CN115975024A - A polyclonal antibody to fucoidan and its application in detecting fucoidan - Google Patents

Abstract

The invention belongs to the technical field of antibody preparation and detection and analysis, and in particular relates to a fucoidan polyclonal antibody and its application in detecting fucoidan. Specifically, the present invention successfully obtains fucoidan polyclonal antibodies by immunizing non-human animals with fucoidan-bovine serum albumin (BSA) covalent complex, and successfully uses it for fucoidan detection. The present invention takes the preparation of fucoidan polyclonal antibody as the entry point, provides method basis for the preparation of polyclonal antibody of carbohydrates through the study of its antibody characteristics, and further provides the establishment of immune method for carbohydrate pharmacokinetics It provides the source material basis, so it has good practical application value.

Description

A polyclonal antibody to fucoidan and its application in detecting fucoidan

technical field

The invention belongs to the technical field of antibody preparation and detection and analysis, and in particular relates to a fucoidan polyclonal antibody and its application in detecting fucoidan.

Background technique

The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

Polysaccharides have various biological activities such as anticoagulant, antiviral, antioxidative, neurotrophic and protective. Therefore, the pharmacological research of polysaccharides has attracted much attention. However, due to the complex chemical structure of polysaccharides, there is no characteristic UV absorption. Therefore, there is currently a lack of a micro-quantitative and specific in vivo detection method. Currently commonly used isotope labeling methods, high performance liquid chromatography and biological assays have some defects and deficiencies. Immunological methods are more and more widely used in pharmacokinetic research because of their good specificity and high sensitivity. The key to the application of immunological methods is to obtain antibodies with good specificity and high affinity. The properties of antibodies directly determine the success of the application of immunological methods.

Fucoidan is a marine sulfated polysaccharide mainly derived from the cell wall of brown algae and some marine invertebrate tissues. It has good biocompatibility and biological activities such as antioxidant, antiviral, immune regulation and anti-inflammatory . However, the structure of fucoidan from different sources is quite different. For example, the structure of fucoidan derived from marine invertebrates is usually relatively simple, generally consisting of regular and simple repetitions of several components. The structure of fucoidan derived from brown algae is relatively complex, usually composed of two chain structures, one is connected by (1→3)-α-L-glucopyranose as the main chain, and the other is α-L-glucopyranose Linked by (1→3) and (1→4) as the main chain. It is worth noting that the biological activity of fucoidan is affected by many factors, which is closely related to its degree of sulfation and molecular weight. For most fucoidan, its structural skeleton is still unclear, and specific sulfate The position of the group and the branching site have not been determined. This brings some difficulties to elucidate its structure-activity relationship. At the same time, it is not conducive to large-scale promotion and research application in academic and commercial fields.

As we all know, since the source of most fucoidan currently on the market is not always consistent, and may be due to differences in manufacturers and batches, the stability of the pharmacological activity of fucoidan cannot be guaranteed. Therefore, there is an urgent need to find an animal, plant or microorganism that can stably produce fucoidan with the same configuration and constant molecular weight. It should be pointed out that compared with animals and plants, microorganisms have lower production costs, are more stable and reliable, and are not easily disturbed by various external environmental factors, so their advantages are more significant. Therefore, it is necessary to establish a method for detecting whether fucoidan is contained in the solution, so as to be used for screening fucoidan-producing bacteria and the like.

Contents of the invention

In view of the prior art, the purpose of the present invention is to provide a fucoidan polyclonal antibody and its application in detecting fucoidan. The present invention takes the preparation of fucoidan polyclonal antibody as the entry point, and provides method basis for the preparation of polyclonal antibody of carbohydrate substances through the study of its antibody characteristics, and further provides the establishment of immune method for carbohydrate pharmacokinetics Provide source material basis. Based on the above research results, the present invention has been accomplished.

In order to realize the above-mentioned technical purpose, the technical scheme provided by the present invention is as follows:

One aspect of the present invention provides a fucoidan polyclonal antibody, which is obtained by subjecting a fucoidan-bovine serum albumin (BSA) covalent complex to a non-human animal. Obtained by immunization; wherein, the fucoidan-bovine serum albumin covalent complex is prepared by the following method: dissolving fucoidan, bovine serum albumin, and NaCNBH ₃ in carbonate buffer solution and reacting at a constant temperature for 2 -4 days, obtained after purification; wherein, the mass ratio of fucoidan, bovine serum albumin, and NaCNBH ₃ is 25-35:5-15:1-5.

The second aspect of the present invention provides a kit for detecting fucoidan, the kit at least containing the above-mentioned fucoidan polyclonal antibody.

The kit may also contain other components for detecting fucoidan, such as buffer, etc., which are not specifically limited here.

The third aspect of the present invention provides the application of the above fucoidan polyclonal antibody or kit in the detection of fucoidan.

The detection can be qualitative or quantitative detection, that is, to determine the concentration or presence of fucoidan. Specifically, the detection can be based on methods such as Western Blot (Western Blot), ELISA, antigen-antibody reaction, fluorescent dyes (such as FITC ), colloidal gold test strips, and protein chips, etc., by any one or more existing detection techniques, and will not be repeated here.

A fourth aspect of the present invention provides a method for detecting fucoidan, the method comprising:

Adding the polyclonal antibody above to the solution to be tested for incubation, then adding fucoidan labeled with a fluorescent dye to the solution for incubation, and judging the concentration or presence of fucoidan in the solution to be tested based on the fluorescence intensity; or,

Add the above polyclonal antibody to the solution to be tested, then add HRP-labeled secondary antibody, add TMB chromogenic solution for color development, and finally add H ₂ SO ₄ to terminate the reaction, and read the OD value at 450nm.

The fifth aspect of the present invention provides the application of the above fucoidan polyclonal antibody, kit or method in any one or more of the following:

(a) screening of fucoidan products;

(b) Basic research on fucoidan;

(c) Fucoidan quality monitoring.

Beneficial technical effects of the above-mentioned one or more technical solutions:

The above technical scheme discloses a fucoidan polyclonal antibody and its application in the detection of fucoidan. Specifically, the above technical scheme uses a fucoidan-bovine serum albumin (BSA) covalent complex to The polyclonal antibody to fucoidan was successfully obtained by immunization of non-human animals, and it was successfully used for the detection of fucoidan.

In summary, the above-mentioned technical scheme takes the preparation of polyclonal antibodies to fucoidan as an entry point, and provides a method basis for the preparation of polyclonal antibodies to carbohydrates through the study of the characteristics of the antibodies, and then provides a basis for the pharmacokinetics of carbohydrates. The establishment of the immune method provides the source material basis, so it has good practical application value.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the present invention. 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.

It should be noted that the terminology used here is only for describing specific implementations, and is not intended to limit the exemplary implementations according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, they mean There are features, steps, operations, means, components and/or combinations thereof.

In a typical embodiment of the present invention, a fucoidan polyclonal antibody is provided, and the fucoidan polyclonal antibody is prepared by pairing a fucoidan-bovine serum albumin (BSA) covalent complex to Obtained by immunization of non-human animals; wherein, the fucoidan-bovine serum albumin covalent complex is prepared by the following method: dissolving fucoidan, bovine serum albumin, and _NaCNBH in carbonate buffer Medium constant temperature reaction (such as 37°C) for 2-4 days (further 3 days), obtained after purification, wherein the mass ratio of fucoidan, bovine serum albumin, and _NaCNBH3 is 25-35:5- 15:1-5 (further 30:10:3). By controlling the dosage ratio of the three, the fucoidan-bovine serum albumin covalent complex is finally obtained.

Wherein, the pH of the carbonate buffer solution is 9.6; the purification is carried out by gel chromatography, specifically, the purification method is as follows: after the reaction is completed, connect the Sephadex G-200 column pair with the AKTA protein purification system The sample is subjected to gel chromatography and detected by ultraviolet light at 280nm to remove unreacted fucoidan, bovine serum albumin and NaCNBH ₃ to obtain the fucoidan-bovine serum albumin covalent complex. In a specific embodiment of the present invention, fucoidan (57.4%) and Protein content (47.8%).

In addition, immunization of non-human animals is a routine method for obtaining polyclonal antibodies. The specific method is well known to those skilled in the art and is not specifically limited. In a specific embodiment of the present invention, the immunization method includes: The fucoidan-bovine serum albumin covalent complex is dissolved in physiological saline, then mixed with complete Freund's adjuvant, and obtained by immunizing non-human animals. Wherein, the salt solution of the fucoidan-bovine serum albumin covalent complex is mixed with Freund's complete adjuvant in equal volumes, and the mixing method can be mixed by a double-push method. The concentration of the fucoidan in the saline solution is 1-10 mg/mL, preferably 8 mg/mL.

The non-human animals include non-human mammals and birds, such as rats, mice, guinea pigs, rabbits, horses, monkeys, orangutans, chickens, ducks, etc. In a specific embodiment of the present invention, the non-human Animals were BABL/c mice.

In yet another specific embodiment of the present invention, a kit for detecting fucoidan is provided, the kit at least containing the above-mentioned fucoidan polyclonal antibody.

The kit may also contain other components for detecting fucoidan, such as buffer, etc., which are not specifically limited here.

In yet another specific embodiment of the present invention, the application of the above fucoidan polyclonal antibody or kit in the detection of fucoidan is provided.

The detection can be qualitative or quantitative detection, that is, to determine the concentration or presence of fucoidan. Specifically, the detection can be based on methods such as Western Blot (Western Blot), ELISA, antigen-antibody reaction, fluorescent dyes (such as FITC ), colloidal gold test strips, and protein chips, etc., by any one or more existing detection techniques, and will not be repeated here.

In yet another specific embodiment of the present invention, a method for detecting fucoidan is provided, the method comprising: adding the above-mentioned polyclonal antibody to the solution to be tested for incubation, and then adding fucoidan labeled with a fluorescent dye Glycans are incubated, and the concentration or presence of fucoidan in the solution to be tested is judged based on the fluorescence intensity;

Wherein, the fluorescent dye may be FITC; the specific condition of the incubation treatment may be incubation at 30-40° C. (further 37° C.) for 4-8 hours. During the fluorescence detection process, the excitation wavelength Ex=485±20nm, and the emission wavelength Em=528±20nm.

Because the fucoidan added first will inhibit the binding of FITC-labeled fucoidan to the antibody, while other polysaccharides hardly affect the binding of FITC-labeled fucoidan to the antibody. And as the concentration of fucoidan increases, the fluorescence intensity gradually weakens, and there is a concentration gradient, so the above method can be used for qualitative and quantitative detection of fucoidan in solution. It should be noted that FITC-labeled fucoidan should not contain free FITC to prevent free FITC from interfering with the test results; in addition, in order to reduce interference, the solution to be tested can also be desalted to remove small molecular substances during the test.

or,

Add the above polyclonal antibody to the solution to be tested, then add HRP-labeled secondary antibody, add TMB chromogenic solution for color development, and finally add H ₂ SO ₄ to terminate the reaction, and read the OD value at 450nm.

The method also includes setting a negative control and a positive control; if the ratio (P/N) of the solution to be tested to the known negative control solution is greater than or equal to 2.1, and the OD value of the target solution to be tested is greater than or equal to 0.4, then it is judged as positive, otherwise it is judged as positive. is negative.

In yet another specific embodiment of the present invention, the application of the above fucoidan polyclonal antibody, kit or method in any one or more of the following is provided:

(a) screening of fucoidan products;

(b) Basic research on fucoidan;

(c) Fucoidan quality monitoring.

Wherein, in (a), the fucoidan-producing products include fucoidan-producing animals, plants or microorganisms, such as fucoidan-producing algae (such as brown algae) and marine invertebrates (such as sea urchins) and sea cucumbers).

The present invention will be described in further detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only used to illustrate the present invention.

Example

1. Antigen preparation

It is known that polysaccharides are T cell-independent antigens, have no immune memory effect, and cannot directly induce the body to produce high-titer specific and high-affinity antibodies. BSA, etc. can increase the immunogenicity of polysaccharides through the carrier effect: Th cells are responsible for recognizing carriers, B cells are responsible for recognizing haptens, and through the regulation of the immune network in the body, B cells are finally differentiated into memory cells, thereby producing high titers against polysaccharides antibodies.

The acetal structure of the end group of fucoidan is opened to connect with the amino group of BSA, and the original conformation of fucoidan is kept as far as possible to prepare the Fucoidan-BSA covalent complex. Methods as below:

Fucoidan (30 mg), BSA (10 mg) and NaCNBH ₃ (3 mg) were dissolved in pH 9.6 carbonate buffer, and placed in a 37°C incubator for 3 days to react. After the reaction was completed, the AKTA protein purification system was connected to the Sephadex G-200 column to perform gel chromatography on the sample, and the ultraviolet 280nm detection was used to remove unreacted Fucoidan, BSA, and NaCNBH3 to obtain Fucoidan-BSA covalent complexes, which were separated after lyophilization. Fucoidan (57.4%) and protein content (47.8%) in the components were determined by phenylcysteine hydrochloride-sulfuric acid method and BCA method.

2. Animal immunity

Experimental animals: BABL/c mice were raised in the animal room for 7 days after purchase to adapt to the surrounding environment. Before immunization, blood was drawn from the tail to obtain non-immune serum as a negative control for subsequent experiments.

Dissolve 4 mg of fucoidan-BSA covalent complex in 5 mL of normal saline, mix with an equal volume of Freund’s complete adjuvant by double-push method, inject 200 μg (0.5 ml) subcutaneously on the back, and inject 200 μg intraperitoneally with incomplete adjuvant one week later (0.5mL), repeated 3 times, once a week. 7 days after the last immunization, blood was collected by clipping the tail to detect serum antibodies by ELISA. For antibody-positive mice (Balb/c), 100 μg of antigen was injected into the tail vein of each mouse.

[Example of double-push method simplified version: take 250 μl of fucoidan mixed solution and complete adjuvant and mix them at a ratio of 1:1. Take 250μl of fucoidan solution (containing 2mg of fucoidan) in a 1.5ml EP tube, add 50μl of complete adjuvant for the first time, pump it with a 2.5ml syringe for 30min to make it fully emulsified, repeat this 5 times, add to 250 μl. About 4 hours, put a drop of the mixed preparation in clear water, and observe whether the milk droplets disperse quickly. If it disappears quickly, continue pumping until the milk droplets persist for a long time (prepared into a "water-in-oil" emulsion). The volume of the final prepared preparation was about 250 μl (considering the loss of half, the amount of fucoidan for each mouse immunization was about 1 mg), and then four to five different parts of the back of the mouse were selected for subcutaneous injection. 】

3. Collection of antibodies

One week before the final immunization, the antibody effect can be verified by taking blood from the tail vein. If the effect is not good, the time can be extended to boost the immunization again. Finally, the eyeballs were removed to take blood, left at room temperature for two hours, centrifuged at 4000r/min at 4°C for 5min to obtain serum, and stored at -20°C.

4. Fluorescence labeling of fucoidan

1. Fluorescently labeled polysaccharides

1.1 Synthesis of Fucoidan-Tyr (reductive amination method)

Dissolve 400mg of fucoidan in 15mL of 0.2mol/L potassium phosphate buffer (pH 8.0), then add 400mg of tyramine (Tyr) and 150mg of sodium cyanoborohydride in sequence, react at 37°C for 96h, and shake occasionally. After the reaction was completed, it was centrifuged, and the supernatant was applied to a Sephadex G-200 column, eluted with water, detected at 210 nm, and the first peak was collected and freeze-dried to obtain Fucoidan-Tyr.

1.2 Fucoidan-Tyr-FITC synthesis

Dissolve 200mg of Fucoidan-Tyr in water, adjust the pH to 8.5 with 0.5mol/L NaHCO ₃ , then add 25mg of FITC, react overnight at room temperature in the dark, add absolute ethanol to the reactant until the final concentration of ethanol is 80%, A large amount of bright yellow-green precipitates were precipitated, and the supernatant was discarded by centrifugation. Precipitate was redissolved with water, ethanol was reprecipitated, and repeated 3 times under the same conditions as above to obtain Fucoidan-Tyr-FITC, which was further purified on a Sephadex G-200 column, eluted with water, detected at 210nm, and the corresponding water-eluted fractions were collected and stored in freeze-dry , to obtain FITC-labeled fucoidan.

5. Antibody verification

1. Determination of antibody dilution

1.1 Dilute the serum in different ratios Dilute the serum at 1:100, 1:500, 1:1000, 1:2000, 1:5000, 1:10000 and 1:20000 respectively.

1.2 Antibody coating Take 100 μl of the above-mentioned diluted serum and 100 μl of negative serum (diluted 1:100), and 100 μl of PBS for the blank control group, add them to a black 96-well high-affinity plate, and incubate in a 37°C incubator for 4-8 hours. Wash 3 times with PBS, 1 min each time.

1.3 Add 100 μl of 5% skimmed milk powder for sealing, and block for 2 hours in a 37°C incubator. Wash 3 times with PBS, 1 min each time.

1.4 Add 100 μl of 200 μg/ml FITC-labeled fucoidan solution and incubate at 37°C for 4-8 hours. Wash with PBST 3 times, 1 min each time.

1.5 Use the cell imaging microplate detection system (Bio-Tek, Cytation 5, USA) to detect the fluorescence intensity (Ex=485±20nm, Em=528±20nm).

The test results are as follows:

Table 1 Detection results of antibody fluorescence intensity at different dilutions (n=3)

Antibody dilution The fluorescence intensity 1:100 176285±884 1:500 89826±918 1:1000 53427±827 1:2000 34927±619 1:5000 22319±583 1:10000 18164±816 1:20000 14645±689 negative serum 13421±752 blank control 4523±157

From the above results, it can be seen that compared with the blank control and negative control, the fluorescence intensity of fucoidan can be detected at different antibody dilutions, and the smaller the antibody dilution, the stronger the fluorescence intensity detected, which has a certain concentration trend. It can be used for the screening of fucoidan. For subsequent experiments, the antibody serum dilution was selected as 1:1000.

2. Make a standard curve

2.1 Make different concentration gradients of FITC-labeled fucoidan at concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μg/ml.

2.2 Antibody coating Dilute the antibody serum at 1:1000, take 100 μl of the above-mentioned diluted serum and 100 μl of negative serum (diluted at 1:1000), and 100 μl of PBS for the blank control group, add them to a black 96-well high-affinity plate, and keep at 37°C Incubate for 4-8 hours in the incubator. Wash 3 times with PBS, 1 min each time.

2.3 Add 100 μl of 5% skimmed milk powder for sealing, and block for 2 hours in a 37°C incubator. Wash 3 times with PBS, 1 min each time.

2.4 Add 100 μl of FITC-labeled fucoidan solution of different concentrations prepared in 2.1, and incubate at 37° C. for 4-8 hours. Wash with PBST 3 times, 1 min each time.

2.5 Use the cell imaging microplate detection system (Bio-Tek, Cytation 5, USA) to detect the fluorescence intensity (Ex=485±20nm, Em=528±20nm).

The test results are as follows:

Table 2 Detection results of binding of different concentrations of FITC-labeled fucoidan to antibody serum (n=3)

As can be seen from the above results, compared with the blank control and negative control, different concentrations of FITC-labeled fucoidin

The fluorescence intensity of fucoidan can be detected after the sugar is combined with the antibody serum, and the fluorescence intensity of 1.56μg/ml FITC-labeled fucoidan has no significant difference from the negative control, while the fluorescence intensity of 200μg/ml FITC-labeled fucoidan There is no significant difference between FITC-labeled fucoidan and 100μg/ml FITC-labeled fucoidan, which indicates that the concentration is too low and this method cannot be detected. After the concentration is too high, only the presence of FITC-labeled fucoidan in the solution can be detected, and its Therefore, the fluorescence intensity has a concentration-dependent enhancement within the concentration range of 3.13-100 μg/ml, which can be used to detect the concentration of FITC-labeled fucoidan.

3. Interference detection of free FITC on detection results

3.1 Prepare 50 μg/ml FITC-labeled fucoidan solution and prepare FITC solution with the same fluorescence intensity.

3.2 Antibody coating Dilute the antibody serum at 1:1000, take 100 μl of the above-mentioned diluted serum and 100 μl of negative serum (1:1000 dilution), and 100 μl of PBS for the blank control group, add them to a black 96-well high-affinity plate, and keep at 37°C Incubate for 4-8 hours in the incubator. Wash 3 times with PBS, 1 min each time.

3.3 Add 100 μl of 5% skimmed milk powder for sealing, and block for 2 hours in a 37°C incubator. Wash 3 times with PBS, 1 min each time.

3.4 Add the 50 μg/ml FITC-labeled fucoidan solution prepared in 3.1, and prepare 100 μl of FITC solutions with the same fluorescence intensity at the same time, and incubate at 37°C for 4-8h. Wash with PBST 3 times, 1 min each time.

3.5 Use the cell imaging microplate detection system (Bio-Tek, Cytation 5, USA) to detect the fluorescence intensity (Ex=485±20nm, Em=528±20nm).

The test results are as follows:

Table 3 free FITC interferes with detection results (n=3)

From the above results, it can be seen that compared with the blank control and negative control, the FITC solution with the same fluorescence intensity has a stronger binding ability than the 50 μg/ml FITC-labeled fucoidan solution, which can seriously interfere with the detection results, so this requires When we prepare FITC-labeled fucoidan, unreacted FITC must be completely removed to prevent free FITC from interfering with the detection results.

4. Determination of the interference of various polysaccharides

4.1 Prepare 100 μg/ml solutions with polysaccharides such as dextran, sodium alginate, hyaluronic acid, heparin, carrageenan, chondroitin sulfate, and fucoidan, respectively.

4.2 Antibody coating Dilute the antibody serum at 1:1000, take 100 μl of the above diluted serum and 100 μl of negative serum (diluted at 1:1000), and 100 μl of PBS for the blank control group, add them to a black 96-well high-affinity plate, and keep at 37°C Incubate for 4-8 hours in the incubator. Wash 3 times with PBS, 1 min each time.

4.3 Add 100 μl of 5% skimmed milk powder for sealing, and block for 2 hours in a 37°C incubator. Wash 3 times with PBS, 1 min each time.

4.4 Add 100 μl of various polysaccharide solutions of 100 μg/ml prepared in 4.1, and incubate at 37° C. for 4-8 hours. Wash 3 times with PBS, 1 min each time. Then, 100 μl of FITC-labeled fucoidan solution was incubated at 37° C. for 4-8 hours. Wash 3 times with PBS, 1 min each time.

4.5 Use the cell imaging microplate detection system (Bio-Tek, Cytation 5, USA) to detect the fluorescence intensity (Ex=485±20nm, Em=528±20nm).

The test results are as follows:

Table 4 Different polysaccharides and antibody serum binding detection results (n=3)

Polysaccharide type The fluorescence intensity Dextran 54550±864 sodium alginate 53904±981 hyaluronic acid 55307±823 heparin 54213±805 carrageenan 55619±737 Chondroitin Sulfate 53608±724 Fucoidan 13526±1026 negative serum 13029±708 blank control 4187±264

It can be seen from the above results that, compared with the blank control and negative control, adding fucoidan first can inhibit the binding of FITC-labeled fucoidan to the antibody, while other polysaccharides hardly affect the binding of FITC-labeled fucoidan to the antibody. The combination of antibodies shows that this detection method can eliminate the interference of various polysaccharides, and it is more specific to detect whether fucoidan is contained in the solution.

5. The method of screening whether fucoidan is contained in the solution

5.1 Prepare 1.56, 3.13, 6.25, 12.5, 25, 50, 100 μg/ml solutions with fucoidan.

5.2 Antibody coating Dilute the antibody serum at 1:1000, take 100 μl of the above-mentioned diluted serum and 100 μl of negative serum (diluted at 1:1000), and 100 μl of PBS for the blank control group, add them to a black 96-well high-affinity plate, and keep at 37°C Incubate for 4-8 hours in the incubator. Wash 3 times with PBS, 1 min each time.

5.3 Add 100 μl of 5% skimmed milk powder for sealing, and block for 2 hours in a 37°C incubator. Wash 3 times with PBS, 1 min each time.

5.4 Add 100 μl of fucoidan solutions with different concentrations prepared in 5.1, and incubate at 37°C for 4-8h. Wash 3 times with PBS, 1 min each time. Then, 100 μl of FITC-labeled fucoidan solution was incubated at 37° C. for 4-8 hours. Wash 3 times with PBS, 1 min each time.

5.5 Use the cell imaging microplate detection system (Bio-Tek, Cytation 5, USA) to detect the fluorescence intensity (Ex=485±20nm, Em=528±20nm).

The test results are as follows:

Table 5 Detection results of binding of different fucoidans to antibody serum (n=3)

Fucoidan concentration The fluorescence intensity 100 14607±925 50 31695±903 25 42601±729 12.5 47028±837 6.25 50149±736 3.13 52033±984 1.56 52304±751 0 54928±832 negative serum 13215±759 blank control 4297±316

It can be seen from the above results that compared with the blank control and negative control, with the increase of the concentration of fucoidan, the fluorescence intensity gradually weakened, and there was a concentration gradient. This method can be used for the detection of fucoidan in solution. In order to reduce interference, the solution can be desalted to remove small molecular substances during detection, and then detected by the above method.

6. Screen for the presence of fucoidan in the unknown bacterial solution

1. Coat 96-well high-affinity plate with PLL (polylysine) 100μg/ml, 100μl

2. Take 100μl of the target solution in the plate

3. Different proportions of BSA are blocked, preferably 0.5% BSA

4. Add 100 μl of the prepared polyclonal antibody and dilute it in different proportions, then add it to the well plate, preferably 1:10000

5. Add 100 μl of HRP-labeled secondary antibody

6. Add TMB100μl

7. Add 2M H ₂ SO ₄ to terminate the reaction

8. 450nm reading OD value

Negative control: replace the target solution with ultrapure water, or other polysaccharide solutions, and keep the other steps the same

Positive control: replace the target solution with fucoidan solution, and the other steps are the same

Result judgment: If the ratio (P/N) of the solution to be tested to the known negative solution is ≥ 2.1, and the OD value of the target solution to be tested is ≥ 0.4, it is judged as positive, otherwise it is judged as negative.

It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or equivalently replace the technical solutions of the present invention as required without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. A fucosan polyclonal antibody is characterized in that the fucosan isThe polyclonal antibody is obtained by immunizing a non-human animal with a fucosan-bovine serum albumin covalent complex; wherein, the fucosan-bovine serum albumin covalent compound is prepared by adopting the following method: fucosan, bovine serum albumin, and NaCNBH ₃ Dissolving in carbonate buffer solution for constant temperature reaction, and purifying to obtain the final product; wherein the fucosan, bovine serum albumin and NaCNBH are added ₃ The mass ratio of (A) to (B) is 25-35.

2. The fucosan polyclonal antibody of claim 1, wherein said carbonate buffer has a ph of 9.6; the purification is carried out by gel chromatography.

3. The fucosan polyclonal antibody of claim 2, wherein said purification method comprises: after the reaction is finished, an AKTA protein purification system is connected with a Sephadex G-200 column to carry out gel chromatography on the sample, ultraviolet 280nm detection is carried out, and unreacted fucosan, bovine serum albumin and NaCNBH are removed ₃ And obtaining the fucosan-bovine serum albumin covalent complex.

4. The fucosan polyclonal antibody of claim 1, wherein said method for immunizing a non-human animal comprises: the fucosan-bovine serum albumin covalent complex is dissolved in physiological saline, and then is uniformly mixed with Freund's complete adjuvant to immunize non-human animals.

5. The fucosan polyclonal antibody of claim 4, wherein the salt solution of the fucosan-bovine serum albumin covalent complex is mixed with Freund's complete adjuvant in equal volume, and the mixing manner is a double-push method; the concentration of the fucosan in the salt solution is 1-10mg/mL.

6. The fucosan polyclonal antibody of claim 1, wherein said non-human animal comprises a non-human mammal and an avian species, further comprising a BABL/c mouse.

7. A kit for detecting fucoidan, wherein said kit comprises at least the fucoidan polyclonal antibody according to any one of claims 1-6.

8. Use of the fucan polyclonal antibody according to any one of claims 1-6 or the kit according to claim 7 for detecting fucan; further, the detection is qualitative or quantitative detection, and the detection is performed based on any one or more detection technologies of western blotting, ELISA, antigen-antibody reaction, fluorescent dye, colloidal gold test paper and protein chip.

9. A method for detecting fucoidan, said method comprising:

adding the fucosan polyclonal antibody of any one of claims 1-6 into a solution to be tested for incubation treatment, adding a fluorochrome-labeled fucosan into the solution to be tested for incubation treatment, and judging the concentration or the presence of the fucosan in the solution to be tested based on fluorescence intensity;

further, the fluorescent dye is FITC; the specific incubation condition is incubation for 4-8h at 30-40 ℃; in the fluorescence detection process, the excitation wavelength Ex =485 +/-20 nm, and the emission wavelength Em =528 +/-20 nm;

or the like, or, alternatively,

adding the fucosan polyclonal antibody of any one of claims 1-6 into the solution to be tested, adding HRP-labeled secondary antibody, adding TMB color developing solution for color development, and adding H ₂ SO ₄ Stopping the reaction, and reading the OD value at 450 nm;

further, the method further comprises setting a negative control and a positive control; if the ratio P/N of the solution to be detected to the known negative control solution is more than or equal to 2.1, and the OD value of the target solution to be detected is more than or equal to 0.4, the solution is judged to be positive, otherwise, the solution is judged to be negative.

10. Use of the fucan polyclonal antibody of any one of claims 1-6, the kit of claim 7 or the method of claim 9 in any one or more of:

(a) Screening a fucosan production product;

(b) A fucosan-related basic study;

(c) Monitoring the quality of fucoidin;

wherein in the (a), the fucoidan-producing substance comprises an animal, a plant or a microorganism producing fucoidan.