CN112083165A - Application of human serum REG I alpha as detection target or standard substance in preparation of reagent or kit for predicting tumor - Google Patents

Abstract

The invention discloses application of human serum pancreatic regeneration protein I alpha (REGI alpha) as a detection target or a standard substance in preparation of a reagent or a kit for predicting tumors. The method finds that the level of the REGI alpha protein in human serum can accurately predict the tumor. The human serum REGI alpha protein which is a brand new marker for predicting tumors and is provided by the invention can be used as a detection target spot for predicting or assisting in judging the occurrence and the development of the tumors of patients. The results of the clinical study showed that: the area under the ROC curve for the serum REGI α test for diagnosis of tumors was 0.764, with a sensitivity of 62.5% and a specificity of 87.5%. The optimal cutoff (cutoff) value for diagnosing tumor resistance was 46.97 ng/ml.

Description

Human serum REG Iα used as a detection target or standard in the preparation of a tumor-predicting reagent or kit application

technical field

The invention relates to the field of diagnosis and detection of tumors, in particular to the application of novel tumor biomarkers.

Background technique

In today's world, with the increasing incidence of tumors, it has caused a huge burden on human health and economy. Therefore, early diagnosis of tumors has always been the focus and hotspot of global research. Tumor biomarkers refer to a substance present or secreted in tumor cells, which can predict the occurrence and development of tumors and monitor the response to tumor therapy, including tumor antigens, hormones, glycoproteins, enzymes and oncogenes. With the development of molecular biology technology, the detection of tumor biomarkers plays an important role in the diagnosis and treatment of various tumors.

The regeneration gene (REG), a gene originally isolated from pancreatic islets, plays an important role in β-cell regeneration. As one of the REG family genes, REG Iα is located on chromosome 2p12, with an amino acid length of 166, including 5 introns and 6 exons. Among malignant tumors, REG Iα has been found to be overexpressed in lung cancer, esophageal cancer, pancreatic cancer, salivary gland cancer, bladder cancer, gastric cancer and colorectal cancer, and is closely related to the occurrence and development of tumors. However, the detection of REG gene often requires pathological samples of patients , invasive and complex. The protein encoded by REG Iα is pancreatic regeneration protein Iα, which is a polypeptide with a molecular weight of 16kDa and belongs to the superfamily of calcium-dependent lectin genes. .

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of a human serum REG Iα protein detection reagent in the preparation of a reagent or a kit for diagnosing tumors, aiming at the above-mentioned deficiencies of the prior art.

The application of human serum pancreatic regeneration protein Iα (REG Iα) as a detection target or standard substance in the preparation of a reagent or kit for predicting tumor.

The application of a reagent for detecting pancreatic regeneration protein Iα in human serum in the preparation of a kit for predicting tumor.

A tumor-predicting kit comprising antibodies to detect REG Iα protein in human serum.

The reagent for detecting REGIα in human serum preferably includes a relevant reagent for detecting REGIα protein in human serum by ELISA.

beneficial effect

The present invention finds that the level of REG Iα protein in human serum can accurately predict tumors (see Figure 1). Based on the above findings, the present invention provides a new marker for predicting tumor human serum REG Iα protein, which can be used as a detection target to predict or assist in judging the occurrence and development of a patient's tumor. The clinical study results showed that the area under the ROC curve of serum REG Iα detection for the diagnosis of tumor was 0.764, the sensitivity was 62.5%, and the specificity was 87.5% (see Figure 2). The best cutoff value for diagnosing tumor was 46.97 ng/ml.

Description of drawings

Figure 1 Quantification of human serum REG Iα protein and its difference between the normal group and tumor group (mean±SEM; 32.93±1.52 vs 60.60±6.87; normal group vs tumor group; **p<0.01).

Figure 2. Human serum REG Iα protein quantification and its differences between normal group and gastrointestinal tumor, breast tumor and respiratory tumor group. (mean ± SEM; gastrointestinal tumor vs normal group 77.41 ± 16.92 vs 32.93 ± 1.52; breast tumor vs normal group 55.03 ± 19.46 vs 32.93 ± 1.52; respiratory tumor vs normal group 41.92 ± 12.64 vs 32.93 ± 1.52; *p<0.05, **p<0.01)

Figure 3. ROC curve of human serum REG Iα protein for diagnosis of tumor.

Detailed ways

Example

The invention discloses that human serum pancreas regeneration protein Iα (REG Iα) is used as a detection target or standard substance to prepare a reagent for predicting tumor.

1. Experimental samples and reagents for detecting the level of REG Iα protein in human serum

1. Collection of experimental samples:

A total of 130 inpatients in internal medicine (Zhongda Hospital Affiliated to Southeast University) were selected, and the inclusion criteria were as follows: patients aged ≥18 years old and ≤90 years old. Exclusion criteria were: pancreatitis; end-stage renal disease; diabetes; active infection and trauma. According to clinical diagnosis, the patients were divided into two groups, normal group and tumor group.

2. Main experimental reagents:

Deionized water

1×TBS (triethanolamine buffered saline solution)

BSA (Bovine Serum Albumin)

100×TBST (TBS+Tween buffer solution)

96-well plates

Orifice plate coating

REG Iα Standard (Pancreatic Surgery Laboratory, University Hospital Zurich, Switzerland)

Coating Antibody (Antibody 1) (BIOTEM)

Biotinylated REG Iα Antibody (Antibody 2) (BIOTEM)

ExtrAvidin alkaline phosphatase (Sigma)

Substrate solution (Sigma)

2. ELISA detection method

1. Serum collection and storage

4 mL of peripheral blood was collected, and after standing, centrifuged for 5 minutes at 4000 rpm, and the upper serum was taken and stored in a sterile test tube at -80°C.

2. Preparation of REG Iα standard curve (prepared within 15 minutes before use):

1) Add 1 mL of 1% BSA (diluted with 1×TBS) to each bottle of REG Iα standard, let stand for 10 min at room temperature, and at the same time repeatedly invert/rub to help dissolve, and vortex to mix.

2) Sequentially dilute 4, 3.5, 2.5, 1.5, 1, 0.5, 0.1, and 0 ng/mL of 8 standard dilutions in sequence (see below), and establish a standard quantitative curve by the standard curve method.

3. Sample processing

1) Melt the serum on ice, shake gently to mix;

2) Take 10 μL serum and add 90 μL TBS to mix well;

3) Take 10 μL of (2) solution, add 490 μL PBS and mix well;

4. Quantification of human serum REG Iα

1) Antibody coating: Dilute Antibody 1 (1:360) with 1×TBS, add 50ul to each well, shake overnight at room temperature.

2) Wash the plate: spin the coated antibody to dry, place absorbent paper on the experimental table, tap the ELISA plate down hard, and repeat the plate washing with 1xTBST for 3 times, 5 minutes each time. After the last wash, the wash solution in the wells should be completely shaken dry.

3) Blocking: 150 ul of 1% BSA/1xTBS was added to each well and blocked for 1 h at room temperature.

4) Add sample (standard or sample): set standard sample wells and test sample wells respectively. Discard the blocking solution, do not wash, add 100 μL of standard samples of different concentrations (see above) and 100 μL of the sample to be tested in turn, cover the ELISA plate, and incubate at 37°C for 1 h.

5) Wash the plate: the steps are the same as the second step.

6) Dilute Antibody 2 (1:830) with 1×TBS, add 50 μL to each well, cover with membrane, and incubate at 37°C for 1 h.

7) Wash the plate: the steps are the same as the second step.

8) Dilute ExtrAvidin alkaline phosphatase (1:10000) with 1% BSA/1xTBS, add 50 μL to each well, add film, room temperature, 45min.

9) Wash the plate: the steps are the same as the second step.

10) Add 100 μL of substrate solution, cover with film, and develop color at room temperature for 8-20 minutes in the dark (the standard turns yellow).

11) Measure the absorbance of each well at a wavelength of 405 nm using a TECAN microplate reader.

12) Calculate the REG Iα concentration of each sample according to the standard curve and the absorbance of each sample well.

3. Effectiveness

The differences in serum REG Iα protein levels between the two groups were detected and compared (see Figure 1); among the three types of tumors with the largest distribution in the tumor group, gastrointestinal tumors, breast tumors and respiratory tract tumors were detected between the serum REG Iα and the normal group. difference (see Figure 2). The area under the ROC curve for diagnosing tumors was 0.764, the sensitivity (true positive rate) was 62.5%, the specificity (true negative rate) was 87.5% (see Figure 3), and the Youden index J was 0.47. The best cutoff value for diagnosing tumor was 46.97 ng/ml.

Example 2

Human serum pancreatic regeneration protein Iα (REG Iα) is used as a detection target or standard to prepare a kit for predicting tumors, including the ELISA reagent for detecting human serum pancreatic regeneration protein Iα (REG Iα) content described in Example 1.

Claims (1)

1. Application of human serum pancreatic regeneration protein I alpha (REGI alpha) as a detection target or standard in preparation of a reagent or a kit for predicting tumors.

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