CN113307863A - Preparation method and application of polyaspartic acid and salt antibody thereof - Google Patents
Preparation method and application of polyaspartic acid and salt antibody thereof Download PDFInfo
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Abstract
The invention discloses a preparation method and application of polyaspartic acid and/or a salt antibody thereof. The invention provides a hapten of polyaspartic acid and/or salts thereof, then the hapten is coupled with carrier protein to obtain artificial antigen and immunize animals to obtain an antibody, the antibody has high sensitivity and high specificity recognition capability on polyaspartic acid and/or salts thereof, and has no cross reaction on free amino acid, which indicates that the antibody has good sensitivity, specificity and sensitivity on polyaspartic acid and/or salts thereof, effectively eliminates the interference of free amino acid, and provides a core material for establishing an immunodetection method of polyaspartic acid and/or salts thereof. The invention also discloses an enzyme-linked immunoassay kit for polyaspartic acid and/or salts thereof, a colloidal gold immunochromatographic test strip and application thereof in food.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to a preparation method and application of polyaspartic acid and a salt antibody thereof.
Background
Potassium polyaspartate is a stabilizer that prevents the growth of potassium bitartrate crystals, and thus some parts or countries of the world allow it to be added to food products such as wine. In 2016, 3, 17 days, the food safety agency of the European Union (EFSA) issues opinions on the safety of the polyaspartic acid potassium as a stabilizer for wine, and approves that the food additive polyaspartic acid is used as a stabilizer for wine, wherein the maximum limit is 300 mg/L; 18.12.2018, and 69-18 announcements issued by the Aus New food Standard office (FSANZ), which approved potassium polyaspartate as a stabilizer for wine with a maximum allowed use limit of 100 mg/L; on 22/2/2021, NOM/ADM-0162 document was issued by the health canada, revising the list of additives allowed to be used, approving the use of potassium polyaspartate in wine. Internationally, although the countries approve the use of the potassium polyaspartate, the countries have different limit specifications, and some countries do not take the potassium polyaspartate as a food additive to be listed in the use standard of the food additive, so that the illegal use risk exists, and the establishment of a corresponding detection method is very necessary in international trade.
At present, no detection method for potassium polyaspartate in food exists in the market, the existing detection method for potassium polyaspartate is spectrophotometry (DB 22/T1851-2014) for detecting the content of polyaspartate in compound fertilizer (compound fertilizer), the standard is abandoned at present, the method cannot detect the potassium polyaspartate in food matrix, and the sensitivity, specificity and the like cannot meet the detection requirements.
Compared with a chromatographic method, the immunodetection method based on antigen-antibody specific molecule recognition has more advantages in the field detection aspect, has the characteristics of rapidness, sensitivity, simplicity and convenience and the like, and is low in cost and low in requirement on skills of operators. The key point of the research and development of the immunoassay method is to design a proper potassium polyaspartate hapten and prepare an antibody with high sensitivity and strong specificity, but reports about the potassium polyaspartate hapten, an artificial antigen, the antibody and application thereof are not found in the prior art.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method and application of an antibody of polyaspartic acid and salts thereof. The antibody of the polyaspartic acid and/or the salt thereof has high sensitivity and high specificity recognition capability on the polyaspartic acid and/or the salt thereof, and provides a core raw material for establishing an immunodetection method of the polyaspartic acid and/or the salt thereof.
The first purpose of the invention is to provide a hapten of polyaspartic acid and/or salts thereof.
The second purpose of the invention is to provide the application of the polyaspartic acid and/or the hapten of the salt thereof in preparing a detection kit of the polyaspartic acid and/or the salt thereof and/or in detecting the polyaspartic acid and/or the salt thereof.
The third purpose of the invention is to provide three complete antigens of polyaspartic acid and/or salts thereof.
The fourth object of the present invention is to provide a complete antigen combination of polyaspartic acid and/or its salts.
The fifth object of the present invention is to provide an antibody against polyaspartic acid and/or a salt thereof.
The sixth purpose of the invention is to provide the application of the complete antigen and/or the antibody of the polyaspartic acid and/or the salt thereof in detecting the polyaspartic acid and/or the salt thereof, and/or the application in preparing a detection kit or a test strip for detecting the polyaspartic acid and/or the salt thereof.
The seventh purpose of the invention is to provide a detection kit for polyaspartic acid and/or salts thereof.
The eighth purpose of the invention is to provide a colloidal gold immunochromatographic strip for polyaspartic acid and/or salts thereof.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention prepares three complete antigens by coupling hapten to carrier protein, respectively uses the three complete antigens for animal immunization and as coating antigen to prepare the antibody of polyaspartic acid and/or salts thereof, and then further establishes the detection method and the detection kit of polyaspartic acid and/or salts thereof by utilizing the coating antigen.
Accordingly, the invention claims the following:
a hapten of polyaspartic acid and/or salts thereof, wherein the structural formula of the hapten is shown as a formula (II):
wherein R is
The application of the polyaspartic acid and/or the salt hapten thereof in preparing a detection kit for the polyaspartic acid and/or the salt thereof and/or the application in detecting the polyaspartic acid and/or the salt thereof.
Preferably, the polyaspartic acid, polyaspartic potassium salt and/or polyaspartic sodium salt.
Most preferably, the polyaspartic acid potassium salt is a mixture of polyaspartic acid hydrolyzed by potassium hydroxide, and the polymerization degree is 6-11; the polyaspartic acid sodium salt is a mixture of polyaspartic acid hydrolyzed by sodium hydroxide, and the polymerization degree is 6-11.
A complete antigen of polyaspartic acid and/or salts thereof has a structural formula shown as a formula (III) or a formula (IV), wherein n is 6-11 (n is an integer),
preferably, the carrier protein in the complete antigen is one or more of Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Lactoferrin (Lactoferrin, LF), or chicken Ovalbumin (OVA).
More preferably, the carrier protein for the complete antigen of formula (III) is Keyhole Limpet Hemocyanin (KLH).
More preferably, the carrier protein for the complete antigen of formula (IV) is Bovine Serum Albumin (BSA).
Most preferably, the complete antigen represented by formula (IV) is prepared by active ester coupling of Bovine Serum Albumin (BSA).
A complete antigen of polyaspartic acid and/or salts thereof, the structural formula of the complete antigen is shown as a formula (V),
wherein R is
Preferably, the carrier protein in the complete antigen is one or more of bovine serum albumin, keyhole limpet hemocyanin, lactoferrin or egg white albumin.
More preferably, the carrier protein for the complete antigen of formula (V) is keyhole limpet hemocyanin.
Most preferably, the complete antigen of formula (V) is prepared by coupling a carrier protein by the SMCC method.
A complete antigen combination of polyaspartic acid and/or salts thereof comprises complete antigens of polyaspartic acid and/or salts thereof shown in formula (III), formula (IV) and formula (V).
Preferably, the carrier protein in the complete antigen combination is one or more of bovine serum albumin, keyhole limpet hemocyanin, lactoferrin or egg white albumin.
More preferably, the carrier protein of the complete antigen represented by formula (III) is Keyhole Limpet Hemocyanin (KLH), the carrier protein of the complete antigen represented by formula (IV) is Bovine Serum Albumin (BSA), and the carrier protein of the complete antigen represented by formula (V) is keyhole limpet hemocyanin.
Preferably, the complete antigen combination comprises the complete antigen shown in the formula (III) or the formula (IV) as a coating antigen and the complete antigen shown in the formula (V) as an immunogen.
More preferably, the complete antigen of formula (IV) is used as a coating antigen and the complete antigen of formula (V) is used as an immunogen.
An antibody of polyaspartic acid and/or its salt is prepared from hapten shown in formula (II) and/or one or two complete antigens in the complete antigen combination.
Preferably, the antibody against polyaspartic acid and/or its salts is prepared from a complete antigen represented by formula (V) as an immunogen.
Preferably, the antibody of polyaspartic acid and/or its salts is any one or more of monoclonal antibody and polyclonal antibody.
More preferably, the antibody of polyaspartic acid and/or its salt is polyclonal antibody of polyaspartic acid and/or its salt.
The complete antigen of the formula (III), the formula (IV) and/or the formula (V), and/or the antibody of the polyaspartic acid and/or the salt thereof is/are applied to detecting the polyaspartic acid and/or the salt thereof, and/or is applied to preparing a detection kit or a test strip for detecting the polyaspartic acid and/or the salt thereof.
Preferably, the detection kit is an enzyme linked immunosorbent assay kit.
Preferably, the test strip is a colloidal gold immunochromatographic test strip.
A detection kit for polyaspartic acid and/or salts thereof is prepared by combining complete antigens of polyaspartic acid and/or salts thereof.
Preferably, the detection kit comprises the complete antigen of formula (III), formula (IV) and/or formula (V), and/or the antibody against polyaspartic acid and/or its salts.
More preferably, the detection kit comprises the complete antigen shown in the formula (IV) and the polyaspartic acid and/or the salt antibody thereof.
Most preferably, the complete antigen represented by formula (IV) in the detection kit is used as a coating antigen.
Preferably, the detection kit is an enzyme linked immunosorbent assay kit.
More preferably, the detection kit further comprises:
an artificial antigen-treated microplate represented by formula (IV): diluting the artificial antigen shown in the formula (IV) to 1 mu g/mL by using a carbonate buffer solution (0.01moL/L, pH 9.6), adding 100 mu L into each hole, incubating overnight at 37 ℃ in a dark place, pouring out liquid in the holes, washing for 2 times by using a washing solution, shaking to dry for 30s each time, adding 120 mu L of a sealing solution into each hole, incubating for 2h at 25 ℃ in a dark place, pouring out liquid in the holes, shaking to dry, drying, and storing in a vacuum seal mode by using an aluminum film;
enzyme conjugate: marking the polyaspartic acid and/or the salt antibody thereof by horseradish peroxidase;
sealing liquid: 1% of fish glue protein (m/v);
substrate color developing solution: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
stopping liquid: 10% sulfuric acid (v/v);
washing liquid: 0.5-1.0% (v/v) of Tween-20 with pH 7.4, 0.01-0.03 thousandths (v/v) of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer with pH 7.4.
More preferably, the use method of the detection kit is as follows:
s1, adding 50 mu L of sample to be detected into an enzyme label plate coated by artificial antigen shown in a formula (IV);
s2, diluting the enzyme conjugate with PBST (0.01M PBS, 0.06% Tween-20(v/v)) according to a volume ratio of 1:11, adding 50 mu L of the diluted enzyme conjugate into micropores corresponding to the enzyme label plate in S1, slightly oscillating and uniformly mixing, and placing the enzyme conjugate in a dark environment at 25 ℃ for reacting for 35min by using a cover plate membrane cover plate;
s3, spin-drying liquid in the holes, adding 300 mu L of washing liquid into the holes, fully washing the holes for 4-5 times at intervals of 10s every time, splashing the washing liquid in the holes of the plates, and patting the plates to be dry by using absorbent paper (bubbles which are not cleaned after patting to be dry can be punctured by using an unused gun head);
s4, adding 50 mu L/hole of substrate color development liquid A, adding 50 mu L/hole of substrate color development liquid B, lightly oscillating and uniformly mixing, and placing the mixture in a dark environment at 25 ℃ for reaction for 10min after covering a plate by a cover plate film;
s5, adding 50 mu L of stop solution into each hole, slightly oscillating and uniformly mixing, setting an enzyme-labeling instrument at 450nm, and measuring the OD value of each hole.
A colloidal gold immunochromatographic test strip of polyaspartic acid and/or salts thereof is prepared by combining the polyaspartic acid and/or salts thereof antibodies and the complete antigen.
Preferably, the colloidal gold immunochromatographic test strip comprises: the kit comprises a gold-labeled antibody of the polyaspartic acid and/or the antibody of the salt thereof, a cellulose membrane coated with the artificial antigen, a water absorption pad, a sample pad and a PVC bottom plate.
More preferably, the cellulose membrane of the colloidal gold immunochromatographic test strip is coated with the artificial antigen shown in the formula (IV).
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention prepares polyaspartic acid and/or hapten of salts thereof, uses hapten coupling carrier protein to obtain artificial antigen, and uses the artificial antigen to prepare polyaspartic acid and/or antibody of salts thereof, the antibody has high sensitivity and high specificity recognition capability to polyaspartic acid and/or salts thereof, and has no cross reaction to free amino acid, which shows that the antibody has good sensitivity, specificity and sensitivity to polyaspartic acid and/or salts thereof, effectively eliminates interference of free amino acid, and provides core material for establishing immunodetection method of polyaspartic acid and/or salts thereof.
(2) The invention utilizes the polyaspartic acid and/or the salt antibody thereof to develop the application of the polyaspartic acid and/or the salt immunoassay kit thereof in the fast immunoassay of the polyaspartic acid and/or the salt thereof in food. The enzyme linked immunosorbent assay kit and the colloidal gold immunochromatographic test strip developed by the invention can specifically recognize the polyaspartic acid and/or the salts thereof, and have high detection sensitivity on the polyaspartic acid and/or the salts thereof.
Drawings
FIG. 1 shows the chromatogram results of the synthesis of P9 hapten.
FIG. 2 shows the result of mass spectrum of synthesized hapten P9 of polyaspartic acid and/or its salt.
FIG. 3 shows the specific structure of P9.
FIG. 4 is a synthetic route of the artificial antigen P9-carrier protein of polyaspartic acid and/or its salts.
FIG. 5 shows the synthetic route of the artificial antigen PASP-EDC-carrier protein of polyaspartic acid and/or its salts
FIG. 6 is a UV spectrum of KLH, P9 and P9-KLH.
FIG. 7 is a UV spectrum of KLH, PASP and PASP-GD-KLH.
FIG. 8 is a UV spectrum of BSA, PASP and PASP-EDC-BSA.
FIG. 9 is a standard inhibition curve of antibody against polyaspartic acid and/or its salts against polyaspartic acid potassium.
FIG. 10 is a graph showing a standard inhibition curve of an antibody against polyaspartic acid and/or a salt thereof.
FIG. 11 is a standard inhibition curve of antibody against polyaspartic acid and/or its salts against sodium polyaspartate.
FIG. 12 is a schematic side view of a test strip for immunochromatography of polyaspartic acid and/or its salts.
FIG. 13 is a diagram showing the result of detection by the test strip of polyaspartic acid and/or its salts using colloidal gold immunochromatography.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 Synthesis and characterization of haptens of polyaspartic acid and/or salts thereof
First, experiment method
(1) The synthesis sequence is as follows: from the C end to the N end of the sequence, the steps are as follows:
a. weighing n equivalents of resin, putting the resin into a reactor, adding DCM (dichloromethane) to swell for half an hour, then pumping off the DCM, adding 2n equivalents of the first amino acid in the sequence, adding 2n equivalents of DIEA (diisopropylethylamine), a proper amount of DMF (dimethylformamide), DCM (a proper amount is that the resin can be fully stirred), DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM and nitrogen for bubbling reaction for 60 min. Then adding about 5n equivalent of methanol, reacting for half an hour, pumping out reaction liquid, and washing with DMF and MEOH;
b. the reactor was charged with the second amino acid in the sequence (also 2N equivalents), 2N equivalents HBTU (benzotriazole-N, N-tetramethyluronium hexafluorophosphate) and DIEA, N2The reaction was bubbled for half an hour, the liquid was washed off, ninhydrin detected, and then capped with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of decapping liquid to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, cleaning, and detecting ninhydrin;
c. adding different amino acids in the sequence in sequence according to the mode of the step b and carrying out various modifications;
d. blowing the resin to dry by using nitrogen, taking the resin out of the reaction column, pouring the resin into a flask, adding a certain amount of cutting fluid (the composition of which is 95% trifluoroacetic acid, 2% ethanedithiol, 2% triisopropylsilane and 1% water) into the flask (the ratio of the cutting fluid to the resin is about 10 ml/g), shaking and filtering the resin;
e. obtaining filtrate, then adding a large amount of ether into the filtrate to separate out a crude product, then centrifuging and cleaning to obtain a crude product of the sequence;
(2) polypeptide purification: the crude product is purified to the required purity by high performance liquid chromatography.
(3) Polypeptide freeze-drying: and (4) putting the purified liquid into a freeze dryer for concentration, and freeze-drying to obtain white powder.
(4) And (5) carrying out polypeptide detection and an experimental report.
Second, experimental results
The results of the synthesis included an HPLC chart (FIG. 1) and a mass spectrum chart (FIG. 2). The structure of the polyaspartic acid and/or the salt hapten (P9) is successfully synthesized according to the analysis of retention time and fragment ion peaks of a mass spectrogram, the structure is shown as a formula (II), the complete structure is shown as a figure 3,
wherein R is
EXAMPLE 2 Synthesis and characterization of Artificial antigen of polyaspartic acid and/or its salts
1. Synthesis of artificial antigen of polyaspartic acid and/or salts thereof
The synthesis of artificial antigens of polyaspartic acid and/or salts thereof was carried out using the hapten of formula (II) successfully prepared in example 1 and a commercially available hapten of formula (I) (CAS:64723-18-8) wherein n is 6-11 and n is an integer,
the specific operation is as follows:
(1) the hapten shown in the formula (II) is coupled with carrier protein by an SMCC method, the specific synthetic route is shown in figure 4, and the specific steps are as follows:
s1, dissolving 10mg of carrier protein in 200-1000 mu L of 99.5% DMF to obtain solution A;
s2, dissolving 4- (N-maleimide methyl) cyclohexane-1-carboxylic sulfonic acid group succinimide ester sodium Salt (SMCC) in 200-1000 mu L of 99.5% DMF to obtain solution B;
s3, slowly dropwise adding the solution B into the solution A under magnetic stirring, and stirring overnight at 0 ℃ in a low-temperature reactor; dialyzing with PBS buffer solution for 2 days to obtain solution C, namely the carrier protein solution modified by SMCC;
s4, dissolving the hapten shown in the formula (II) in 500-1000 mu L of phosphate buffer (0.01moL/L, pH 7.4) to obtain solution D;
s5, slowly dripping the solution D into the solution C (SMCC-carrier protein) under magnetic stirring, and stirring for 2 hours at room temperature; dialyzing with PBS buffer solution for two days, 4 times per day, collecting protein solution into centrifuge tube after dialysis to obtain protein conjugate, i.e. polyaspartic acid and/or its salt artificial antigen P9-carrier protein, whose structure is shown in formula (V), subpackaging in centrifuge tube, storing at-20 deg.C for use,
wherein R is
(2) Coupling hapten shown in a formula (I) to carrier protein by a glutaraldehyde method, and specifically comprising the following steps:
s1, dissolving a hapten shown in a formula (I) in 50-200 mu L of phosphate buffer (0.01moL/L, pH 7.4), adding 50-100 mu L of 25% Glutaraldehyde (GA) (v/v), stirring at room temperature in a dark place, and reacting for 1-3 hours to obtain an activation solution;
s2, adding 10mg of carrier protein into a phosphate buffer (0.01moL/L, pH 7.4) to obtain a carrier protein solution;
s3, slowly dropwise adding the activating solution in the S1 into the carrier protein solution in the S2 under magnetic stirring at room temperature, and reacting for 24 hours; dialyzing for two days with PBS buffer solution for 4 times per day, collecting the protein solution into a centrifuge tube after dialysis to obtain a protein conjugate, namely polyaspartic acid and/or salt artificial antigen PASP-GD-carrier protein shown in formula (III) (n is 6-11, n is an integer), subpackaging into centrifuge tubes, storing at-20 ℃ for later use,
(3) the hapten shown in the formula (I) is coupled with carrier protein by an active ester method, the specific synthetic route is shown in figure 5, and the specific steps are as follows:
s1, weighing 1moL of hapten shown in a formula (I), 1.4moL of NHS and 1.6moL of 99% EDC, dissolving in 50-200 mu L of 99.5% DMF, and stirring at room temperature in a dark place for 2-4 h to obtain polyaspartic acid and/or a salt hapten activating solution thereof;
s2, dissolving 10mg of carrier protein in 1mL of PBS buffer solution (0.01moL/L, pH 7.4), slowly and dropwise adding polyaspartic acid and/or hapten activated solution of salts thereof in S1 at room temperature, and reacting for 12h at 4 ℃; dialyzing with PBS buffer solution for 2 days for 4 times per day, and collecting polyaspartic acid and/or its salt artificial antigen PASP-EDC-carrier protein (n is 6-11, n is integer) as shown in formula (IV), subpackaging in centrifuge tube, storing at-20 deg.C for use,
in the synthesis of the artificial antigen, the formula of the PBS buffer solution is as follows: na (Na)2HPO4·12H2O 2.90g，NaCl 8.50g，KCl 0.20g，KH2PO40.20g, adding distilled water to a constant volume of 1000 mL; the carrier protein is Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH).
2. Identification of artificial antigen of polyaspartic acid and/or salts thereof
Six artificial antigens were successfully synthesized by this example: the results of UV scanning of P9-KLH, P9-BSA, PASP-GD-KLH, PASP-GD-BSA, PASP-EDC-KLH, and the artificial antigens P9-KLH, PASP-GD-KLH, and PASP-EDC-BSA, among others, are shown in FIGS. 6, 7, and 8, indicating the successful synthesis of the artificial antigens P9-KLH, PASP-GD-KLH, and PASP-EDC-BSA.
EXAMPLE 3 preparation of antibody against polyaspartic acid and/or its salt
The artificial antigens PASP-EDC-KLH, PASP-GD-KLH and P9-KLH prepared in example 2 were emulsified with immune adjuvants (Freund's adjuvant for the first immunization and Freund's incomplete adjuvant for the subsequent booster immunization) in a volume ratio of 1:1, respectively, to immunize white New Zealand rabbits.
The weight of the New Zealand white rabbit is 2.5-3 kg, subcutaneous multipoint injection is adopted on the neck and the back, the second immunization is carried out after 4 weeks, and the boosting immunization is carried out once every 3 weeks later. Blood was taken from the ear peripheral vein 1 week after the third booster immunization and serum titers were determined using indirect competition ELISA. When the titer no longer increased, the marginal ear vein was used for boosting. Blood was collected from the heart one week later, and the manner in which the collected blood was used to obtain serum was: and (3) carrying out warm bath on the collected heart blood at 37 ℃ for 0.5-1 h, standing overnight at 4 ℃, sucking the precipitated serum by using a suction tube, centrifuging at 3000-5000 rpm at 4 ℃ for 10min, and taking the supernatant to obtain the antiserum. The antiserum is purified by adopting an octanoic acid-ammonium sulfate precipitation method to obtain three artificial antigens which are used as polyaspartic acid prepared by immunogen and/or a polyclonal antibody of the salt thereof, and the polyaspartic acid and/or the polyclonal antibody of the salt thereof are frozen and stored at the temperature of minus 20 ℃ for standby.
EXAMPLE 4 optimization of combinations of immunogens and coating antigens of polyaspartic acid and/or salts thereof
First, experiment method
The three antibodies prepared in example 3 were screened for the antigen by ELISA using the artificial antigens PASP-EDC-BSA, PASP-GD-BSA and P9-BSA prepared in example 2 as the antigens, respectively.
The specific operation steps are as follows:
(1) the three antibodies of polyaspartic acid and/or its salts in example 3 were diluted to 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000 with PBST (0.01M PBS, 0.06% Tween-20(v/v)), respectively, while blank control wells were provided (PBST was used instead of the antibody dilution);
(2) coating: respectively diluting polyaspartic acid and/or its salt artificial antigens PASP-EDC-BSA, PASP-GD-BSA and P9-BSA to the concentration of 1 μ g/mL by using coating solution (0.05M phosphate buffer solution, pH 9.6), respectively coating on a 96-well enzyme label plate, adding 100 μ L of each well, incubating overnight in a constant temperature water bath box at 37 ℃, discarding the coating solution, and washing for 2 times by using PBST (0.01M PBS, 0.06% Tween-20 (v/v));
adding 120 μ L of blocking solution (1% fish glue protein (m/v)) into each well, blocking at 37 deg.C for 3 hr, discarding the blocking solution, clapping, and oven drying at 37 deg.C in drying oven for use;
(3) PBST is used for diluting 1mg/mL of polyaspartic acid potassium by 1000 times, and the solution is 1 microgram/mL of polyaspartic acid potassium diluent;
(4) adding 50 mu L of 1 mu g/mL potassium polyaspartate diluent (three groups are parallel) into each line of the enzyme label plate, adding 50 mu L/hole of the potassium polyaspartate antibody diluent in the step (1), incubating for 40min at 37 ℃, and washing for 5 times;
(5) adding goat anti-rabbit secondary antibody IgG-HRP (5000-fold dilution), incubating for 30min at 37 ℃, washing for 5 times, and clapping;
(6) adding 50 mu L/hole of the color development liquid A, adding 50 mu L/hole of the substrate color development liquid B, lightly oscillating and uniformly mixing, and placing the mixture in a dark environment at 37 ℃ for reaction for 10min after a cover plate is covered by a cover plate film;
(7) add 50. mu.L of 10% H2SO4The reaction was stopped and the OD read at 450 nm;
the color development liquid consists of a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine.
The method for screening the coating source is the same as the method for screening the polyaspartic acid and the sodium polyaspartate respectively.
Second, experimental results
The titer and inhibition by ELISA are shown in tables 1 and 2. After screening different immunogen and coating antigen combinations, the optimal combination is as follows: the artificial antigen P9-KLH is used as immunogen, and the artificial antigen PASP-EDC-BSA/P9-BSA is used as coating antigen. Under the combination, the antibody can specifically recognize target analytes such as polyaspartic acid, potassium polyaspartate and sodium polyaspartate, and the sensitivity of the antibody is good.
TABLE 1 potency and inhibition data for combinations of potassium polyaspartate 4 group immunogens and coatants
TABLE 2 potency and inhibition data for polyaspartic acid and its sodium salt
EXAMPLE 5 ELISA kit of polyaspartic acid and/or its salts
Enzyme linked immunosorbent assay kit composition containing polyaspartic acid and/or salts thereof
An enzyme linked immunosorbent assay kit for detecting polyaspartic acid and/or salts thereof comprises the following components:
(1) the artificial antigen PASP-EDC-BSA treated ELISA plate prepared in example 2: diluting the artificial antigen PASP-EDC-BSA to 1 mu g/mL by using a coating solution (0.05M phosphate buffer solution, pH 9.6), adding 100 mu L of the artificial antigen PASP-EDC-BSA into each hole, incubating overnight at 37 ℃ in a dark place, pouring off liquid in the holes, washing for 2 times by using a washing solution, shaking to dry for 30s each time, then adding 120 mu L of a sealing solution (1% of fish glue protein (M/v)) into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, shaking to dry, drying, and storing by using an aluminum film in a vacuum seal manner;
(2) enzyme conjugate: labeling the polyaspartic acid and/or the salt antibody thereof prepared in example 3 with horseradish peroxidase;
(3) substrate color developing solution: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(4) stopping liquid: 10% sulfuric acid (v/v);
(5) washing liquid: 0.5-1.0% (v/v) of Tween-20 with pH 7.4, 0.01-0.03 thousandths (v/v) of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer with pH 7.4.
Use of enzyme linked immunosorbent assay kit containing poly-aspartic acid and/or salts thereof
S1, adding 50 mu L of a sample to be detected into an enzyme label plate treated by an artificial antigen PASP-EDC-BSA;
s2, diluting the enzyme conjugate by using PBST (0.01M PBS, 0.06% Tween-20(v/v)) according to a volume ratio of 1:11 (namely adding 11 parts of PBST into 1 part of the enzyme conjugate for use when being prepared), adding 50 mu L of enzyme conjugate into micropores corresponding to the enzyme label plate in S1, lightly shaking and uniformly mixing, and placing the enzyme conjugate in a dark environment at 25 ℃ for reacting for 35min by using a cover plate membrane cover plate;
s3, spin-drying liquid in the holes, adding 300 mu L of washing liquid into the holes, fully washing the holes for 4-5 times at intervals of 10s every time, splashing the washing liquid in the holes of the plates, and patting the plates to be dry by using absorbent paper (bubbles which are not cleaned after patting to be dry can be punctured by using an unused gun head);
s4, adding 50 mu L/hole of substrate color development liquid A, adding 50 mu L/hole of substrate color development liquid B, lightly oscillating and uniformly mixing, and placing the mixture in a dark environment at 25 ℃ for reaction for 10min after covering a plate by a cover plate film;
s5, adding 50 mu L of stop solution into each hole, slightly oscillating and uniformly mixing, setting an enzyme-labeling instrument at 450nm, and measuring the OD value of each hole.
EXAMPLE 6 establishment of Standard Curve for ELISA kit of polyaspartic acid and/or its salts
First, experiment method
Using the ELISA kit of example 5, polyaspartic acid standard solutions (concentrations of 0. mu.g/L, 1.25. mu.g/L, 25. mu.g/L, 500. mu.g/L, 10000. mu.g/L, 200000. mu.g/L, respectively), potassium polyaspartate standard solutions (concentrations of 0. mu.g/L, 1.25. mu.g/L, 25. mu.g/L, 500. mu.g/L, 10000. mu.g/L, 200000. mu.g/L, respectively), and sodium polyaspartate standard solutions (concentrations of 0. mu.g/L, 1.25. mu.g/L, 25. mu.g/L, 500. mu.g/L, 10000. mu.g/L, 200000. mu.g/L, respectively) were assayed and their OD values per well at 450nm were determined, respectively. And respectively drawing standard curve graphs of the polyaspartic acid, the polyaspartic acid potassium and the polyaspartic acid sodium by adopting a four-parameter curve fitting method.
Second, experimental results
FIG. 10 shows the standard curve of the concentration of the antibody half-Inhibitory (IC) against polyaspartic acid and/or its salts50) The detection limit is 0.6ng/mL and is 13.94 ng/mL; the standard curve for potassium polyaspartate is shown in FIG. 9, the semi-Inhibitory Concentration (IC) of the antibody50) 13.58ng/mL and the detection limit is 0.4 ng/mL; the standard curve for sodium polyaspartate is shown in FIG. 11, the semi-Inhibitory Concentration (IC) of the antibody50) The concentration was 14.47ng/mL, and the detection limit was 0.9 ng/mL. The antibody and the kit prepared by the invention have high-sensitivity recognition capability on the polyaspartic acid and/or the salts thereofThe detection sensitivity is high, and the detection requirement of the potassium aspartate is met.
EXAMPLE 7 specific assay of an enzyme-linked immunosorbent kit for polyaspartic acid and/or its salts
First, experiment method
Using the immunoenzyme-linked kit of example 5, potassium polyaspartate and polyaspartic acid, sodium polyaspartate and other analogs (free amino acids) were tested for specificity as represented by the cross-reactivity ratio (CR), with the smaller the cross-reactivity, the better the specificity. The cross-reactivity (CR) of polyaspartic acid and its salts and various analogs was calculated using the following formula:
second, experimental results
The results of the cross-reaction experiments of potassium polyaspartate with polyaspartate, sodium polyaspartate and other analogues (free amino acids) are shown in table 3, and it was found that: the cross reaction rate of the kit on the potassium polyaspartate is 100 percent, and IC50The value was 13.6 ng/mL; the cross reaction rate of the kit to the sodium polyaspartate is 93.85%; the cross reaction rate of the kit to the polyaspartic acid is 97.42 percent; there was no cross-reaction to its analogue (free amino acids). The enzyme-linked immunoassay kit has high specificity to the polyaspartic acid and the salts thereof, and effectively eliminates the interference of free amino acid.
TABLE 3 Cross-reactivity test results of potassium polyaspartate with polyaspartate, sodium polyaspartate and other analogs
Note: NR indicates no reaction.
EXAMPLE 8 experiment for adding and recovering polyaspartic acid and/or its salt in ELISA kit
First, experiment method
Selecting food (including red wine and white wine) such as wine as a standard sample, adding potassium polyaspartate standard substances with 3 concentrations of 100mg/L, 200mg/L and 400mg/L respectively, and setting non-standard samples without poly-potassium aspartate according to the verification respectively. The treatment method of the sample to be detected is 50 times dilution extraction, and the specific operation is as follows: taking a sample of 2g (accurate to 0.001g) in a 250mL dry beaker, accurately adding 98g of ultrapure water, shaking up, then standing for 10min, and taking the supernatant as a sample to be detected for later use.
Calculated according to the following recovery formula: the recovery rate (detected concentration of sample polyaspartic acid potassium-detected concentration of non-labeled sample polyaspartic acid potassium)/standard concentration × 100%.
Second, experimental results
The results of the additive recovery experiments are shown in table 3. The results show that the adding recovery rates of the red wine and the white wine are both 80-120%, and the coefficient of variation is less than 15%, so that the kit is proved to have high precision and good stability, and can meet the detection requirements of the potassium polyaspartate.
Table 3 sample addition recovery test results
Example 9 Immunochromatographic test strip for detecting polyaspartic acid and/or salts thereof
Preparation of immunochromatographic test strip of polyaspartic acid and/or salts thereof
1. Preparation of gold-labeled antibody and preparation of conjugate pad
Colloidal gold suspension with average diameter of 40nm was prepared by reducing chloroauric acid with trisodium citrate.
Firstly, 0.2mol of K is used for the colloidal gold2CO3The pH of the solution is adjusted to 8.2, then the labeling amount of the antibody is determined by a classical NaCl titration method, and finally 30 mu g of the polyaspartic acid and/or the salt antibody prepared in the embodiment 3 and 1mL of colloidal gold solution are selected to be labeled to obtain a gold-labeled antibody which is stored at 4 ℃.
Spraying 4% BSA (m/v) solution onto glass wool at 8 μ L/cm with XYZ-3000 three-dimensional film spraying instrument, drying at 42 deg.C for 50min, spraying the gold-labeled antibody onto glass wool at 6 μ L/cm, drying at 42 deg.C for 50min, and vacuum drying for storage.
2. Preparation of cellulose membranes
An artificial antigen PASP-EDC-BSA (coating antigen) at a concentration of 1mg/mL was sprayed on one side of a cellulose membrane as a detection line (T line) in an amount of 1.2. mu.L/cm using an XYZ-3000 three-dimensional spray coater. Goat anti-rabbit IgG at a concentration of 120. mu.g/L was sprayed onto the other side of the cellulose membrane as a control line (C line) in an amount of 1.2. mu.L/cm using an XYZ-3000 three-dimensional spray coater, with a space of 8mm between the two lines.
3. Assembly of immunochromatographic test strip
Sticking a cellulose membrane on the middle part of a PVC bottom plate, sticking one end of a water absorption pad (chromatographic water absorption paper) on the upper side of the cellulose membrane, and overlapping the cellulose membrane by 1 mm; the bonding pad is stuck at one end of the cellulose membrane and overlapped by 1 mm; the sample pad (blood filtering membrane) is stuck to the other end of the combination pad and overlapped by 2 mm; the assembled test paper board was cut into test paper strips 3.05mm wide with a cutter.
FIG. 12 is a schematic side view of a test strip for immunochromatography of polyaspartic acid and/or its salts, in which 1 is a PVC substrate; 2 is a sample pad; 3 is a bonding pad; 4 is a cellulose membrane; 5 is a detection line (T line); 6 is control line (line C); and 7 is a water absorption pad.
Immune chromatography test paper strip of di, polyaspartic acid and/or its salt
The immunochromatographic test strip for polyaspartic acid and/or salts thereof comprises: a sample pad (blood filtering film) arranged on the PVC bottom plate, the prepared combination pad, the prepared cellulose film and the water absorption pad in sequence.
Third, use of immunochromatographic test strip
1. Sample pretreatment
Liquid sample 1 (red wine) and liquid sample 2 (white wine)
Weighing 2g (accurate to 0.001g) of sample in a 250mL dry beaker, accurately adding 98g of water, shaking up, then standing for 10min, and taking supernatant for testing.
2. Rapid test strip detection and judgment
(1) Detection of sample 1 to be tested
And (3) sucking the solution of the sample 1 to be detected on the sample pad of the immunochromatography test strip by using a pipette, reacting at room temperature, and judging the result according to the color development condition of the immunochromatography test strip.
(2) Detection of sample 2 to be tested
And (3) detecting the sample 2 to be detected by using an immunochromatographic test strip, wherein the specific method is as described in the step (1).
(3) Results of detection and determination of sample 1 (sample 2)
The detection result of the polyaspartic acid and/or the salt thereof by the colloidal gold immunochromatographic test strip is shown in figure 13,
negative (-): the quality control line (C line) is colored, if the detection line (T line) is also colored, the sample does not contain polyaspartic acid and salts thereof or the content of polyaspartic acid and salts thereof is lower than 8 mu g/g (6 mu g/g), and the sample is judged to be negative;
positive (+): the quality control line (C line) is colored, if the detection line (T line) is not colored, the sample contains polyaspartic acid and/or salts thereof, the content of the polyaspartic acid and/or the salts thereof is higher than 8 mu g/g (6 mu g/g), and the polyaspartic acid and/or the salts thereof is judged to be positive;
and (4) invalidation: when the quality control line (C line) is not colored, whether the detection line (T line) is colored or not indicates that the experimental result is invalid.
Example 10 determination of detection Limit of Immunochromatographic test strip for polyaspartic acid and/or salts thereof
First, experiment method
Taking a liquid sample 1 (red wine) and a liquid sample 2 (white wine), respectively adding a series of standard medicines with concentration into the liquid sample 1 and the liquid sample 2, carrying out sample pretreatment in the embodiment 9, detecting the samples by using the immunochromatographic test strip in the embodiment 9, and determining the visible detection limit through naked eye qualitative judgment.
Second, experimental results
Specific results are shown in the following table.
TABLE 4 determination of test strip detection limits
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A hapten for polyaspartic acid and/or salts thereof, wherein the hapten has a structural formula as shown in formula (II):
wherein R is
2. Use of the hapten of claim 1 for preparing a kit for detecting polyaspartic acid and/or salts thereof, and/or for detecting polyaspartic acid and/or salts thereof.
3. A complete antigen of polyaspartic acid and/or salts thereof, which is characterized in that the structural formula of the complete antigen is shown as a formula (III) or a formula (IV); wherein n is 6-11;
4. a complete antigen of polyaspartic acid and/or salts thereof is characterized in that the structural formula of the complete antigen is shown as a formula (V),
wherein R is
5. A complete antigen combination of polyaspartic acid and/or salts thereof, comprising the complete antigen of claim 3 and claim 4.
6. The complete antigen combination of claim 5, wherein the complete antigen of claim 3 is used as a coating antigen and the complete antigen of claim 4 is used as an immunogen.
7. An antibody against polyaspartic acid and/or its salts, which is produced using the hapten of claim 1 and/or the complete antigen combination of claim 5, or both.
8. Use of the complete antigen of claim 3, the complete antigen of claim 4 and/or the antibody of claim 7 for detecting polyaspartic acid and/or salts thereof, and/or for preparing a detection kit or test strip for detecting polyaspartic acid and/or salts thereof.
9. A kit for detecting polyaspartic acid and/or salts thereof, which is prepared from the complete antigen combination of polyaspartic acid and/or salts thereof according to claim 5.
10. A colloidal gold immunochromatographic strip of polyaspartic acid and/or salts thereof, which is prepared by combining the antibody of claim 7 and the complete antigen of claim 5.
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Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0133833A1 (en) * | 1983-08-01 | 1985-03-06 | ANVAR Agence Nationale de Valorisation de la Recherche | Compositions of vaccines conditioned for the vaccination of immunitarily non-naive subjects against a predetermined pathogenic agent and containing a haptene itself comprising an antigenic site characteristic of said pathogenic agent or an oligomer of this haptene |
| US4762781A (en) * | 1983-12-30 | 1988-08-09 | Centre National De La Recherche Scientifique (Cnrs) | Antibodies capable of specifically identifying haptenic groups, their preparation and their use and to new antigens permitting their preparation |
| WO1989010961A1 (en) * | 1988-05-04 | 1989-11-16 | Igen, Inc. | Peptide analogs and their use as haptens to elicit catalytic antibodies |
| WO1998032469A2 (en) * | 1997-01-29 | 1998-07-30 | Nycomed Imaging As | Polymers |
| WO2000040973A1 (en) * | 1999-01-05 | 2000-07-13 | Axis-Shield Asa | Assay for homocysteine |
| US6423547B1 (en) * | 1998-04-03 | 2002-07-23 | Mallinckrodt Inc. | Non-covalent bioconjugates useful for diagnosis and therapy |
| US20040057958A1 (en) * | 2002-05-17 | 2004-03-25 | Waggoner David W. | Immunogenicity-enhancing carriers and compositions thereof and methods of using the same |
| JP2008094729A (en) * | 2006-10-06 | 2008-04-24 | Osaka Prefecture Univ | Hapten compound and antibody |
| US20080305497A1 (en) * | 2007-05-23 | 2008-12-11 | Ventana Medical Systems, Inc. | Polymeric carriers for immunohistochemistry and in situ hybridization |
| US20120295287A1 (en) * | 2009-11-06 | 2012-11-22 | Pharmasan Labs, Inc. | Preparing hapten-specific antibodies and their application for immunodiagnostics and research |
| CN103665286A (en) * | 2012-09-21 | 2014-03-26 | 王玉麒 | Method for polymerizing hapten into immunogen |
| CN104987366A (en) * | 2014-08-21 | 2015-10-21 | 上海市东方医院 | Rheumatoid arthritis autoantibody conjugated antigen and applications thereof |
| US20160291038A1 (en) * | 2013-11-13 | 2016-10-06 | Sk Telecom Co., Ltd. | Immunological measurement method using hapten and antibody binding thereto as reference antibody, and immunological measurement apparatus using reference antibody |
| CN106366021A (en) * | 2016-08-16 | 2017-02-01 | 华南农业大学 | Urethane hapten composition and artificial antigen composition, and preparation methods and application thereof |
| US20170049883A1 (en) * | 2014-04-25 | 2017-02-23 | Tria Bioscience Corp. | Synthetic hapten carrier compositions and methods |
| WO2018032359A1 (en) * | 2016-08-16 | 2018-02-22 | 华南农业大学 | Ethyl carbamate hapten combination, artificial antigen combination, and method for preparing same and application of same |
| CN108152499A (en) * | 2017-12-06 | 2018-06-12 | 华南农业大学 | A kind of antigen of amantadine, antibody and its enzyme-linked immunologic detecting kit |
| CN108164697A (en) * | 2017-09-21 | 2018-06-15 | 郭文祥 | Asparagus fern carbamide resin new synthetic method |
| WO2018107984A1 (en) * | 2016-12-14 | 2018-06-21 | 中国药科大学 | Polypeptide as an immunostimulant of a b-cell epitope or peptide hapten and polypeptide immunogen consitituted thereby and use thereof |
| CN109369913A (en) * | 2018-09-13 | 2019-02-22 | 天津大学 | PH-responsive polyaspartic acid grafted with hydrophobic amino acid and preparation method thereof |
| CN110865182A (en) * | 2019-11-19 | 2020-03-06 | 东莞市东阳光诊断产品有限公司 | Blocking agent and application thereof in immunodetection |
-
2021
- 2021-05-25 CN CN202110569378.3A patent/CN113307863B/en active Active
Patent Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0133833A1 (en) * | 1983-08-01 | 1985-03-06 | ANVAR Agence Nationale de Valorisation de la Recherche | Compositions of vaccines conditioned for the vaccination of immunitarily non-naive subjects against a predetermined pathogenic agent and containing a haptene itself comprising an antigenic site characteristic of said pathogenic agent or an oligomer of this haptene |
| US4762781A (en) * | 1983-12-30 | 1988-08-09 | Centre National De La Recherche Scientifique (Cnrs) | Antibodies capable of specifically identifying haptenic groups, their preparation and their use and to new antigens permitting their preparation |
| WO1989010961A1 (en) * | 1988-05-04 | 1989-11-16 | Igen, Inc. | Peptide analogs and their use as haptens to elicit catalytic antibodies |
| WO1998032469A2 (en) * | 1997-01-29 | 1998-07-30 | Nycomed Imaging As | Polymers |
| US6423547B1 (en) * | 1998-04-03 | 2002-07-23 | Mallinckrodt Inc. | Non-covalent bioconjugates useful for diagnosis and therapy |
| WO2000040973A1 (en) * | 1999-01-05 | 2000-07-13 | Axis-Shield Asa | Assay for homocysteine |
| US20040057958A1 (en) * | 2002-05-17 | 2004-03-25 | Waggoner David W. | Immunogenicity-enhancing carriers and compositions thereof and methods of using the same |
| JP2008094729A (en) * | 2006-10-06 | 2008-04-24 | Osaka Prefecture Univ | Hapten compound and antibody |
| US20080305497A1 (en) * | 2007-05-23 | 2008-12-11 | Ventana Medical Systems, Inc. | Polymeric carriers for immunohistochemistry and in situ hybridization |
| US20120295287A1 (en) * | 2009-11-06 | 2012-11-22 | Pharmasan Labs, Inc. | Preparing hapten-specific antibodies and their application for immunodiagnostics and research |
| CN103665286A (en) * | 2012-09-21 | 2014-03-26 | 王玉麒 | Method for polymerizing hapten into immunogen |
| US20160291038A1 (en) * | 2013-11-13 | 2016-10-06 | Sk Telecom Co., Ltd. | Immunological measurement method using hapten and antibody binding thereto as reference antibody, and immunological measurement apparatus using reference antibody |
| US20170049883A1 (en) * | 2014-04-25 | 2017-02-23 | Tria Bioscience Corp. | Synthetic hapten carrier compositions and methods |
| CN104987366A (en) * | 2014-08-21 | 2015-10-21 | 上海市东方医院 | Rheumatoid arthritis autoantibody conjugated antigen and applications thereof |
| CN106366021A (en) * | 2016-08-16 | 2017-02-01 | 华南农业大学 | Urethane hapten composition and artificial antigen composition, and preparation methods and application thereof |
| WO2018032359A1 (en) * | 2016-08-16 | 2018-02-22 | 华南农业大学 | Ethyl carbamate hapten combination, artificial antigen combination, and method for preparing same and application of same |
| WO2018107984A1 (en) * | 2016-12-14 | 2018-06-21 | 中国药科大学 | Polypeptide as an immunostimulant of a b-cell epitope or peptide hapten and polypeptide immunogen consitituted thereby and use thereof |
| CN108164697A (en) * | 2017-09-21 | 2018-06-15 | 郭文祥 | Asparagus fern carbamide resin new synthetic method |
| CN108152499A (en) * | 2017-12-06 | 2018-06-12 | 华南农业大学 | A kind of antigen of amantadine, antibody and its enzyme-linked immunologic detecting kit |
| CN109369913A (en) * | 2018-09-13 | 2019-02-22 | 天津大学 | PH-responsive polyaspartic acid grafted with hydrophobic amino acid and preparation method thereof |
| CN110865182A (en) * | 2019-11-19 | 2020-03-06 | 东莞市东阳光诊断产品有限公司 | Blocking agent and application thereof in immunodetection |
Non-Patent Citations (5)
| Title |
|---|
| GAO: "Water-soluble dendritic polyaspartic porphyrins:potential photosensitizers for photodynamic therapy", 《NEW JOURNAL OF CHEMISTRY》 * |
| NIKHIL R JANA: "Cysteine-functionalized polyaspartic acid: a polymer for coating and bioconjugation of nanoparticles and quantum dots", 《LANGMUIR THE AXA JOURNAL OF SURFACES AND COLLOIDS》 * |
| 姚日生: "《药用高分子材料 第二版》", 30 April 2008, 化学工业出版社 * |
| 沈关心: "《现代免疫学实验技术》", 31 October 1998, 湖北科学技术出版社 * |
| 陈新建: "《免疫学技术在植物科学中的应用》", 31 May 1998, 中国农业大学出版社 * |
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