WO2021057986A1

WO2021057986A1 - Use of reagent for detecting content of faecal calprotectin in preparation of kit for screening ovarian lesions

Info

Publication number: WO2021057986A1
Application number: PCT/CN2020/118200
Authority: WO
Inventors: 陆华; 李方远; 刘芊辰; 廖睿
Original assignee: 成都中医药大学
Priority date: 2019-09-27
Filing date: 2020-09-27
Publication date: 2021-04-01
Also published as: CN112578125A; CN112578125B

Abstract

Provided in the present invention is the use of a reagent for detecting the content of faecal calprotectin in the preparation of a kit for screening ovarian lesions.

Description

Use of reagent for detecting fecal calprotectin content in preparing ovarian lesion screening kit

Technical field

The invention belongs to the field of disease screening kits.

Background technique

Calprotectin was first isolated from neutrophils by Fagerhol in 1980 and named L1 protein. It was named after the discovery that its structure contains calcium and has antimicrobial properties. It has a history of 39 years as of today. In 1988, Wilkinson et al. named calgranulin based on this antigenic characteristic. Dorin and Freemont et al. further confirmed that these proteins have the structural characteristics of S-100 protein. Later, they named this protective calcium-binding protein with multiple functions. It is calprotectin.

Calprotectin is a heterodimeric complex composed of S100A8 and S100A9 in the S100 calcium binding protein family. It is a calcium-zinc binding protein derived from neutrophils and macrophages. It can be detected in serum, body fluids or feces. It has been identified as a marker of inflammatory bowel disease IBD. At present, elevated levels of calprotectin (S100A8/S100A9) can be detected in people with inflammation, tumor cells and cancer.

In the field of gynecology, studies have found that calprotectin is related to the occurrence of inflammation-related endometrial cancer, and it plays an important role in the production of inflammatory factors, tumor occurrence and metastasis.

However, there is currently no relationship between ovarian lesions and calprotectin, and no relationship between ovarian lesions and calprotectin (faecal calprotectin, FC) in stool.

Summary of the invention

The purpose of the present invention is to provide the use of the reagent for detecting the content of fecal calprotectin in the preparation of a screening kit for ovarian lesions, and a new screening kit for ovarian lesions.

The technical scheme of the present invention includes:

Use of a reagent for detecting calprotectin in feces in preparing a screening kit for ovarian lesions.

As for the aforementioned purposes, the reagents for detecting calprotectin in feces are: reagents for immunohistochemical detection methods, reagents for Western blot detection methods, reagents for colloidal gold methods, or reagents for ELISA detection methods.

As for the aforementioned use, the reagents also include protein purification reagents.

As for the aforementioned use, the ovarian disease is premature ovarian failure, decreased ovarian reserve, polycystic ovary syndrome, adnexal cystic space and/or cyst.

As for the aforementioned purposes, if the kit detects that the content of calprotectin in the stool of a female subject is higher than that of a normal female, the female subject is at high risk of ovarian disease.

An ovarian lesion screening kit, which includes a reagent for detecting calprotectin in feces.

As in the aforementioned kit, the reagents for detecting calprotectin in feces are: reagents for immunohistochemical detection methods, reagents for Western blot detection methods, reagents for colloidal gold methods, or reagents for ELISA detection methods.

Like the aforementioned kit, it also includes protein purification reagents.

As in the aforementioned kit, the ovarian disease is premature ovarian failure, decreased ovarian reserve, polycystic ovary syndrome, adnexal cystic space and/or cyst.

As with the aforementioned kit, if the kit detects that the content of calprotectin in the stool of a female subject is higher than that of a normal woman, the female subject is at high risk of ovarian disease.

By detecting the content of fecal calprotectin in different populations, the present invention finds that the content is significantly associated with ovarian lesions. With the help of this principle, a kit is developed, which can quickly and non-invasively screen ovarian lesions, and has good application prospects.

Obviously, according to the above-mentioned content of the present invention, according to common technical knowledge and conventional means in the field, various other modifications, substitutions or alterations can be made without departing from the above-mentioned basic technical idea of the present invention.

Hereinafter, the above-mentioned content of the present invention will be further described in detail through specific implementations in the form of examples. However, it should not be understood that the scope of the above-mentioned subject of the present invention is limited to the following examples. All technologies implemented based on the foregoing content of the present invention belong to the scope of the present invention.

Description of the drawings

Figure 1: The ratio of fecal calprotectin greater than 15ug/g in patients with various ovarian diseases.

Figure 2: The ratio of fecal calprotectin in patients with various ovarian diseases is greater than or equal to 60ug/g.

Figure 3: Statistics of the distribution interval of fecal calprotectin in patients with various ovarian diseases.

Figure 4: Statistics of the distribution interval of fecal calprotectin in patients with ovarian disease.

In the figure, FC refers to fecal calprotectin.

detailed description

Example 1 Screening kit of the present invention (ELISA)

1. Composition

(1) Pre-coated plate: Ninety-six-well plate coated with anti-human calprotectin rabbit IgG antibody on the inner wall and bottom of the well.

(2) Enzyme-labeled antibody: (30 times concentrated) HRP-labeled anti-human calprotectin rabbit IgG antibody.

(3) Standard: Calprotectin.

(4) Buffer: PBS containing 1% BSA and 0.05% Tween 20.

(5) Chromogenic agent: TMB substrate solution.

(6) Stop solution: 1N sulfuric acid.

2. Sample preparation

(1) Use the special adhesive device for sample sampling to collect the specimen, and discharge the stool in the center of the unfolded adhesive device. Pay attention that the stool cannot be stained with urine, blood, toilet water, toilet paper and other contaminants;

(2) Insert the sampling rod into the stool sample, then put the sampling rod back into the stool sampling tube containing the sample diluent, screw it tightly and shake it well, repeat the above actions 3 times;

(3) Samples are taken at several different parts of the same stool sample each time, with a total sampling volume of about 50 mg (approximately the size of a match head);

(4) Tighten the sampling tube, shake well and set aside;

(5) If the patient has diarrhea, use a disposable straw to suck thin stool, collect about 100uL (3 drops) into the stool sampling tube, shake it well for use, and perform the test as soon as possible.

3. Use

(1) Add 0.1 mL of serum sample (standard substance) to the wells of the pre-coated plate, incubate at 37°C for 30 minutes, pour out the liquid in the wells, tap 3 times to reduce liquid adhesion;

(2) Add 0.15mL buffer to wash the protein that is not bound to the pre-coated plate, repeat 5 times;

(3) Add 0.1 mL of enzyme-labeled antibody, incubate at 37°C for 30 min, discard the enzyme-labeled antibody, and wash 5 times with 0.15 mL of buffer;

(4) Add 0.1 mL of chromogenic reagent and incubate at 37°C for 10 min;

(5) Add 0.05 mL of stop solution;

(6) Judgment of the result: Observe the color depth with naked eyes, the darker the color, the stronger the positive degree; or measure the OD value (450nm).

The standard curve can be prepared in advance with gradient dilution standards according to the aforementioned steps, and the sample concentration can be adjusted when the sample is formally tested, so that the concentration falls within the linear interval (the curve of concentration and OD value).

The screening kit of the present invention can also be prepared as follows:

Replace the corresponding protein standard in any commercial protein detection kit with a calprotectin solution, and replace the antibody with an anti-calprotectin antibody.

Example 2 The relationship between ovarian lesions and the content of fecal calprotectin (FC)

The inventor performed a fecal calprotectin test on some infertility patients in the outpatient clinic of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine from 2019.06 to 2019.09 (using the calprotectin test kit (colloidal gold method) of Xiamen Weizheng Biotechnology Co., Ltd. ), of which 32 patients were diagnosed with ovarian disease by ultrasound and sex hormone examinations (including 4 cases of premature ovarian failure (POF), 6 cases of decreased ovarian reserve (DOR), and 16 cases of polycystic ovary syndrome (PCOS). Annex 4 cases of cystic space, 2 cases of cystic space). And monitor the fecal calprotectin of healthy adult women as a control.

The detection method is as follows:

1. Preparation for patient testing

(1) For the first test, patients must collect test samples before taking medicine and enema;

(2) Recheck patients should take samples at least 24 hours after stopping the drug;

(3) The 24-hour diet before sampling is best to be similar to the usual diet, avoid overeating or taking too much spicy food, and drinking alcohol is prohibited;

(4) Don't stay up the night before sampling (guarantee sleep duration ≥ 6 hours);

(5) Sampling should not be taken during menstruation;

(6) If conditions permit, try to collect stool samples from the patient's first defecation in the morning for testing;

(7) Patients who cannot be sampled in accordance with the sampling requirements should not be tested.

2. Sample collection and testing

(4) Tighten the sampling tube, shake well and set aside;

(5) If the patient has diarrhea, use a disposable straw to suck thin stool, collect about 100uL (3 drops) into the stool sampling tube, shake it well for later use, and perform the test as soon as possible;

(6) Take out the matching test card from the aluminum foil bag, mark it and place it flat on a horizontal workbench;

(7) Unscrew the cap of the sampling tube, discard the first two drops of the diluted sample, slowly and vertically drop 100uL (3 drops) of the non-bubble diluted sample in the center of the sample hole of the test card, and insert the test card into the fecal calprotectin test Meter, and start timing;

(8) Interpret the results within 10-15 minutes, and the test results are invalid after 15 minutes.

The results are as follows:

By comparing the positive rates of various types of ovarian lesions (Figure 1 and Figure 2), it is found that if FC>15ug/g is the positive predictive value, 4 cases of POF patients with FC-positive are detected, and the positive rate is 100%; FC-positive DOR There were 4 patients, with a positive rate of 66.7%; 12 patients with FC-positive PCOS, with a positive rate of 75%; 3 patients with FC-positive adnexal cystic occupying patients, with a positive rate of 75%, and FC-positive patients with cystic cysts 0 For example, the positive rate is 0%. If FC≥60ug/g is taken as the positive predictive value, there are 2 DOR patients with FC positive, the positive rate is 33.33%; 5 PCOS patients with FC positive, the positive rate is 31%; FC-positive accessory cystic space-occupying patients One case has a positive rate of 25%; so far no FC-positive POF and cystic patients have been detected.

Distribution range of FC value of ovarian disease (Figure 3, Figure 4): The FC value of patients with ovarian disease is mainly concentrated in the range of 15-60ug/g (15,46.875%). The lowest value of FC positive in POF patients is 17ug/g and the highest value is 57ug/g; the lowest value of FC positive in DOR patients is 24ug/g, and the highest value is more than 1000ug/g; the lowest value of FC positive in PCOS patients is 34ug/g and the highest value is 432ug/g; accessories The lowest value of FC positive in patients with cystic space was 50ug/g and the highest value was 138ug/g.

If the FC content is greater than or equal to 15ug/g, it is positive. 40 healthy women were (31.272±6.604) years old, of which 28 had calprotectin FC<15 and 12 had FC ≥15. The highest positive FC value was 56ug/g, and the positive FC content was (34.927±9.134) ug/g; 32 patients with ovarian disease, age (32.955±7.006) years old, 9 patients with ovarian disease with FC<15, and 23 with FC≥15 had a positive rate of 71.8%; positive FC content (218.677±173.623) ug/g, the highest positive value>1000ug/g, the difference in the positive value of FC between the two groups (15-1000ug/g) was statistically significant (P<0.05). The details are shown in Table 1 and Table 2 below.

Table 1 FC positive rate of ovarian disease (FC content ≥15ug/g)

Table 2 Comparison of FC-positive ovarian lesions and healthy women

Considering the overall situation (including FC<15ug/g and FC≥15ug/g), the mean FC of healthy women and patients with ovarian disease will be lowered because of the partial data of FC negative (FC<15ug/g). However, the proportion of healthy women with FC<15ug/g is significantly greater than that of patients with ovarian disease. The average FC value of healthy women will be lower in the FC negative part, and the difference between healthy women and patients with ovarian disease will be greater. It can be seen that the FC values of healthy women and patients with ovarian disease are very different from a global perspective, and the FC value can be used to distinguish between healthy women and women with ovarian disease.

This example shows that the FC content of patients with ovarian disease is significantly higher than that of healthy women, and women with high FC content are at higher risk of suffering from ovarian disease.

In summary, ovarian disease is related to calprotectin in stool. The kit of the present invention can quickly screen for ovarian disease by detecting calprotectin in stool samples, and judge the risk of ovarian disease in the target population. If the FC content is high, The risk is high, and if the FC content is low, the risk is low.

Claims

Use of a reagent for detecting calprotectin in feces in preparing a screening kit for ovarian lesions.
The use according to claim 1, wherein the reagents for detecting calprotectin in feces are: reagents for immunohistochemical detection methods, reagents for Western blot detection methods, reagents for colloidal gold methods, or ELISA detection methods Use reagents.
The use according to claim 1, wherein the reagents further comprise protein purification reagents.
The use according to claim 1, wherein the ovarian disease is premature ovarian failure, decreased ovarian reserve, polycystic ovary syndrome, adnexal cystic space and/or cyst.
The use according to any one of claims 1 to 4, wherein if the kit detects that the content of calprotectin in the stool of a female subject is higher than that of a normal female, the female subject is at high risk of ovarian disease .
A screening kit for ovarian lesions is characterized in that the kit includes a reagent for detecting calprotectin in feces.
The kit according to claim 6, wherein the reagents for detecting calprotectin in feces are: reagents for immunohistochemical detection methods, reagents for Western blot detection methods, reagents for colloidal gold methods, or ELISA detection Method with reagents.
The kit according to claim 6, characterized in that it further comprises a protein purification reagent.
The kit according to claim 6, wherein the ovarian disease is premature ovarian failure, decreased ovarian reserve, polycystic ovary syndrome, adnexal cystic space and/or cyst.
The kit according to any one of claims 6-9, wherein if the kit detects that the content of calprotectin in the stool of a female subject is higher than that of a normal female, then the female subject is at risk of ovarian disease high.