WO2018157831A1

WO2018157831A1 - Lung cancer monitoring kit and application method thereof

Info

Publication number: WO2018157831A1
Application number: PCT/CN2018/077713
Authority: WO
Inventors: 陈翠英
Original assignee: 江苏先思达生物科技有限公司; 先思达（南京）生物科技有限公司; 常州吉泰生物科技有限公司
Priority date: 2017-03-02
Filing date: 2018-03-01
Publication date: 2018-09-07
Also published as: CN106950379B; CN106950379A

Abstract

A lung cancer monitoring kit and an application method thereof. The monitoring kit comprises: a reagent A prepared by adding 5% of SDS into an ammonium bicarbonate solution having a concentration of 10 mM; a reagent B prepared by adding not less than 2.2 unit/μL of an exoglycosidase into NP40 having a concentration of 3.3%; a reagent C comprising a mixed solution of equal volumes of 20 mM of APTS in 1.2 M of a citric acid and 1 M of an organic reducing agent in DMSO; and a reagent D comprising 0.25 μL NH4AC having a concentration of 100 mM and pH 5, 0.2 μL of a neuraminidase, and 2.55 μL hydrogen peroxide. The kit is used to evaluate a lung cancer patient on the basis of a diagnostic indicator of a fingerprinting pattern generated by detecting oligosaccharide chains in a blood glycoprotein, so as to diagnose a tumor stage of a lung cancer, implementing regular and non-invasive detection for a lung cancer patient, and monitoring the progress of the disease in real time.

Description

Lung cancer monitoring kit and using method thereof

Technical field

The invention belongs to the technical field of biomedicine, and particularly relates to a lung cancer monitoring kit and a using method thereof.

Background technique

Lung cancer has become the leading cause of cancer deaths worldwide, both male and female. In 2005, there were estimated 500,000 new cases of lung cancer in China (about 330,000 in men and 170,000 in women). In 2007, there were an estimated 213,380 new cases of lung cancer in the United States and 160,390 deaths worldwide. There are two main reasons for the high mortality rate of lung cancer: First, lung cancer lacks effective early diagnosis methods, more than 70% of patients are in advanced stage at the time of diagnosis; second, advanced lung cancer (especially non-small cell lung cancer, NSCLC) is sensitive to chemotherapy. Poor sex, the overall efficiency of the existing chemotherapy regimen for NSCLC is only about 30%.

Lung cancer can be divided into two categories according to anatomical classification: central lung cancer and peripheral lung cancer. Central lung cancer occurs in the bronchus above the lung segment or above the lung segment, mainly in the form of squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and carcinoid. Peripheral lung cancer occurs in the bronchi below the lung segment and is found in a variety of histological types. The basic gross pathological form is an intrapulmonary nodule or mass. The histopathology of lung cancer is classified into two basic types, the first being non-small cell lung cancer and the other being small cell lung cancer. The diagnosis of lung cancer is mainly through the following methods. Chest imaging is one of the most important methods for diagnosing tumors, and it is of great significance for early diagnosis of lung cancer. Bronchoscopy can directly observe the morphological features of the lesion and bronchoconstriction. It can also directly select the tissue for pathological examination by bronchoscopy lung biopsy and bronchoscopy needle aspiration cytology (TBNA) to improve the diagnosis rate of lung cancer. It also plays an important guiding role in the staging of lung cancer (especially NSCLC). Endobronchial ultrasonography (EBUS) is a method of examining and treating the bronchial lumen, trachea and surrounding tissues through an ultrasound probe under the guidance of a bronchoscope. Autofluorescence bronchoscopy (AFB) is a new type of fiberoptic bronchoscope developed by using cell spontaneous fluorescence and computer image analysis technology, which can significantly improve the sensitivity of bronchoscopy for early diagnosis of lung cancer and precancerous lesions. Electromagnetic navigation bronchoscopy (ENB) combined with the advantages of traditional bronchoscopy and spiral CT to simulate bronchoscopy can improve the positive detection rate of peripheral bronchoscopy on peripheral lung lesions. Optical interference tomography (OCT) is the separation of infrared light into two beams, one beam is directly irradiated, and the other beam is reflected and then irradiated. The two beams overlap to generate light interference phenomenon, and the intensity and time delay of light reflection of each layer are detected. After computer processing, a quadratic tomographic image similar to B-mode ultrasound and CT is obtained, which obtains a reflection image higher than that of any previous method.痰 exfoliated cell examination is an important means of diagnosing lung cancer. For example, sputum specimen collection method is appropriate. Three consecutive sputum specimen examinations can improve the diagnosis rate of central lung cancer by 80%, and the diagnosis rate of peripheral lung cancer is 50%. Tumor markers are simple, minimally invasive, and reproducible. They are the preferred method for clinical screening and early diagnosis of tumors. Their expression in specific parts of the body is relatively specific for tumor diagnosis. Commonly used tumor markers for the diagnosis of lung cancer include carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cytokeratin 19 fragment (CYFRA21-1), CA-125, lung cancer-associated antigen, vascular endothelial growth factor, and proliferation. Nuclear antigen, epidermal growth factor receptor, and the like.

A glycoprotein molecule consists of a polypeptide chain and a sugar moiety. Any change in serum glycoproteins can reflect physiological changes in the body. Recent studies in glycomics have also shown that changes in oligosaccharide chains are closely related to the occurrence and development of various tumors (References: Liu, XE, Desmyter, L, Gao, CF, Laroy, W, Dewaele, S, Vanhooren, V, Wang, L, Zhuang, H, Callewaert, N, Libert, C, Contreras, R, Chen, CN-Glycomic changes in hepatocell μLar carcinoma patients with liver cirrhosis induced by hepatitis B virus. Hepatology, 46, 1426- 1435, 2007.).

In view of the uncertainty of lung cancer surveillance, there is an urgent need for a kit and monitoring and evaluation method that can effectively monitor lung cancer.

Summary of the invention

Technical Problem to be Solved: The object of the present invention is to solve the uncertainty of lung cancer monitoring by the existing detection method, and to provide a lung cancer monitoring kit and a method for using the same, by detecting N-glycosidic linkage of carbohydrates of blood glycoprotein (N Changes in the amount of oligosaccharides) to assess the progression of lung cancer and the recurrence of tumors.

Technical Solution: A lung cancer monitoring kit consisting of the following reagents:

Reagent A: a solution of 10% by mass of ammonium bicarbonate in a solution of 5% by mass of SDS;

Reagent B: a concentration concentration of not less than 3.3% NP40 is added to not less than 2.2 units / μL of glycoside exonuclease;

Reagent C: a mixture of 1.2 M citric acid in an equal volume of 20 mM APTS and 1 M organic reducing agent in DMSO;

Reagent D: not less than 0.25 μL of NH ₄ AC at a concentration of 100 mM, not less than 0.2 μL of sialidase, and 2.55 μL of hydrogen peroxide.

Wherein the organic reducing agent is NaCNBH ₃ .

The volume of the reagent A may be 2 μL, the volume of the reagent B may be 4 μL, the volume of the reagent C may be 2 μL, and the volume of the reagent D may be 2 μL.

The method for using the above lung cancer monitoring kit includes the following steps:

Step 1, adding 2 μL of reagent A to 4 μL of diluted serum to carry out a denaturation reaction, adding 4 μL of reagent B, reacting at 37 ° C for 3 hours, and then drying to obtain a sample;

Step 2, adding 2 μL of reagent C to the obtained sample, performing fluorescent labeling, and then adding 200 μL of water to terminate the labeling reaction to obtain a fluorescently labeled sample;

Step 3, adding 2 μL of reagent D to 2 μL of the fluorescently labeled sample, performing a de-terminal sialic acid reaction, and adding 200 μL of water to terminate the labeling reaction to obtain a sample of the terminal sialic acid reaction;

In step 4, 10 μL of the sample subjected to the terminal sialic acid reaction was taken, and the oligosaccharide chain was separated by an ABI sequencer to obtain a map.

Further, the condition of the step 1 denaturation reaction is heating not lower than 95 °C.

Further, the condition of fluorescent labeling in step 2 is 60-70 ° C heating.

Further, the condition of the terminal sialic acid reaction in the step 3 is heating not higher than 45 °C.

The lung cancer monitoring kit described above can be used to detect blood or all body fluids containing oligosaccharide chain components.

Advantageous Effects: The lung cancer monitoring kit of the present invention evaluates lung cancer patients based on the detection of a fingerprint of oligosaccharide chains in blood glycoproteins as a diagnostic index to diagnose tumor staging of lung cancer. This test method allows many lung cancer patients to undergo routine, non-invasive testing, to help doctors detect lung cancer, and to monitor disease progression in a timely manner.

DRAWINGS

1 is a schematic flow chart of the analysis of the N-oligosaccharide chain fingerprinting technique using serum in Example 1;

2 is a G-Test map of human serum used in Example 1;

Figure 3 is a serum G-Test spectrum of the healthy control group in Example 1;

4 is a serum G-Test chart of a lung cancer patient in Example 1.

detailed description

The invention will be further described in detail below. It should be noted that the following description is merely illustrative of the technical solutions claimed in the present invention, and is not intended to limit the technical solutions. The scope of the invention is defined by the appended claims.

The experimental methods in which the specific experimental conditions are not indicated in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

The study found that there is a significant correlation between the change in glycoside-linked carbohydrate content of blood glycoproteins and the histology of lung cancer patients. Further, the clinical monitoring method for assessing lung cancer can effectively screen and regularly evaluate the progress of lung cancer and the recurrence of tumor after treatment.

In the present invention, serum oligosaccharide chain fingerprinting technology (G-Test method for short) is used as a diagnostic index to evaluate lung cancer patients. The main steps of the method are: releasing and fluorescently labeling the oligosaccharide chain of the glycoprotein in the serum or plasma sample; separating the content or fingerprint of the fluorescently labeled oligosaccharide chain in the measurement sample (referred to as G-Test map); analyzing and comparing the oligosaccharide Fingerprint map, get the test index parameters, the specific process is shown in Figure 1. This method allows many lung cancer patients to receive routine, non-invasive testing, to help doctors detect lung cancer, and to monitor the occurrence and progression of the disease in a timely manner.

Specifically, the composition for monitoring the risk of lung cancer or lung cancer is selected from the group consisting of oligosaccharide chains: NA3F, NA2F, NA2FB, NGA2F, NGA2FB, and NA3. In the following examples, the ratio of (NA3F+NA2FB)/NA3 was used to diagnose lung cancer detection. As shown in Figure 2, the G-Test spectrum of human serum probably shows nearly 10 N-oligosaccharide chain peaks. Different oligosaccharide chains exhibit different mobility due to different molecular sizes, that is, they are expressed in G-Test. The different peaks on the map represent different oligosaccharide chains; the relative concentration of oligosaccharide chains is expressed in the measured peak height.

The above oligosaccharide abbreviations are respectively represented as: NGA2F, galactose-deficient two antennas containing core fucose (α1,6Fuc); NGA2FB, galactose-deficient core fucose modified with two aliquots of acetylglucosamine (G1cNAc) (α1,6Fuc) two antennas; NG1A2F, galactose single deletion core fucose (α1,6Fuc) two antennas (single agalacto, core-α-1,6-fucosylated biantennary); NA2, two antennas (bigalacto, biantennary NA2F, core fucose (α1,6Fuc) two antennas; NA2FB, core fucose (α1,6Fuc) antenna with halved acetylglucosamine (GlcNAc) modification; NA3, three antennas; NA3F a three-antenna modified by branched fucose (α1,3/1,2Fuc).

The invention provides a lung cancer monitoring kit consisting of the following reagents:

Reagent A (denaturation buffer): 10 mM ammonium bicarbonate 5% SDS;

Reagent B (glycosidase reaction buffer): final concentration of exoglycosidase 2.2 units / μL at 3.33% NP40;

Reagent C (APTS Labeling Buffer): Mix equal volumes of 20 mM APTS (dissolved in 1.2 M citric acid) and 1 M organic reducing agent (dissolved in DMSO);

Reagent D (sialidase reaction solution): 0.25 μL of 100 mM NH ₄ AC, pH 5; 0.2 μL of sialidase, 2.55 μL of hydrogen peroxide.

In the present invention, the organic reducing agent is preferably NaCNBH ₃ .

Example 1

First, the test sample

A total of 21 patients with lung cancer were collected from the serum of this patient. The serum was collected from Zhenjiang First People's Hospital. The serum of 57 healthy controls was from Zhenjiang First People's Hospital.

All of the above 21 patients underwent clinical laboratory analysis and histological examination and met the following selection criteria: (a) patients with lung cancer infection; (b) exclusion of other viral infections such as human immunodeficiency virus (HIV); (c) Serum collection Before the patient did not receive any treatment, clinical blood routine, biochemical, tumor markers, DNA load and other data were collected simultaneously with the serum. The above data is used to assess the extent of lung function impairment in a patient.

Second, equipment and reagents

The equipment is mainly ABI sequencer (Applied Biosystems), and the same capillary electrophoresis system.

The reagent mainly contains reagent A (denaturation buffer): 10 mM ammonium hydrogencarbonate 5% SDS; reagent B (glycosidase reaction buffer): final concentration of exoglycosidase 2.2 units / μL at 3.33% NP40; reagent C ( APTS labeling buffer): Mix equal volumes of 20 mM APTS (dissolved in 1.2 M citric acid) and 1 M NaCNBH ₃ (dissolved in DMSO); Reagent D (sialidase reaction solution): 0.25 μL 100 mM NH ₄ AC; 0.2 μL saliva Acidase, 2.55 μL hydrogen peroxide.

Third, the steps to detect the G-Test map in the serum sample

The G-Test map analysis procedure consists of four steps:

Step 1. Prepare a free oligosaccharide chain with a specific N-glycosidic bond hydrolase: add 2 μL of reagent A to 4 μL of diluted serum, and denature at 95 ° C for 5 minutes, then equal volume (4 μL) of reagent B. , reacted at 37 ° C for 3 hours and then dried;

Step 2, fluorescently labeling the free oligosaccharide chain: adding 2 μL of reagent C to the liquid of step 1, heating at 65 ° C for 3 hours for fluorescent labeling, and then adding 200 μL of water to terminate the labeling reaction;

Step 3, remove the terminal sialic acid: take 2 μL of the fluorescently labeled liquid of step 2, then add 2 μL of reagent D, heat at 45 ° C for 3 hours to carry out the terminal sialic acid reaction, and then add 200 μL of water to terminate the labeling reaction;

Step 4. Isolation and analysis of the fluorescently labeled N-oligosaccharide chain: 10 μL of the end-to-sialic acid reaction liquid obtained in the step 3 was taken, and the N-oligosaccharide chain fragment was separated by an ABI 3500 dx sequencer to obtain a G-Test spectrum.

Detection analysis:

Quantify each peak in the measured G-Test spectrum, expressed as the relative content of each peak (%): quantify the sum of the peak height of each peak divided by the height of all peaks. Calculate and calculate the G-TEST value. The differences in relative content between lung cancer patients and healthy controls were compared statistically. The specific results are shown in the following table:

The G-Test map of human serum represents different N-oligosaccharide chain peaks, showing different mobility due to different molecular sizes, ie different peaks on the G-Test map represent different oligosaccharide chains. The relative concentration of the oligosaccharide chain is expressed in the measured peak height. As shown in Fig. 3 and Fig. 4, NA2FB of lung cancer group and normal control group, with two oligoacetylglucosamine (GlcNAc) modified core fucose (α1,6Fuc) antennas; NA3, three antennas; NA3F The three antennas modified by branched fucose (α1,3/1,2Fuc) have a significant gap, the change of N-glycosidically linked carbohydrate (N-oligosaccharide) content of blood glycoprotein and the histology of lung cancer patients There is a significant correlation between them. The study found that the ratio of (NA3F+NA2FB)/NA3 was used to diagnose lung cancer. The effect of setting (NA3F+NA2FB)/NA3 was less than 1 for the normal group, and forty of the normal group, 49 were satisfactory. It is 85.9%. When the threshold of (NA3F+NA2FB)/NA3 was greater than 1.5, it was the lung cancer group, and in the lung cancer group, 19 cases met the requirement, and the accuracy was 90.5%. The results indicate that there is a significant correlation between the change in the N-glycosidically linked carbohydrate (N-oligosaccharide) content of the blood glycoprotein and the histology of lung cancer patients.

Claims

A lung cancer monitoring kit characterized by: consisting of the following reagents:

Reagent A: a solution of 10% by mass of ammonium bicarbonate in a solution of 5% by mass of SDS;

Reagent B: a concentration concentration of not less than 3.3% NP40 is added to not less than 2.2 units / μL of glycoside exonuclease;

Reagent C: a mixture of 1.2 M citric acid in an equal volume of 20 mM APTS and 1 M organic reducing agent in DMSO;

Reagent D: not less than 0.25 μL of NH 4 AC at a concentration of 100 mM, not less than 0.2 μL of sialidase, and 2.55 μL of hydrogen peroxide.
The lung cancer monitoring kit according to claim 1, wherein the organic reducing agent is NaCNBH 3 .
A method of using the lung cancer monitoring kit according to claim 1, comprising the steps of:

Step 1, adding 2 μL of reagent A to 4 μL of diluted serum to carry out a denaturation reaction, adding 4 μL of reagent B, reacting at 37 ° C for 3 hours, and then drying to obtain a sample;

Step 2, adding 2 μL of reagent C to the obtained sample, performing fluorescent labeling, and then adding 200 μL of water to terminate the labeling reaction to obtain a fluorescently labeled sample;

Step 3, adding 2 μL of reagent D to 2 μL of the fluorescently labeled sample, performing a de-terminal sialic acid reaction, and adding 200 μL of water to terminate the labeling reaction to obtain a sample of the terminal sialic acid reaction;

In step 4, 10 μL of the sample subjected to the terminal sialic acid reaction was taken, and the oligosaccharide chain was separated by an ABI sequencer to obtain a map.
The method of using the lung cancer monitoring kit according to claim 3, wherein the condition of the denaturation reaction of the step 1 is heating not lower than 95 °C.
The method of using a lung cancer monitoring kit according to claim 3, wherein the condition of fluorescent labeling in step 2 is 60-70 ° C heating.
The method of using the lung cancer monitoring kit according to claim 3, wherein the condition of the terminal sialic acid reaction in the step 3 is heating not higher than 45 °C.
The lung cancer monitoring kit according to claim 1 or 2 is for detecting blood or all human body fluids containing an oligosaccharide chain component.