本文選題：磺胺類藥物　切入點：氟喹諾酮類藥物　出處：《中國農(nóng)業(yè)大學》2016年博士論文

【摘要】：動物性食品中化學性危害物的殘留問題是食品安全重要的組成部分,發(fā)展高效、靈敏、快速、多殘留的免疫分析方法是保障食品安全的有效途徑。免疫分析中傳統(tǒng)的多克隆抗體和單克隆抗體,一經(jīng)制備,后期改造的可能性很小。而重組抗體可從基因水平上對抗體進行修飾、改造、定向標記、體外表達,而且其單價結合特性,靈敏度更高。熒光素酶由于其發(fā)光強度高,發(fā)光迅速,適合于原核表達,以其為基礎的生物發(fā)光技術由于靈敏度高,光譜范圍廣等優(yōu)點在獸藥殘留檢測領域已經(jīng)引起關注。本論文開展了動物性食品中磺胺類和氟喹諾酮類藥物殘留的生物發(fā)光免疫分析方法研究,旨在為獸藥殘留監(jiān)控提供快速、靈敏、高通量的檢測手段。以1：1融合表達的方式分別將堿性磷酸酶(Alkaline phosphatase, ALP)和海腎熒光素酶(Renilla luciferase, Rluc)與氟喹諾酮類藥物(Fluoroquinolones, FQs)的單鏈抗體(single chain variable fragment, scFv)進行定向標記,構建了FQs-scFv-ALP和FQs-scFv-Rluc融合蛋白。分別建立了可檢測20種FQs的化學發(fā)光直接競爭免疫分析方法(CL-cdELISAFQs-scFv-ALP)和可檢測20種FQs的生物發(fā)光直接競爭免疫分析方法(BL-cdELIS AFQs-scFv-Rluc)。以諾氟沙星(Norfloxacin,NOR)分別建立標準曲線,CL-cdELISAFQs-scFv-ALP中NOR的IC50值為0.15μg/L,線性范圍為0.04-1.08μg/L,牛奶樣品采用乙酸乙酯提取,氮吹,PBS復溶的方法處理后可消除基質效應,檢測限(Limit of detection, LOD)為 0.021μg/L,與其他19種氟喹諾酮類藥物的交叉反應率介于0.43-133.89%; BL-cdELISAFQs-scFv-Rluc中NOR的IC50值為0.031 μg/L,線性范圍為0.006-0.16μg/L,牛奶樣品采用直接稀釋40倍的方法可消除基質效應,LOD為0.124μg/L,與其他19種氟喹諾酮類藥物的交叉反應率介于0.45-129.7%。以歐盟規(guī)定牛奶中必檢的五種氟喹諾酮類藥物,環(huán)丙沙星(Ciprofloxacin, CIP)、恩諾沙星(Enrofloxacin, ENR)、馬波沙星(Marbofloxacin, MAR)達氟沙星(Danofloxacin, DAN)以及氟甲喹(Flumequine, FLU)進行添加回收試驗,其中CL-cdELISAFQs-scFv-ALP的批內(nèi)和批間添加回收率分別為78.2-119.4%和79.6-117.4%,變異系數(shù)分別為3.1-11.4和4.4-10.3%。BL-cdELISAFQs-scFv-Rluc的批內(nèi)和批間添加回收率分別為78.4-117.2%和77.8-117.9%,變異系數(shù)分別為6.9-13.4%和7.4-12.9%。對比結果顯示,本研究建立的兩種方法的IC50值比傳統(tǒng)ELISA方法分別降低了約2倍和10倍,生物發(fā)光的IC50值比化學發(fā)光的IC50值低大約5倍,而且耗時短,牛奶樣品處理簡單,雖然生物發(fā)光的變異系數(shù)大于化學發(fā)光,但是回收率均介于75-120%,變異系數(shù)小于15%,均達到了獸藥殘留分析的要求。構建了磺胺類藥物(S ulfonamides, SAs)單鏈抗體與螢火蟲熒光素酶(Firefly luciferase, Fluc)的融合蛋白(SAs-scFv-Fluc)�；贔Qs-scFv-Rluc 和 SAs-scFv-Fluc,建立了同時檢測牛奶中21種磺胺類和20種氟喹諾酮類藥物的多殘留直接競爭生物發(fā)光免疫分析方法(DBL-cdELISA)。以NOR和磺胺二甲基嘧啶(Sulfamethazine, SMZ)分別建立標準曲線,NOR和SMZ的IC50值分別為0.051μg/L和0.211μg/L。與其他19種氟喹諾酮類藥物的交叉反應率介于0.56-130.46%,與其他20種磺胺類藥物的交叉反應率介于2.25-1406.67%。牛奶稀釋80倍可消除基質效應,NOR和SMZ的LOD分別為0.232μg/L和1.894μg/L。分別選取五種氟喹諾酮類藥物(CIP, ENR, MAR, DAN以及FLU)和五種磺胺類藥物(SMZ,磺胺二甲氧嘧啶(Sulfadimethoxine, SDM),磺胺喹VA林(Sulfaquinoxaline, SQX),磺胺間甲氧嘧啶(Sulfamonomethoxine, SMM)以及磺胺甲基異VA唑(Sulfamethoxazole, SMX))進行添加回收試驗。五種FQs的添加回收率為76.6-118.4%,變異系數(shù)為5.7-9.7%五種SAs的添加回收率為79.2-118.6%,變異系數(shù)為5.4-9.6%,滿足獸藥殘留檢測的相關要求。雙熒光素酶的使用可使底物同時加入,信號同時采集,實現(xiàn)了基于雙酶標記的固相免疫分析方法中兩類物質的同時檢測。以海腎熒光素酶和腔腸素為能量供體,量子點為能量受體,氟喹諾酮類藥物為模式藥物,建立了基于單鏈抗體的生物發(fā)光能量共振轉移(QD-BRET)均相免疫分析體系。海腎熒光素酶的最適底物為腔腸素h, QD-BRET的福斯特距離為7.86nm,供體與受體之間的距離為8.9nm。以ENR建立標準曲線,其IC50值為0.782μg/L,線性范圍為0.023-25.60μg/L, LOD為2.54 ng/L。與7種代表性的氟喹諾酮類藥物的交叉反應率介于2.55-111.6%,與第二章第三章的交叉反應率一致。五種氟喹諾酮類藥物(CIP、ENR、MAR、DAN以及FLU)批內(nèi)和批間的添加回收率分別為78.5-118.2%和79.3-116.6%,變異系數(shù)均小于10%。雖然本方法靈敏度不及DBL-cdELISA,但本方法是一個均相的分析方法,省去了包被、洗滌等步驟,耗時短,操作簡單,并且可以滿足氟喹諾酮類藥物的殘留檢測要求。綜上所述,本研究建立的基于單鏈抗體和熒光素酶融合蛋白的均相和非均相生物發(fā)光免疫分析方法均可用于牛奶中磺胺類和氟喹諾酮類藥物的殘留檢測,在最大殘留限量之下可檢測21種磺胺類藥物和20種氟喹諾酮類藥物。與化學發(fā)光相比,生物發(fā)光靈敏度高,耗時短,且牛奶樣品處理簡單,不同熒光素酶的聯(lián)合應用,可實現(xiàn)多種物質的同時檢測。此外本研究初步證明,基于量子點的QD-BRET體系可用于小分子物質的檢測,可作為獸藥殘留監(jiān)控中快速、靈敏、高通量的檢測手段。
[Abstract]:The residual chemical hazards in animal food is an important part of food safety, the development of efficient, sensitive, rapid, multi residue immunoassay method is an effective way to ensure food safety. The traditional immunoassay of polyclonal antibodies and monoclonal antibodies, the preparation, the possibility of reform is very small. The recombinant antibody can be modified, the antibody from gene level transformation, directional marker, and its expression in vitro, monovalent binding properties, higher sensitivity. Luciferase because of its high luminous intensity, luminous rapidly, suitable for prokaryotic expression, based on the bioluminescence technique because of its high sensitivity, wide spectrum etc. in the field of veterinary drug residues have been paid close attention. This thesis has carried out the analysis of immune sulfonamides and Fluoroquinolones Residues in animal food bioluminescence, intended for animal drug residues Keep monitoring to provide rapid, sensitive and high-throughput detection methods. The expression of 1:1 fusion method respectively, alkaline phosphatase (Alkaline phosphatase, ALP) and Renilla luciferase (Renilla luciferase, Rluc) and fluoroquinolones (Fluoroquinolones, FQs) single chain antibody (single chain variable fragment, scFv) directional markers the FQs-scFv-ALP and FQs-scFv-Rluc fusion protein were established. The chemical can detect 20 kinds of luminescence of FQs direct competitive immunoassay method (CL-cdELISAFQs-scFv-ALP) and can detect 20 kinds of FQs bio Luminescent Immunoassay direct competition (BL-cdELIS AFQs-scFv-Rluc). With norfloxacin (Norfloxacin, NOR) respectively to establish the standard curve in CL-cdELISAFQs-scFv-ALP NOR IC50. The value is 0.15 g/L, the linear range is 0.04-1.08 ~ g/L, milk samples using ethyl acetate extraction, nitrogen blowing, the processing method of PBS complex solution After the elimination of matrix effect, the limit of detection (Limit of detection, LOD) for 0.021 g/L, between 0.43-133.89% and other cross reaction of 19 fluoroquinolones in BL-cdELISAFQs-scFv-Rluc NOR rate; IC50 value is 0.031 g/L, the linear range of 0.006-0.16 g/L, the milk sample by direct diluted 40 times can be eliminated matrix effects, LOD is 0.124 g/L, the rate between 0.45-129.7%. and other cross reaction of 19 fluoroquinolones with EU regulations of five fluoroquinolones in milk will be seized, ciprofloxacin (Ciprofloxacin, CIP), enrofloxacin (Enrofloxacin, ENR), marbofloxacin (Marbofloxacin, Danofloxacin, DAN (MAR) of danofloxacin flumequine) and (Flumequine, FLU) add recovery test, the CL-cdELISAFQs-scFv-ALP intra batch and inter batch recoveries were 78.2-119.4% and 79.6-117.4% respectively, the coefficient of variation 3.1-11.4 and 4.4-10.3%.BL-cdELISAFQs-scFv-Rluc in intra and inter batch recoveries were 78.4-117.2% and 77.8-117.9%, 6.9-13.4% and 7.4-12.9%. respectively. The coefficient of variation of the results showed that two kinds of the method of IC50 value than the traditional ELISA method were reduced by about 2 times and 10 times, IC50 bioluminescence value than Cl IC50 value low of about 5 times, and short time, the milk sample, while the coefficient of variation is greater than the bioluminescence chemiluminescence, but the recovery rate is 75-120%, the coefficient of variation is less than 15%, reached for the analysis of veterinary drug residues. Construction of sulfonamides (S ulfonamides, SAs) scFv and firefly luciferase (Firefly luciferase, Fluc) fusion protein (SAs-scFv-Fluc). FQs-scFv-Rluc and SAs-scFv-Fluc based on the simultaneous detection of 21 kinds of sulfonyl amine in milk and 20 kinds of fluoroquinolonic Nobel Quinolones residues in direct competition bioluminescence immunoassay (DBL-cdELISA). The NOR (Sulfamethazine, SMZ) and sulfamethazine respectively to establish the standard curve, NOR and SMZ IC50 were cross reaction of 0.051 g/L and 0.211 g/L. and 19 other fluoroquinolones ranged between 0.56-130.46%, between 2.25-1406.67%. milk diluted 80 times can eliminate the matrix effect and cross reaction of other 20 sulfonamides rate, NOR and SMZ LOD were 0.232 g/L and 1.894 g/L. were selected five fluoroquinolones (CIP, ENR, MAR, DAN and FLU) and five sulfonamides (SMZ, sulfadimethoxine (Sulfadimethoxine, SDM VA Lin), Sulfaquinoxaline (Sulfaquinoxaline, SQX), sulfamonomethoxine (Sulfamonomethoxine, SMM) and sulfamethoxazole (Sulfamethoxazole, SMX) of VA) to add five FQs recovery test. Add the recovery rate was 76.6-118.4%, the coefficient of variation was five SAs 5.7-9.7% recovery rate is 79.2-118.6%, the coefficient of variation is 5.4-9.6%, to meet the relevant requirements of veterinary drug residues detection. Using the dual luciferase substrate can be added at the same time, the signal collected at the same time, the double solid phase immune enzyme labeled detection and analysis of two kinds of material and method based on to Renilla luciferase and coelenterazine as energy donor, quantum dots as energy acceptor, fluoroquinolones as a model drug, a single chain antibody based on bioluminescence resonance energy transfer (QD-BRET) homogeneous immunoassay system. Renilla luciferase was the optimal substrate coelenterazine h QD-BRET, the Forster distance is 7.86nm and the distance between the donor and acceptor for 8.9nm. ENR to establish the standard curve, the IC50 value is 0.782 g/L, the linear range of 0.023-25.60 g/L, LOD 2.54 ng /L. and 7 generation The cross reaction of fluoroquinolones rate is 2.55-111.6%, consistent with the cross reaction rate. The third chapter second chapter five fluoroquinolones (CIP, ENR, MAR, DAN and FLU) intra and inter the recoveries were 78.5-118.2% and 79.3-116.6%, the coefficient of variation was less than 10%. while the sensitivity method less than DBL-cdELISA, but this method is a homogeneous analysis method, eliminating the coated, washing, short time, simple operation, and can meet the requirements of detection of residues of fluoroquinolones. In summary, this study established the homogeneous and single chain antibody and luciferase fusion protein based on heterogeneous biological immunoassay the analysis method can be used to detection of residues in milk of sulfonamides and fluoroquinolones, the maximum residue limits under the detection of 21 sulfonamides and 20 kinds of fluoroquinolones and. Chemiluminescence, bioluminescence, high sensitivity, short time, and the milk sample, combined application of different luciferase, can detect a variety of substances at the same time. In addition this study proved that quantum dots, QD-BRET system can be used for the detection of small molecule based, can be used as a veterinary drug residue monitoring in rapid, sensitive detection method high throughput.

【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S859.84
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本文編號：1667556