CN115975024A - Fucosan polyclonal antibody and application thereof in detecting fucosan - Google Patents
Fucosan polyclonal antibody and application thereof in detecting fucosan Download PDFInfo
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Abstract
The invention belongs to the technical field of antibody preparation and detection analysis, and particularly relates to a fucosan polyclonal antibody and application thereof in detecting fucosan. Specifically, the invention successfully obtains the fucosan polyclonal antibody by immunizing a non-human animal with a fucosan-Bovine Serum Albumin (BSA) covalent complex, and successfully uses the fucosan polyclonal antibody for detecting the fucosan. The invention takes the preparation of the fucosan polyclonal antibody as an entry point, provides a method basis for the preparation of the carbohydrate polyclonal antibody through the research on the antibody characteristics of the fucosan polyclonal antibody, and further provides a source material basis for the establishment of an immunization method of carbohydrate pharmacokinetics, so the invention has good value of practical application.
Description
Technical Field
The invention belongs to the technical field of antibody preparation and detection analysis, and particularly relates to a fucosan polyclonal antibody and application thereof in detecting fucosan.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The polysaccharide has anticoagulant, antiviral, antioxidant, neurotrophic and protective biological activities. Pharmaceutical research of polysaccharides is therefore of great interest. However, because of the complex chemical structure of polysaccharides, there is no characteristic UV absorption. Therefore, a trace quantitative in vivo detection method with strong specificity is lacked at present. The current common isotope labeling method, high performance liquid chromatography, biological determination method and the like have some defects and shortcomings. The immunological method is widely applied to pharmacokinetic research due to good specificity and high sensitivity. The key to the application of immunological methods is to obtain antibodies with good specificity and high affinity. The nature of the antibody directly determines the success of the immunological method.
Fucoidan (Fucoidan) is a marine sulfated polysaccharide mainly derived from the cell wall of brown algae and some marine invertebrate tissues, and has good biocompatibility and biological activities of resisting oxidation, virus, immune regulation, inflammation and the like. However, fucoidans of different origin differ greatly in structure. For example, fucoidan derived from marine invertebrates is generally simple in structural composition, typically with regular simple repeats of several components. Fucoidan derived from brown algae is complex in structure and generally consists of two chain structures, one of which is composed of (1 → 3) - α -L-glucopyranose as a main chain, and the other of which is composed of α -L-glucopyranose as a main chain via (1 → 3) and (1 → 4) linkages. It is noteworthy that the biological activity of fucoidan is influenced by a number of factors, which are closely related to its sulfation degree and molecular weight, while for most fucoidans the structural skeleton is still unclear and the position and branching site of a particular sulfate group has not been determined. This poses certain difficulties in elucidating their structure-activity relationship. Meanwhile, the method is not beneficial to large-scale popularization and research application in academics and businesses.
It is known that the source of most of the fucoidans currently on the market is not always consistent and that the stability of the pharmacological activity of fucoidans cannot be ensured, possibly due to differences in manufacturers and batches. Therefore, there is a need to find an animal, plant or microorganism that can stably produce fucoidan with the same configuration and a definite molecular weight. It should be noted that, compared with animals and plants, the microorganism has the advantages of low production cost, stability, reliability and less interference from various external environmental factors, so the microorganism has more remarkable advantages. Therefore, it is necessary to establish a method for detecting whether or not fucoidan is contained in a solution, and the method is useful for screening of fucoidan-producing bacteria and the like.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a fucosan polyclonal antibody and application thereof in detecting fucosan. The invention takes the preparation of the fucosan polyclonal antibody as an entry point, provides a method basis for the preparation of the carbohydrate polyclonal antibody through the research on the antibody characteristics of the fucosan polyclonal antibody, and further provides a source material basis for the establishment of an immunization method of carbohydrate pharmacokinetics. The present invention has been completed based on the above results.
In order to achieve the technical purpose, the technical scheme provided by the invention is as follows:
in one aspect of the present invention, there is provided a fucosan polyclonal antibody obtained by immunizing a non-human animal with a fucosan-Bovine Serum Albumin (BSA) covalent complex; wherein, the fucosan-bovine serum albumin covalent compound is prepared by the following method: fucosan, bovine serum albumin, naCNBH 3 Dissolving in carbonate buffer solution, reacting at constant temperature for 2-4 days, 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.
In a second aspect of the present invention, there is provided a kit for detecting fucosan, said kit comprising at least the above-described fucosan polyclonal antibody.
The kit may further contain other components for detecting fucoidan, such as buffer, and the like, and is not particularly limited herein.
In a third aspect of the invention, the application of the fucosan polyclonal antibody or the kit in detecting fucosan is provided.
The detection may be qualitative or quantitative detection, that is, determining the concentration or presence of fucosan, and specifically, the detection may be performed based on any one or more existing detection technologies, such as Western blotting (Western Blot), ELISA, antigen-antibody reaction, fluorescent dye (e.g., FITC), colloidal gold test strips, and protein chips, and will not be described herein again.
In a fourth aspect of the present invention, there is provided a method for detecting fucosan, the method comprising:
adding the polyclonal antibody into a solution to be tested for incubation treatment, then adding fucosan marked by a fluorescent dye into the polyclonal antibody for incubation treatment, and judging the concentration or the existence of the fucosan in the solution to be tested based on the fluorescence intensity; or the like, or, alternatively,
adding the polyclonal antibody into a solution to be detected, adding HRP-labeled secondary antibody, adding TMB color development liquid for color development, and finally adding H 2 SO 4 The reaction was stopped and the OD was read at 450 nm.
In a fifth aspect of the present invention, there is provided the use of the above-described fucosan polyclonal antibody, kit or method in any one or more of:
(a) Screening a fucosan production product;
(b) A fucosan-related basic study;
(c) And (5) monitoring the quality of the fucoidin.
The beneficial technical effects of one or more technical schemes are as follows:
the technical scheme discloses a fucosan polyclonal antibody and application thereof in detecting fucosan, and particularly, the technical scheme successfully obtains the fucosan polyclonal antibody by immunizing a non-human animal with a fucosan-Bovine Serum Albumin (BSA) covalent complex, and successfully uses the fucosan polyclonal antibody in detecting the fucosan.
In conclusion, the technical scheme takes the preparation of the fucosan polyclonal antibody as an entry point, provides a method basis for the preparation of the carbohydrate polyclonal antibody through the research on the antibody characteristics of the fucosan polyclonal antibody, and further provides a source material basis for the establishment of an immunization method of carbohydrate pharmacokinetics, so that the fucosan polyclonal antibody has good practical application value.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In one exemplary embodiment of the present invention, there is provided a fucosan polyclonal antibody obtained by immunizing a non-human animal with a fucosan-Bovine Serum Albumin (BSA) covalent complex; wherein, the fucosan-bovine serum albumin covalent compound is prepared by the following method: fucosan, bovine serum albumin, naCNBH 3 Dissolving in carbonate buffer solution, reacting at constant temperature (such as 37 deg.C) for 2-4 days (further 3 days), and purifying to obtain the final product, wherein the fucosan, bovine serum albumin, and NaCNBH 3 The mass ratio of (A) is 25-35. And finally obtaining the fucosan-bovine serum albumin covalent compound by controlling the dosage proportion of the three components.
Wherein the carbonate buffer has a pH of 9.6; the purification is carried out by adopting a gel chromatography method, and specifically, the purification method comprises the following steps: 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 and bovine serum albumin are removedWhite and NaCNBH 3 And obtaining the fucosan-bovine serum albumin covalent complex. In one embodiment of the invention, fucosan (57.4%) and protein content (47.8%) in the fucosan-bovine serum albumin covalent complex were measured using the phenylcysteine hydrochloride-sulfuric acid method and the BCA method, respectively.
Furthermore, immunization of non-human animals is a routine method for obtaining polyclonal antibodies, and specific methods are well known to those skilled in the art and are not specifically limited, and in one embodiment of the present invention, the method for immunization 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. Wherein, the salt solution of the fucosan-bovine serum albumin covalent complex and Freund's complete adjuvant are mixed in equal volume, and the mixing mode can adopt a double-pushing method to mix uniformly. The concentration of the fucosan in the salt solution is 1-10mg/mL, and the concentration is preferably 8mg/mL.
The non-human animal includes non-human mammals and birds, such as rats, mice, guinea pigs, rabbits, horses, monkeys, chimpanzees, chickens, ducks, and the like, and in one embodiment of the invention, the non-human animal is a BABL/c mouse.
In another embodiment of the present invention, a kit for detecting fucosan is provided, which comprises at least the above-described fucosan polyclonal antibody.
The kit may further contain other components for detecting fucosan, such as a buffer, and the like, and is not particularly limited herein.
In another embodiment of the present invention, the fucosan polyclonal antibody or the kit is used for detecting fucosan.
The detection may be qualitative or quantitative detection, that is, determining the concentration or presence of fucosan, and specifically, the detection may be performed based on any one or more existing detection technologies, such as Western blotting (Western Blot), ELISA, antigen-antibody reaction, fluorescent dye (e.g., FITC), colloidal gold test strips, and protein chips, and will not be described herein again.
In another embodiment of the present invention, there is provided a method for detecting fucoidan, the method comprising: adding the polyclonal antibody into a solution to be tested for incubation treatment, then adding fucosan marked by a fluorescent dye into the solution for incubation treatment, and judging the concentration or the existence of the fucosan in the solution to be tested based on the fluorescence intensity;
wherein the fluorescent dye may be FITC; the incubation treatment may be carried out under conditions of 30-40 deg.C (further 37 deg.C) for 4-8h. In the fluorescence detection process, the excitation wavelength Ex =485 +/-20 nm, and the emission wavelength Em =528 +/-20 nm.
Since the fucoidan added first inhibits the binding of FITC-labeled fucoidan to the antibody, the other polysaccharides hardly affect the binding of FITC-labeled fucoidan to the antibody. And the fluorescence intensity gradually weakens along with the increase of the concentration of the fucosan, and the method has a concentration gradient, so the method can be used for qualitative and quantitative detection of the fucosan in the solution. And it should be noted that the FITC-labeled fucoidan cannot contain free FITC, so as to prevent the free FITC from interfering with the detection result; in addition, in order to reduce interference, the solution to be detected can be desalted to remove small molecular substances during detection.
Or the like, or, alternatively,
adding the polyclonal antibody into a solution to be detected, adding an HRP-labeled secondary antibody, adding a TMB color development solution for color development, and finally adding H 2 SO 4 The reaction was stopped and OD was read at 450 nm.
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, judging the target solution to be detected to be positive, otherwise, judging the target solution to be negative.
In another embodiment of the present invention, there is provided a use of the above-described fucosan polyclonal antibody, kit or method in any one or more of:
(a) Screening a fucosan production product;
(b) A fucosan-related basic study;
(c) And (5) monitoring the quality of the fucoidin.
Wherein, in the (a), the fucoidan-producing substance comprises animal, plant or microorganism producing fucoidan, such as algae (such as brown algae) producing fucoidan and marine invertebrate (such as sea urchin and sea cucumber).
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof.
Examples
1. Antigen preparation
It is known that polysaccharose T cell independent antigen has no immunological memory effect and cannot directly induce body to produce high titer specificity and high affinity antibody. BSA, etc. can increase the immunogenicity of polysaccharides by carrier effects: th cells are responsible for recognizing vectors, B cells are responsible for recognizing haptens, and the B cells are finally differentiated to form memory cells through in vivo immune network regulation, so that high-titer antibodies against polysaccharides are generated.
The terminal acetal structure of fucosan is opened to connect with the amino group of BSA, and the original conformation of fucosan is maintained as much as possible to prepare Fucoidan-BSA covalent complex. The method comprises the following steps:
fucoidan (30 mg), BSA (10 mg), naCNBH 3 (3 mg) was dissolved in a carbonate buffer solution of pH9.6, and the mixture was placed in an incubator at 37 ℃ for 3 days. 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, unreacted Fucoidan, BSA and NaCNBH3 are removed, a Fucoidan-BSA covalent complex is obtained, and after freeze-drying, fucoidan (57.4%) and protein content (47.8%) in the component are determined by a benzene cysteine hydrochloride-sulfuric acid method and a BCA method respectively.
2. Animal immunization
Experimental animals: BABL/c mice after purchase were first raised in the animal room for 7 days, acclimated, tail bled before immunization, and non-immune sera obtained as negative controls for subsequent experiments.
Fucoidan-BSA covalent complex 4mg was dissolved in physiological saline 5mL, mixed with equal volume of Freund's complete adjuvant by double push, injected subcutaneously at the back with 200. Mu.g (0.5 mL), and after one week, the incomplete adjuvant was intraperitoneally injected with 200. Mu.g (0.5 mL) 3 times a week. And finally, after 1 immunization for 7d, cutting the tail and taking blood to measure the serum antibody by an ELISA method. For antibody-positive mice (Balb/c), 100. Mu.g of antigen was injected per tail vein.
An example of a simple operation of the double push method: and taking 250 ul of the fucosan mixed solution and the complete adjuvant, and mixing uniformly according to the weight ratio of 1. Mu.l of fucoidan solution (containing 2mg of fucoidan) was taken in a 1.5ml EP tube, 50. Mu.l of complete adjuvant was added for the first time, and the mixture was thoroughly emulsified by pipetting with a 2.5ml syringe for 30min, and this was repeated 5 times to add 250. Mu.l. About 4h, adding one drop of the mixed preparation into clear water, observing whether the emulsion drops rapidly disperse, and if the emulsion drops rapidly fade, continuing to suck until the emulsion drops are not dispersed for a long time (prepared into water-in-oil emulsion). The final preparation was made in a volume of about 250. Mu.l (approximately 1mg of fucoidan per immunized mouse, considering half the loss), and then four to five different sites on the back of the mouse were selected for subcutaneous injection. Therein for teaching
3. Collection of antibodies
One week before final immunization, the effect of the antibody can be verified by taking blood from the tail vein, and if the effect is not good, the immunization can be strengthened again for a prolonged period. And finally, taking blood from eyeballs, standing for two hours at normal temperature, centrifuging for 5min at 4 ℃ at 4000r/min, taking serum, and storing at-20 ℃.
4. Fluorescent labeling of fucoidan
1. Fluorescent-labeled polysaccharide
1.1 Synthesis of Fucoidan-Tyr (reductive amination)
400mg of fucosan was dissolved in 15mL of 0.2mol/L potassium phosphate buffer (pH 8.0), and then 400mg of tyramine (Tyr) and 150mg of sodium cyanoborohydride were added in this order to react at 37 ℃ for 96 hours with occasional shaking. And after the reaction is finished, performing centrifugal separation, putting the supernatant on a Sephadex G-200 column, eluting with water, detecting at 210nm, collecting a first peak, and freeze-drying to obtain the Fucoidan-Tyr.
1.2Fucoidan-Tyr-FITC Synthesis
200mg of Fucoidan-Tyr are dissolved in water and 0.5mol/L NaHCO is used 3 Adjusting pH to 8.5, adding 25mg FITC, reacting at room temperature under dark condition overnight, adding anhydrous ethanol to the reaction product until the final concentration of ethanol is 80%, a large amount of bright yellow green precipitate is separated out, and the supernatant is discarded by centrifugation. Adding water to the precipitate for redissolving, precipitating with ethanol, repeating for 3 times under the same conditions to obtain Fucoidan-Tyr-FITC, further purifying with Sephadex G-200 column, eluting with water, detecting at 210nm, collecting corresponding water eluate components, and freeze-drying for storage to obtain FITC-labeled fucosan.
5. Antibody validation
1. Determination of antibody dilution
1.1 dilution of serum in different ratios serum was diluted in 1:100,1:500,1:1000,1:2000,1:5000,1:10000 and 1:20000 to dilute.
1.2 antibody coating 100. Mu.l of the diluted serum and 100. Mu.l of the negative serum (1. PBS wash 3 times, each time for 1min.
Adding 100 mul of 5% skimmed milk powder into the container under 1.3 sealing condition, and sealing the container in an incubator at 37 ℃ for 2h. PBS wash 3 times, each time for 1min.
1.4 Add 200. Mu.g/ml FITC labeled fucoidan solution 100. Mu.l and incubate at 37 ℃ for 4-8h. PBST washing 3 times, each time 1min.
1.5 fluorescence intensity was measured using cell imaging microplate detection system (Bio-Tek, catalysis 5, USA) (Ex = 485. + -.20nm, em = 528. + -.20 nm).
The detection results are as follows:
table 1 results of fluorescence intensity measurements of different dilutions of antibody (n = 3)
| Dilution of antibody | Intensity of fluorescence |
| 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 results, compared with a blank control and a negative control, the fluorescence intensity of the fucosan can be detected by different antibody dilutions, and the fluorescence intensity detected by the smaller the antibody dilution is, the stronger the fluorescence intensity is, the concentration trend is certain, and the method can be used for screening the fucosan. Subsequent experiments selected antibody serum dilutions of 1:1000.
2. making a standard curve
2.1 fucoidan labeled with FITC different concentration gradients were made at concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200. Mu.g/ml.
2.2 antibody coating antibody sera were expressed as 1:1000 dilution, 100. Mu.l of the above diluted serum and 100. Mu.l of the negative serum (1 dilution 1000) and 100. Mu.l of PBS as a blank control were added to a black 96-well high affinity plate and incubated at 37 ℃ for 4-8 hours in an incubator. PBS wash 3 times, each time for 1min.
2.3 sealing, adding 100 mul of 5% skimmed milk powder, and sealing at 37 ℃ for 2h. PBS wash 3 times, each time for 1min.
2.4 Add 100. Mu.l of FITC labeled fucoidan solution prepared at 2.1 different concentrations and incubate at 37 ℃ for 4-8h. PBST was washed 3 times for 1min each.
2.5 fluorescence intensity was measured using a cell imaging microplate detection system (Bio-Tek, catalysis 5, USA) (Ex = 485. + -.20nm, em = 528. + -.20 nm).
The detection results are as follows:
TABLE 2 detection of FITC-labeled fucoidan binding to antibody serum at various concentrations (n = 3)
As can be seen from the above results, FITC labeled fucoidan was present at different concentrations compared to the blank control and the negative control
After the sugar is combined with antibody serum, the fluorescence intensity of the fucosan can be detected, the fluorescence intensity of 1.56 mu g/ml FITC marked fucosan has no significant difference with a negative control, and the fluorescence intensity of 200 mu g/ml FITC marked fucosan and 100 mu g/ml FITC marked fucosan also have no significant difference, which indicates that the method cannot detect the fucosan with too low concentration, and only can detect the existence of FITC marked fucosan in the solution after the concentration is too high, so that the concentration of the FITC marked fucosan cannot be measured, and the fluorescence intensity has concentration-dependent enhancement in the concentration range of 3.13-100 mu g/ml, and can be used for detecting the concentration of FITC marked fucosan.
3. Interference detection of free FITC on detection result
3.1 prepare FITC labeled fucosan solution of 50. Mu.g/ml, and prepare FITC solution of the same fluorescence intensity.
3.2 antibody coating antibody sera were expressed as 1:1000 dilution, 100. Mu.l of the above diluted serum and 100. Mu.l of the negative serum (1 dilution 1000) and 100. Mu.l of PBS as a blank control were added to a black 96-well high affinity plate and incubated at 37 ℃ for 4-8 hours in an incubator. PBS wash 3 times, each time for 1min.
3.3 sealing, adding 100 mul of 5% skimmed milk powder, and sealing in an incubator at 37 ℃ for 2h. PBS wash 3 times, each time for 1min.
3.4 Add 3.1 preparation of 50. Mu.g/ml FITC labeled fucoidan solution, while preparing 100. Mu.l each of FITC solution of the same fluorescence intensity, incubate at 37 ℃ for 4-8h. PBST was washed 3 times for 1min each.
3.5 fluorescence intensity was measured using cell imaging microplate detection system (Bio-Tek, catalysis 5, USA) (Ex = 485. + -.20nm, em = 528. + -.20 nm).
The detection results are as follows:
table 3 interference detection of free FITC on detection (n = 3)
From the above results, it can be seen that the FITC solution having the same fluorescence intensity has stronger binding ability than the FITC-labeled fucosan solution of 50. Mu.g/ml and can seriously interfere with the detection result, compared to the blank control and the negative control, and therefore this requires us to completely remove the unreacted FITC when preparing FITC-labeled fucosan in order to prevent the free FITC from interfering with the detection result.
4. Determination of interference of various polysaccharides
4.1 preparing 100 microgram/ml solutions by using polysaccharide such as dextran, sodium alginate, hyaluronic acid, heparin, carrageenan, chondroitin sulfate, fucosan, etc.
4.2 antibody coating antibody sera were expressed as 1:1000 dilution, 100. Mu.l of the above diluted serum and 100. Mu.l of the negative serum (1 dilution 1000) and 100. Mu.l of PBS as a blank control were added to a black 96-well high affinity plate and incubated at 37 ℃ for 4-8 hours in an incubator. PBS wash 3 times, each time for 1min.
4.3 sealing, adding 100 mul of 5% skimmed milk powder, and sealing at 37 ℃ for 2h. PBS wash 3 times, each time for 1min.
4.4 Add 4.1 100. Mu.l of each polysaccharide solution prepared at 100. Mu.g/ml and incubate at 37 ℃ for 4-8h. PBS wash 3 times, each time for 1min. Then, 100. Mu.l of FITC-labeled fucosan solution was incubated at 37 ℃ for 4 to 8 hours. PBS wash 3 times, each time for 1min.
4.5 fluorescence intensity was measured using a cell imaging microplate detection system (Bio-Tek, catalysis 5, USA) (Ex = 485. + -.20nm, em = 528. + -.20 nm).
The detection results are as follows:
TABLE 4 results of detection of binding of different polysaccharides to antibody sera (n = 3)
| Classes of polysaccharides | Intensity of fluorescence |
| Dextran | 54550±864 |
| Sodium alginate | 53904±981 |
| Hyaluronic acid | 55307±823 |
| Heparin | 54213±805 |
| Carrageenan | 55619±737 |
| Chondroitin sulfate | 53608±724 |
| Fucosan | 13526±1026 |
| Negative serum | 13029±708 |
| Blank control | 4187±264 |
From the results, compared with a blank control and a negative control, adding the fucosan at first can inhibit the combination of the FITC-labeled fucosan and the antibody, and other polysaccharides hardly influence the combination of the FITC-labeled fucosan and the antibody, which shows that the detection method can eliminate the interference of various polysaccharides and can detect whether the solution contains the fucosan more specifically.
5. Method for screening fucosan in solution
5.1 fucosan is used to prepare solutions of 1.56, 3.13, 6.25, 12.5, 25, 50, 100. Mu.g/ml.
5.2 antibody coating antibody sera were expressed as 1:1000 dilution, 100. Mu.l of the above diluted serum and 100. Mu.l of the negative serum (1 dilution 1000) and 100. Mu.l of PBS as a blank control were added to a black 96-well high affinity plate and incubated at 37 ℃ for 4-8 hours in an incubator. PBS wash 3 times, each time for 1min.
5.3 sealing, adding 100 mul of 5% skimmed milk powder, and sealing at 37 deg.C for 2h. PBS wash 3 times, each time for 1min.
5.4 Add 5.1 prepare different concentrations of fucoidan solution 100 u l,37 degrees C were incubated for 4-8h. PBS wash 3 times, each time for 1min. Then, 100. Mu.l of FITC-labeled fucosan solution was incubated at 37 ℃ for 4 to 8 hours. PBS wash 3 times, each time for 1min.
5.5 fluorescence intensity was measured using a cell imaging microplate detection system (Bio-Tek, catalysis 5, USA) (Ex = 485. + -.20nm, em = 528. + -.20 nm).
The detection results are as follows:
TABLE 5 results of binding assay of different fucoidans to antibody sera (n = 3)
| Fucosan concentration | Intensity of fluorescence |
| 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 |
From the above results, it can be seen that, compared with the blank control and the negative control, the fluorescence intensity gradually decreases with the increase of the fucosan concentration, and the method has a concentration gradient, and can be used for detecting the fucosan in the solution. In order to reduce interference, the solution can be desalted to remove small molecular substances during detection, and then the detection method is adopted.
6. Screening unknown bacteria solution for the presence of fucoidan
1. 96-well high affinity plates were coated with 100. Mu.l of PLL (polylysine) 100. Mu.g/ml
2. 100 μ l of the target solution was placed in a plate
3. Blocking with different proportions of BSA, preferably 0.5% BSA
4. After adding 100. Mu.l of the prepared polyclonal antibody at various ratios, the mixture is added to a well plate, preferably 1:10000
5. Add HRP-labeled Secondary antibody 100. Mu.l
6. Adding 100. Mu.l of TMB
7. Addition of 2M H 2 SO 4 Termination of the reaction
OD value read at 8.450nm
Negative control: the target solution is changed into ultrapure water or other polysaccharide solution, and other steps are kept consistent
Positive control: the target solution is changed into the fucosan solution, and other steps are consistent
And (4) judging a result: if the ratio (P/N) of the solution to be detected to the known negative 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, judging the solution to be detected to be positive, otherwise, judging the solution to be negative.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution 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.
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