CN110609143A - Calprotectin heterodimer detection kit and application thereof - Google Patents

Abstract

The invention provides a calprotectin heterodimer detection kit and application thereof, wherein the kit comprises a coating antibody, and the coating antibody comprises an anti-myeloid-related protein 8/14 antibody. The kit provided by the invention adopts an anti-myeloid related protein 8/14 antibody as a coating antibody, realizes enrichment effect on trace calprotectin heterodimers in a sample, and then adopts a calprotectin monoclonal antibody as a detection antibody to detect calprotectin, so that interference of other types of calprotectin antigens on results is basically eliminated, and the detection sensitivity and the result accuracy are improved.

Description

Calprotectin heterodimer detection kit and application thereof

Technical Field

The invention belongs to the technical field of medical inspection, and relates to a calprotectin heterodimer detection kit and application thereof.

Background

Calprotectin (also called calprotectin) is a heterodimeric protein derived from neutrophils and macrophages, and has the functions of regulating immunity, inducing apoptosis, inhibiting bacterial growth and the like, and recently, research on the relationship between calprotectin and Ulcerative Colitis (UC), Inflammatory Bowel Disease (IBD) and tumors is also becoming a focus. The study finds that the content of calprotectin in body fluid and excrement of patients with ulcerative colitis, inflammatory bowel disease and tumors is changed, and the calprotectin can be used as a tumor marker for early diagnosis of tumors.

Calprotectin (calprotectin) forms a heterodimer by non-covalent association of 2 heavy chain MRP14 and 1 light chain MRP8, and exists in different forms at different stages of differentiation, expressing MRP8 and MRP14 monomers in the cytoplasm of neutrophils and monocytes. During the random transformation of circulating monocytes into macrophages, the expression levels of MRP8 and MRP14 were down-regulated. In the early inflammatory infiltration stage, the heteropolymer MRP8/14 of MRP8 and MRP14, calprotectin, appears in the cell membrane and cytoplasm of activated neutrophils and monocytes. Calprotectin is an important inflammatory reactive protein, which is stably present in feces. Research shows that the content change of calprotectin can dynamically reflect the activity of ulcerative colitis. UC is a chronic nonspecific colon inflammation, the disease cause is not clear, the disease course is long, the disease condition is mild and serious, the UC attacks frequently and repeatedly, and the diagnosis rate in China is increased continuously. Therefore, maintenance therapy and prevention of relapse become key to treatment.

CN108333368A discloses a kit for detecting calprotectin in human feces and a preparation method thereof, wherein the kit comprises a kit body and a detection test strip positioned in the kit; the detection test strip comprises a bottom plate, and a sample pad, a combination pad 1, a combination pad 2, a reaction membrane and absorbent paper which are sequentially arranged on the bottom plate; the conjugate pad 1 is coated with a first anti-calprotectin antibody-fluorescent microsphere conjugate and a biotin-labelled second anti-calprotectin antibody; the binding pad 2 is coated with avidin; a detection T line and a quality control C line are sequentially arranged on the reaction membrane according to the chromatography direction, the detection T line is coated with a biotin-bovine serum albumin conjugate or a biotin-chicken egg albumin conjugate at the position of the T line on the reaction membrane, and a goat anti-mouse IgG antibody is coated on the quality control C line; the first calprotectin antibody fluorescent microsphere conjugate is prepared by the following method: coupling the activated fluorescent microspheres with a first anti-calprotectin antibody, and then blocking with a blocking agent to obtain the first anti-calprotectin antibody fluorescent microspheresThe conjugate comprises 1-3 wt% of gelatin and 2-4 wt% of NH as a sealing agent₂PEG and 0.5 to 1 wt% trehalose in 25 to 50mM MES buffer. However, the kit cannot be used for quantitative or semi-quantitative detection of calprotectin.

Therefore, the method for detecting the calprotectin and the related kit are provided to realize the purpose of quickly, accurately and sensitively diagnosing the calprotectin, and have great application value and market prospect.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a calprotectin heterodimer detection kit and application thereof, wherein the kit adopts an anti-myeloid related protein 8/14 antibody as a coating antibody, realizes the specific detection of calprotectin heterodimer, and has the advantages of high detection sensitivity and simple and rapid operation.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a kit for detecting calprotectin heterodimers, the kit comprising a coated antibody, the coated antibody comprising an anti-myeloid related protein 8/14 antibody.

In the invention, an anti-myeloid-related protein 8/14 antibody (anti-MRP 8/MRP14 antibody) is used as a coating antibody, an enrichment effect is realized on trace calprotectin heterodimers in a sample, and a calprotectin monoclonal antibody is subsequently used as a detection antibody for calprotectin detection, so that the interference of other types of calprotectin antigens on the result is basically eliminated, the accuracy is higher, and a false negative result is avoided.

Preferably, the coating concentration of the anti-myeloid-related protein 8/14 antibody is 0.01-5 μ g/mL, and may be, for example, 0.01 μ g/mL, 0.1 μ g/mL, 0.5 μ g/mL, 1 μ g/mL, 1.5 μ g/mL, 2 μ g/mL, 2.5 μ g/mL, 3 μ g/mL, 3.5 μ g/mL, 4 μ g/mL, 4.5 μ g/mL, or 5 μ g/mL.

Preferably, the coated antibody is coated directly and/or indirectly, preferably indirectly, on a solid support.

According to the invention, the indirect coating is the immobilization of the coated antibody on a solid support by means of a specific affinity reaction between biotin and streptavidin.

Preferably, the solid phase carrier is any one or a combination of at least two of an elisa plate, a magnetic bead, a microsphere, an affinity membrane or a liquid phase chip, and is preferably a streptavidin elisa plate.

Preferably, the anti-myeloid-related protein 8/14 antibody is labeled with biotin.

In the invention, the enzyme label plate is coated by a coating antibody anti-MRP 8/MRP14 antibody, and the method comprises the following steps: adding coating solution into an ELISA plate with at least 8 holes, wherein each hole is 100 mu L, the concentration of an antibody contained in the coating solution is 0.01-5 mu g/mL, and coating overnight at 4 ℃; washing with PBST (250 μ L/well) for 3 times, draining, sealing with sealing solution (150 μ L/well) at 37 deg.C for 1h, washing with PBST (250 μ L/well) for 3 times, draining, placing into vacuum bag, adding desiccant, vacuumizing, and storing at 4 deg.C; the preparation method of the coating liquid comprises the following steps: 1.5g of Na₂CO₃And 2.92g NaHCO₃Dissolving in 800mL double distilled water, adjusting pH to 9.6, and diluting to 1000 mL.

Preferably, the kit further comprises a first antibody.

Preferably, the first antibody is a calprotectin monoclonal antibody.

Preferably, the concentration of the first antibody is 0.01 to 5. mu.g/mL, and may be, for example, 0.01. mu.g/mL, 0.1. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL, 2. mu.g/mL, 2.5. mu.g/mL, 3. mu.g/mL, 3.5. mu.g/mL, 4. mu.g/mL, 4.5. mu.g/mL, or 5. mu.g/mL.

Preferably, the kit further comprises an enzyme-labeled secondary antibody.

Preferably, the enzyme-labelled secondary antibody comprises HRP-labelled goat anti-mouse IgG1 and/or HRP-labelled goat anti-rabbit IgG 1.

Preferably, the concentration of the enzyme-labeled secondary antibody is 0.01-5. mu.g/mL, for example, 0.01. mu.g/mL, 0.1. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL, 2. mu.g/mL, 2.5. mu.g/mL, 3. mu.g/mL, 3.5. mu.g/mL, 4. mu.g/mL, 4.5. mu.g/mL, or 5. mu.g/mL. .

In the invention, an enzyme-labeled secondary antibody is diluted by adopting HRP protective solution according to the volume ratio of the enzyme-labeled secondary antibody to the protective solution of 1 (2000-10000), and the specification of the enzyme-labeled secondary antibody is 12mL per bottle; the preparation method of the HRP protective solution comprises the following steps: to 1g BSA and 2.5g EDTA was added 800mL 1 XPBS to bring the volume to 1000 mL.

Preferably, the kit further comprises any one or a combination of at least two of a positive control, a negative control, an antibody diluent, a developing solution, a stop solution, a blocking solution or a washing solution.

Preferably, the positive control is a calprotectin standard.

Preferably, the concentration of the positive control is 50-100 ng/mL, for example, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.

Preferably, the negative control is bovine serum albumin.

Preferably, the concentration of the negative control is 50-100 ng/mL, for example, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.

In the invention, the positive control adopts an antibody diluent to dilute the calprotectin standard substance according to the volume ratio of 1 (20-100), and the specification is 1mL per tube; the negative control substance adopts an antibody diluent to dilute the negative protein according to the volume ratio of 1 (20-100), and the specification is 1mL per tube.

In the invention, the preparation method of the antibody diluent comprises the following steps: adding 800mL of 1 XPBS into 1g of BSA, and fixing the volume to 1000mL, wherein the specification of the antibody diluent is 12mL per bottle; the preparation method of the color developing solution comprises the following steps: to 50mg of 3,3',5,5' -Tetramethylbenzidine (TMB), 10mL of DMSO, 9.8g of citric acid, and 1.2mL of 30% H₂O₂Adding 800mL of 1 XPBS (phosphate buffer solution) into the solution, and fixing the volume to 1000mL, wherein the specification of the color development solution is 12mL per bottle; the preparation method of the stop solution comprises the following steps: adding 60mL of double distilled water into 10mL of 98% concentrated sulfuric acid, and fixing the volume to 100mL, wherein the specification of the stop solution is 6mL per bottle; the preparation method of the washing liquid comprises the following steps: mixing 0.2g KH₂PO₄、2.9g Na₂HPO₄·12H₂O, 8.0g NaCl, 0.2g KCl and 0.5mL tween 20 (0.05% V/V) were added to 180mL double distilled water, the pH was adjusted to 7.4, and the volume was adjusted to 100 mL.

In a second aspect, the present invention provides a method of using a kit as described in the first aspect, the method comprising the steps of:

(1) adding a sample to be detected into the ELISA plate, setting a positive control group and a negative control group at the same time, incubating and washing;

(2) adding a first antibody, incubating and washing;

(3) adding enzyme-labeled secondary antibody, incubating and washing;

(4) adding color development liquid, incubating, adding stop solution, and detecting the light absorption value at 450 nm.

Preferably, the sample to be tested is derived from any one or a combination of at least two of feces, body fluid, blood or tissue, preferably feces.

Preferably, the result of the sample to be tested is determined as:

the light absorption value of the sample to be detected/the light absorption value of the positive sample is less than 0.20, and the sample is judged to be calprotectin negative;

the light absorption value of the sample to be detected/the light absorption value of the positive sample is between 0.20 and 0.25, and the sample is judged to be a suspicious sample;

and the light absorption value of the sample to be detected/the light absorption value of the positive sample is more than 0.25, and the sample is judged to be positive.

In a third aspect, the present invention provides a use of the kit according to the first aspect in the preparation of a reagent and/or a medicament for detecting a bowel disease.

Preferably, the intestinal disease comprises any one of ulcerative colitis, inflammatory bowel disease or colorectal cancer or a combination of at least two of them.

Compared with the prior art, the invention has the following beneficial effects:

(1) the calprotectin heterodimer detection kit selects the anti-MRP 8/MRP14 antibody as the coating antibody, realizes the enrichment effect on trace calprotectin heterodimer in a sample, obviously improves the sensitivity and specificity of detection, has the detection rate of the calprotectin heterodimer in a human excrement sample as high as 90 percent, and provides a sensitive and accurate detection tool for the clinical diagnosis of intestinal diseases;

(2) the kit has the advantages of simple and convenient use method, high speed and efficiency, wide application range and great market value.

Detailed Description

To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

EXAMPLE 1 kit Assembly

1. Primary reagent

(1) Coating liquid: 1.5g of Na₂CO₃And 2.92g NaHCO₃Dissolving in 800mL double distilled water, adjusting the pH value to 9.6, and metering the volume to 1000 mL;

(2) enzyme-labeled secondary antibody: the enzyme-labeled secondary antibody is diluted by HRP protective solution according to the volume ratio of the enzyme-labeled secondary antibody to the protective solution of 1 (2000-10000), and the specification of the enzyme-labeled secondary antibody is 12mL per bottle;

(3) positive control: diluting a calprotectin standard substance by using an antibody diluent according to the volume ratio of 1 (20-100), wherein the specification is 1mL per tube;

(4) negative control: diluting the negative protein by using an antibody diluent according to the volume ratio of 1 (20-100), wherein the specification is 1mL per tube;

(5) antibody diluent; adding 800mL of 1 XPBS into 1g of BSA, and fixing the volume to 1000mL, wherein the specification of the antibody diluent is 12mL per bottle;

(6) HRP protective solution: adding 800mL of 1 XPBS into 1g of BSA and 2.5g of EDTA, and fixing the volume to 1000 mL;

(7) color development liquid: to 50mg of 3,3',5,5' -Tetramethylbenzidine (TMB), 10mL of DMSO, 9.8g of citric acid, and 1.2mL of 30% H₂O₂Adding 800mL of 1 XPBS (phosphate buffer solution) into the solution, and fixing the volume to 1000mL, wherein the specification of the color development solution is 12mL per bottle;

(8) stopping liquid: adding 60mL of double distilled water into 10mL of 98% concentrated sulfuric acid, and fixing the volume to 100mL, wherein the specification of the stop solution is 6mL per bottle;

(9) washing liquid: mixing 0.2g KH₂PO₄、2.9g Na₂HPO₄·12H₂O, 8.0g NaCl, 0.2g KCl and 0.5mL tween 20 (0.05% V/V) were added to 180mL double distilled water, the pH was adjusted to 7.4, and the volume was adjusted to 100 mL.

2. Preparation of ELISA plates

The enzyme label plate is coated by a coating antibody anti-MRP 8/MRP14 antibody, and the method comprises the following steps: adding coating solution into an ELISA plate with at least 8 holes, wherein each hole is 100 mu L, the concentration of an antibody contained in the coating solution is 0.01-5 mu g/mL, and coating overnight at 4 ℃; washing with PBST (250 μ L/well) for 3 times, draining, sealing with sealing solution (150 μ L/well) at 37 deg.C for 1h, washing with PBST (250 μ L/well) for 3 times, draining, placing into vacuum bag, adding desiccant, vacuumizing, and storing at 4 deg.C; the preparation method of the coating liquid comprises the following steps: 1.5g of Na₂CO₃And 2.92g NaHCO₃Dissolving in 800mL double distilled water, adjusting pH to 9.6, and diluting to 1000 mL.

3. Biotin labeling of coated antibodies

Purified anti-MRP 8/MRP14 antibodies were selected for biotin labeling, as follows: dissolving long-chain activated biotin in dimethyl sulfoxide according to the concentration of 1 mg/mL; the antibody to be coupled and purified was dissolved in a 0.1mol/L sodium bicarbonate solution at pH 9.0 at a concentration of 5 μ g/mL; mixing the activated biotin solution with an antibody solution to be coupled according to a ratio of 1:8, and incubating for 4 hours at room temperature; dialyzed at 4 ℃ for 24h against 0.05mol/L PBS buffer pH 7.2, with 4 changes to remove unbound free biotin and the labeled antibodies mixed in proportion to form the coated antibody.

Example 2 ELISA detection of calprotectin heterodimers

(1) The kit is balanced for 0.5 to 1 hour at room temperature, and calprotectin is diluted by sample diluent;

(2) adding 100 μ L diluted sample to be tested, negative reference substance and positive reference substance into corresponding wells of 96-well enzyme label plate, sealing plate, incubating at 37 deg.C for 30min, and removing supernatant;

(3) washing 96-pore plates with the washing liquid at a concentration of 250 mu L/pore for 3 times, discarding the washing liquid, and patting the washing liquid on absorbent paper with force to avoid drying the coating pores;

(4) adding 100 mu L of biotin-labeled calprotectin monoclonal antibody into an enzyme label plate, closing the plate, incubating for 60min at 37 ℃, washing for 3 times by using a washing solution, and then patting the plate on absorbent paper by force so as to avoid drying of a coating hole;

(5) adding 100 mu L of HRP-labeled goat anti-mouse IgG1 into an ELISA plate, and incubating for 30min at 37 ℃;

(6) adding 100 mu L of TMB solution, developing for 10min in a dark place at 37 ℃, adding 50 mu L of stop solution, stopping the reaction, gently shaking the ELISA plate, and uniformly mixing the reaction solution;

(7) the absorbance of the sample was measured and recorded using a microplate reader, and the measurement was completed within 5 minutes.

The detection result meets the following conditions:

the mean value (P) of the calprotectin positive controls minus the mean value (N) of the negative controls must be greater than 0.15, the mean value (N) of the negative controls must be less than or equal to 0.15, the positivity/negativity of the calprotectin sample is judged by calculating the ratio of the sample to the positive control (S/P), the specific calculation method being as follows:

1) if the S/P value is less than 0.20, the sample is judged to be calprotectin-negative (-);

2) if the S/P value is between 0.20 and 0.25, the sample is judged to be suspicious (+/-), and if the S/P value is greater than 0.25, the sample is judged to be positive (+/-).

Comparative example 1

The microplate was coated with calprotectin monoclonal antibody as compared to example 1, and the other conditions were the same as in example 1.

Comparative example 2

The microplate was coated with anti-MRP 8 antibody as compared with example 1, and the other conditions were the same as in example 1.

Comparative example 3

The microplate was coated with anti-MRP 14 antibody as compared with example 1, and the other conditions were the same as in example 1.

Example 3 accuracy test

Calprotectin heterodimer measurements were performed on faeces of 623 healthy donors using the kits of example 1 and comparative examples 1-3, and the results are shown in table 1.

TABLE 1 statistical table of clinical diagnosis results of kit

Numbering	Positive for	Negative of	Accuracy (%)
				Example 1	6	617	99.0
Comparative example 1	17	606	97.3
				Comparative example 2	45	578	92.8
Comparative example 3	41	582	93.4

It can be seen that the kit prepared by using the anti-MRP 8/MRP14 antibody as the coating antibody of the present application is superior to the kit of the comparative example in terms of specificity and sensitivity in terms of accuracy due to the other kits prepared by using a single antibody as the coating antibody.

Example 4 sensitivity test

The kit is adopted to carry out 20 times of repeated tests on calprotectin heterodimer zero standard solution, and the measurement result is the sensitivity of the kit by taking the average value plus 2 times of standard deviation.

The results show that: the sensitivity of the kit to calprotectin heterodimers was 0.35. mu.g/mL.

Example 5

The kit is adopted to carry out calprotectin heterodimer determination on feces samples of 100 cases of Inflammatory Bowel Disease (IBD) patients, 91 cases are calprotectin positive, and the clinical sensitivity is 91 percent, which shows that the kit has strong sensitivity and can be used for clinical sample detection.

In conclusion, the calprotectin heterodimer detection kit selects the anti-MRP 8/MRP14 antibody as the coating antibody, realizes the enrichment effect on trace calprotectin heterodimer in a sample, obviously improves the sensitivity and specificity of detection, has the detection rate of the calprotectin heterodimer in a human fecal sample as high as 90 percent, and provides a sensitive and accurate detection tool for the clinical diagnosis of intestinal diseases; the kit is simple and convenient in use method, rapid and efficient, wide in application range and high in market value.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (10)

1. A kit for the detection of calprotectin heterodimers, said kit comprising a coated antibody, said coated antibody comprising an anti-myeloid related protein 8/14 antibody.

2. The kit according to claim 1, wherein the coating concentration of the anti-myeloid-related protein 8/14 antibody is 0.01-5 μ g/mL.

3. The kit of claim 1 or 2, wherein the coated antibody is coated directly and/or indirectly on a solid support;

preferably, the solid phase carrier is any one or a combination of at least two of an enzyme label plate, a magnetic bead, a microsphere, an affinity membrane or a liquid phase chip.

4. The kit of any one of claims 1 to 3, wherein the anti-myeloid-related protein 8/14 antibody is labeled with biotin.

5. The kit of any one of claims 1-4, wherein the kit further comprises a first antibody;

preferably, the first antibody is a calprotectin monoclonal antibody;

preferably, the concentration of the first antibody is 0.01-5 mug/mL.

6. The kit of any one of claims 1 to 5, wherein the kit further comprises an enzyme-labeled secondary antibody;

preferably, the enzyme-labeled secondary antibody comprises HRP-labeled goat anti-mouse IgG1 and/or HRP-labeled goat anti-rabbit IgG 1;

preferably, the concentration of the enzyme-labeled secondary antibody is 0.01-5 mu g/mL.

7. The kit according to any one of claims 1 to 6, wherein the kit further comprises any one of a positive control, a negative control, an antibody diluent, a developing solution, a stopping solution, a blocking solution, or a washing solution, or a combination of at least two thereof.

8. A kit as claimed in any one of claims 1 to 7 wherein the positive control is a calprotectin standard;

preferably, the concentration of the positive control substance is 50-100 ng/mL;

preferably, the negative control is bovine serum albumin;

preferably, the concentration of the negative control substance is 50-100 ng/mL.

9. A method of using the kit of any one of claims 1 to 8, comprising the steps of:

(1) adding a sample to be detected into the ELISA plate, setting a positive control group and a negative control group at the same time, incubating and washing;

(2) adding a first antibody, incubating and washing;

(3) adding enzyme-labeled secondary antibody, incubating and washing;

(4) adding a color development solution, incubating, adding a stop solution, and detecting the light absorption value at 450 nm;

preferably, the sample to be tested is derived from any one or a combination of at least two of feces, body fluid, blood or tissue, preferably feces;

preferably, the result of the sample to be tested is determined as:

the light absorption value of the sample to be detected/the light absorption value of the positive sample is less than 0.20, and the sample is judged to be calprotectin negative;

the light absorption value of the sample to be detected/the light absorption value of the positive sample is between 0.20 and 0.25, and the sample is judged to be a suspicious sample;

and the light absorption value of the sample to be detected/the light absorption value of the positive sample is more than 0.25, and the sample is judged to be positive.

10. Use of a kit according to any one of claims 1 to 8 for the preparation of a reagent for the detection of intestinal disorders and/or a medicament for the detection of intestinal disorders;

preferably, the intestinal disease comprises any one of ulcerative colitis, inflammatory bowel disease or colorectal cancer or a combination of at least two of them.