CN112394168A

CN112394168A - Application of IgA2 detection reagent in preparation of KRAS mutant colorectal cancer diagnostic agent

Info

Publication number: CN112394168A
Application number: CN201910754156.1A
Authority: CN
Inventors: 刘瑞贤; 杨湘玲; 陈骏雄; 温创宇; 韦丽丽; 谭会柳; 李伟倩; 徐重; 刘焕亮
Original assignee: Sixth Affiliated Hospital of Sun Yat Sen University; National Sun Yat Sen University
Current assignee: Sixth Affiliated Hospital of Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2019-08-15
Filing date: 2019-08-15
Publication date: 2021-02-23
Anticipated expiration: 2039-08-15
Also published as: CN112394168B

Abstract

The invention belongs to the field of biological medicines, and relates to application of an IgA2 detection reagent in an immunoglobulin subtype in preparation of a KRAS mutant colorectal cancer diagnosis or prognosis reagent/kit. The research of the invention finds that IgA2 in immunoglobulin subtype is low expressed in KRAS mutant colorectal cancer, and has great difference and reliability. The IgA2 is used as a colorectal cancer diagnosis biomarker, has the advantages of strong specificity and high sensitivity, is beneficial to improving the diagnosis and treatment level of colorectal cancer, and effectively prevents and treats colorectal cancer.

Description

Application of IgA2 detection reagent in preparation of KRAS mutant colorectal cancer diagnostic agent

Technical Field

The invention belongs to the field of biological medicines, and relates to application of an IgA2 detection reagent in preparation of a KRAS mutation diagnosis reagent/kit and a colorectal cancer diagnosis reagent/kit.

Background

Colorectal cancer (CRC) is the most common malignancy of the digestive tract, the second largest cancer in the world, with high morbidity and mortality at the third and fourth sites, respectively. In recent years, with the improvement of living standard of people in China, the change of living style and the aging of population, the incidence of colorectal cancer in China shows a trend of gradually rising, and the third incidence and the fifth incidence of malignant tumor in China seriously threaten the health and life of people. Early discovery, early diagnosis and early treatment are the key points for treating colorectal cancer. However, most colorectal cancers have no obvious symptoms at the initial stage, and are diagnosed by enteritis as the disease progresses, so that most colorectal cancer patients are in the tumor progression stage when diagnosed, the optimal treatment time is lost, the five-year survival rate is less than 20%, and the prognosis is poor. Therefore, exploring an early, rapid and large-scale diagnosis method for colorectal cancer and improving the diagnosis and treatment level are problems to be solved urgently for preventing and treating colorectal cancer at present.

KRAS belongs to a member of the RAS gene family, encodes a P21 protein located inside the cell membrane, belongs to a Guanosine Triphosphate (GTP) binding protein, and is an important "switch" in intracellular signaling pathways. Normally, the cell has vital positive effects on cell proliferation, survival and differentiation; however, when KRAS is mutated, it cannot be inactivated by hydrolysis of hydrolase, and is in a state of continuous activation, so that RAF/MAPK is up-regulated, and cells are overgrown and proliferated, thereby generating various malignant tumors. Studies have shown that approximately 30% to 45% of colorectal cancer patients carry mutations in the proto-oncogene KRAS.

The United states national cancer combination therapy alliance (NCCN) clinical practice guidelines for colorectal cancer (V1.2015) clearly states that: (1) all metastatic colorectal cancer patients should detect the KRAS gene status; (2) only KRAS wild-type patients are advised to receive treatment with EGFR inhibitors (e.g., erbitux and panitumumab). The colorectal cancer diagnosis and treatment regulations (2010 edition) promulgated by the Ministry of health also explicitly indicate that: when a relapse or metastatic colorectal cancer is determined, the KRAS gene status in the tumor tissue is examined to determine an appropriate treatment regimen. Therefore, monitoring the KRAS mutation level of the colorectal cancer patient has important clinical significance. At present, biopsy tissues of colorectal cancer patients are widely used clinically to determine whether KRAS gene is mutated or not through sequencing, and although the method is accurate, the method is limited by a detection method, and large-scale screening cannot be carried out in the mode. These tests all require sequencing of tumor biopsies and sequencing of tumors in situ. The in vivo detection wound easily causes tumor metastasis, and the problem that the dynamic multiple detection cannot be realized exists. The serum tumor marker detection is a common means for early diagnosis, identification and staging guidance of tumors and judgment of tumor recurrence and metastasis because of quick, convenient and safe sampling.

Human serum contains a large amount of Immunoglobulin (Ig), a large Y-shaped protein that is secreted mainly by plasma cells and used by the immune system to identify and neutralize foreign substances such as pathogens like bacteria, viruses, etc., and has various Immunoglobulin subtypes; more importantly, different subtypes of immunoglobulins fluctuate when the body is infected; compared with other protein molecules, the immunoglobulin has more stable property and longer half-life, and the characteristics ensure that the expression levels of different immunoglobulin subtypes in blood plasma have wide prospects in the diagnosis and application of diseases such as infection, tumor and the like.

IgA is a highly glycosylated protein, which exists mainly in both forms IgA1 and IgA 2. More than 80% of serum IgA belongs to IgA1, IgA1 in the exudate accounts for 50% -74% of total IgA; IgA2 produced in larger amounts in secretory Gut Associated Lymphoid Tissues (GALT) than in non-secretory lymphoid organs (spleen and peripheral lymph nodes) and was resistant to the breakdown of part of the corresponding proteases. IgA plays a role in protecting mucosa through two modes of physical protection and immune response. Its main role is by reducing the direct contact of the intestinal mucosa with pathogens. IgA participates in the formation of intestinal biological barriers, and resists the invasion of pathogens in physical modes such as promotion of intestinal peristalsis, villus movement and intestinal loose mucus layer flowing after being combined with the pathogens; or through chemical neutralization, the bacterial suspension is mutually neutralized with enzymes, enterotoxins and the like in the intestinal tract, and the pathogens are doubly hindered from contacting the mucous membrane in space and time; in addition, IgA-activated macrophages kill enteric pathogens via an APC-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. IgA is a pleiotropic antibody, and its content is relatively stable. When the amount changes significantly, it suggests that it may be related to the occurrence of certain diseases in the intestinal tract.

Disclosure of Invention

The invention aims to provide a KRAS mutant molecular marker with strong specificity and high sensitivity, a diagnostic reagent of the molecular marker and application of the molecular marker.

The invention also aims to provide a molecular marker of colorectal cancer with strong specificity and high sensitivity, a diagnostic reagent of the molecular marker and application thereof.

The invention also aims to provide a molecular marker of KRAS mutant colorectal cancer with strong specificity and high sensitivity, a diagnostic reagent of the molecular marker and application of the diagnostic reagent.

The above object of the present invention is achieved by the following technical means:

in one aspect, the invention provides the use of a reagent for detecting IgA2 in the preparation of a KRAS mutation diagnostic reagent or kit.

In another aspect, the invention provides the use of a reagent for the detection of IgA2 in the preparation of a KRAS mutant colorectal cancer diagnostic reagent or kit.

The inventor collects blood of healthy people (preferably adopting a plasma sample) and plasma samples of colorectal cancer patients (KRAS wild type and mutant type), obtains the normalized abundance of different plasma proinflammatory/anti-inflammation factors through data processing analysis, and obviously discovers that the immunoglobulin subtype IgA2 is differentially expressed and can be used as a KRAS mutant type colorectal cancer diagnosis biomarker. The inventors have non-apparently found that IgA2 shows the ability to better discriminate KRAS mutant colorectal cancers from healthy and colorectal cancer groups.

The IgA2 detection reagent is used for detecting the expression level of mRNA of IgA 2; or detecting the expression level of the IgA2 protein or detecting one or more reagents for detecting the biological activity of the IgA2 protein. In a preferred embodiment of the present invention, the reagent for detecting IgA2 is a reagent for detecting the expression level of IgA2 protein.

In a preferred embodiment, the expression level of IgA2 is the concentration of IgA2 in the test sample; a more preferred embodiment is the plasma concentration of IgA 2.

In a preferred embodiment, the detection reagent is selected from one or more of an antibody that detects IgA2, a functional fragment of an antibody, or a conjugated antibody.

In a preferred embodiment, the conjugated antibody is selected from one or two of a fluorescein conjugated antibody or a bio-enzyme conjugated antibody.

The antibody may be a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a multispecific antibody (e.g., bispecific antibody), and/or an antibody fragment linked to a proteasome. The antibody may be a chimeric antibody, a humanized antibody, a CDR-grafted antibody or a human-type antibody. Antibody fragments may be, for example, Fab, Fab ', F (ab') 2, Fv, Fd, single chain Fv (scFv), Fv with disulfide bonds (sdFv), or VL, VH domains. The antibody may be in a conjugated form, e.g., conjugated to a label, a detectable label, or a cytotoxic agent.

In a preferred embodiment, the kit is an ELISA kit, and further, an ELISA kit based on an anti-human IgA 2-conjugated antibody, and the detection kit is used for detecting the expression level of IgA2 protein.

In a preferred embodiment, the detection method of the detection reagent is any one or more of an ELISA method, a protein chip method, a liquid chromatography method, an immunoturbidimetry method and a flow cytometry method; in a preferred embodiment, the detection method of the detection reagent is one or more of a protein chip method, an ELISA method and an immunoturbidimetry method.

In a preferred embodiment, the critical value of the expression level of IgA2 protein for judging high risk or low risk of KRAS mutant colorectal cancer by the detection reagent is 1994.05pg/ml to 3409.95 pg/ml.

In a more preferred embodiment, the critical value of the expression level of the IgA2 protein is 2194.25 pg/ml; and when the expression amount of the IgA2 protein is less than or equal to 2194.25pg/ml, the KRAS mutant colorectal cancer is high in risk, and when the expression amount of the IgA2 protein is more than 2194.25pg/ml, the KRAS mutant colorectal cancer is low in risk.

As an exemplary embodiment of the present invention, a standard curve method may be used for the calculation method of the expression level of IgA 2.

In a preferred embodiment, the sample to be tested by the test reagent is blood; in a preferred embodiment, the sample to be detected by the detection reagent is plasma.

In another aspect, the invention also provides a reagent or a kit for KRAS mutant colorectal cancer diagnosis, wherein the reagent or the kit contains a detection reagent for IgA 2. Detection reagents for IgA2 are described above.

In another aspect, the present invention provides a KRAS mutant colorectal cancer diagnosis chip, which comprises a solid phase carrier and a probe of biomarker IgA2 immobilized on the solid phase carrier. In a preferred embodiment, the chip is a protein chip.

In another aspect, the present invention provides a KRAS mutant colorectal cancer diagnostic system, comprising:

a) a detection means: the detection means is used for detecting the expression level of IgA2 in a diagnosis object;

b) a result judgment means: and the result judging component is used for obtaining the possibility or risk value of the KRAS mutant colorectal cancer according to the expression quantity of the IgA2 detected by the detection component.

In a preferred embodiment, the detection component is one or more of a microplate reader, a laser scanner, a flow cytometer and a liquid chromatograph; in a preferred embodiment, the detection member is one or two of a microplate reader and a laser scanner.

In a preferred embodiment, the result judging component comprises an input module, an analysis module and an output module; the input module is used for inputting the expression quantity of IgA 2; the analysis module is used for analyzing the colorectal cancer morbidity possibility or risk value according to the IgA2 expression amount; the output module is used for outputting the analysis result of the analysis module.

Wherein in the system, the expression level of IgA2 is the expression level of mRNA of IgA 2; or the expression level of IgA2 protein. In a preferred embodiment, the detection means detects the expression level of IgA2 protein.

In a preferred embodiment, the critical value of the expression level of IgA2 protein for determining high risk or low risk of KRAS mutant rectal cancer in the structural judgment component is 1994.05pg/ml to 3409.95 pg/ml.

In a preferred embodiment, the critical value of the expression level of IgA2 protein in the structural judgment means is 2194.25 pg/ml; and when the expression amount of the IgA2 protein is less than or equal to 2194.25pg/ml, the KRAS mutant colorectal cancer is high in risk, and when the expression amount of the IgA2 protein is more than 2194.25pg/ml, the KRAS mutant colorectal cancer is low in risk.

Wherein the diagnostic sample of the diagnostic system is blood; more preferably plasma.

At the same time, the inventors have also non-obviously found that IgA2 also showed the ability to distinguish KRAS mutant colorectal cancer from KRAS wild type colorectal cancer in diagnosed colorectal cancer patients.

In a preferred embodiment, the value of the critical value of the IgA2 protein expression level for judging high risk or low risk of KRAS mutant colorectal cancer from colorectal cancer patients by using the detection reagent is in the range of 2778.00pg/ml to 5456.60 pg/ml.

In a more preferable embodiment, the detection reagent judges that the critical value of the IgA2 protein expression quantity of KRAS mutant colorectal cancer with high risk or low risk is 3459.55pg/ml from a colorectal cancer patient; and when the expression amount of the IgA2 protein is less than or equal to 3459.55pg/ml, the KRAS mutant colorectal cancer is high in risk, and when the expression amount of the IgA2 protein is more than 3459.55pg/ml, the KRAS mutant colorectal cancer is low in risk.

The invention has the beneficial effects that:

(1) the invention discovers that IgA2 is differentially expressed in a plurality of immunoglobulin subtypes, can be used as a KRAS mutant colorectal biomarker, and has good specificity and sensitivity.

(2) KRAS mutation is monitored clinically mainly by gene sequencing through biopsy tissues at present, and although the method is accurate, the method is limited by a detection method and cannot be used for large-scale screening in such a way. The detection object of the detection reagent is blood, and dynamic and large-scale screening can be realized.

(3) Compared with other proteins in plasma, the immunoglobulin has longer half-life and stable property, and is more suitable to be used as a marker for disease diagnosis.

Drawings

FIG. 1 is a volcano of differential immunoglobulin subtypes;

FIG. 2 is a comparison of IgG3 and IgA2 concentrations in plasma of 49 healthy persons, 32 KRAS wild-type colorectal cancer patients, and 24 KRAS mutant colorectal cancer patients;

FIG. 3 is a ROC curve analysis of single indices IgG3 and IgA2 to differentiate KRAS mutant colorectal cancers;

FIG. 4 is a ROC curve analysis of IgA2 single markers to distinguish KRAS mutant colorectal cancers among colorectal cancer patients;

FIG. 5 is a graph of the gradient dilution of an immunoglobulin standard.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Example 1 expression analysis of differential immunoglobulin subtypes

The experimental method comprises the following steps:

the invention firstly collects the plasma of 49 healthy people and 56 colorectal cancer patients (32 of them are KRAS wild type and 24 are KRAS mutant) in the sixth hospital affiliated to Zhongshan university, and obtains and screens differential immunoglobulin subtypes by the following steps:

1. collecting 3-5cm of whole blood of healthy people or patients with colorectal cancer through a sodium citrate anticoagulation blood collection tube;

centrifuging at 2.500g for 10 min, precipitating blood cells, and collecting the upper layer of blood plasma;

3. collecting supernatant of the plasma, and centrifuging for 15min at 2000g rotating speed until the plasma sample after sediment removal;

4. subpackaging and freezing in a refrigerator at-80 ℃;

5. carrying out Riboao protein chip (model QAH-ISO-1) detection on the plasma sample;

(1) complete drying of slide chips

Taking out the slide chip from the box, balancing at room temperature for 20-30min, opening the packaging bag, uncovering the sealing strip, and then placing the chip in a vacuum drier or drying at room temperature for 1-2 hours.

(2) Configuration of standards

The immunoglobulin subtype standards were diluted in gradient as shown in FIG. 3.

Add 500. mu.L of sample diluent (sample diluent) to the vial of the immunoglobulin subtype standard mixture and redissolve the standard. Before opening the vial, the vial was rapidly centrifuged and gently blown up and down to dissolve the powder, and the vial was labeled Std 1.

The 6 clean centrifuge tubes were labeled Std2, Std3 to Std7, respectively, and 200 μ L of sample diluent was added to each vial.

Std1, 100. mu.L, was added to Std2, gently mixed, and then 100. mu.L was extracted from Std2 and added to Std3, thus gradient-diluted to Std 7.

Draw 100 μ L of sample dilution into another new centrifuge tube, labeled CNTRL, as a negative control.

Note: since the initial concentration of each immune globulin subtype was different, the serial concentrations of each cytokine were different after gradient dilution from Std1 to Std 7.

(3) Chip operation process

(a) Add 100. mu.L of sample dilution to each well, incubate for 1h on a shaker at room temperature, and block the quantitative antibody chip.

(b) Buffer was removed from each well, 100. mu.L of standard and sample were added to the wells and incubated overnight at 4 ℃. (sample 1 ten thousand fold dilution)

(c) The slide was washed with a Thermo Scientific Wellwash Versa chip washer in two steps, first with 1 Xwash I, 250. mu.L of 1 Xwash I per well for 10 times with 10s shaking each time with high shaking intensity, and 20 Xwash I was diluted with deionized water. Then, the washing is carried out by changing to 1 Xwashing liquid II channel, 250 mu L of 1 Xwashing liquid II is washed for 6 times, each time the washing is carried out for 10s, the shaking intensity is selected to be high, and 20 Xwashing liquid II is diluted by deionized water.

(d) Incubation of the detection antibody mixture, centrifugation of the detection antibody mixture vial, then addition of 1.4ml of sample diluent, mixing well and then rapid centrifugation again. Add 80. mu.L of detection antibody to each well and incubate for 2 hours on an RT shaker.

(e) Cleaning, same step (c)

(f) Cy 3-streptavidin incubation, centrifugation of Cy 3-streptavidin tube, then adding 1.4ml sample dilution, mixing uniformly and again rapid centrifugation. Add 80. mu.L of Cy 3-streptavidin to each well, wrap the slide with aluminum foil paper and incubate in the dark for 1 hour on an RT shaker.

(g) Cleaning, same step (c)

(h) Fluorescence detection, signals were scanned using a laser scanner such as InnoScan 300, using Cy3 or green channel (excitation frequency of 532nm), instrument model: innoscan 300Microarray Scanner, manufacturer: innopsys, scan parameters: WaveLengh: 532 nm; resolution: 10 μm, and data analysis was performed by using QAH-ISO-1 data analysis software.

6. The test data were normalized and compared to analyze the expression of each immunoglobulin subtype in three groups of samples.

Analyzing data:

1. differential immunoglobulin volcano plot analysis

The results of the differential immunoglobulin volcano plot analysis are shown in table 1 and figure 1.

Differentially Expressed Proteins (DEPs).

To detect differentially expressed proteins, 10000-fold dilutions of samples were compared to concentrations of different antibody subtypes, and the fold change of each antibody subtype (log2 fold change) and the P-value after T-test (-log10P value) were obtained between the two groups.

Differentially expressed proteins refer to proteins with a P value of less than 0.05(-log10P value greater than 1.3) and a fold change (log2 fold change) of greater than +0.7 or less than-0.7.

TABLE 1 differential immunoglobulin comparison

KRAS mutant colorectal cancer versus healthy cohorts

	IgM	IgD	IgE	IgG1	IgG2	IgG3	IgG4	IgA1	IgA2
										log2 fold change	0.0181	0.4150	-0.3826	0.2728	0.0024	0.8677	0.4952	-0.0084	-0.7076
Log10P value	0.0589	0.5610	1.8792	0.9638	0.0054	2.1866	0.7311	0.0276	1.6682

Illustrated by table 1 and fig. 1: compared to the healthy human group, the IgG3 immunoglobulin subtype was significantly highly expressed in the KRAS mutant colorectal cancer patient group, and the IgA2 immunoglobulin subtype was significantly underexpressed in the KRAS mutant colorectal cancer patient group.

TABLE 2 differential immunoglobulin contrast

KRAS mutant colorectal cancer versus KRAS wild-type colorectal cancer

	IgM	IgD	IgE	IgG1	IgG2	IgG3	IgG4	IgA1	IgA2
										log2 fold change	0.0233	-0.6053	0.1126	0.1711	-0.0450	0.3389	-0.0295	-0.0244	-1.0358
Log10P value	0.0790	0.9068	0.2961	0.5151	0.1068	0.6230	0.0280	0.0821	2.3619

As can be seen from table 2 and fig. 1, plasma concentrations of IgA2 immunoglobulin subtype among the various immunoglobulin subtypes vary significantly between KRAS mutant and wild-type colorectal cancer patients.

Combining the results in table 1 and table 2 and fig. 1, we screened IgG3 and IgA2 as difference indicators among healthy human group, KRAS wild type colorectal cancer group, and KRAS mutant colorectal cancer group, and further analyzed them.

2. Comparison of plasma IgG3 and IgA2 concentrations in healthy humans, KRAS wild-type colon cancer patients, and KRAS mutant colorectal cancer patients

49 healthy persons, 32 KRAS wild type colorectal cancer patients, and 24 KRAS mutant colorectal cancer patients were compared for plasma IgA2 concentration (see fig. 2).

IgG3 differed greatly between KRAS mutant colorectal cancer groups and healthy persons (P0.0065), but did not differ significantly between KRAS wild-type and mutant patients (P0.2382).

IgA2 differed between KRAS mutant colorectal cancer groups and healthy people (P ═ 0.021), and there was a more significant difference between KRAS wild-type and mutant patients (P ═ 0.0043).

ROC curve analysis of individual indices IgG3, IgA2

1 and 2 results suggest that both IgG3 and IgA2 have potential as differential indicators to differentiate KRAS mutant colorectal cancers. To assess the ability of a single index of IgG3 or IgA2 to specifically distinguish KRAS mutant colorectal cancer from healthy persons and colorectal cancer patients, we performed ROC curve analysis on both indices separately.

As shown in fig. 3, IgA2 showed better ability to distinguish KRAS mutant colorectal cancers from healthy and colorectal cancer groups (area under curve 0.693, P0.004).

Furthermore, the results in 2 suggest that IgA2 in the colorectal cancer group had significant differences between KRAS wild-type and KRAS mutant patients, and to further evaluate the ability of IgA2 single index to screen for KRAS mutant patients from colorectal cancer patients, we performed ROC curve analysis on IgA 2.

As shown in fig. 4, IgA2 also showed the ability to distinguish KRAS mutant colorectal cancer from KRAS wild-type colorectal cancer in confirmed colorectal cancer patients (area under curve 0.693, P0.014).

Combining the above results, IgA2 can be used not only as an indicator of differences in screening KRAS mutant colorectal cancers from healthy persons and colorectal cancers, but also as an indicator of differences in distinguishing KRAS wild and KRAS mutations from colorectal cancer patients.

Selection of optimal diagnostic threshold for IgA2

We rank the diagnostic limits in the ROC curve analysis of FIG. 3 by Youden's index, and select the first 10 limits of Youden index for analysis as shown in Table 3: the highest ability to discriminate KRAS mutant colorectal cancers was shown when the critical value was 2194.25pg/ml IgA2 concentration (sensitivity 0.77, specificity 0.61).

TABLE 3 optimal diagnostic threshold for IgA2 (healthy and colorectal cancer groups)

Variable of test result IgA2

From the results in table 3 and fig. 2, the critical value of IgA2 protein expression level for judging high risk or low risk of KRAS mutant colorectal cancer from the healthy group and the colorectal cancer group ranged from 1994.05pg/ml to 3409.95 pg/ml.

Combining the results of fig. 2, the plasma concentration IgA2 after 10000-fold dilution is 2194.25pg/ml as a critical value, and the expression level of IgA2 protein is less than or equal to 2194.25pg/ml, so that KRAS mutant colorectal cancer is at high risk; the detection result of the detection reagent is as follows: and the expression amount of the IgA2 protein is more than 2194.25pg/ml, and the KRAS mutant colorectal cancer is low in risk.

In addition, we rank the diagnosis limits in the ROC curve analysis of fig. 4 by jorden's index (Youden's index), and as shown in table 4, select the limit at the top 10 of the jorden's index for analysis, and find that: the highest ability to discriminate KRAS mutant colorectal cancer was shown when the critical value was 3459.55pg/ml IgA2 concentration (sensitivity 0.69, specificity 0.75).

TABLE 4 IgA2 optimal diagnostic threshold values (colorectal cancer group)

Variable of test result IgA2

From the results in table 4 and fig. 2, the critical value of IgA2 protein expression level for judging high risk or low risk of KRAS mutant colorectal cancer in colorectal cancer patients ranged from 2778.00pg/ml to 5456.60 pg/ml.

Combining the results in fig. 2, the plasma concentration IgA2 of a colorectal cancer patient after 10000-fold dilution is 3459.55pg/ml as a critical value, and the expression amount of IgA2 protein is less than or equal to 3459.55pg/ml, so that KRAS mutant colorectal cancer is at high risk; the detection result of the detection reagent is as follows: and the expression amount of the IgA2 protein is more than 3459.55pg/ml, and the KRAS mutant colorectal cancer is low in risk.

The invention finds that IgA2 can be used as a marker existing in plasma, and a good pre-diagnosis effect can be realized in a blood sampling mode, so that the method has great significance for relieving the sampling pain of people to be tested and realizing more popular pre-diagnosis screening.

Claims

The application of the IgA2 detection reagent in preparing KRAS mutation diagnosis reagent or kit; preferably, use in the manufacture of a KRAS mutant colorectal cancer diagnostic reagent or kit.
2. The use according to claim 1, wherein the IgA2 detection reagent is selected from one or more of a reagent that detects the amount of expression of mRNA for IgA2, or that detects the amount of expression of IgA2 protein, or that detects the biological activity of IgA2 protein; preferably, the reagent for detecting IgA2 is selected from the group consisting of reagents for detecting the expression level of IgA2 protein.
3. The use of claim 1, wherein the detection reagent is selected from one or more of an antibody that detects IgA2, a functional fragment of an antibody, or a conjugated antibody; preferably, the conjugated antibody is selected from one or two of fluorescein conjugated antibody and biological enzyme conjugated antibody.
4. The use of claim 1, wherein the kit is an ELISA kit.
5. The use of claim 1, wherein the detection method of the detection reagent is any one or more selected from the group consisting of an ELISA method, a protein chip method, a liquid chromatography method, an immunoturbidimetry method, and a flow cytometry method; preferably, it is one or more of a protein chip method, an ELISA method or an immunoturbidimetric method.
6. The use of claim 1, wherein the detection reagent determines that the critical value of the expression level of IgA2 protein with high risk or low risk of KRAS mutant colorectal cancer is 1994.05pg/ml to 3409.95 pg/ml;

preferably, the critical value of the IgA2 protein expression amount is 2194.25 pg/ml; when the expression quantity of the IgA2 protein is less than or equal to 2194.25pg/ml, the KRAS mutant colorectal cancer is high in risk; the detection result of the detection reagent is as follows: and the expression amount of the IgA2 protein is more than 2194.25pg/ml, and the KRAS mutant colorectal cancer is low in risk.
7. The use of claim 1, wherein the test sample of the test agent is blood; preferably plasma.
8. A reagent or kit for the diagnosis of KRAS mutant colorectal cancer comprising a reagent for the detection of IgA 2;

preferably, the IgA2 detection reagent is selected from one or more of reagents for detecting the expression level of mRNA of IgA2, or detecting the expression level of IgA2 protein, or detecting the biological activity of IgA2 protein;

preferably, the reagent is selected from reagents for detecting the expression level of IgA2 protein;

preferably, the reagent or kit comprises an antibody, antibody functional fragment or conjugated antibody for detecting IgA 2; more preferably, the conjugated antibody is selected from one or more of a fluorescein conjugated antibody or a bio-enzyme conjugated antibody;

preferably, the kit is an ELISA kit;

preferably, the detection method of the detection reagent is selected from any one or more of an ELISA method, a protein chip method, a liquid chromatography method, an immunoturbidimetry method and a flow cytometry method;

preferably, the detection method of the detection reagent is one or more of a protein chip method, an ELISA method or an immunoturbidimetry method;

preferably, the detection reagent judges the critical value of the IgA2 protein expression level of KRAS mutant colorectal cancer with high risk or low risk to have the value range of 1994.05pg/ml to 3409.95 pg/ml;

preferably, the critical value of the IgA2 protein expression amount is 2194.25 pg/ml; when the expression quantity of the IgA2 protein is less than or equal to 2194.25pg/ml, the KRAS mutant colorectal cancer is high in risk; the detection result of the detection reagent is as follows: the expression amount of IgA2 protein is more than 2194.25pg/ml, and the KRAS mutant colorectal cancer is low in risk;

preferably, the detection sample of the detection reagent is blood; preferably plasma.
9. A KRAS mutant colorectal cancer diagnosis chip is characterized by comprising a solid phase carrier and a probe of a biomarker IgA2 fixed on the solid phase carrier; preferably, the chip is a protein chip.
10. A KRAS mutant colorectal cancer diagnostic system, wherein the detection system comprises:

a) a detection means: the detection means is used for detecting the expression level of IgA2 in a diagnosis object;

b) a result judgment means: the result judging component is used for obtaining the possibility or risk value of the KRAS mutant colorectal cancer according to the expression quantity of IgA2 detected by the detection component;

preferably, the detection component is one or more of a microplate reader, a laser scanner, a flow cytometer and a liquid chromatograph; more preferably, the detection component is one or two of a laser scanner and a microplate reader;

preferably, the result judging component comprises an input module, an analysis module and an output module; the input module is used for inputting the expression quantity of IgA 2; the analysis module is used for analyzing the KRAS mutant colorectal cancer morbidity possibility or risk value according to the IgA2 expression quantity; the output module is used for outputting the analysis result of the analysis module;

preferably, the expression amount of IgA2 is the expression amount of mRNA of IgA 2; or the amount of IgA2 protein expressed; more preferably the expression level of IgA2 protein;

preferably, the diagnostic sample of the diagnostic system is a blood sample; more preferably a plasma sample;

preferably, in the structural judgment component, the critical value of the expression quantity of the IgA2 protein for judging the high risk or the low risk of the KRAS mutant colorectal cancer ranges from 1994.05pg/ml to 3409.95 pg/ml;

more preferably, the critical value of the IgA2 protein expression amount is 2194.25 pg/ml; and when the expression amount of the IgA2 protein is less than or equal to 2194.25pg/ml, the KRAS mutant colorectal cancer is high in risk, and when the expression amount of the IgA2 protein is more than 2194.25pg/ml, the KRAS mutant colorectal cancer is low in risk.