A sea cucumber saponin hapten, artificial antigen, antibody and preparation method and application thereof

Abstract

The present invention discloses a hapten, an artificial antigen, an antibody and a preparation method and application thereof. The structural formula of the hapten is shown in formula (I), and the hapten is used to prepare artificial antigens and antibodies for detecting lipopolysaccharide saponins. The enzyme-linked immunosorbent assay kit prepared by using the artificial antigen and antibody for lipopolysaccharide saponins provided by the present invention has a detection sensitivity of 0.0103 ng / mL for lipopolysaccharide saponins, and IC 50 The linear range is 0.01-10 ng / mL. The enzyme-linked immunosorbent assay kit is easy to use, has low detection cost, and is fast, efficient, and accurate. It is suitable for the quality control of sea cucumber saponins in sea cucumber tissues or the on-site rapid detection of sea cucumber saponin residues and the screening of a large number of samples, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of natural active ingredient detection, and more specifically to a sea cucumber saponin hapten, an artificial antigen, an antibody and a preparation method and application thereof. Background Art

[0002] Apostichopus japonicus belongs to the class Holothuroidea of ​​the phylum Echinodermata, the order Apostichopus, and the family Stichopidae. It has high edible and medicinal value and is a precious marine tonic. my country is a major country in sea cucumber breeding and processing. The main processed sea cucumber products in China are dried sea cucumbers, water-soaked sea cucumbers, and deep-processed sea cucumber products. Deep-processed sea cucumber products have high added value and increased scientific and technological content, but lack quality control indicators for raw materials and products. As an important nutrient component of Apostichopus japonicus, how to establish a rapid and accurate detection of Apostichopus japonicus saponins has become a limiting factor in the development of the sea cucumber industry.

[0003] The earliest report on sea cucumber saponins began in the 1950s. In 1952, Nigrelli isolated a sea cucumber saponin from the Cuvierian tubules of Actinopyga agassizi. Since then, the research on sea cucumber saponins has become increasingly active. With the maturity of mass spectrometry technology, the determination technology of sea cucumber saponins has developed rapidly. So far, the structures of more than 100 sea cucumber saponins have been elucidated. Sea cucumber saponins are composed of two parts: aglycones and oligosaccharide chains. The 3-position of aglycones is substituted with a hydroxyl group, which is combined with sugars through β-O-glycosidic bonds to form glycosides.

[0004] At present, there are few literature reports on the determination methods of sea cucumber saponins, including thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and chromatography-mass spectrometry (LC-MS / MS). TLC is mainly used for the separation and purification of sea cucumber saponins, and the detection sensitivity is low. Due to the interference of matrix effect, the HPLC-FLD method has problems in sensitivity and selectivity using fluorescence detection. The use of chromatography-mass spectrometry (LC-MS / MS) detection can improve selectivity and sensitivity, but the equipment is expensive and requires high professional and technical personnel. Immunoassay detection technology is based on the principle of antigen-antibody reaction, and uses the competitive binding of the analyte with the labeled analyte to detect the analyte with the antibody, which can be used for screening tests of certain drugs. Immunoassay detection has the characteristics of simple sample pretreatment, simple operation, rapid, sensitive and high throughput, and has broad application prospects in the field of food quality control. However, for immunoassay, antibodies are the core raw materials, and the effect of antibodies depends largely on the antigen structure that causes the corresponding animals to have an immune response. Therefore, in order to ensure the quality of food and the development of foreign export trade, it is necessary to establish an accurate, reliable, highly sensitive, low-requirement instrument and personnel-based immunoassay detection technology for sea cucumber saponins, and then a qualitative and quantitative method suitable for on-site monitoring and large-scale sample screening. The key to the establishment of an immunoassay method is to design a suitable artificial antigen for sea cucumber saponins and obtain antibodies with high sensitivity and strong specificity. However, there are no reports on haptens, artificial antigens, antibodies, etc. for sea cucumber saponins. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects and shortcomings of the prior art, which lack sea cucumber saponin hapten, artificial antigen and antibody, and to provide a sea cucumber saponin hapten, artificial antigen and antibody and a preparation method and application thereof.

[0006] The first object of the present invention is to provide a sea cucumber saponin hapten imitating a sea cucumber.

[0007] The second object of the present invention is to provide a method for preparing the sea cucumber saponin hapten imitating sea cucumber saponin.

[0008] The third object of the present invention is to provide an artificial antigen imitating sea cucumber saponin.

[0009] The fourth object of the present invention is to provide a method for preparing the artificial antigen of sea cucumber saponin imitating sea cucumber.

[0010] The fifth object of the present invention is to provide an antibody imitating sea cucumber saponin.

[0011] The sixth object of the present invention is to provide a kit for detecting sea cucumber saponins from Apostichopus japonicus.

[0012] The seventh object of the present invention is to provide an immunoassay method for detecting sea cucumber saponins from Apostichopus japonicus.

[0013] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0014] A hapten imitating sea cucumber saponin, the structural formula of the hapten is shown in formula (I):

[0015]

[0016] The sea cucumber saponin hapten of formula (I) provided by the present invention has an active -Br group, which can be combined with the amino groups on the molecules of carrier protein, antibody, horseradish peroxidase, alkaline phosphatase, etc. to prepare various detection reagents. The sea cucumber saponin hapten removes the oligosaccharide chain part of the sea cucumber saponin, retains the aglycone molecular skeleton structure of the sea cucumber saponin to the greatest extent, is conducive to the preparation of antibodies with high cross-reaction rate with multiple components of the sea cucumber saponin, and retains the characteristics of inducing high-affinity antibodies.

[0017] The present invention also provides a method for preparing the sea cucumber saponin hapten imitating sea cucumber, comprising the following steps:

[0018] S1. Dissolve the sea cucumber saponin Holotoxin A1 in a buffer solution, add a cosolvent, add naringinase after dissolution, mix well, and heat to react; extract, combine the extracts, wash, dry, filter, remove the solvent, and dry to obtain a light yellow solid; the sea cucumber saponin Holotoxin A1 has the structural formula: (CAS: 85344-35-0);

[0019] S2. The light yellow solid obtained in step S1 is dissolved in an anhydrous alcohol solution, and a bromine-containing alcohol solution is added. After the reaction is completed, the solvent and excess bromine are removed to obtain a yellow paste, which is then recrystallized several times and dried to obtain a yellow solid, which is the sea cucumber saponin hapten shown in formula (I).

[0020] Preferably, the buffer in step S1 is a 0.1 M phosphate-acetate buffer with a pH of 4.4.

[0021] Preferably, the co-solvent described in step S1 is ethanol, and the volume ratio of ethanol to phosphate-acetate buffer is 1:9.

[0022] Preferably, the reaction in step S1 is carried out at 38-42°C for 4-6 days, and TLC is used to indicate whether the reaction is complete. More preferably, the reaction is carried out at 40°C for 5 days.

[0023] Preferably, the extraction solvent used in step S1 is ethyl acetate and n-butanol.

[0024] Preferably, the washing in step S1 is performed using dilute hydrochloric acid, and the drying is performed using anhydrous sodium sulfate.

[0025] Preferably, the solvent removal in step S1 is carried out by distilling the solvent under reduced pressure.

[0026] Preferably, the mass ratio of the sea cucumber saponin to naringinase is 0.5 to 10:1.

[0027] More preferably, the mass ratio of the sea cucumber saponin to naringinase is 2-4:1; and even more preferably, the mass ratio of the sea cucumber saponin to naringinase is 3:1.

[0028] Preferably, the reaction in step S2 is carried out at a constant temperature of 4° C. for 3 h, and TLC is used to indicate whether the reaction is complete.

[0029] Preferably, the removal of the solvent in step S2 is carried out by rotary evaporation.

[0030] Preferably, the recrystallization uses a mixed solvent of methanol and n-hexane in a volume ratio of 1:1.

[0031] Preferably, the molar ratio of the sea cucumber saponin to bromine is 1:3-17.

[0032] More preferably, the molar ratio of the sea cucumber saponin to bromine is 1:5-15; even more preferably, the molar ratio of the sea cucumber saponin to bromine is 1::10-11.

[0033] The present invention also provides application of the sea cucumber saponin-imitating hapten in preparing sea cucumber saponin-imitating artificial antigen.

[0034] An artificial antigen imitating sea cucumber saponin, the structural formula of the artificial antigen imitating sea cucumber saponin is shown in formula (II):

[0035]

[0036] Preferably, the carrier protein is selected from bovine serum albumin (BSA) or chicken ovalbumin (OVA).

[0037] The present invention also provides a method for preparing the sea cucumber saponin-like artificial antigen, which comprises dissolving the sea cucumber saponin-like hapten of formula (I) in a solvent, adding the hapten dropwise to a buffer solution containing a carrier protein for reaction, adjusting the pH to 9.6-10, reacting at 4-6°C, adding glycine for blocking, and dialysis purification to obtain the sea cucumber saponin-like artificial antigen of formula (II).

[0038] Preferably, the molar ratio of the sea cucumber saponin hapten to the carrier protein is 5 to 100:1.

[0039] More preferably, the molar ratio of the sea cucumber saponin hapten to the carrier protein is 30 to 60:1; and even more preferably, the molar ratio of the sea cucumber saponin hapten to the carrier protein is 40 to 50:1.

[0040] Preferably, the molar ratio of the sea cucumber saponin hapten to glycine is 1:10-60.

[0041] More preferably, the molar ratio of the sea cucumber saponin hapten to glycine is 1:20-50; and even more preferably, the molar ratio of the sea cucumber saponin hapten to glycine is 1:30-40.

[0042] As a preferred implementation method, the method for preparing the artificial antigen of sea cucumber saponin imitating sea cucumber comprises the following steps:

[0043] Using methanol as solvent, dissolve the sea cucumber saponin hapten, add it dropwise to 10-20 mg / mL carrier protein solution, stir, adjust pH to 9.6-10, and react at room temperature for 3 hours; then add glycine to block, adjust pH to 7.4, and stir overnight at 4°C; dialyze against 0.01M, pH 7.2 phosphate buffer 6 times to obtain the sea cucumber saponin artificial antigen.

[0044] The present invention also provides the use of any of the above-mentioned sea cucumber saponin-imitating artificial antigens in the preparation of sea cucumber saponin-imitating antibodies.

[0045] The present invention also provides an Apostichopus japonicus saponin antibody, which is prepared by immunizing an animal with the Apostichopus japonicus saponin artificial antigen shown in the above formula (II) as an immunogen.

[0046] Preferably, the sea cucumber saponin-like antibody is a sea cucumber saponin-like rabbit-derived polyclonal antibody.

[0047] Furthermore, the immunogen is an artificial antigen that is a sea cucumber saponin hapten coupled with bovine serum albumin.

[0048] The present invention also provides the use of any of the above-mentioned sea cucumber saponin artificial antigens and sea cucumber saponin antibodies in the preparation of a kit for detecting sea cucumber saponin residues in sea cucumber tissues.

[0049] The present invention also provides a kit for immunoassay of detecting sea cucumber saponins for non-disease diagnosis purposes, comprising any of the above-mentioned sea cucumber saponin artificial antigens as a coating source and any of the above-mentioned sea cucumber saponin antibodies as a detection antibody.

[0050] Preferably, the kit is an enzyme-linked immunosorbent assay kit.

[0051] More preferably, the kit further comprises enzyme-linked immunosorbent assay-related reagents.

[0052] The present invention also provides an immunoassay method for detecting sea cucumber saponins, which uses any of the above-mentioned sea cucumber saponins artificial antigens as a coating source and any of the above-mentioned sea cucumber saponins antibodies as a detection antibody for detection. The immunoassay method includes but is not limited to enzyme immunoassay, immunochromatography, immunosensor, immunocolloidal gold, etc.

[0053] Furthermore, the coating is originally an artificial antigen that is a sea cucumber saponin hapten coupled with chicken ovalbumin.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The present invention provides a hapten for sea cucumber saponins imitating sea cucumbers. The hapten for sea cucumber saponins imitating sea cucumbers has an active -Br group, which can be combined with amino groups on molecules such as carrier proteins, antibodies, horseradish peroxidase, and alkaline phosphatase to prepare a variety of detection reagents. The hapten for sea cucumber saponins imitating sea cucumbers removes the oligosaccharide chain part of sea cucumber saponins imitating sea cucumbers, and retains the aglycone molecular skeleton structure of sea cucumber saponins imitating sea cucumbers to the greatest extent, which is conducive to the preparation of antibodies with high cross-reaction rate with multiple components of sea cucumber saponins imitating sea cucumbers, while retaining the characteristics of inducing high-affinity antibodies. At the same time, the present invention provides an artificial antigen obtained by directly coupling sea cucumber saponins imitating sea cucumbers with a carrier protein, which has a simple synthesis method and high purity and yield. After immunizing experimental animals with the artificial antigen for sea cucumber saponins imitating sea cucumbers, the body produces antibodies against sea cucumber saponins imitating sea cucumbers. The polyclonal antibody prepared by the artificial antigen of the imitation sea cucumber saponin provided by the present invention has the characteristics of high titer, strong affinity, high cross-reaction rate, etc., and has a high cross-reaction with other components of the imitation sea cucumber saponin, that is, the components of the imitation sea cucumber saponin can be detected during the immunoassay process, and the total content of the imitation sea cucumber saponin in the imitation sea cucumber can be accurately evaluated. The enzyme-linked immunosorbent assay kit prepared by using the imitation sea cucumber saponin antigen antibody provided by the present invention has a detection sensitivity of 0.0103ng / mL for sea cucumber saponin, and IC 50 The linear range is 0.01-10 ng / mL. The enzyme-linked immunosorbent assay kit is easy to use, has low detection cost, and is fast, efficient, and accurate. It is suitable for the quality control of sea cucumber saponins or the on-site rapid detection of sea cucumber saponin residues and the screening of a large number of samples, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a synthetic route for sea cucumber saponin haptens and artificial antigens.

[0057] Figure 2 This is an ultraviolet scanning image of the artificial antigen imitating sea cucumber saponin.

[0058] Figure 3 It is an ELISA standard curve for sea cucumber saponins. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0060] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0061] Holotoxin A1 (CAS 85344-35-0) was purchased from Shanghai Taiyu Biotechnology Co., Ltd.

[0062] Holotoxin B1 (CAS 55892-88-1) was purchased from Shanghai Taiyu Biotechnology Co., Ltd.

[0063] Holothurin C (CAS 70726-48-6) was purchased from Shanghai Taiyu Biotechnology Co., Ltd.

[0064] Echinoside A (CAS 75410-53-6) was purchased from Shanghai Taiyu Biotechnology Co., Ltd.

[0065] Example 1 Synthesis and identification of sea cucumber saponin hapten

[0066] 1. Synthesis of sea cucumber saponin hapten imitating sea cucumber, the synthesis route is as follows Figure 1 As shown, the specific steps include:

[0067] (1) 50 mg of sea cucumber saponin Holotoxin A1 was added to 4.5 mL of 0.1 M phosphate-acetate buffer at pH 4.0, and 0.5 mL of ethanol was added to a final concentration of 10%. Then 16.7 mg of naringinase was added and reacted at 40° C. for 5 days. After TLC showed that the reaction was complete, 4.5 mL of ethyl acetate and 0.5 mL of n-butanol were added for extraction three times. The extracts were combined, washed with dilute hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to remove the solvent under reduced pressure. The filtrate was dried at 60° C. to obtain a yellow solid, which was product 1.

[0068] (2) Dissolve 50 μL of bromine in 10 mL of anhydrous methanol. Take 150 μL of the above solution and add it dropwise to a 15 mg / mL anhydrous methanol solution of the product 1. React at 4°C in the dark for 3 h. Blow dry the solution with nitrogen gas to remove excess methanol and bromine. Redissolve the above solution in methanol, recrystallize it several times, and dry it to obtain a yellow solid, which is the sea cucumber saponin hapten of formula (I).

[0069] 2. Identification of sea cucumber saponin haptens

[0070] The product 1 prepared above was sent to the Testing and Analysis Center of Shenyang Agricultural University for NMR identification. The specific results are as follows: 1 H NMR: δ0.85-0.98(6H,0.90(s),0.93(s)),1.07-1.21(6H,1.12(s),1.16(s)),1.22-1.80(24H,1.2 7(s),1.33(s),1.42(tt,J=7.3,6.7Hz),1.42(tt,J=7.3,6.7Hz),1.44(t,J=7.3Hz),1.44(t,J=7. 3Hz),1.48(dddd,J=13.1,9.4,4.4,4.2Hz),1.47(dd,J=4.4,1.9Hz),1.55(dtd,J=13.2,2.9,2.6H z),1.57(dtd,J=13.6,4.2,2.1Hz),1.55(s),1.61(dddd,J=13.1,4.3,2.1,1.9Hz),1.65(ddd,J=13 .1,2.9,2.8Hz),1.65(t,J=6.7Hz),1.65(t,J=6.7Hz),1.68(ddd,J=13.1,10.3,2.9Hz),1.70(ddd d,J=13.6,9.6,9.4,4.3Hz),1.70(dtd,J=13.2,10.3,2.8Hz)),1.95(1H,dd,J=9.6,4.2Hz),2.43-2 .75(4H,2.51(dd,J=17.0,8.9Hz),2.59(d,J=17.2Hz),2.64(d,J=17.2Hz),2.67(dd,J=17.0,7.2H z)),3.17(1H,dd,J=10.3,2.6Hz),3.37-3.47(2H,3.42(s),3.42(s)),5.45(1H,dd,J=8.9,7.2Hz).

[0071] 1H NMR: δ0.85-0.98(6H,0.90(s),0.93(s)),1.12(3H,s),1.22-1.80(25H,1.27(s),1.33(s),1.44(tt, J=7.4,7.3Hz),1.44(tt,J=7.4,7.3Hz),1.48(dddd,J=13.1,9.4,4.4,4.2Hz),1.47(dd,J=4.4,1.9H z),1.55(dtd,J=13.2,2.9,2.6Hz),1.54(s),1.57(dtd,J=13.6,4.2,2.1Hz),1.56(s),1.61(dddd,J =13.1,4.3,2.1,1.9Hz),1.65(ddd,J=13.1,2.9,2.8Hz),1.64(t,J=7.3Hz),1.64(t,J=7.3Hz),1.68( dddd,J=13.1,10.3,2.9Hz),1.70(dddd,J=13.6,9.6,9.4,4.3Hz),1.70(dtd,J=13.2,10.3,2.8Hz)), 1.80-2.02(3H,1.86(t,J=7.4Hz),1.86(t,J=7.4Hz),1.95(dd,J=9.6,4.2Hz)),2.42-2.72(4H,2.50( dd,J=17.0,8.9Hz),2.59(d,J=17.2Hz),2.64(dd,J=17.0,7.2Hz),2.64(d,J=17.2Hz)),3.17(1H,dd ,J=10.3,2.6Hz),4.88-4.98(2H,4.93(d,J=1.3Hz),4.93(d,J=1.3Hz)),5.43(1H,dd,J=8.9,7.2Hz).

[0072] It can be seen that the structural formula of the sea cucumber saponin hapten is as shown in formula (I):

[0073]

[0074] Example 2 Synthesis of artificial antigens imitating sea cucumber saponins

[0075] 1. Dissolve 10 mg of the sea cucumber saponin hapten synthesized in Example 1 with 0.2 mL of DMF, add dropwise to 1 mL of 13 mg / mL bovine serum albumin (BSA) (or chicken ovalbumin, OVA) phosphate buffer (0.1 M, pH 7.4), stir, and react at room temperature for 18 h; then add 15 mg of glycine to block, adjust the pH to 7.4, and stir overnight at 4 ° C; dialyze 6 times against 0.01 M, pH 7.2 phosphate buffer to obtain the sea cucumber saponin artificial antigen, which is packaged and stored at -20 ° C.

[0076] 2. UV scanning to determine the conjugate

[0077] Using 0.01mol / L, pH7.4 PBS as diluent, prepare 0.2mg / mL antigen solution, 0.02mg / mL sea cucumber saponin hapten, and 0.2mg / mL BSA solution, and use variable wavelength UV spectrophotometer to scan sea cucumber saponin hapten, BSA and conjugate respectively. Compare the differences in their UV absorption curves to determine whether the conjugation is successful.

[0078] 3. Determine the binding ratio

[0079] Select a suitable wavelength from the UV absorption curves of the hapten, BSA, and conjugate. Measure the molar absorption coefficients ε of the hapten, BSA, and conjugate at this wavelength. H ,ε BSA ,ε 偶 , the binding ratio is calculated according to the following formula:

[0080] Binding ratio = (ε 偶 —ε BSA ) / ε H

[0081] The conjugate was scanned by UV spectrophotometer, and the results were as follows: Figure 2 As shown, the absorption curve of the ultraviolet absorption spectrum of the sea cucumber saponin-BSA antigen has changed compared with BSA and the sea cucumber saponin hapten. The absorbance value of the sea cucumber saponin-BSA antigen at the 265nm absorption peak of the sea cucumber saponin has increased significantly, and it has both the ultraviolet absorption characteristics of the hapten and the ultraviolet absorption characteristics of BSA. This shows that the sea cucumber saponin hapten has been successfully coupled with BSA.

[0082] According to the formula: Binding ratio = (ε 偶 -ε BSA ) / ε HCalculate the binding ratio of hapten to carrier protein in each conjugate. The binding ratio of the conjugate is between 22:1. Many data on small molecule immunochemistry show that a high binding ratio of hapten to carrier protein is prone to produce immune tolerance, while a low binding ratio is not easy to produce high titer antibodies. Generally, it is appropriate to choose a binding ratio between 10 and 30:1.

[0083] Example 3 Preparation of polyclonal antibodies against sea cucumber saponins

[0084] Six male New Zealand white rabbits were purchased from the Animal Experimental Center of China Medical University and numbered 1#, 2#, 3#, 4#, 5#, and 6#. They were about 2.5 months old and weighed 1.8-2.2 kg. They were raised in the standard rabbit house of Shenyang Agricultural University and observed for six days. They were injected with the triple vaccine of rabbit plague-Patternella-Weigeniasis, and serum was collected as negative control 8-10 days later.

[0085] The first immunization uses Freund's complete adjuvant and the sea cucumber saponin artificial antigen solution prepared in Example 2 to emulsify, and each subsequent booster immunization uses Freund's incomplete adjuvant and the antigen solution to emulsify. Dilute the artificial antigen to 0.3 mg / mL with sterile physiological saline, and add an equal amount of adjuvant solution. Inhale the pre-calculated amount into a sterilized syringe, connect another syringe with a rubber tube, push both hands together, mix and emulsify to a W / O state, that is, drop it into ice water without diffusion. Remove the rubber tube and inject the immunized animal according to the scheme described in Table 1.

[0086] Table 1 Immunization scheme of artificial antigens imitating sea cucumber saponins

[0087]

[0088] Blood was collected from the animals before immunization as a negative control. After three immunizations, blood was collected from the ear vein on the seventh day after each immunization. The titer of the antiserum was determined by the agar diffusion method. When the titer reached the requirement (>1:32), blood was removed from the carotid artery.

[0089] When collecting blood, use a 5mL glass syringe and a No. 7 needle to trim the edge of the rabbit's ear, disinfect with an alcohol cotton ball, press the proximal end of the ear vein with your fingers, scrub the ear vein repeatedly to fill it, then place the needle bevel upward, flatten the rabbit's ear with your hand, and insert the needle into the blood vessel at a 30° angle to the skin. When blood returns, lower the needle parallel to the skin and slowly withdraw the syringe. After collecting blood, remove the needle, slowly squeeze the blood along the wall of the test tube, inject it into the test tube, and press the rabbit's ear to stop bleeding. Blood can be collected alternately from both ears. The collected blood is first left at room temperature for 1 hour, and then placed in a 4°C refrigerator overnight to fully condense. The next day, absorb the naturally precipitated serum, then centrifuge at 1000rpm for 20 minutes, aspirate the serum, freeze it at -20°C, and determine the titer of the antiserum.

[0090] Example 4 Specificity test of polyclonal antibody against sea cucumber saponin

[0091] (1) Preparation of Sea Cucumber Saponin ELISA Standard Curve

[0092] The sea cucumber saponin-chicken egg white protein coating was diluted to a concentration of 0.5 μg / mL with carbonate buffer, added to the ELISA plate, and coated overnight at 4°C. The sea cucumber saponin standard (purchased from Shanghai Aladdin Reagent Co., Ltd., item number 9-340) was serially diluted with 0.01M, pH 7.2 phosphate buffer (containing 15% methanol), and 50 μL was added to each well, and then the anti-sea cucumber saponin polyclonal antibody prepared in Example 3 was appropriately diluted, and the plate was heated at 37°C for 30 minutes, washed, and sheep anti-rabbit mouse enzyme-labeled secondary antibody was added for 30 minutes. After washing the ELISA plate, TMB-H2O2 substrate was added, and 2M sulfuric acid was added to terminate the reaction after color development, and the OD value was tested by the ELISA instrument. The absorbance value obtained by dividing the absorbance value (B0) of the first standard solution (0 standard) with each concentration of the standard solution by 100% to obtain the percentage absorbance value. The standard curve was drawn with the logarithm of the concentration of the sea cucumber saponin standard (ng / mL) as the X-axis and the percentage absorbance as the Y-axis. Figure 3 shown.

[0093] Percent absorbance value % = B / B0×100%

[0094] After calculation, the regression equation is: I = 49.21 + 14.71LogC, r = 0.9973, IC 50 1.135 ng / mL, IC 20 The value was 0.0103 ng / mL, RSD = 5.93% (n = 5). In the range of 0.0103-10 ng / mL, I and LogC were linearly correlated, with good detection sensitivity.

[0095] (2) Cross-reaction rate of anti-sea cucumber saponin antibodies

[0096] The cross-reaction rate of the antibody was tested by indirect competitive ELISA, and other drugs were serially diluted, and the drug to be tested was substituted for the sea cucumber saponin tested in step (1). The specific steps are as follows:

[0097] The sea cucumber saponin-chicken ovalbumin coating was diluted to a concentration of 0.25 μg / mL with carbonate buffer, added to the ELISA plate, and coated overnight at 4°C. The sea cucumber saponin structural analog standard was serially diluted with 0.01M, pH 7.2 phosphate buffer (containing 10% methanol), and 50 μL was added to each well, followed by the addition of appropriately diluted sea cucumber saponin polyclonal antibody (rabbit No. 2, the same below), incubated at 37°C for 30 minutes, washed, added with goat anti-rabbit enzyme-labeled secondary antibody for 30 minutes, washed the ELISA plate, added TMB substrate, and after color development, 2M sulfuric acid was added to terminate the reaction, and the OD value was tested by an ELISA instrument.

[0098] Draw a concentration-inhibition rate curve based on the test results and calculate the IC of each competitor 50 The cross-reaction rate between each competitor and the polyclonal antibody against sea cucumber saponins was calculated using the following formula.

[0099] Cross-reactivity rate = [IC 50 (Imitation of sea cucumber saponins) / IC 50 (Competitor with similar structure to sea cucumber saponin)] × 100%

[0100] The cross-reaction rates of the sea cucumber saponin polyclonal antibody and the sea cucumber saponin analogs and other sea cucumber saponins are shown in Table 2. As can be seen from Table 2, the sea cucumber saponin polyclonal antibody prepared by the present invention has a high cross-reaction with the sea cucumber saponin analogs, and the sea cucumber saponin analogs can be detected during the detection process, indicating that the sea cucumber saponin polyclonal antibody prepared by the present invention has a strong broad spectrum, and the sea cucumber saponin components can be detected during the immunoassay process. The content of the sea cucumber saponin in the sea cucumber can be accurately quantified, and then the content of the sea cucumber saponin can be comprehensively evaluated, which has a high application prospect.

[0101] Table 2 Cross-reaction rate of polyclonal antibodies against sea cucumber saponins

[0102]

[0103] Example 5 Accuracy test of the sea cucumber saponin enzyme-linked test kit

[0104] Pretreatment of the sea cucumber sample: Wash and remove the internal organs of the sea cucumber sample, accurately weigh 5g of sea cucumber tissue, add 20mL, 80% methanol aqueous solution, 10000rpm, homogenize for 2min, dilute to 25mL, centrifuge at 5000rpm for 20min, remove the precipitate, take 100μL of supernatant and add 900μL of sample diluent, which can be used for enzyme-linked immunosorbent assay, and the sample dilution factor is 100. The ELISA plate prepared by the method in Example 4 was taken with three concentrations of sea cucumber saponin standard solutions, namely 20μg / kg, 50μg / kg, and 100μg / kg, and the samples were subjected to the addition recovery test, and 3 parallels were made for each concentration, and the recovery rates were calculated respectively.

[0105] The results are shown in Table 3. The recovery rate of sea cucumber saponins added to sea cucumber samples is between 85.7% and 92.7%, indicating that the sea cucumber saponin detection kit prepared by the sea cucumber saponin antigen and antibody provided by the present invention has high accuracy.

[0106] Table 3 Recovery test of sea cucumber saponin ELISA

[0107]

[0108]

[0109] Example 6 Precision test of the sea cucumber saponin enzyme-linked test kit

[0110] Ten microwells were extracted from each batch of ELISA plates prepared by the method in Example 4, and the absorbance value (OD value) of 1 ng / mL standard solution was measured. The measurement was repeated three times, and the coefficient of variation CV% was calculated (Table 3).

[0111] As shown in Table 4, the intra-batch coefficient of variation ranges from 12.3% to 15.6%, which meets the requirement of a coefficient of variation of less than 20%, indicating that the precision of the standard product of this kit has reached the standard.

[0112] Table 4 Precision test of ELISA kit for sea cucumber saponin

[0113]

[0114] Experimental Example 7 Stability test of sea cucumber saponin enzyme-linked reagent kit

[0115] The kit is stored at 2-8°C. After 6 months of testing, the maximum absorbance value (zero standard), 50% inhibition concentration, and actual measured value of the kit's sea cucumber saponin addition are all within the normal range. The results of the 37°C accelerated aging experiment show that the kit's indicators meet the requirements.

[0116] The description presented in the above exemplary embodiments is only used to illustrate the technical solution of the present invention, and is not intended to be exhaustive, nor is it intended to limit the present invention to the precise form described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described to explain the specific principles of the present invention and its practical application, so that other technicians in the field can easily understand, implement and use the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be defined by the attached claims and their equivalent forms.

Claims

1. A sea cucumber saponin hapten imitating sea cucumber saponin, characterized in that: The structural formula of the hapten is shown in formula (I):

2. The method for preparing the sea cucumber saponin hapten according to claim 1, characterized in that: The steps include: S1. Dissolve the sea cucumber saponin Holotoxin A1 in a buffer solution, add a cosolvent, add naringinase after dissolution, mix, and heat to react; extract, combine the extracts, wash, dry, filter, remove the solvent, and dry to obtain a light yellow solid; S2. The light yellow solid obtained in step S1 is dissolved in an anhydrous alcohol solution, and a bromine-containing alcohol solution is added. After the reaction is completed, the solvent and excess bromine are removed to obtain a yellow paste, which is then recrystallized several times and dried to obtain a yellow solid, that is, the sea cucumber saponin hapten shown in formula (I) is obtained.

3. Use of the sea cucumber saponin hapten described in claim 1 in the preparation of sea cucumber saponin artificial antigen.

4. An artificial antigen imitating sea cucumber saponin, characterized in that: The structural formula of the artificial antigen imitating sea cucumber saponin is shown in formula (II):

5. The artificial antigen of sea cucumber saponin imitating sea cucumber according to claim 4, characterized in that: The carrier protein is selected from bovine serum albumin or chicken ovalbumin.

6. The method for preparing the artificial antigen of sea cucumber saponin imitating sea cucumber according to claim 4 or 5, characterized in that: The sea cucumber saponin hapten described in claim 1 is dissolved in a solvent, and then added dropwise to a buffer solution containing a carrier protein for reaction, the pH is adjusted to 9.6-10, the reaction is carried out at 4-6°C, the reaction is sealed, and the sea cucumber saponin artificial antigen shown in formula (II) is obtained.

7. Use of the artificial antigen of sea cucumber saponin imitating sea cucumber according to claim 4 or 5 in the preparation of sea cucumber saponin imitating sea cucumber saponin antibodies.

8. A sea cucumber saponin antibody, characterized in that: The method is prepared by immunizing an animal with the artificial antigen of sea cucumber saponin imitating sea cucumber as claimed in claim 4 or 5 as an immunogen.

9. A kit for detecting sea cucumber saponins from Apostichopus japonicus, characterized in that: It comprises the artificial antigen of sea cucumber saponin imitating sea cucumber described in claim 4 or 5 and the antibody of sea cucumber saponin imitating sea cucumber saponin described in claim 8.

10. An immunoassay method for detecting sea cucumber saponins for non-disease diagnosis purposes, characterized in that: The detection is carried out using the sea cucumber saponin artificial antigen described in claim 4 or 5 as the coating source and the sea cucumber saponin antibody described in claim 8 as the detection antibody.

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