CN113845596A - Antibody for resisting low molecular weight xanthan gum and application thereof - Google Patents
Antibody for resisting low molecular weight xanthan gum and application thereof Download PDFInfo
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Abstract
The invention provides a monoclonal antibody for specifically recognizing low-molecular-weight xanthan gum (the relative molecular mass is 100 ten thousand), and a preparation method and application thereof. Combining protein informatics and immunology, the low molecular weight xanthan gum is coupled to KLH carrier protein to be used as immunogen, and monoclonal antibody capable of specifically recognizing the low molecular weight xanthan gum is obtained after mice are immunized. The monoclonal antibody has no cross reaction with high molecular weight xanthan gum and other antigens, has the advantages of high affinity, high sensitivity and high specificity when being used for detection, and can be widely applied to product quality inspection and clinical research.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a monoclonal antibody of low-molecular-weight xanthan gum and application of the monoclonal antibody.
Background
Xanthan Gum (XG) is a biological macromolecular polysaccharide, is produced by fermenting Xanthomonas campestris (Xanthomonas campestris), and has a basic structure consisting of a D-glucose main chain and a trisaccharide side chain which are connected by beta-1, 4 bonds, wherein the side chain is formed by alternately connecting D-mannose and D-glucuronic acid. The xanthan gum has a relative molecular mass (Mr) of 2 × 106～2×107. The basic structure of the xanthan gum molecule is composed of pentose repeating units, a main chain is D-glucose connected by beta-1, 4 bonds, a side chain is trisaccharide formed by alternately connecting D-mannose and D-glucuronic acid, wherein the C position 6 of mannose connected with the main chain is partially acetylated, and the C positions 4 and 6 of mannose at the tail end of partial side chain are connected with 1 pyruvate group; the secondary structure is a double-helix or multi-helix structure formed by reversely winding the trisaccharide side chain on the main chain and maintaining the trisaccharide side chain through a hydrogen bond; the tertiary structure is a network structure formed by the secondary helical structure through non-covalent bonds.
The structure of xanthan gum gives it a number of excellent properties: (1) pseudoplastic rheology: the viscosity of the solution decreases with the shearing rate; (2) stability: the main chain is protected by the spiral structure and the reticular structure of the xanthan gum molecule, and the xanthan gum has the capabilities of resisting enzymolysis, high temperature and acid and alkali; (3) thickening property: xanthan gum has a higher viscosity at low concentrations, and the viscosity of its aqueous solution is about 100 times the same mass of gelatin; (4) suspension and emulsification properties: by means of the thickening effect of xanthan gum on the water phase, the incompatibility of oil phase and water phase is weakened, and stable oil-water dynamic balance is formed; (5) biocompatibility: the research shows that the xanthan gum has no acute toxicity, no growth inhibition effect and no irritation to eyes and skin for dogs and rats. Long term feeding had no significant effect on blood, survival and growth rate in dogs and rats. Xanthan gum was first approved by the FDA in the united states for use in food and medicine in 1969 and is now incorporated as a pharmaceutical excipient in the 2010 edition of the chinese pharmacopoeia. At present, xanthan gum is widely used as a food additive and a pharmaceutical adjuvant. In the field of pharmaceutical preparations, xanthan gum has been widely studied as sustained and controlled release preparations for oral administration, mucosal administration preparations, and sustained and controlled release materials for mucosal administration preparations and implant preparations.
Although xanthan gum is widely used as a food additive, a pharmaceutical adjuvant and the like, reports on metabolism in organisms are less because a better method for measuring the content of a medicament in the organisms is lacked. Oral xanthan gum is hardly absorbed and utilized by the body. Mixing into feed for rat14C-labeled xanthan gum with dosage of 50mg for 100 hr metabolism of small amount of radioactive substance to CO2Most of the radioactivity was recovered by excretion and fecal excretion, and no tissue accumulation was observed.
The monoclonal antibody obtained by the invention specifically recognizes the low molecular weight xanthan gum, has the advantages of high affinity, high sensitivity and high specificity when being used for detection, and is widely applied to xanthan gum product quality inspection and in vivo drug content determination clinical research.
Disclosure of Invention
The invention provides an antibody against low molecular weight xanthan gum, which is obtained by immunizing a mouse to prepare hybridoma cells by coupling the low molecular weight xanthan gum to carrier protein as an immunogen. In a specific embodiment, the carrier protein is KLH. The antibody is capable of specifically recognizing low molecular weight xanthan gum.
In one aspect, the invention provides an antibody against xanthan gum, which has a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 2.
In one embodiment, the xanthan gum is a low molecular weight xanthan gum; in one embodiment, the low molecular weight xanthan gum has a relative molecular mass of from 10 to 200 million, and in other embodiments, the low molecular weight xanthan gum has a relative molecular mass of from 50 to 150 million, for example, 100 million.
In one embodiment, the antibody is a recombinant antibody, a monoclonal antibody or a polyclonal antibody, preferably, a monoclonal antibody.
In one embodiment, the antibody is a murine antibody, a human antibody, or a chimeric antibody.
In a preferred embodiment, the antibody is a mouse antibody.
In one embodiment, the antibody further comprises a heavy chain constant region comprising an IgG selected from IgG1, IgG2, IgG3, or IgG 4.
In another aspect, the invention also provides hybridoma cells producing the above antibodies.
On the other hand, the invention also provides the application of the antibody in detecting xanthan gum; in one embodiment, the xanthan gum is a low molecular weight xanthan gum.
In another aspect, the present invention also provides a method for detecting xanthan gum, which comprises the step of detecting xanthan gum by using the above antibody.
In one embodiment, the method is an indirect immunoenzyme-linked immunosorbent (ELISA) method or an Immunohistochemistry (ICH) method.
In another aspect, the present invention also provides a reagent for detecting xanthan gum, which comprises the above antibody.
In another aspect, the present invention also provides a method for detecting xanthan gum in a sample, the method comprising the step of detecting xanthan gum using the above antibody.
In one embodiment, the sample is selected from the group consisting of food, drugs, metabolites of human or other animals such as plasma, tissue, joint synovial fluid, and the like.
The invention uses low molecular weight xanthan gum which can induce an organism to generate immunoreaction and is coupled to KLH to serve as immunogen to immunize a mouse, a hybridoma cell strain which can continuously and stably secrete the low molecular weight xanthan gum monoclonal antibody is obtained by cell fusion and screening through a hybridoma technology, and the monoclonal antibody is obtained by the secretion of the cell strain. The monoclonal antibody prepared by coupling low molecular weight xanthan gum with carrier protein as immunogen can specifically identify low molecular weight xanthan gum, 40 positive clones are screened out again, 6 positive clones are further screened out, and the monoclonal antibody is respectively named as 3A 7. The monoclonal antibody 3A7 has good affinity, and experiments show that the antibody titer can reach more than 0.001ug/mL in the experiment of combining 3A7 with low molecular weight xanthan gum ELISA, and the monoclonal antibody has no obvious specific cross with other related polysaccharide molecules; and 3A7 can be used for detecting xanthan gum drug metabolism and distribution in vivo by immunohistochemical method
Drawings
FIG. 1 shows that positive clones for identifying low molecular weight xanthan gum are screened by indirect ELISA method, wherein 600 clones are screened, 40 positive clones are screened, and better 10 positive clones are selected for further re-screening.
FIG. 2 shows an SDS-PAGE electrophoresis of mAb 3A7, wherein M is a protein molecular weight standard (kDa) and 100/10/1 is the concentration of mAb in μ g/mL;
FIG. 3. results of Clone3A7 used in immunohistochemical method to detect xanthan gum drug metabolism and distribution in vivo.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Example 1 establishment of hybridoma cell lines
First, experimental material
1. Immunogen: the low molecular weight xanthan gum with the relative molecular weight of 100 ten thousand is coupled to carrier protein to be used as immunogen, and the hybridoma is prepared by immunizing mice.
2. Culture medium: DMEM medium was purchased from Hyclone; HAT, HT selection medium, pristane were purchased from Sigma.
3. Experimental animals: BALB/c mice, 8-12 weeks old, female, SPF-grade animals.
4. Other materials: freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma company; PEG4000 from Fluka; HRP-goat anti-mouse IgG antibody was purchased from Jackson Immune; the other reagents are all domestic analytical pure products.
Second, establishment of hybridoma cell line
1. Animal immunization
1) Basic immunity: the antigen was mixed with Freund's complete adjuvant in equal volume and emulsified thoroughly, and injected subcutaneously in divided portions, each BALB/c mouse being injected in an amount of 100. mu.g per time.
2) And (3) boosting immunity: the boosting immunity adopts emulsion of antigen and Freund's incomplete adjuvant. 3 days before cell fusion, a physiological saline solution containing 150ug of antigen was intraperitoneally injected.
2. Preparation of hybridoma cells
Spleen cells from mice were harvested conventionally and fused with SP2/0 cells at a 10:1 ratio with 500g/L PEG 4000. Selectively culturing with HAT culture solution, taking supernatant, and screening hybridoma cell strains secreting low molecular weight xanthan gum antigen by adopting an indirect ELISA method 10-15 days after fusion. The obtained positive clones were subcloned by limiting dilution method. The indirect ELISA method was performed as follows: the plate was coated with 200. mu.L of low molecular weight xanthan gum, using immune mouse serum 1:2000 as a positive control, culture medium supernatant without clonal growth and normal mouse serum as negative controls, adding 1:2000 HRP-goat anti-mouse IgG 100. mu.L per well, and finally determining the OD at 450 nm. When the OD450 value is more than 2 times of that of the negative control, the positive clone can be judged initially, 40 positive clones are obtained in total, and 10 better positive clones are selected from the positive clones, as shown in figure 1.
3. Establishment of hybridoma cell lines
After 4 times of subcloning and indirect ELISA or cell ELISA screening, 1 hybridoma cell line which stably secretes the monoclonal antibody aiming at the low molecular weight xanthan gum is obtained.
TABLE 1 rescreening positive clones (remaining clones underlined)
| Clone# | 1E4 | 3A6 | 3A7 | 3C5 | 3G2 | 4C10 | 4D8 | 5A7 | 6G8 | NC | PC |
| OD | 0.112 | 0.244 | 1.879 | 0.113 | 0.072 | 0.084 | 0.138 | 0.085 | 0.098 | 0.072 | 0.689 |
4. Potency assay for monoclonal antibodies using the hybridoma cell lines
1) And (3) measuring ascites titer of the mice: the ascites titer prepared by detecting the 3A7 hybridoma cells by an indirect ELISA method is as follows: >1:20000000
2) And (3) measuring the purified antibody titer: the titer of the purified antibody prepared by detecting the hybridoma cells by an indirect ELISA method is as follows: 0.05 ng/mL.
5. Subculturing of hybridoma cell lines
And (2) continuously culturing and passaging the hybridoma cell line in a DMEM culture medium containing 10% fetal calf serum, wherein after 10 generations of culture, the hybridoma cell line can still grow well and stably passaged, and the titer of the supernatant of the culture solution can still reach more than 1: 10000.
The results show that the obtained hybridoma cell line can be stably passaged, and can continuously and stably secrete the monoclonal antibody against the low-molecular-weight xanthan gum.
After obtaining hybridoma cells producing the desired monoclonal antibody, a portion of the hybridoma cells must be preserved, otherwise, during serial passages, mutations or chromosomal shifts may occur such that the intrinsic properties are lost or antibody-producing properties are lost. In addition, in the long-term culture process, pollution and destruction cannot be avoided. A part must be kept refrigerated. The preservation method comprises the following steps:
1. material
(1) Cell: cells were taken at logarithmic growth phase.
(2) 10% dimethyl sulfoxide protective solution (dimethyl sulfoxide can damage the filter, and is damaged by high pressure, so that it can not be filtered or sterilized by high pressure, and is itself a drug, and is sterile): contains 10% of dimethyl sulfoxide, 20% of inactivated fetal bovine serum and 70% of RPMI-1640 liquid.
(3) 20% FCS-1640 medium: contains 100U/mL of penicillin and 100 mu g/mL of streptomycin.
(4) Sterilized 2mL ampoule or the like
2. Method of operation
(1) The old culture medium was removed from the cell culture flask, and 10% FCS-1640 solution was added to suspend the cells.
(3) Centrifuging at 1000 r/min for 10min, and removing supernatant. The cell sediment is 10%Preparing dimethyl sulfoxide protective solution into suspension to 1.0 × 107cells/mL.
(3) Sampling, trypan blue staining, and counting viable cells, which should be above 95%.
(4) The cells are subpackaged into ampoules with 0.5-1.0 mL of each ampoule by an injector, and the ampoules are sealed by melting.
(5) Standing at 4 deg.C for 2 h.
(6) Placing in liquid nitrogen tank to obtain gaseous part (-70 deg.C) for 15 hr.
(7) Transferring into liquid nitrogen part.
Example 2 preparation of monoclonal antibodies against Low molecular weight Xanthan Gum
First, antibody preparation
Adult BALB/c mice were selected and intraperitoneally inoculated with 0.5mL of pristane per mouse. The 16 th generation of 3A7 hybridoma cells are inoculated to the abdominal cavity after 7 to 10 days, and the cell size of each mouse is 1 multiplied by 106～2×106And (4) respectively. After 5 days, when the abdomen is obviously enlarged and the hand touches the abdomen, the skin is tense, and the ascites can be collected by using the 9-gauge needle.
The ascites fluid was centrifuged (13000r/min for 30 minutes), the cell fraction and other precipitates were removed, and the supernatant was collected. Purifying with Protein G-Sepharose CL-4B, wherein the upper column liquid is 20mM PBS buffer solution, and the column chromatography eluent is: obtaining the monoclonal antibody of the low molecular weight xanthan gum by using 20mM glycine buffer solution with the pH value of 2.7.
Identification of antibodies
1. And (3) identifying the purity of the antibody:
the purity was above 95% as determined by SDS-PAGE, as shown in FIG. 2.
2. Identification of antibody class and subclass:
the Ig subtypes of the antibody produced by the hybridoma cells were identified by indirect ELISA using antibodies against various Ig subtypes of mice, and the results showed that Clone3A7 was IgG2a, and as shown in Table 2, Clone3A7 showed the strongest signal of IgG2a, and Clone3A7 was IgG2a subtype according to the judgment criteria of the subtype identification results.
TABLE 2 subtype identification of mAb 3A7
| 3A7 | |
| IgG1 | 0.0725 |
| IgG2a | 0.5172 |
| IgG2b | 0.0703 |
| IgG3 | 0.0744 |
| IgA | 0.0696 |
| IgM | 0.0725 |
3.Clone3A7 variable region sequencing
mRNA is extracted from two cloned cells, reverse transcription is carried out to obtain cDNA, high fidelity PCR amplification is carried out by using variable region universal primer, PCR product fragment is inserted into T vector for DNA sequence determination, and obtained sequence is translated into amino acid sequence of protein. Variable region amino acid sequence of the antibody of Clone3a 7: the light chain variable region amino acid sequence is shown as SEQ ID No.1, and the heavy chain variable region amino acid sequence is shown as SEQ ID No. 2. The alignment of the sequences did not show the same sequence, indicating that the sequence obtained was clone-specific and the monoclonal antibody was designated 3A 7.
Example 3 validation of affinity and specificity Using purified antibodies
The monoclonal antibody 3A7 was subjected to a cell affinity assay by ELISA to determine its binding titer to low molecular weight xanthan gum and related polysaccharide molecules having a relative molecular weight of 100 ten thousand.
The detection method comprises the following steps: plating preparations of low molecular weight xanthan gum were performed on the first day, 200uL per well in 96 wells, overnight. The following day, after removing the supernatant, the cells were washed 3 times with PBS and blocked for 1h by adding 200uL/well of 5% skim milk. After PBS is washed for three times, gradient diluted Clone3A7 is added, after incubation for 1h at 37 ℃, HRP (horse radish peroxidase) marked mouse secondary antibody (1:2000) is added after PBS is washed for three times, after incubation for 1h, PBS is washed for 5 times (the first three times for 5min, and the last 2 times for 10min), color developing agent is added for developing for 15min, and OD450 is detected on a computer.
The formula of the developer A liquid is that 1g of carbamide peroxide, 10.3g of citric acid and 35.8g of Na are added into 1000mL of water2HPO4·12H2O, Tween-20100. mu.L, pH 5; the formulation of solution B is prepared by adding 700mg of Tetramethylbenzidine (TMB) (40mL dissolved in DMSO), 10.3g citric acid, pH2.4 per 1000mL of distilled water.
TABLE 3 results of ELISA assay
As shown in Table 3, the titer of 3A7 reaches more than 0.001ug/mL, shows higher affinity, has no obvious specific cross with other related polysaccharide molecules such as hyaluronic acid, sclerotium rolfsii and the like, and shows the characteristics of high affinity, high specificity and high sensitivity.
Example 4 in vivo use of monoclonal antibody 3A7
In this embodiment, the in vivo application of mab 3a7 was examined.
The test is carried out by adopting an immunohistochemical experiment method, which comprises the following steps:
1. embedding tissues: adding liquid paraffin into the iron mould, slightly cooling, placing the tissue to be embedded into the paraffin, arranging the tissue in order, covering the plastic mould box, adding a little liquid paraffin, and freezing to make the paraffin become solid.
2. Slicing: the embedded tissue is taken down from the mould, placed on a paraffin slicer for slicing, and the glass slide containing the complete tissue is taken out after being placed in warm water at 40 ℃ and dried in an oven at 37 ℃.
3. Dewaxing: placing the glass slide into xylene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol in sequence, and placing each reagent for 10 min.
4. Antigen retrieval: washed in clear water with 3% H after dewaxing2O2Soaking for 10min, washing with water twice, adding citric acid buffer solution, cooking with microwave oven for 3min (middle fire) to boil, cooling to room temperature, cooking again, and cooling to room temperature.
5. Serum blocking: after cooling to room temperature, the citric acid buffer was poured off, washed with water 2 times, the slides were placed in PBS for 5min, washed 2 times, wiped to dry the PBS surrounding the tissue, immediately sealed with serum, and incubated at 37 ℃ for 30 min. Serum was diluted 10-fold (900. mu.L PBS: 100. mu.L serum blocking solution).
6. Adding a primary antibody: the slide glass in the incubator is taken out, the serum is sucked dry by absorbent paper, and primary antibody is added to be stored in the incubator at 4 ℃ overnight.
7. Adding a secondary antibody: the slide was removed from the refrigerator, washed 3 times with PBS for 5min each, wiped dry with PBS and then added with secondary antibody in an incubator at 37 ℃ for 30 min.
8. Adding SABC: taking out the slices from the incubator, washing with PBS for 3 times, each for 5min, wiping off PBS, adding SABC, and incubating at 37 deg.C for 30 min. SABC was diluted 100-fold (990. mu. LPBS: 10. mu. LSABC).
9. Adding a color developing agent: taking out the slices from the incubator, washing with PBS for 3 times, each for 5min, wiping off PBS, and adding color-developing agent. (color developer preparation: 1 drop of color developer A in 1mL of water, shaking up, then 1 drop of color developer B, shaking up, then 1 drop of color developer C, shaking up) A: DAB; b: h2O2(ii) a C: phosphate buffer.
10. Counterdyeing: washing the developed slices with clear water for a period of time, and soaking in hematoxylin for staining for 30 s.
11. And (3) dehydrating: after the counterdyed slices are placed in water for washing, the glass slides are sequentially placed in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene. Each reagent is placed for 2min, and finally soaked in xylene and moved to a fume hood.
12. Sealing: drop the gel on the tissue side with neutral gum, cover with a glass cover, and air dry in a fume hood.
The results are shown in fig. 3, 3a7 can be used to detect xanthan gum drug metabolism and distribution in vivo using immunohistochemical methods, wherein the brown region is a positive reaction.
SEQUENCE LISTING
<110> Shandong province scientific college of pharmacy
<120> antibody against low molecular weight xanthan gum and application thereof
<130> 111
<160> 2
<170> PatentIn version 3.5
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<213> Artificial Sequence
<220>
<223> light
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Glu Val Gln Leu Arg Gln Ser Gly Ala Asp Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Ile Cys Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Tyr Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ala
<210> 2
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<212> PRT
<213> Artificial Sequence
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Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
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Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Asn
100 105 110
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln
115 120 125
Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly
130 135 140
Asn Ser Phe Met His Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys
145 150 155 160
Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg
165 170 175
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro
180 185 190
Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Glu
195 200 205
Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
210 215 220
Claims (10)
1. An antibody against xanthan gum, the antibody having a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 2.
2. The antibody of claim 1, wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody.
3. The antibody of claim 1 or 2, wherein the antibody is a murine, human or chimeric antibody.
4. The antibody of claim 1, further comprising a heavy chain constant region comprising an IgG selected from the group consisting of IgG1, IgG2, IgG3, and IgG 4.
5. Use of an antibody according to any one of claims 1 to 4 for detecting xanthan gum.
6. A method for detecting xanthan gum, said method comprising the step of detecting xanthan gum using the antibody of any one of claims 1-4.
7. The antibody of claim 6, wherein the method is an indirect immunoenzyme-linked immunosorbent (ELISA) method or an Immunohistochemistry (ICH) method.
8. A reagent for detecting xanthan gum, said reagent comprising the antibody of any one of claims 1-4.
9. A method of detecting xanthan gum in a sample, said method comprising the step of detecting xanthan gum using an antibody of any one of claims 1-4.
10. The method of claim 9, wherein the sample is selected from the group consisting of food, pharmaceuticals, and metabolites of humans and other animals.
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Citations (2)
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| US20130266930A1 (en) * | 2010-12-15 | 2013-10-10 | Cytosed, Inc. | Antibody-linked immuno-sedimentation agent and method of isolating a target from a sample using same |
| CN113933498A (en) * | 2021-09-18 | 2022-01-14 | 山东大学 | Double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) method for detecting xanthan gum |
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