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Immunogenic antigen (spinosin-BSA) and coating antigen (spinosin-OVA) of spinosin were synthesized by sodium periodate oxidation method. UV scanning analysis method showed that these two spinosins were successfully conjugated with carrier protein and the coupling ratio was 17 and 13.7, respectively. Meanwhile, when immunized by spinosin-BSA，the mice can produce anti-spinosin antibodies with the high titer (1:32 000),specificity (IC₅₀ 211.6 μg·L⁻¹) and low cross-reaction rate measured by ELISA tests. The artificial antigen of spinosin was successfully synthesized, which can be applied for preparation of monoclonal antibodies and establishment of appropriate immune method.
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Objective To develop a method for the synthesis of poly [lactic-co-(glycolic acid)] (PLGA) wrinkled microparticles,and to investigate their immobilization on model protein,i.e.bovine serum albumin (BSA),so as to provide a scientific basis for the preparation of artificial antigen-presenting cells (aAPCs).PLGA wrinkled microparticles were prepared by double emulsion-solvent evaporation method combined with porogen NH4HCO3.The effects of PLGA relative molecular mass,porogen mass concentration and double emulsion stirring speed on the morphology of PLGA micro particles were investigated.The PLGA wrinkled particles were incubated with different concentrations of FITC-conjugated BSA (FITC-BSA).The levels of BGA loaded with PLGA wrinkled particles were detected by laser scanning confocal microscopy,flow cytometry and diquinoline formic acid.The effect of the loading process on the BSA structure was analyzed by circular dichroism spectrometer.Results The molecular weight of 5 000 was the optimized parameters for PLGA wrinkled microparticles preparation.The morphology of PLGA wrinkled particles could be maintained when the mass concentration of porogen NH4HCO3 was less than 10 g/L.When the mixing speed of multiple emulsion increased from 400 r/min to 3 600 r/min,the average particle size of PLGA wrinkled particles decreased from 35 μm to 9 μm,which meets the size requirement of artificial aAPCs.The fluorescence intensity of PLGA wrinkled particles was directly proportional to the concentration of BSA,and the BSA structure was not affected by the adsorption of BSA by the PLGA wrinkled particles.Conclusion The relative molecular mass of PLGA has an important influence on the morphology of the particles.PLGA with a relative molecular mass of 5 000 can be used to prepare particles with a wrinkled topology,which can load proteinaceous macromolecules and maintain protein activity.This result has potential applications in artificial aAPCs.
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In the present study, 2-methyl-4-( trifluoromethyl) thiaZole-5-carboxylic acid ( MTCA) was acted as hapten of thifluZamide to synthesiZe artificial antigens.Immunogen MTCA-bovine serum albumin ( BSA ) was synthesiZed by active ester method.Meanwhile, three coating antigens, MTCA-OVA-1, MTCA-OVA-2 and MTCA-OVA-3 were synthesiZed by active ester method, mixed anhydride method and N, N-carbonyldiimidaZole/4-dimethylaminopyridine ( CDI/DMAP ) method, respectively.Anti-thifluZamide polyclonal antibody with high specificity was obtained from the immuniZed mice, then indirect competitive enZyme linked immunosorbent assay ( ic-ELISA ) method for thifluZamide detection was developed by using MTCA-OVA-3 and polyclonal antibody.The linear detection range in ic-ELISA was 0.08-10 mg/L with an IC50 of 1.39 mg/L and a LOD (IC10) of 0.08 mg/L.The recoveries for spiked samples including tap water, lake water and wheat ranged from 72.0%-128.3% in ic-ELISA method.Good correlation (R2=0.9994) was obtained between the results of ic-ELISA and those of HPLC analysis.The proposed ic-ELSA was promising for rapid detection of thifluZamide in water and agricultural products.
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Immunogenic antigen (jujuboside A-BSA) and coating antigen (jujuboside A-OVA) of jujuboside A were synthesized by sodium periodate oxidation method for the first time. Jujuboside A artificial antigen was confirmed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). The titer and specificity of the antibody in serum of immunized mice were detected by enzyme-linked immunosorbent assay (ELISA). The corrected relation curve of inhibition rate showed that the antibody against Jujuboside A obtained from immunized mice could bind to jujuboside A and the titer was up to 1∶4 000. The jujuboside A artificial antigen was synthesized, which can be used further to preparation of monoclonal antibody and the pharmacokinetics study of jujuboside A in laboratory animals.
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Objective:On the basis of synthetic antigens on minoxidil ,we prepared its polyclonal antibody and established a ELISA method to detect minoxidil .Methods:We synthesized minoxidil artificial antigen with glutaraldehyde and identified by UV scan -ning.We had established a detection of minoxidil competition ELISA .Results: The UV scan showed that the minoxidil successfully coupled to a carrier protein.The conjugate of minoxidil-BSA was used to immunize BALB/c mice.And the produced antiserum showed high titer of 1:12 800 in the indirect ELISA.Its ELISA curve equation:y=-0.082 3 x+0.938 ( R2 =0.9811 ) , minoxidil mass concentration and the absorbance in 0.001-10 μg/ml showed a good linearity .Conclusion: This method has been successfully preparing minoxidil artificial antigen and polyclonal antibodies and established ELISA detection method for minoxidil ,which provides a basis for the practical application of minoxidil immunoassay method .
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Objective:To study feasibility of preparing artificial antigen by membrane coated with hapten-carrier.To compare the Emodin-BSA membrane antigen immunogenicity and specificity against the liquid antigen.Methods:Emodin-BSA-PVDF membrane was prepared by the method that BSA was coated on PVDF membrane and the BSA was coupled with Emodin-couplint agent derivative.Rats were immunized by subcutaneous implantation.The immunogenicity and antibody specificity were characterized using Emodin-CA or Chrysophanol-CA or Physcion-CA membrane immunoassay. Results: The immunogenicity of Emodin-BSA coated membrane antigen was higher than Emodin-BSA liquid antigen;the specificity for three anthraquinones was almost the same(P>0.05). Conclusion:Emodin antiserum generated using Emodin-BSA coated membrane antigen has a high immunogenicity and specificity to Emodin.The results show it is feasible that membrane coated with hapten-carrier is used as artificial antigen.
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Objective: To synthesize and identify the artificial antigen of loganin for the first time, and provide a foundation for the preparation of specific monoclonal antibody and establishment of immunoassay method. Methods: Sodium periodate oxidation method was used to synthesize immunogenic antigen (loganin-BSA) and coating antigen (loganin-OVA) of loganin. Whether loganin was conjugated with BSA and OVA or not was confirmed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). The titer and specificity of the antibody in serum of immunised mice were detected by enzyme-linked immunosorbent assay (ELISA). Results: The results of MALDI-TOF-MS indicated that loganin was conjugated to BSA. The antibody against loganin obtained from immunised-mice could bind to loganin and the titer was up to 1:40 000. Conclusion: The artificial antigen of loganin was synthesized, which can be used further in the preparation of monoclonal antibody, application in quality control of Chinese materia medica and the pharmacokinetic study of loganin in laboratory animals.
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The objective of this study was to produce artificial antigens for astragaloside IV that could be used to prepare antibodies against astragaloside IV screened in Radix astragali (Astragalus membranaceus (Fisch) Bunge, Fabaceae) and its preparations, using an indirect ELISA. Astragaloside IV was coupled to carrier proteins, bovine serum albumin and ovalbumin using the sodium periodate method and was then evaluated using SDS-PAGE, MALDI-TOF MS and animal immunizations. The coupling ratio of astragaloside IV to bovine serum albumin ratio was determined to be thirteen, and the indirect ELISA demonstrated that three groups of mice immunized with astragaloside IV-bovine serum albumin produced anti-astragaloside IV- bovine serum albumin-specific antibody, with a minimum serum titer of 1:9600. A method for synthesizing highly immunogenic astragaloside IV artificial antigens was successfully developed thus indicating its feasibility in the establishment of a fast immunoassay for astragaloside IV content determination in Radix astragali and its products.
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Alkaloids are a kind of natural nitrogen compounds which are widespread in the nature, with special and significant activities in pharmacology. In recent years, immunoassay techniques have been applied for the qualitative and quantitative analysis of alkaloids from traditional Chinese medicine (TCM). However, preparation and identifica-tion of artificial antigen with good immunogenicity are necessary and important for establishment of immunoassay for alkaloids from TCM. This paper summarized the designing ideas and procedures for preparation of artificial antigen and identification for the conjugates of alkaloids and carrier protein, in order to provide reference point for establish-ment and application of immunoassay techniques.
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Objective:To compare the immunogenity of the AST artificial antigen with two different carrier proteins, bovine serum albumin(BSA) and keyhole limpet hemocyanin(KLH).Methods: The hapten Astragaloside Ⅳ(AST) was linked to carrier protein BSA or KLH as immunogen,and linked ovalbumin(OVA) as coating antigen by the sodium periodate method.Then the 6-8 weeks BALB/c mice were immunized with AST-BSA or AST-KLH.The titer and specificity of the antiserum were detected by indirect enzyme-linked immunosorbent assay ( iELISA ) and indirect competitive enzyme-linked immunosorbent assay ( icELISA ) respectively.Results:The antiserum of the mice immunized with AST-KLH had better specificity than which immunized with AST-BSA.The IC50 of AST-KLH antiserum was about 5 μg/ml in the detection by icELISA.The high titer of both antiserums was about 1∶51 200 after immunizing mouse.Conclusion:The conjugate of AST-KLH was testified to have a better immunogenity in comparison with AST-BSA,this investigation would be the foundation of the further research of AST immunoassay.
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Objective:AFM1-BSA and AFM1-OVA were synthesized and then identified in this experiment.Methods: Using oximation method ,AFM1 was transformed to oxime compounds while the reaction process was monitored via TLC method aiming to identify the compounds.Coupled with carrier protein BSA and OVA respectively , we obtained AFM1-BSA and AFM1-OVA, then identified synthetic antigen via UV spectrophotometry and SDS-PAGE.Antigens were injected into experimental animals , finally obtaining the murine multi-antiserum.Eventually , the multi-antiserums were detected via indirect inhibition ELISA method to judge whether the antigens were effectively or not.Results:After oximation reaction ,the migration distance of oxime compounds in the thin layer plate was shorter.The maximum absorption peak of AFM1-BSA occurred in 274 nm,and was inconsistent with both UV absorption peaks of BSA and AFM 1.The electrophoretic velocity of AFM 1-BSA was less than that of BSA.All the titers of three immunized mice were 1×10-4 approximately;the multi-antiserum from No.3 sample had the best sensitivity ,its IC50 was 359.9 ng/ml.Conclusion:In this study,we obtained AFM1 artificial antigen and murine multi-antiserum of high sensitivity.
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Objective: To synthesize and identify the hyodeoxycholic acid (HDCA) artificial antigen and to lay a foundation for the preparation of its specific monoclonal antibody, establishment of immunoassay method, and application in the pharmacological researches. Methods: The HDCA artificial immunogen (HDCA-BSA) and coating antigen (HDCA-OVA) were synthesized by mixed anhydride method and then examined by TLC method. BALB/c mice were immunized with the emulgator containing the prepared HDCA-BSA conjugate and Freund's adjuvant by the back sc multipoint injection for three times following with a booster immunization by the prepared HDCA-BSA conjugate alone. Then the blood of mice was obtained from tail vein 24 h after the booster, and the sera were isolated for the titer and specificity test by ELISA. Results: According to TLC, HDCA was successfully conjugated to BSA. The antibody against HDCA obtained from immunized-mice could bind to HDCA and the titer was over 1:8 000. As shown in the competitive inhibition curves, the antibodies in the serum were specifically bound to HDCA without cross-relativities with glycyrrhizic acid, baicalin, geniposide, etc, except cholic acid (90%). Conclusion: The HDCA-BSA is successfully synthesized, which could be used for the preparation of anti-HDCA monoclonal antibody and the research on pharmacological targets of HDCA.
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Objective: To synthesize artificial chlorogenic acid (CHA) antigen, to prepare CHA polyclonal antibody, and to lay the foundation for the preparation of immune chip for allergic component analysis. Methods: CHA-bovine serum albumin (CHA-BSA) and CHA-ovalbumin (CHA-OVA) were synthesized by carbodiimide method. The characterization of the synthesis was examined by UV spectrometry and TLC method. The titer and specificity of the antibody in serum of immunised mice were detected by indirect enzyme-linked immunosorbent assay (I-ELISA) and indirect competitive enzyme-linked immunosorbent assay (I-CELISA), respectively. Results: According to the UV and TLC determination, the CHA was successfully conjugated with BSA. The serum antibody titer was 1:128 000 with the linear range of 15.6-250 ng/mL, R2 = 0.995, and Y = -0.10 lnX+1.158. No cross-reaction was detected with glycyrrhizic acid, parietic acid and so on, which were commonly used in the combination with CHA. Conclusion: The preparation of multi-clonal antibody with good sensitivity and specificity against CHA is successful, which provides a foundation for the trace detection and allergy research of CHA.
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OBJECTIVE: To synthesize conjugates as artificial complete antigen and coated antigen of icariin to establish an enzyme linked immunosorbent assay (ELISA).
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OBJECTIVE: To synthesize and identify a polyclonal or monoclonal antibody against ginseonside Re (GRe).
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Objective: To synthesize the artificial antigen of ginsenoside Rg1-bovine serum albumin (Rg1-BSA) and the artificial coated antigen of ginsenoside Rg1-polylysine (Rg1-PLL), and to provide the basis for the preparation of monoclonal antibody (MAb) and the establishment of immunoassay method. Methods: Rg1-BSA and Rg1-PLL were synthesized by sodium periodate oxidation method. The characterization of the synthesis was examined by UV spectrometry and TLC method. The titer and specificity of the antibody in serum of immunised mice were detected by indirect enzyme-linked immunosorbent assay (I-ELISA) and indirect competitive enzyme-linked immunosorbent assay (I-CELISA), respectively. Results: According to the UV and TLC, the Rg1 was successfully conjugated with BSA and PLL. I-ELISA and IC-ELISA methods were developed using Rg1-PLL. The anti-Rg1 antibody obtained from immunized mice could bind to Rg1 specially and the titer was up to 1:80000. Conclusion: The artificial immunogen Rg1-BSA and coated antigen Rg1-PLL are successfully synthesized, which could be used to prepare the MAb of Rg1 and establish the immunoassay method.
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Under low temperature conditions, the hapten carboxyl-norketamine was synthesized by reacting norketamine and succinaldehyde acid.Identification result using electrospray ionization mass showed the hap ten was successfully synthesized.The artificial antigen confirmed by infrared spectroscopy was developed by conjugating hapten to carrier proteins with carbodiimide(EDC) method.Matrix-assisted laser desorption ioni zation time of flight mass spectrometry(MALDI-TOF-MS) showed that the ratio of hapten to BSA was 11:1.The antibody with high titer(5.12 × 10~4) was produced after immuning to rabbits.
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Artificial antigen Cd-IEDTA-BSA (HSA) was successfully synthesized by the bifunctional chelating agent IEDTA (isothiocyanobenzylethyl enediamine tetraacetic acid) coupling with cadmium and protein carrier. The fluorescence characteristics of antigen were determined by fluorescence spectrophotometry and the coupling ratio 11∶ 1-16∶ 1 was obtained for the coupling reaction. The folding status of serum albumin during the synthesis process of artificial antigen Cd-IEDTA-BSA (HSA) was analyzed using "fluorescence phase diagram", and the result shows that it was conformed to the "all-or-none" pattern. The polarization fluorescence spectra show that the microenvironments of tryptophan residues in carrier protein have some changes to cause changes in protein conformation.
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Objective:To explore the possibility of immune prevention and cure for tetrodotoxin (TTX) intoxication and develop an antitoxin vaccine against TTX.Methods:TTX was conjugated to Tachypleus tridentatus hemocyanin(TTH) in presence of formaldehyde and this conjugate(TTX-TTH) was used as an immunogen to immunize Balb/C mice.The quality of antisera in animal was detected on schedule by enzyme linked immunosorbent assay(ELISA) and competition-inhibited enzyme immunoassay(CIEIA).Mice immunized with TTX-TTH were challenged intraperitoneally with lethal doses of TTX(1LD=13.5 ?g/kg).Results:The high titer and affinity of antisera could last for a time as long as more than one year.The immunized mice were ip challenged with 1?LD of TTX once and again at a fixed period,there was a affirmative antitoxic effect in about 12 months(total 15?LD),and a partial effect in following time;about one fourth of animal survived till 24 months post initial immunization(total 26?LD),and which was at a stage of senescence in mice.The anti-TTX poisoning effect of animal was approximately consistent with the antisera quality tested.Conclusion:The experimental vaccine of TTX could effectively protect animal from TTX intoxication and its effect was of longer duration of validity.Immunoprophylaxis would be the hopeful means for detoxification of TTX. [
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Tetrodotoxin(TTX) was coupled to Tachypleus tridentatus hemocyanin (TTH) chemically to form an artificial antigen.Balb/c mice were immunized with TTX TTH. The immunologic effect of different immunization schedules [14～20d for longer interval (group L) and 7～10d for shorter interval (group S)] of experimental anti TTX vaccine was compared. The quality of antisera and effect of vaccine on TTX poisoning were detected. The antibody quality of serum was increased more quickly in group S than that in group L after immunization. The average survival rates were 99 3% and 92 9%, respectively ( P