抗AMP单抗的制备及牛奶中AMP残留ELISA检测方法的建立
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摘要
氨苄青霉素(简称AMP)属于β-内酰胺类抗生素，是广谱半合成青霉素，抗菌谱比青霉素G广，对G~+菌和G~-菌均有较高的抗菌活性，是禽畜等农产品养殖中常用的抗生素类药物之一，在奶牛的饲养过程中青霉素药渣常被作为饲料添加剂和广泛应用于治疗奶牛的乳腺炎，尿道、生殖道、胃肠道呼吸系统等感染，从而造成牛奶中的残留，会给人们的健康和奶类发酵品的生产带来很大危害。因此，建立牛奶中氨苄青霉素残留快速检测方法具有重要的现实意义。本研究在制备AMP人工抗原的基础上，研制抗AMP单克隆抗体，建立AMP残留检测酶联免疫检测方法(ELISA)。主要实验结果如下：
(1)采用戊二醛法和物理法将氨苄青霉素分别与牛血清白蛋白(BSA)、卵清蛋白(OVA)两种载体蛋白偶联，制备免疫用和ELISA检测用抗原。以紫外扫描和SDS-PAGE电泳鉴定偶联成功。
应用L9(3~4)正交试验确定戊二醛法的最佳制备条件为：AMP与BSA的起始摩尔比40：1，25％戊二醛量10μl，反应时间4h；物理法的AMP与BSA最适起始摩尔比为20：1，反应时间3h。
采用自行改良的BCA—紫外结合法测定最佳条件下制备的AMP人工抗原的偶联比，结果为戊二醛法制备的AMP人工抗原平均偶联比为10.23，物理法制备的AMP人工抗原平均偶联比为17.68。物理法制备的AMP人工抗原的偶联比要明显高于戊二醛法。
(2)将两种方法制备的人工抗原各免疫3只小鼠，四次免疫之后通过间接ELISA法测定各小鼠的效价达到1～5×10~4，以100ng／mL的AMP标品进行间接竞争ELISA实验，测定各血清各小鼠均呈现不同程度的阻断抑制，其中②号小鼠对的抑制率达到93.4％，敏感性最高。采用物理法制备的AMP人工抗原免疫小鼠后的抗血清对AMP标品的抑制率高于戊二醛法。
运用杂交瘤技术选用②号小鼠的脾细胞与SP2／0骨髓瘤细胞进行细胞融合，融合孔率为21％，阳性孔率为5.4％。应用有限稀释法进行亚克隆，筛选到3个稳定分泌抗氨苄青霉素单抗的克隆，依次是X.5.2，X.16.3.5和X.16.4.8。挑选阳性最高、敏感性也最好的X.16.3.5克隆注射到小鼠体内制备腹水。
采用辛酸—饱和硫酸铵法纯化腹水，纯化后抗体蛋白浓度为5.85mg／ml。间接ELISA测定腹水效价为3.2×10~5，IC_(50)值约为12.5ng／ml，与载体蛋白OVA、BSA无交叉反应，与抗生素类药物庆大霉素、四环素、卡拉霉素、链霉素无交叉反应。筛选出的单克隆敏感性和特异性较高。
(3)对ELISA各个条件进行了选择和优化。以PH8.0的Tris—HCL缓冲液为基质，应用间接竞争ELISA方法建立了检测AMP的标准曲线，检测线性范围为：0.5～500 ng／ml，回归方程为Y=32.539X+13.651，相关系数R~2=0.982，灵敏度和最低检测线分别为13.09 ng／ml和0.77 ng／ml，批内和批间的变异系数分别为4.45％和5.80％，重现性较好。
以缓冲液溶解的5％的脱脂牛奶为基质，成功建立了牛奶中检测AMP残留的标准曲线，得到的回归方程为Y=30.427X+13.773，线性范围为0.5～500ng／ml，相关系数R~2=0.9904，灵敏度和最低检测线分别为15.51 ng／ml和0.75ng／ml。建立该检测方法的精密度实验，批内变异系数为4.35％＜5％、批间变异系数5.70％＜10％，对牛奶的5个水平的加标实验的平均回收率为93.64％，该方法的最低检测限能达到欧盟标准，重现性和准确性均较好。
制备的抗AMP单克隆抗体和建立的ELISA检测方法可用于牛奶中AMP残留检测。
Ampicillin, a kind ofβ-lactam antibiotics, is a semi-synthetic broad-spectrumpenicillin which has a wider sterilizing range than penicillin G and is active to bothG~+ and G~-. It is one of the antibiotics commonly used in breeding agricultural animalssuch as birds and livestock. In the process of feeding cows, ampicillin is often addedto the feed and heavy used in the treatment of inflammation and infections of cowssuch as mastitis, urethra and reproductive tract infections, which causes penicillinantibiotics residues in milk. Long-term consumption of the milk containingpenicillin antibiotics residues could lead to body resistance and hypersusceptibilityand do harm to human beings. Therefore, it is very meaningful to establish a rapidmeasurement for detecting penicillin residue in milk. In this paper we have producedanti-ampicillin monoclonal antibody on the basis of preparation of AMP artificialantigen,and then established ELISA detection method. The main results of our studyare as follows:
(1) In this paper, ampicillin was coupled with carrier proteins bovine serumalbumin (BSA) and ovalbumin (OVA) by methods of physiology and glutaraldehydeto prepare artificial antigen for immunization and ELISA. Identified by UV scan andSDS-PAGE electrophoresis the couplings were successful.
The best preparation conditions were optimized by L9(3~4) orthogonal test,glutaraldehyde method: the initial molar ratio of AMP and BSA is 40:1, 25%glutaraldehyde 10μ1, reaction time 4 hours; physiological method: the initial molarratio of AMP and BSA is 20:1, reaction time 3 hours. And their coupling rates were10.23 and 17.68 respectively, which were measured by the improved BCA method
(2) Each artificial antigens immunised 3 mice, after the fourthimmunization, these mice's antiserum were measured, the titer were reached 1～5×10~4 by indirect ELISA,and all mice present inhibition to Standard AMP inindirect competitive ELISA with 100ng/mL. The best mouse's number is②whoseinhibition rate rezched 93.4 percent. The inhibition rate of the antiserum of mouseimmunized by artificial antigen prepared by method of physiology to standard AMPwas higher than that of glutaraldehyde.
Spleen cells of mouse②were fused with SP2/0 myeloma cells according toHybridoma Technology. The ratio of fused hole and positive hole were 21% and 5.4% respetively.Limited dilution method was employed to do subclone, threeClones named X.5.2, X.16.3.5 and X.16.4.8 stablely producing anti- ampicillinmonoclonal antibody were finally selected. The clone X.16.3.5 with the highestpositive and the best sensitivity was selected to inject into mice for preparing ascites.
The ascites with antibody were purified by caprylic acid-saturated ammoniumsulphate method. Pure antibody's protein concentration was 5.85mg/ml. The titer ofmouse ascites was 3.2×10~5 determined by indirect ELISA, IC_(50) value was about12.5ng/ml, had no cross-reaction with the carrier protein OVA, BSA and antibioticssuch as gentamicin, tetracycline, karaoke adriamycin and streptavidin. The selectedmonoclonal antibody had high sensitivity and specificity.
(3) The conditions of ELISA experiment were optimized. The standard curve ofdetecting AMP was established by indirect competitive ELISA in Tris-HCL bufferbuffer. The linear range of detection was between 0.5 and 500 ng / ml, the equationwas Y=32.539X+13.651, the correlation coefficient was 0.982, the sensitivity anddetection limit were 13.09ng/ml and 0.77ng/ml respectively, the coefficients ofvariation of ELISA in intra-assay and inter-assay were 4.45% and 5.80%respectively with good reproducibility.
The standard curve of detecting AMP residues in milk was successfullyestablished by 5% defatted milk as buffer. The equation was Y=30.427X+13.773,the linear range was between 0.5 and 500ng/ml, the correlation coefficient was0.9904, the sensitivity and detection limit were 15.51ng/ml and 0.75ng/mlrespectively. The precision experiment of this detection method was established. Thecoefficient of variation in intra-assay and inter-assay were 4.35 %＜5% and 5.70%＜10% respectively. The average recoveries of standard addition of the five levels ofmilk was 93.64 %. The detection limit of this method achieved to EU standards withgood reproducibility and accuracy.
The anti-AMP monoclonal antibody and the ELISA method can be used todetect AMP residues in milk.
(1)采用戊二醛法和物理法将氨苄青霉素分别与牛血清白蛋白(BSA)、卵清蛋白(OVA)两种载体蛋白偶联，制备免疫用和ELISA检测用抗原。以紫外扫描和SDS-PAGE电泳鉴定偶联成功。
应用L9(3~4)正交试验确定戊二醛法的最佳制备条件为：AMP与BSA的起始摩尔比40：1，25％戊二醛量10μl，反应时间4h；物理法的AMP与BSA最适起始摩尔比为20：1，反应时间3h。
采用自行改良的BCA—紫外结合法测定最佳条件下制备的AMP人工抗原的偶联比，结果为戊二醛法制备的AMP人工抗原平均偶联比为10.23，物理法制备的AMP人工抗原平均偶联比为17.68。物理法制备的AMP人工抗原的偶联比要明显高于戊二醛法。
(2)将两种方法制备的人工抗原各免疫3只小鼠，四次免疫之后通过间接ELISA法测定各小鼠的效价达到1～5×10~4，以100ng／mL的AMP标品进行间接竞争ELISA实验，测定各血清各小鼠均呈现不同程度的阻断抑制，其中②号小鼠对的抑制率达到93.4％，敏感性最高。采用物理法制备的AMP人工抗原免疫小鼠后的抗血清对AMP标品的抑制率高于戊二醛法。
运用杂交瘤技术选用②号小鼠的脾细胞与SP2／0骨髓瘤细胞进行细胞融合，融合孔率为21％，阳性孔率为5.4％。应用有限稀释法进行亚克隆，筛选到3个稳定分泌抗氨苄青霉素单抗的克隆，依次是X.5.2，X.16.3.5和X.16.4.8。挑选阳性最高、敏感性也最好的X.16.3.5克隆注射到小鼠体内制备腹水。
采用辛酸—饱和硫酸铵法纯化腹水，纯化后抗体蛋白浓度为5.85mg／ml。间接ELISA测定腹水效价为3.2×10~5，IC_(50)值约为12.5ng／ml，与载体蛋白OVA、BSA无交叉反应，与抗生素类药物庆大霉素、四环素、卡拉霉素、链霉素无交叉反应。筛选出的单克隆敏感性和特异性较高。
(3)对ELISA各个条件进行了选择和优化。以PH8.0的Tris—HCL缓冲液为基质，应用间接竞争ELISA方法建立了检测AMP的标准曲线，检测线性范围为：0.5～500 ng／ml，回归方程为Y=32.539X+13.651，相关系数R~2=0.982，灵敏度和最低检测线分别为13.09 ng／ml和0.77 ng／ml，批内和批间的变异系数分别为4.45％和5.80％，重现性较好。
以缓冲液溶解的5％的脱脂牛奶为基质，成功建立了牛奶中检测AMP残留的标准曲线，得到的回归方程为Y=30.427X+13.773，线性范围为0.5～500ng／ml，相关系数R~2=0.9904，灵敏度和最低检测线分别为15.51 ng／ml和0.75ng／ml。建立该检测方法的精密度实验，批内变异系数为4.35％＜5％、批间变异系数5.70％＜10％，对牛奶的5个水平的加标实验的平均回收率为93.64％，该方法的最低检测限能达到欧盟标准，重现性和准确性均较好。
制备的抗AMP单克隆抗体和建立的ELISA检测方法可用于牛奶中AMP残留检测。
Ampicillin, a kind ofβ-lactam antibiotics, is a semi-synthetic broad-spectrumpenicillin which has a wider sterilizing range than penicillin G and is active to bothG~+ and G~-. It is one of the antibiotics commonly used in breeding agricultural animalssuch as birds and livestock. In the process of feeding cows, ampicillin is often addedto the feed and heavy used in the treatment of inflammation and infections of cowssuch as mastitis, urethra and reproductive tract infections, which causes penicillinantibiotics residues in milk. Long-term consumption of the milk containingpenicillin antibiotics residues could lead to body resistance and hypersusceptibilityand do harm to human beings. Therefore, it is very meaningful to establish a rapidmeasurement for detecting penicillin residue in milk. In this paper we have producedanti-ampicillin monoclonal antibody on the basis of preparation of AMP artificialantigen,and then established ELISA detection method. The main results of our studyare as follows:
(1) In this paper, ampicillin was coupled with carrier proteins bovine serumalbumin (BSA) and ovalbumin (OVA) by methods of physiology and glutaraldehydeto prepare artificial antigen for immunization and ELISA. Identified by UV scan andSDS-PAGE electrophoresis the couplings were successful.
The best preparation conditions were optimized by L9(3~4) orthogonal test,glutaraldehyde method: the initial molar ratio of AMP and BSA is 40:1, 25%glutaraldehyde 10μ1, reaction time 4 hours; physiological method: the initial molarratio of AMP and BSA is 20:1, reaction time 3 hours. And their coupling rates were10.23 and 17.68 respectively, which were measured by the improved BCA method
(2) Each artificial antigens immunised 3 mice, after the fourthimmunization, these mice's antiserum were measured, the titer were reached 1～5×10~4 by indirect ELISA,and all mice present inhibition to Standard AMP inindirect competitive ELISA with 100ng/mL. The best mouse's number is②whoseinhibition rate rezched 93.4 percent. The inhibition rate of the antiserum of mouseimmunized by artificial antigen prepared by method of physiology to standard AMPwas higher than that of glutaraldehyde.
Spleen cells of mouse②were fused with SP2/0 myeloma cells according toHybridoma Technology. The ratio of fused hole and positive hole were 21% and 5.4% respetively.Limited dilution method was employed to do subclone, threeClones named X.5.2, X.16.3.5 and X.16.4.8 stablely producing anti- ampicillinmonoclonal antibody were finally selected. The clone X.16.3.5 with the highestpositive and the best sensitivity was selected to inject into mice for preparing ascites.
The ascites with antibody were purified by caprylic acid-saturated ammoniumsulphate method. Pure antibody's protein concentration was 5.85mg/ml. The titer ofmouse ascites was 3.2×10~5 determined by indirect ELISA, IC_(50) value was about12.5ng/ml, had no cross-reaction with the carrier protein OVA, BSA and antibioticssuch as gentamicin, tetracycline, karaoke adriamycin and streptavidin. The selectedmonoclonal antibody had high sensitivity and specificity.
(3) The conditions of ELISA experiment were optimized. The standard curve ofdetecting AMP was established by indirect competitive ELISA in Tris-HCL bufferbuffer. The linear range of detection was between 0.5 and 500 ng / ml, the equationwas Y=32.539X+13.651, the correlation coefficient was 0.982, the sensitivity anddetection limit were 13.09ng/ml and 0.77ng/ml respectively, the coefficients ofvariation of ELISA in intra-assay and inter-assay were 4.45% and 5.80%respectively with good reproducibility.
The standard curve of detecting AMP residues in milk was successfullyestablished by 5% defatted milk as buffer. The equation was Y=30.427X+13.773,the linear range was between 0.5 and 500ng/ml, the correlation coefficient was0.9904, the sensitivity and detection limit were 15.51ng/ml and 0.75ng/mlrespectively. The precision experiment of this detection method was established. Thecoefficient of variation in intra-assay and inter-assay were 4.35 %＜5% and 5.70%＜10% respectively. The average recoveries of standard addition of the five levels ofmilk was 93.64 %. The detection limit of this method achieved to EU standards withgood reproducibility and accuracy.
The anti-AMP monoclonal antibody and the ELISA method can be used todetect AMP residues in milk.
引文
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