摘要
金黄色葡萄球菌(Staphylococcus aureus)是一种重要的人畜共患致病菌,广泛存在于自然界中,其产生的肠毒素(staphylococcal enterotoxins, SE)可通过污染食物而导致食物中毒。目前,随着人们对食品安全重视度的加深,国际上已将肠毒素的检测列入食品检验法规,因此建立灵敏、快速的肠毒素检测方法,是食品安全检测的一项重要研究内容。
肠毒素的检测方法主要有动物学试验、免疫学方法、分子生物学方法、生物传感器技术和超抗原技术。在这些方法中,免疫学方法因其具有简便、快速、灵敏、特异性高等特点,是检测肠毒素广泛采用的一项技术。单克隆抗体(monoclonal anibody, McAb)纯度高,特异性强,一经研制出,便可大批量生产,将其用于肠毒素的免疫学检测将大大提高检测的特异性和灵敏度。商品化肠毒素ELISA检测试剂盒中采用的单克隆抗体主要是通过细胞融合技术,对实验动物单独免疫一种抗原所产生的单抗,一次实验只能检测一种肠毒素。已知肠毒素种类多达20种,葡萄球菌食物中毒往往也不是由单一肠毒素引起的,在实际检测过程中,有时不需要对肠毒素血清型作出鉴定,只要判断有没有肠毒素存在即可,因此如果一次反应能同时检测多种肠毒素将大大减少葡萄球菌食物中毒分析的工作量。为此,采用混合免疫法,筛选制备能同时针对多种肠毒素抗原的共同决定簇的单克隆抗体,有可能实现一次反应检测多种肠毒素。
本课题以引起葡萄球菌食物中毒比较常见的金黄色葡萄球菌A型和B型肠毒素(staphylococcal enterotoxin A, SEA和staphylococcal enterotoxin B, SEB)作为免疫原,采用混合免疫方法,运用杂交瘤技术制备得到同时针对SEA和SEB的单克隆抗体；同时以SEA单独免疫BALB/c小鼠为对照,经同样的方法,制备得到相应的抗SEA单克隆抗体。对两种免疫方法制备的单克隆抗体的性质进行了一系列的分析和比较,并采用间接竞争ELISA对混合免疫制备的单抗在金黄色葡萄球菌肠毒素污染的乳制品中肠毒素的检测进行了初步的应用评价。主要研究结果如下：
1抗SEA-SEB多克隆抗体的制备及ELISA分析
采用SEA和SEB两种抗原等量混合免疫BALB/c小鼠(编号1～3),免疫剂量为20μg/次/只(SEA和SEB各10μg),在每次免疫后的第7d进行采血,得到抗血清。间接ELISA实验分析表明,免疫小鼠从第21d开始有明显抗体产生,第49d抗体的效价达到较高水平,其中2号小鼠第四次免疫后的抗血清针对SEA的效价为4.5×104,针对SEB的效价为5.0×104；抗血清对SEA的灵敏度为81.3 ng/mL,对SEB的灵敏度为62.4 ng/mL。抗血清对SEA和SEB都有很强的特异性,与金黄色葡萄球菌C1型肠毒素(SEC1)、牛血清白蛋白(BSA)和卵清白蛋白(OVA)均无交叉反应。
同时,采用SEA单独免疫BALB/c小鼠(编号4～6),免疫剂量为10μg/次/只,免疫小鼠从第21d开始有明显抗体产生,第49d抗体的效价达到较高水平,其中4号小鼠第四次免疫后的抗血清针对SEA的效价为5.0×104；抗血清对SEA的灵敏度为73.9 ng/mL。抗血清对SEA有很强的特异性,与SEB、SEC1、BSA和OVA均无交叉反应。
2抗SEA-SEB单克隆抗体的制备及性质分析
采用SEA和SEB混合免疫的2号小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA筛选和有限稀释法亚克隆,获得1株能同时分泌抗SEA和SEB的杂交瘤细胞3F2。采用SEA单独免疫的4号小鼠,经同样的方法,获得1株分泌抗SEA的杂交瘤细胞3C12。
将2株杂交瘤细胞分别注射于BALB/c小鼠体内生产腹水抗体,用间接ELISA方法对收集到的抗体的效价、灵敏度、特异性、亲和力、杂交瘤细胞的染色体数目和分泌抗体的稳定性等性质进行了一系列的分析。结果表明,杂交瘤细胞3F2所产腹水抗体针对SEA的效价为4×105,灵敏度为133.2 ng/mL,亲和力常数为8.18×107L/mol;针对SEB的效价为1×105,灵敏度为82.5 ng/mL,亲和力常数为5.32×107L/mol,与SEC1、BSA、OVA等均无交叉反应。杂交瘤细胞3C12所产腹水抗体针对SEA的效价为2×105,灵敏度为115.9 ng/mL,亲和力常数为6.76×107 L/mol,与SEC1、BSA、OVA等均无交叉反应,与SEB仅有很微弱的交叉反应。
通过对两株杂交瘤细胞的染色体数目分析表明,杂交瘤细胞3F2和3C12的染色体数都在100条左右,近似等于两亲本细胞染色体数目之和,说明得到的杂交瘤细胞是亲本细胞的融合。2株杂交瘤细胞经连续传代6周和三个月的反复冻融后,分泌抗体的能力及效价水平变化不大,表明这两株杂交瘤细胞株比较稳定。
3抗SEA-SEB单克隆抗体在乳制品检测中的初步应用
选取易受肠毒素污染的乳制品(牛奶和酸奶)为研究对象,对混合免疫制备的单抗3F2在肠毒素检测中的应用效果进行了评价。(1)将单一肠毒素SEA或SEB添加到不含肠毒素的牛奶样品中,分别用单抗3F2、单抗3C12作为检测抗体,采用间接竞争ELISA法对样品中肠毒素的回收率进行了测定。3F2对SEA和SEB都有较好的回收率,对SEA的平均回收率在88.3%～103.5%之间,对SEB的平均回收率在86.2%～101.6%之间；3C12对SEA的平均回收率在86.7%～98.1%之间。(2)将三种不同型别的肠毒素(SEA、SEB和SEC1)进行组合,分别添加到不含肠毒素的牛奶样品中,用混合免疫制备的单抗3F2对牛奶样品中的SEA和SEB进行检测,发现3F2不受SEC1的干扰,能够准确的检测到SEA和SEB,并且对SEA和SEB的平均回收率都在70%～120%之间。(3)将产SEA和SEB的金黄色葡萄球菌菌株,以单一接种和混合接种的两种方式,接种于不含肠毒素的牛奶和酸奶,放置到37℃条件下培养36～48 h后,用混合免疫制备的单抗3F2检测其产生的肠毒素,发现人工污染样品中均能检测到SEA和SEB。
Staphylococcus aureus is a major human and zoonotic pathogen widely spreading in the nature. It can produce staphylococcal enterotoxins (SEs) and induce food poisoning. Detection of SEs is listed in food safety inspection regulations with the improving of people's awareness about food safety. It is necessary to establish a rapid and sensitive method for detection of SEs to insure food safety.
Methods for detection of SEs have been mainly based on zoology test, immunological methods, polymerase chain reaction, biosensors, and technique of superantigens. Among these methods, immunological methods are convenient, rapid, sensitive and specific. It is a widely-used technology for detection of SEs. Monoclonal antibody (McAb) has the benefit of high purity and specificity, and can be put into full scale production. If it is used for immunological detection of SEs, it can improve the level of specificity and sensitivity in detection. Monoclonal anibodies used in commercial ELISA kits for detecting SEs are mainly pepared by hybridomas technique and immunized with single antigen, and it can only detect its related toxin. At present, serotypes of enterotoxin are above 20 types, and staphylococcal food poisoning is not usually only caused by a single toxin. In actual testing process, sometimes there is no need to identify serotypes of enterotoxin, it is important to determine whether enterotoxins exist. So it will greatly reduce detecting workload when a variety of toxins can be simultaneously determined in one reaction. Therefore, if monoclonal antibody against common antigen determinants shared by multiple SEs can be prepared by the method of simultaneous immunization, it is possible to detect a variety of enterotoxins in one reaction.
In this paper, through hybridomas technique, one hybridomas cell, designated 3F2, was obtained when Staphylococcal enterotoxin A (SEA) and B (SEB) were used as immunogen by the method of simultaneous immunization. Hybridomas cell 3F2 can produce antibody against both SEA and SEB. At the same time, the other hybridoma cell was obtained following the same procedure with mouse immunized by SEA, which was designated 3C12. Two monoclonal antibodies were obtained by injection 3F2 and 3C12 into BALB/c mice, respectively. Characteristics of two antibodies were determined by ELISA method. Potential application for 3F2 to detection SEs in dairy products was evaluated by indirect competitive ELISA. Results were as followed.
1 Preparation and analysis of polyclonal antibody against SEA-SEB
BALB/c mice (No.1～3) were simultaneously immunized with equal amount of SEA and SEB, immunizing dose of each mouse was 20μg every time (SEA and SEB were 10μg, respectively). After a week' immunization, mice were bled and antisera were obtained. Characteristics of antisera were analyzed by an indirect ELISA. There were obvious antibodies produced at 21st day, and mice had the highest titer of antibodies at 49th day. After the fourth immunization, the titer of antisera from mouse No.2 was 4.5×104 for SEA, and 5.0×104 for SEB; sensitivity of antisera was 81.3 ng/mL for SEA and 62.4 ng/mL for SEB. Antisera had high specificity against both SEA and SEB. It had no cross reaction with staphylococcal enterotoxin Cl (SEC1), bovine serum albumin (BSA) and ovalbumin (OVA).
BALB/c mice (No.4～6) immunized with SEA followed the same procedure, and immunizing dose of each mouse was 10μg every time. Characteristics of antisera were analyzed by an indirect ELISA. There were obvious antibodies produced at 21st day, and mice had the highest titer of antibodies at 49th day. After the fourth immunization, the titer of antisera from mouse No.4 was 5.0×104 for SEA; sensitivity of antisera was 73.9 ng/mL for SEA. Antisera had high specificity against SEA. It had no cross reaction with SEB, SEC1, BSA and OVA.
2 Preparation and analysis of monoclonal antibody against SEA-SEB
McAb was produced by fusing the spleen cells taken from mouse No.2 with SP2/0 myeloma cells after the mouse was immunized by multi-antigen (SEA and SEB). By screened using the method of an indirect ELISA and subcloned by limiting dilution assay, one hybridoma cell was obtaind, designated 3F2. It secreted stable antibodies and reacted with both SEA and SEB. The other hybridoma cell, designated 3C12, was obtained following the same procedure with mouse No.4 immunized by SEA.
Two monoclonal antibodies were obtained by injection of 3F2 and 3C12 hybridoma cells into BALB/c mice, respectively. To ananlyze and contrast characteristics between two antibodies, the McAb's titer, sensitivities, specificities, affinities, the chromosomal number of hybridoma and stability of antibody were determined by ELISA method, respectively. Results indicated that the titer of ascites from hybridoma 3F2 against SEA was 4×105, sensitivity was 133.2 ng/mL, affinity was 8.18×107 L/mol; the titer of 3F2 against SEB was 1×105, sensitivity was 82.5 ng/mL, affinity was 5.32×107 L/mol. It had no cross reaction with SEC1, BSA and OVA. The titer of ascites from hybridoma 3C12 against SEA was 2×105, sensitivity was 115.9 ng/mL, and affinity was 6.76×107 L/mol. It had no cross reaction with SEC1, BSA and OVA and its cross reaction with SEB was weak.
Both hybridoma 3F2 and 3C12 had chromosomal number of about 100 after chromosome analysis, and it is similar with the number of mixture of spleen cells and SP2/0 myeloma cells. This indicated that two hybridoma cells obtained were fusion of parent cells. Two hybridoma cells had stable antibody titer when they had passed down serial generations, frozen and thawn in three months, which idicated that they had a good stability.
3 Initial application of McAb 3F2 against SEA-SEB for SEs detecion in dairy products
Dairy products easily contaminated with SEs were selected for this study. Effect of detection SEs in food using McAb 3F2 obtained by simultaneous immunization was evaluated. (1) Dairy products uncontaminated with SEs were spiked with SEA or SEB, respectively. McAb 3F2 and 3C12 were used for detection SEs in these samples. Average recoveries of spiked samples were assayed by indirect competitive ELISA. McAb 3F2 had good recoveries for both SEA and SEB, average recoveries of spiked milk were from 88.3% to 103.5% for SEA and from 86.2% to 101.6% for SEB. Average recoveries of spiked milk were from 86.7% to 98.1% for SEA with McAb 3C12. (2) SEA, SEB and SEC1 were mixed with various amounts, spiked into milk samples, and then SEA and SEB were determined using McAb 3F2 by icELISA. It showed that both SEA and SEB could be determined without interfered by SEC1. Average recoveries of spiked milk were from 70% to 120% for both SEA and SEB. (3) Milk and yogurt samples uncontaminated by SEs were inoculated with SEA or SEB secreting strains in manner of single and simultaneous inoculation. After incubated 36～48 hours at 37℃, SEs were detected using McAb 3F2 by icELISA, both SEA and SEB could be detected.
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