CN112578125A - Application of reagent for detecting content of calprotectin in preparation of ovarian lesion screening kit - Google Patents
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Abstract
The invention provides application of a reagent for detecting the content of calprotectin (calprotectin) in excrement in preparing an ovarian lesion screening kit, belonging to the field of disease detection kits. According to the invention, the correlation between the content and ovarian lesion is obvious through detecting the content of the calprotectin in different groups of people, and the kit developed by the principle can be used for quickly and non-invasively screening the ovarian lesion, so that the application prospect is good.
Description
Technical Field
The invention belongs to the field of disease screening kits.
Background
Calprotectin was first isolated from neutrophils by Fagerhol in 1980 and named L1 protein, after which it was named for its structural calcium-containing and antimicrobial properties, which has been known for 39 years to date. Wilkinson et al further confirmed that these proteins have the structural characteristics of the S-100 protein in 1988 based on the antigenic characteristics, also designated calgranulin, Dorin and Freemont et al, later designated calprotectin as a multifunctional and calbindin protein.
Calprotectin is a heterodimeric complex consisting of S100A8 and S100a9 in the S100 calbindin family, a calcium-zinc binding protein from neutrophils and macrophages, detectable in serum, body fluids or feces, has been identified as a marker of IBD in inflammatory bowel disease, and elevated calprotectin levels (S100A8/S100a9) are currently detectable in inflammatory, tumor cell and cancer populations.
In the gynecological field, researches show that calprotectin has a certain relation with the occurrence of endometrial cancer related to inflammation, and plays an important role in the generation of inflammatory factors, the occurrence of tumors and the metastasis.
However, no relationship between ovarian lesions and calprotectin is found at present, and no relationship between ovarian lesions and calprotectin (FC) in feces is found.
Disclosure of Invention
The invention aims to provide application of a reagent for detecting the content of calprotectin in preparation of an ovarian lesion screening kit and a novel ovarian lesion screening kit.
The technical scheme of the invention comprises the following steps:
use of a reagent for detecting calprotectin in feces in the preparation of an ovarian lesion screening kit.
As for the aforementioned use, the reagent for detecting calprotectin in feces is: a reagent for immunohistochemical detection, a reagent for Western Blot detection, a reagent for colloidal gold detection or a reagent for ELISA detection.
The reagent also includes a protein purification reagent for use as previously described.
The ovarian disease is premature ovarian failure, diminished ovarian reserve function, polycystic ovarian syndrome, accessory cystic space occupation and/or bursa as described above.
As with the aforementioned use, if the kit detects a higher content of calprotectin in the stool of a female subject than in a normal female, the risk of ovarian disease in the female subject is high.
A ovarian lesion screening kit, comprising a reagent for detecting calprotectin in feces.
As with the kit described above, the reagents for detecting calprotectin in feces are: a reagent for immunohistochemical detection, a reagent for Western Blot detection, a reagent for colloidal gold detection or a reagent for ELISA detection.
Such as the aforementioned kit, which further comprises a protein purification reagent.
The kit as described above, wherein the ovarian disease is premature ovarian failure, diminished ovarian reserve function, polycystic ovarian syndrome, accessory cystic space occupation, and/or bursa.
As with the kit described above, if the kit detects a higher calprotectin content in the feces of a female subject than that of a normal female, the risk of ovarian disease in the female subject is high.
According to the invention, the correlation between the content and ovarian lesion is obvious through detecting the content of the calprotectin in different groups of people, and the kit developed by the principle can be used for quickly and non-invasively screening the ovarian lesion, so that the application prospect is good.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: the proportion of the faecal calprotectin in patients with various ovarian diseases is more than 15 ug/g.
FIG. 2: the proportion of the faecal calprotectin in patients with various ovarian diseases is more than or equal to 60 ug/g.
FIG. 3: counting distribution intervals of the content of the calprotectin in patients with various ovarian diseases.
FIG. 4: and (5) counting distribution intervals of the content of the calprotectin in the patients with ovarian diseases.
In the drawings, FC means calprotectin.
Detailed Description
Example 1 screening kit (ELISA) of the invention
1. Composition of
(1) Pre-coating a plate: ninety-six well plates coated with anti-human calprotectin rabbit IgG antibody on the inner wall and bottom of the well.
(2) Enzyme-labeled antibody: (30-fold concentration) HRP-labeled anti-human calprotectin rabbit IgG antibody.
(3) And (3) standard substance: calprotectin.
(4) Buffer PBS containing 1% BSA, 0.05% Tween 20.
(5) The color developing agent is TMB substrate solution.
(6) Stopping liquid: 1N sulfuric acid.
2. Sample preparation
(1) Collecting a sample by using a special sample sampling adhesive device, and discharging excrement in the center of the unfolded adhesive device, wherein the excrement cannot be stained with pollutants such as urine, blood, toilet water, toilet paper and the like;
(2) inserting a sampling rod into the excrement sample, then putting the sampling rod back into an excrement sampling tube filled with sample diluent, screwing and shaking uniformly, and repeating the actions for 3 times;
(3) sampling at a plurality of different points of the same stool sample each time, wherein the total sampling amount is about 50mg (similar to the size of a match head);
(4) screwing the sampling tube and shaking uniformly for later use;
(5) if the patient with diarrhea absorbs the thin excrement by a disposable suction tube, about 100uL (3 drops) of the thin excrement is collected into an excrement sampling tube, the thin excrement is fully shaken up for standby, and the detection is carried out as soon as possible.
3. Use of
(1) Adding 0.1mL of serum sample (standard product) into the pre-coated plate hole, incubating for 30min at 37 ℃, pouring off liquid in the hole, beating for 3 times, and reducing liquid adhesion;
(2) add 0.15mL buffer to wash proteins not bound to pre-coated plate, repeat 5 times;
(3) adding 0.1mL of enzyme-labeled antibody, incubating for 30min at 37 ℃, pouring off the enzyme-labeled antibody, and adding 0.15mL of buffer solution for washing for 5 times;
(4) adding 0.1mL of color developing agent, and incubating for 10min at 37 ℃;
(5) adding 0.05mL of stop solution;
(6) and (4) judging a result: the color depth is observed by naked eyes, and the deeper the color is, the stronger the positive degree is; or measuring the OD (450 nm).
The standard curve can be prepared by the steps of diluting the standard substance with gradient in advance, and the concentration of the sample is adjusted when the sample is formally detected so that the concentration falls in a linear interval (a curve of the concentration and the OD value).
The screening kit can also be prepared by the following method:
and (3) replacing the corresponding protein standard in any commercial protein detection kit with a calprotectin solution, and replacing the antibody with an anti-calprotectin antibody.
Example 2 relationship between ovarian lesions and the content of calprotectin (FC)
The inventor carried out fecal calprotectin detection (using calprotectin detection kit (colloidal gold method) of Xiamen, Zhengzhi Biotechnology GmbH) at 2019.06-2019.09 for partial infertility patients diagnosed by the clinic of the affiliated university of Chinese medicine, wherein 32 patients were diagnosed with ovarian disease by ultrasound and sex hormone examination (4 cases of Premature Ovarian Failure (POF), 6 cases of ovarian reserve function Decline (DOR), 16 cases of polycystic ovarian syndrome (PCOS), 4 cases of accessory cystic space occupation, and 2 cases of bursa). And calprotectin was monitored as a control in healthy adult women.
The detection method comprises the following steps:
1. patient pre-test preparation
(1) For the first detection, a patient needs to collect a detection sample before taking medicine and clysis;
(2) the patient needs to be checked and sampled at least 24 hours after stopping taking the medicine;
(3) the diet 24 hours before sampling is preferably similar to that of the ordinary diet, so that overeating or taking of excessive spicy and pungent foods is avoided, and drinking is forbidden;
(4) the patient can not stay up night before sampling (the sleeping time is guaranteed to be more than or equal to 6 hours);
(5) during menstruation, sampling is not suitable;
(6) if the condition allows the feces sample of the first defecation of the patient in the early morning to be collected as much as possible for detection;
(7) patients who cannot be sampled according to the sampling requirements are not suitable for testing.
2. Sample collection and detection
(1) Collecting a sample by using a special sample sampling adhesive device, and discharging excrement in the center of the unfolded adhesive device, wherein the excrement cannot be stained with pollutants such as urine, blood, toilet water, toilet paper and the like;
(2) inserting a sampling rod into the excrement sample, then putting the sampling rod back into an excrement sampling tube filled with sample diluent, screwing and shaking uniformly, and repeating the actions for 3 times;
(3) sampling at a plurality of different points of the same stool sample each time, wherein the total sampling amount is about 50mg (similar to the size of a match head);
(4) screwing the sampling tube and shaking uniformly for later use;
(5) if a patient with diarrhea absorbs the thin excrement by using a disposable suction tube, collecting about 100uL (3 drops) of the thin excrement into an excrement sampling tube, fully shaking the thin excrement into a uniform state for standby, and detecting as soon as possible;
(6) taking out the matched detection card from the aluminum foil bag, marking and horizontally placing the detection card on a horizontal workbench;
(7) unscrewing a cap of the sampling tube, discarding two drops of diluted samples at the head, slowly and vertically dripping 100uL (3 drops) of bubble-free diluted samples at the center of a sample adding hole of a detection card, inserting the detection card into a calprotectin detector, and starting timing;
(8) the result is interpreted within 10-15min, and the test result is invalid after 15 min.
The results are as follows:
by comparing the positive rates of various ovarian lesions (as shown in figure 1 and figure 2), the positive rate is 100% if FC > 15ug/g is taken as a positive predictive value, 4 POF patients with FC positive are detected; 4 cases of FC-positive DOR patients with a positive rate of 66.7%; 12 PCOS patients with FC-positive had a 75% positive rate; FC-positive annex cystic occupancy patients in 3 cases showed 75% positive, and FC-positive chocolate capsular patients in 0 cases showed 0% positive. If FC is more than or equal to 60ug/g as a positive predicted value, the positive rate is 33.33 percent in 2 cases of FC-positive DOR patients; 5 patients with FC-positive PCOS had a positive rate of 31%; FC-positive annex cystic occupancy of 1 patient with a positive rate of 25%; FC-positive POF and bursal patients have not been detected.
FC value distribution interval of ovarian lesion (fig. 3, fig. 4): FC values for patients with ovarian lesions were mainly concentrated in the 15-60ug/g interval (15, 46.875%). The FC positive minimum value of the POF patient is 17ug/g, and the maximum value is 57 ug/g; the FC positive minimum value of the DOR patient is 24ug/g, and the maximum value is more than 1000 ug/g; the FC positive of the PCOS patient has the lowest value of 34ug/g and the highest value of 432 ug/g; the accessory cystic occupancy patient has a FC positive minimum of 50ug/g and a maximum of 138 ug/g.
If the FC content is more than or equal to 15ug/g, the positive result is obtained. 40 healthy women are aged (31.272 +/-6.604), wherein 28 calprotectin FC <15, 12 FC > 15, the highest positive FC value is 56ug/g, and the FC content of the positive person is 34.927 +/-9.134 ug/g; 32 ovarian disease patients, wherein the patients are aged (32.955 +/-7.006) and 9 ovarian disease patients with FC <15, and 23 ovarian disease patients with FC > 15 have a positive rate of 71.8%; the FC content (218.677 +/-173.623) ug/g is positive, the highest positive value is more than 1000ug/g, and the difference of the positive values (15-1000ug/g) of the two groups of FCs has statistical significance (P is less than 0.05). Specifically, the following tables 1 and 2 show the results.
TABLE 1 ovarian lesion FC Positive Rate (FC content ≥ 15ug/g)
Table 2 FC positive ovarian lesions in comparison to healthy women
From global considerations (including FC <15ug/g and FC ≧ 15ug/g), both healthy women and patients with ovarian lesions will have FC means that are pulled low by the FC-negative (FC <15ug/g) segment of the data. But the proportion of FC <15ug/g of healthy women is obviously larger than that of ovarian disease patients, the FC mean value of the healthy women is pulled lower in the FC negative part data, and the FC value difference of the healthy women and the ovarian disease patients is larger. It can be seen that FC values of healthy women and ovarian disease patients are very different from each other globally, and healthy women and women with ovarian disease patients can be distinguished by FC values.
This example shows that the FC content of patients with ovarian disorders is significantly higher than that of healthy women, and women with high FC content are at higher risk of suffering from ovarian disorders.
In conclusion, the ovarian lesion is related to calprotectin in excrement, the kit disclosed by the invention can be used for rapidly screening the ovarian lesion by detecting calprotectin in an excrement sample, judging the risk of the ovarian lesion of a target population, wherein the risk is high if the FC content is high, and the risk is low if the FC content is low.
Claims (10)
1. Use of a reagent for detecting calprotectin in feces in the preparation of an ovarian lesion screening kit.
2. The use of claim 1, wherein the agent for detecting calprotectin in faeces is: a reagent for immunohistochemical detection, a reagent for Western Blot detection, a reagent for colloidal gold detection or a reagent for ELISA detection.
3. Use according to claim 1, characterized in that: the reagents also include protein purification reagents.
4. The use of claim 1, wherein the ovarian disorder is premature ovarian failure, decreased ovarian reserve function, polycystic ovarian syndrome, adnexal cystic space occupation, and/or bursa.
5. The use of any one of claims 1 to 4, wherein the female subject is at increased risk of developing ovarian disease if the kit detects a higher calprotectin content in the faeces than in a normal female subject.
6. An ovarian lesion screening kit, which is characterized in that: the kit comprises a reagent for detecting calprotectin in excrement.
7. The kit of claim 6, wherein: the reagent for detecting calprotectin in excrement is as follows: a reagent for immunohistochemical detection, a reagent for Western Blot detection, a reagent for colloidal gold detection or a reagent for ELISA detection.
8. The kit of claim 6, wherein: it also includes protein purification reagents.
9. The kit of claim 6, wherein the ovarian disorder is premature ovarian failure, decreased ovarian reserve function, polycystic ovary syndrome, accessory cystic space occupation, and/or bursa.
10. A kit as claimed in any one of claims 6 to 9 wherein the female subject is at increased risk of developing ovarian disease if the kit detects a higher calprotectin content in the faeces than in a normal female subject.
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| 杨学斌 等: ""镉对去卵巢大鼠钙代谢的影响"", 《中国公共卫生》 * |
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| CN112578125B (en) | 2022-10-25 |
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