiac cancer.

Claims (10)

1. The application of the combination of tumor-associated antigens CD38, CCND1, RB1, KMT2D, FAT1 and EP300 in preparing an early cardiac cancer screening kit.

2. The ELISA kit for screening the early cardiac cancer is characterized by comprising a solid phase carrier and tumor-associated antigens coated on the solid phase carrier, wherein the tumor-associated antigens are CD38, CCND1, RB1, KMT2D, FAT1 and EP 300.

3. The ELISA kit for screening of early cardiac cancer according to claim 2, wherein the kit further comprises a sample diluent, a second antibody diluent, a positive control serum, a negative control serum, a developing solution, a stopping solution and a washing solution; both the sample diluent and the second antibody diluent were PBST buffer containing 1% (W/V) BSA; the color development liquid consists of a color development liquid A and a color development liquid B, wherein the color development liquid A is 0.02% (W/V) TMB, and the color development liquid B is 0.006% (W/V) urea peroxide; the termination solution is concentrated sulfuric acid with the weight percent of 10 percent; the wash was PBST buffered solution pH =7.4 and containing 0.05 v% tween-20.

4. The ELISA kit for screening of early cardiac cancer as claimed in claim 3 wherein the second antibody is labeled with a detectable marker.

5. The ELISA kit for screening early cardiac cancer according to claim 4 wherein the marker is horseradish peroxidase.

6. The ELISA kit for screening of early cardiac cancer according to claim 5 wherein the second antibody is RecA protein.

7. The ELISA kit for screening of early cardiac cancer as claimed in claim 6 wherein the positive control serum is KMT2D positive control serum and the negative control serum is KMT2D negative control serum.

8. The ELISA kit for screening of early cardiac cancer as claimed in claim 7 wherein the KMT2D positive control serum is cardiac cancer patient serum positive for KMT2D antibody by indirect ELISA and Western blot method; the KMT2D negative control serum is normal human serum with KMT2D antibody expression level detected by indirect ELISA and Western blot method as the average content of antibody in normal human serum.

9. The ELISA kit for screening of early cardiac cancer according to claims 3 to 8 wherein the solid support is an ELISA plate.

10. The ELISA kit for screening of early cardiac cancer as claimed in claim 9 wherein the detection object of the ELISA kit is human serum.

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