1. An ELISA fluorescence detection kit for detecting the content of human-derived Galectin-3 is characterized by comprising a Galectin-3 standard substance, a human serum Galectin-3 positive control, a reaction strip plate coated by a Galectin-3 capture antibody, a specific detection antibody for resisting the Galectin-3, an enzyme-labeled secondary antibody, a biological sample diluent, a washing liquid and a fluorescence luminescent liquid.

2. The ELISA fluorescence detection kit for detecting the amount of human Galectin-3 as claimed in claim 1, wherein the Galectin-3 standard substance is human Galectin-3.

3. The ELISA fluorescence detection kit for detecting the amount of human Galectin-3 in claim 1, wherein neither the Galectin-3 capture antibody coated on the reaction strip nor the specific detection antibody against Galectin-3 react with human Galectin-1, Galectin-2, Galectin-4, Galectin-7, Galectin-8 or Galectin-10.

4. The ELISA fluorescence detection kit for detecting the amount of human Galectin-3 as claimed in claim 1, wherein the enzyme-labeled secondary antibody is HRP-labeled streptavidin.

5. The ELISA fluorescence detection kit for detecting the amount of human Galectin-3 as claimed in claim 1, wherein the antibody specific for detection against galectitn-3 comprises a biotin label, and the signal is amplified by streptavidin-peroxidase and detected by a fluorescent light-emitting solution.

6. The ELISA fluorescence detection kit for detecting the amount of human Galectin-3 as claimed in claim 1, further comprising an ELISA plate coated with a specific antibody for capturing human Galectin-3.

7. The use of the ELISA fluorescence detection kit of any one of claims 1-6 for detecting the amount of human Galectin-3 in a biological sample, wherein the ELISA fluorescence detection of the amount of human Galectin-3 is performed by a double antibody sandwich method.

8. The use of the ELISA fluorescence detection kit of claim 7 for detecting the amount of Galectin-3 in a biological sample, wherein the biological sample is derived from a cell culture fluid, a cell lysate, and human plasma or serum.

9. The method for detecting the amount of Galectin-3 in a biological sample by using the ELISA fluorescent assay kit as claimed in any one of claims 1 to 6, which comprises the following steps:

step 1: coating the Galectin-3 capture antibody on a high-adsorbability enzyme label plate;

step 2: contacting the sample with a specific detection antibody against Galectin-3 to form a Galectin-3: Galectin-3 antibody complex;

and step 3: detecting the binding condition of the Galectin-3 molecule and the antibody in the sample;

and 4, step 4: and comparing with the control sample, and determining the content of Galectin-3 in the biological sample.

Background

Galectin-3 belongs to the Galectin family. At present, 14 members of this family have been reported, all of which possess at least one conserved galactose-binding carbohydrate recognition domain. Galectin-3 is a more specific one of this family, consisting of an N-terminal collagen-like sequence domain free of structural domains and a C-terminal Carbohydrate Recognition Domain (CRD). Galectin-3 is located on chromosome 14, and Galectin-3 can guide polymerization of Galectin-3 monomers through an N-terminal structural domain to form a self-multimer or form a multimeric lattice with other glycoproteins or glycolipids. The N-terminal domain can also assist Galectin-3 to enter a cell nucleus, and through phosphorylation of serine at the 6 th site, the N-terminal domain can influence the combination of the CRD domain on a sugar chain; galectin-3 can also enter other organelles such as nucleus or mitochondria.

The C-terminal CRD domain of Galectin-3 is a domain which functions to bind a sugar chain. CRD domains can bind to a variety of glycoproteins, thereby affecting the function of these target proteins. For example, Galectin3 can bind to insulin receptor through CRD domain, inhibit downstream Akt phosphorylation and further inhibit downstream pathway of insulin receptor, resulting in insulin resistance. Galectin-3 can also be combined with proteins such as EGFR, KRAS and the like to influence the development process of various tumors.

Galectin-3 is widely expressed in various tissues in humans, but its major source is macrophages. Galectin-3 can be secreted extracellularly, enter the blood circulation and reach various parts of the body through the blood circulation. Many studies have shown that Galectin-3 levels in human blood are associated with a variety of diseases. At present, Galectin-3 has been used as a biomarker for heart failure. In a variety of other diseases, elevated levels of Galectin-3 are found in the blood of patients. For example, in pancreatic cancer, the amount of Galectin-3 in blood in combination with the amount of CEA can be more effective in determining the degree of onset of pancreatic cancer. The expression level of Galectin-3 can be up-regulated in the blood of patients with cancers such as cervical cancer, colon cancer, liver cancer and the like. Therefore, the detection of the Galectin-3 content in human blood has important significance for auxiliary judgment of some diseases.

At present, some human Galectin-3ELISA detection kits are on the market at home and abroad, but all have certain defects, such as: the detection concentration range of the kit k093758 is 1.4-94.8ng/mL, and the error is larger when the kit is detected below 10 ng/mL; the detection range is large, and in most diseases, the blood concentration of Galectin-3 is between 5 and 15ng/mL, so that the detection is not suitable. For another example: both the R & D company and the abcam company have Galectin-3 detection kits, the detection range is 0.16-10ng/mL, but the kits adopt an absorbance detection method, so that the absorbance cannot be obtained easily because the sample concentration exceeds 10ng/mL, and the kits are not suitable for large-scale human blood sample detection. Therefore, a kit with strong specificity, wide application range, high accuracy and simple operation is needed, which is very important for solving the existing problems.

Disclosure of Invention

The invention aims to provide an ELISA fluorescence detection kit for detecting the content of human Galectin-3, which solves the problems in the prior art, adopts a double-antibody sandwich method, takes a human Galectin-3 specific monoclonal antibody as an antibody for capturing the Galectin-3, can ensure the specificity of a detection result, and can reduce test errors.

The invention also aims to provide the application of the ELISA fluorescence detection kit in the aspect of detecting the content of Galectin-3 in biological samples, and the kit can be used for accurately detecting the content of the Galectin-3 in different biological samples and providing data support for clinical medicine.

The invention also aims to provide a method for detecting the Galectin-3 content in a biological sample by using the ELISA fluorescence detection kit, which is simple and easy to operate, can reduce test errors and improve the accuracy of test results.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an ELISA fluorescence detection kit for detecting the content of human Galectin-3, which comprises a Galectin-3 standard substance, a human serum Galectin-3 positive control, a reaction ribbon board coated by a Galectin-3 capture antibody, a specific detection antibody against galectitn-3, an enzyme-labeled secondary antibody, a biological sample diluent, a washing liquid and a fluorescence liquid.

Preferably, the Galectin-3 standard is human Galectin-3.

Preferably, neither the Galectin-3 capture antibody coated on the reaction strip nor the anti-Galectin-3 specific detection antibody can react with human Galectin-1, Galectin-2, Galectin-4, Galectin-7, Galectin-8 and Galectin-10.

Preferably, the enzyme-labeled secondary antibody is HRP-labeled streptavidin.

Preferably, the antibody for detecting specificity to galectitn-3 contains a biotin label, and the signal is amplified by streptavidin-peroxidase and detected by a fluorescent light-emitting solution.

Preferably, the kit further comprises an ELISA plate, and the specific antibody for capturing the human Galectin-3 is coated on the ELISA plate.

The invention also provides application of the ELISA fluorescence detection kit in detecting the Galectin-3 content in a biological sample, and the ELISA fluorescence detection of the Galectin-3 content is carried out by adopting a double-antibody sandwich method.

Preferably, the biological sample is derived from cell culture fluid, cell lysate, and human plasma or serum.

The invention also provides a method for detecting the Galectin-3 content in a biological sample by using the ELISA fluorescence detection kit, which comprises the following steps:

step 1: coating the Galectin-3 capture antibody on a high-adsorbability enzyme label plate;

step 2: contacting the sample with a specific detection antibody against Galectin-3 to form a Galectin-3: Galectin-3 antibody complex;

and step 3: detecting the binding condition of the Galectin-3 molecule and the antibody in the sample;

and 4, step 4: and comparing with the control sample, and determining the content of Galectin-3 in the biological sample.

The invention discloses the following beneficial technical effects:

the ELISA fluorescence detection kit for detecting the content of human Galectin-3 specifically adopts a double-antibody sandwich method, and uses a human Galectin-3 specific monoclonal antibody as an antibody for capturing Galectin-3, so that the specificity of a detection result is ensured; then, biotin-labeled specific polyclonal antibody is used as a detection antibody, and streptavidin-labeled horse radish reactive oxidase is used for forming an antibody detection complex, so that false positives are further eliminated, and a 4-component sandwich-shaped conjugate is formed; and finally, adding ECL fluorescent luminous liquid, effectively amplifying the obtained correct signal, comparing the standard substance and the internal reference experiment which are synchronously and parallelly carried out, further eliminating reagents or operation errors in the experiment, and further improving the accuracy of the test result.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1, a human Galectin-3ELISA detection standard curve;

FIG. 2 shows the results of testing the stability of the sample in the ELISA test plate for human Galectin-3 serum samples;

FIG. 3 shows the results of stability measurement of a sample in a human Galectin-3ELISA assay plate in the supernatant of a cell culture medium;

FIG. 4 shows the results of measuring the stability of a sample in a human Galectin-3ELISA assay plate in a cell lysate;

FIG. 5 shows the results of ELISA testing of the stability of samples between plates for human Galectin-3 serum samples.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Composition and configuration of ELISA kit

1.1 ELISA fluorescence detection kit for detecting the content of human Galectin-3, which comprises a Galectin-3 standard substance, a human serum Galectin-3 positive control, a reaction ribbon board coated by a Galectin-3 capture antibody, a specific detection antibody against Galectin-3, an enzyme-labeled secondary antibody, a biological sample diluent, a washing liquid and a fluorescent light-emitting liquid.

The following were the formulations of the reagents:

(1) galectin-3 standard substance

Galectin-3 protein was dissolved in 0.01mol/L PBS, pH7.4, 0.1% BSA, and Galectin-3 was 100. mu.g/mL, and diluted with standard formulation to seven gradients of 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, and 0.3125ng/mL at the time of use.

(2) Galectin-3 positive control sample

Human serum at known concentrations.

(3) Biological sample diluent

Serum dilution, biochemistry technology product number 649.

(4) Standard substance diluent

In the detection of human serum and plasma samples, 20% FBS diluted in PBS was used.

In cell culture supernatant sample assays, cell culture broth was used.

In the detection of a cell lysate sample, a cell lysate is used.

(5) Antibody diluent

BSA was dissolved in PBS solution to prepare BSA at a mass concentration of 5%.

(6) Washing liquid (PBST)

8g NaCl、0.2g KCl、1.44g Na₂HPO₄And 0.24g KH₂PO₄Tween-200.5 mL, double distilled water is added to 800mL, the pH is adjusted to 7.4, the volume is adjusted to 1L, and PBST with the concentration of 0.01mol/L is prepared.

(7) Galectin-3 antibody mixture

The Galectin-3 detection antibody is BAF-1154 from R & D company, and is labeled with biotin, and the working concentration is 50 ng/mL;

the HRP-conjugated streptavidin was DY998 from R & D, diluted at a working concentration of 1:200, prepared with antibody diluent prior to use and used immediately.

(8) ECL color developing liquid

The ECL color developing solution is from an ECL hypersensitivity kit of a domestic Tanon company.

(9) Enzyme label plate

The enzyme-linked immunosorbent assay kit is a white enzyme-linked immunosorbent assay plate which is embedded with antibodies in advance and sealed with serum, is dried on a clean bench, is packaged in a vacuum sealing way, is taken out from a sealing bag when in use, and the rest enzyme-linked immunosorbent assay strips are put back into the sealing bag for storage at 4 ℃.

The Galectin-3 capture antibody is MAB-11541 from R & D company, the final concentration of the monoclonal antibody is 500ug/mL, and the monoclonal antibody is diluted to 4ug/mL by using a coating solution when in use.

The antibody coating solution is 0.05M NaHCO₃/Na₂CO₃The buffer solution has pH of 9.6, and the preparation method comprises the following steps: 1.46g NaHCO₃And 0.75g of Na₂CO₃Dissolved in 500mL ddH₂And (4) in O. The coating liquid needs to be placed in a refrigerator at 4 ℃ for use within one week. After dilution of the antibody, 60. mu.L of antibody was added to each well of the microplate to ensure that the antibody was spread over the entire bottom of the well. Sealing with sealing plate film, placing into 4 deg.C refrigerator, and coating for 14-16 h.

And taking out the enzyme label plate, throwing the residual antibody coating solution to dry, and patting the antibody coating solution on absorbent paper to dry. Washing with washing solution for 2min for three times. Blocking with 200. mu.L PBS diluted 10% FBS overnight. After washing with the washing solution for four times, the washing solution was removed and air-dried.

1.2 preparation of biological samples

1.2.1 preparation of human serum samples

Centrifuging the serum sample under the conditions of 1000g of centrifugal force and 15min of centrifugation, and immediately analyzing the obtained serum sample; if the sample cannot be used immediately, the sample can be subpackaged and frozen, and stored at-80 ℃. It should be noted that all blood components should be handled as potentially dangerous substances while avoiding contamination of the sample in the reagent bottle.

1.2.2 preparation of cell lysate samples

(1) Culturing cells, washing the cells for two times by PBS when the cells cover more than 85% of the culture surface, and then sucking the PBS;

(2) adding Cell Extraction Buffer solution (Cell Extraction Buffer) with a ratio of 1 × 10⁷Adding 1mL of cell extraction buffer solution into each cell, collecting the cells by using a cell scraper, and transferring the cells into a microcentrifuge tube;

(3) after ice-bath for 10min, the cells are crushed by an ultrasonic crusher, mixed evenly for 10min at 4 ℃ on a mixing instrument, and then centrifuged for 10min at 13000rpm at 4 ℃, and the supernatant is taken as cell lysate or protein extract of the cells. The cell lysate can be stored at-80 ℃ and multiple freeze-thaw cycles are avoided during use.

Cell extraction buffer composition: 50mM Tris, 150mM NaCl, 1mM EDTA, 1% NP-40, protease inhibitor cocktail phosphatase inhibitor. It should be noted that: phosphatase inhibitors and protease inhibitors are added before use.

Experimental example 1

The composition of the ELISA kit and the method for detecting the content of Galectin-3 in a biological sample by using the ELISA fluorescent detection kit are specifically as follows:

(1) after the serum sample to be detected, the standard sample and the positive control sample are placed at room temperature for temperature balancing in advance, the serum sample is diluted by one half by using a biological sample diluent (if a sample with very high concentration is encountered, the serum sample can be diluted by one quarter or one eighth); the standard substance is diluted to seven gradients of 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL and 0.3125 ng/mL; directly selecting standard product diluent as negative control;

(2) adding 100uL of biological sample diluent into an ELISA plate, sequentially adding the above samples into micropores of the ELISA plate, and repeating the experiment for 2 samples, wherein each pore is 50 uL. After incubation for 3h at 25 ℃, Galectin-3 will bind to the capture antibody on the surface of the solid phase carrier, the antibody pre-coated in the wells.

(3) And preparing an antibody compound by using the antibody diluent. The final concentration of the detection antibody of Galectin-3 is 50ng/mL, and the dilution ratio of HRP-streptavidin is 1: 200. The sample in the microplate was removed and patted dry on absorbent paper. The microplate was washed 5 times with PBST, and freshly prepared antibody complex was added to the microplate at 80. mu.L per well. The second incubation was performed at 25 ℃ for 2 h. The Galectin-3 detection antibody can be combined with the Galectin-3 bound by the capture antibody in the first incubation, and the HRP-streptavidin can be combined with the biotin coupled on the detection antibody to form a 4-component antigen-antibody complex.

(4) Washing with PSBT 6 times, washing off the unbound enzyme-labeled secondary antibody, adding ECL chemochromic solution, and reading the fluorescence intensity on an enzyme-labeling instrument after 30 s.

(5) The level of the fluorescence intensity reading is in positive correlation with the Galectin-3 content in the sample, and a standard curve with the standard concentration as the X axis and the fluorescence intensity value as the Y axis is prepared (see figure 1).

(6) Calculating the concentration of Galectin-3 in the sample: if the measured value of the positive control sample is compared with the marked value, the coefficient of variation is less than 10 percent, which indicates that the measuring process is reliable, and the concentration of the human Galectin-3 in the measured sample can be calculated according to the standard curve obtained in the previous step.

Experimental example 2

The method for detecting the content of Galectin-3 in a biological sample by using the ELISA fluorescent detection kit in the experimental example 1 specifically determines the stability of samples in ELISA detection plates of human Galectin-3 from different sources of serum samples, cell culture medium supernatant and cell lysate, and the stability of samples between ELISA detection plates of human Galectin-3 serum samples.

3.1 stability of human Galectin-3 serum sample in ELISA assay plate

The samples were tested in parallel 20 times in the same 96-well plate by the ELISA method of example 2, and the stability of in-plate serum sample testing was obtained by calculating the mean, standard deviation, CV of the 20 results (as shown in FIG. 2 and Table 1), which resulted in high stability of human Galectin-3 in different serum samples in the plate.

TABLE 1 in-plate serum sample stability test results

3.2 stability of samples in human Galectin-3ELISA assay plates in cell culture supernatant

Human Galectin-3 standards were prepared in complete medium (cell culture medium + 10% FBS) for standard curve generation. Human Galectin-3 is dissolved in the same culture medium to prepare Galectin-3 detection samples with high, medium and low concentration gradients, and the samples are parallelly detected 20 times in the same 96-well plate. The stability of the detection of the in-plate cell culture fluid sample is obtained by calculating the average value, standard deviation and CV of the 20 results (as shown in figure 3 and table 2), and the results show that the stability of the human Galectin-3 in the supernatant of different in-plate cell culture media is high.

TABLE 2 stability results of in-plate cell culture supernatant samples

3.3 stability of samples in human Galectin-3ELISA assay plates in cell lysates

Preparing a human Galectin-3 standard substance by using the cell lysate for preparing a standard curve. Collecting cells, resuspending the cells with cell lysate, and mixing gently at 4 deg.C for 10min to lyse the cells completely. The centrifuge was precooled to 4 ℃ and centrifuged at 12000rpm for 10min to remove cell debris. The supernatant was transferred to a clean centrifuge tube for detection. The lysate is diluted to within the standard curve, if necessary. Samples were tested in parallel 20 times in the same 96-well plate by ELISA as described previously. The stability of in-plate cell culture fluid sample detection is obtained by calculating the average value, standard deviation and CV of 20 results (as shown in figure 4 and table 3), and the results show that the stability of the human Galectin-3 in different cell lysates is high.

TABLE 3 stability results of in-plate cell lysate samples

4. Stability of human Galectin-3 serum sample ELISA detection plate sample

The samples were independently tested 20 times by the ELISA method of example 2, and the stability of the inter-plate serum sample test was obtained by calculating the average, standard deviation and CV of the 20 results (as shown in FIG. 5 and Table 4), which indicates that the stability of the human Galectin-3 derived from different serum sample sources between plates was high.

TABLE 4 stability results of serum sample testing between plates

The above calculation formulas for the average value, standard deviation and CV% are as follows:

mean value calculation formula mean:

standard deviation calculation formula:

CV calculation formula: CV (stdev/mean)

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.