金黄色葡萄球菌B型肠毒素单克隆抗体的制备及初步应用研究
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摘要
目的 制备金黄色葡萄球菌B型肠毒素(staphylococcus aureusenterotoxin B, SEB)的单克隆抗体(Monoclonal antibody, McAb)，建立检测金葡菌B型肠毒素的ELISA检测方法，并进行初步的临床应用。
方法 以天然纯化的SEB免疫BALB／C小鼠，采用细胞杂交瘤技术，筛选出抗SEB的McAb，在此基础上建立双抗体夹心ELISA方法，用标准菌株和临床分离株进行评价，并初步应用于烧伤病人血清中SEB的检测。
结果 制备了抗SEB-McAb共六株，其中1C_8、1A_5、1D_1、3D_6分泌的McAb类型为Ig G1，2C_(11)、3A_9分泌的McAb类型为Ig G2a。6株中有4株仅与SEB发生反应，有两株与SEC_1有弱交叉反应，与大肠埃希菌、粪肠球菌、绿脓假单胞菌、腐生葡萄球菌均不发生反应。同时制备了抗SEB多克隆抗体1株。SEB-McAb诱导小鼠腹水纯化后效价为1：12800-1：25600，抗体亲和力常数为5×10~7～2×10~9。Western-blot实验显示出一条特异性条带，位于分子量28.5KDa处，证明了其特异性。用特异McAb建立了双抗体夹心ELISA法检测SEB，其敏感性高，达0.078μg/L，高于目前国内报道的水平。特异性强，重复性和稳定性均较好。双抗体夹心ELISA方法检测临床分离金葡菌肠毒素结果显示，耐甲氧西林金葡菌(MRSA)中肠毒素的总带毒率为93.9％，其中B型肠毒素带毒率最高，占检出的53.3％，其次为C型(28.5％)。甲氧西林敏感金葡菌的带毒率仅为28.6％，两者之间差异有统计学意义。重度烧伤病人血清中SEB水平明显高于对照组及轻度烧伤组，差异有统计学意义。
结论 制备了抗SEB-McAb，其效价较高，亲和力较强，特异性强。在此基础上建立双抗体夹心ELISA法检测SEB，灵敏性达0.078μg/L，特异性和重复性均较好，可用于临床分离菌株SEB的检测以及检测烧伤病人血清中的SEB。本方法为临床金葡菌感染的诊断和分型，为阐明SEB的临床意义提供了方法。
Objective Monoclonal antibodies were prepared according to the protocol of hybridoma technique. It was aimed to investigate the prevalence of Staphylococcus aureus enterotoxins isolated from the hospital infections strains and SEB in Burn infections groups using ELISA which was newly developed by McAbs .
Methods BALB/C Mice were immunized with purified Staphylococcus aureus enterotoxinB and McAbs against SEB were obtained according to hybridoma technique, Subsequently, A sandwich ELISA technique was developed and used to detect Staphylococcus aureus standard strain and hospital infections strains respectively, Furthermore to investigate the prevalence of Staphylococcus aureus enterotoxins and SEB in Burn infection groups.
Results McAbs against SEB were obtained successfully,The titers of McAbs were between 1:12800 ~ 1:25600 in the pruified ascites ,The subclasses of these McAbs were IgG1 except IgG2(two strains)and the main immunological properties of these McAbs were Characterized, It showed that these McAbs had an identical epitopic specificity except two strains (3D6 and lDl).Cross-reactivity experiments showed that they had no reactivity with SEA, Escherichia coli Pseudomonas aeruginosa, Staphylococcus saprophticus and
Streptococcus intestinalis. but had two strains cross-reactivity with SEC1 and one strain (polyclonal antibody).subsequenctly ,A11 McAbs recognized a 28.5KDa protein which was definded as SEB by Western-blot analysis. These data indicated that high titer of McAbs against SEB with high specificities and high stability were successfully established in this study.They also indicated these McAbs with high sensitivity (0.078 u g/L).
The newly developed double sandwich ELISA could be used to detect SEB .These results showed the prevalence of SE in MRSA were
significant(93.9%)compared with the MSSA group(28.6%).The prevalence of SE in SEB group(53.3%)were higher than SEC1 group(28.5%)and SEA(12.2%)group. Datas showed that SEB levels after burn followed by Staphylococcus aureus infection were significantly higher than those of normal controls and slight burn control group(p<0.01).
Conclusions McAbs against SEB were successfully obtained. Subsequently ,A sandwich ELISA technique was developed and used to detect SEB .Datas showed this methods with high specificity n stability and sensitivity(0.078 U g/L) and could be used to detect SEB in hospital infection strains and in burn infection groups which might play an important role in the early diagnosis and cure of Staphylococcus aureus infection.
方法 以天然纯化的SEB免疫BALB／C小鼠，采用细胞杂交瘤技术，筛选出抗SEB的McAb，在此基础上建立双抗体夹心ELISA方法，用标准菌株和临床分离株进行评价，并初步应用于烧伤病人血清中SEB的检测。
结果 制备了抗SEB-McAb共六株，其中1C_8、1A_5、1D_1、3D_6分泌的McAb类型为Ig G1，2C_(11)、3A_9分泌的McAb类型为Ig G2a。6株中有4株仅与SEB发生反应，有两株与SEC_1有弱交叉反应，与大肠埃希菌、粪肠球菌、绿脓假单胞菌、腐生葡萄球菌均不发生反应。同时制备了抗SEB多克隆抗体1株。SEB-McAb诱导小鼠腹水纯化后效价为1：12800-1：25600，抗体亲和力常数为5×10~7～2×10~9。Western-blot实验显示出一条特异性条带，位于分子量28.5KDa处，证明了其特异性。用特异McAb建立了双抗体夹心ELISA法检测SEB，其敏感性高，达0.078μg/L，高于目前国内报道的水平。特异性强，重复性和稳定性均较好。双抗体夹心ELISA方法检测临床分离金葡菌肠毒素结果显示，耐甲氧西林金葡菌(MRSA)中肠毒素的总带毒率为93.9％，其中B型肠毒素带毒率最高，占检出的53.3％，其次为C型(28.5％)。甲氧西林敏感金葡菌的带毒率仅为28.6％，两者之间差异有统计学意义。重度烧伤病人血清中SEB水平明显高于对照组及轻度烧伤组，差异有统计学意义。
结论 制备了抗SEB-McAb，其效价较高，亲和力较强，特异性强。在此基础上建立双抗体夹心ELISA法检测SEB，灵敏性达0.078μg/L，特异性和重复性均较好，可用于临床分离菌株SEB的检测以及检测烧伤病人血清中的SEB。本方法为临床金葡菌感染的诊断和分型，为阐明SEB的临床意义提供了方法。
Objective Monoclonal antibodies were prepared according to the protocol of hybridoma technique. It was aimed to investigate the prevalence of Staphylococcus aureus enterotoxins isolated from the hospital infections strains and SEB in Burn infections groups using ELISA which was newly developed by McAbs .
Methods BALB/C Mice were immunized with purified Staphylococcus aureus enterotoxinB and McAbs against SEB were obtained according to hybridoma technique, Subsequently, A sandwich ELISA technique was developed and used to detect Staphylococcus aureus standard strain and hospital infections strains respectively, Furthermore to investigate the prevalence of Staphylococcus aureus enterotoxins and SEB in Burn infection groups.
Results McAbs against SEB were obtained successfully,The titers of McAbs were between 1:12800 ~ 1:25600 in the pruified ascites ,The subclasses of these McAbs were IgG1 except IgG2(two strains)and the main immunological properties of these McAbs were Characterized, It showed that these McAbs had an identical epitopic specificity except two strains (3D6 and lDl).Cross-reactivity experiments showed that they had no reactivity with SEA, Escherichia coli Pseudomonas aeruginosa, Staphylococcus saprophticus and
Streptococcus intestinalis. but had two strains cross-reactivity with SEC1 and one strain (polyclonal antibody).subsequenctly ,A11 McAbs recognized a 28.5KDa protein which was definded as SEB by Western-blot analysis. These data indicated that high titer of McAbs against SEB with high specificities and high stability were successfully established in this study.They also indicated these McAbs with high sensitivity (0.078 u g/L).
The newly developed double sandwich ELISA could be used to detect SEB .These results showed the prevalence of SE in MRSA were
significant(93.9%)compared with the MSSA group(28.6%).The prevalence of SE in SEB group(53.3%)were higher than SEC1 group(28.5%)and SEA(12.2%)group. Datas showed that SEB levels after burn followed by Staphylococcus aureus infection were significantly higher than those of normal controls and slight burn control group(p<0.01).
Conclusions McAbs against SEB were successfully obtained. Subsequently ,A sandwich ELISA technique was developed and used to detect SEB .Datas showed this methods with high specificity n stability and sensitivity(0.078 U g/L) and could be used to detect SEB in hospital infection strains and in burn infection groups which might play an important role in the early diagnosis and cure of Staphylococcus aureus infection.
引文
1 Bergdoll,MS, M.J Surgalla, G.M.Dack, et al. Staphylococcus enterotoxin:identification of specific precipitating antibody with enterotoxin neutralizing property.J Immunol, 1959, 83:334-338.
2 Casman ,EP Further serological studies of staphylococcal enterotoxin. J Bacteriol, 1960,79:849-856.
3 Bergdoll, M.S, CR, Borja, R.M.Arena, et al. Identification of a enterotoxin as enterotoxin C. J Bacteriol, 1967,90:1481-1485.
4 Avena,RM and MS.Bergdoll. Parification and some physicoclical properties of enterotoxin C. J Biochistry, 1967,6:1474-1480.
5 Bergdoll, MS. Enterotoxins P.559-598 InC. Adlam and C.S EasMon(ed.),staphylococcal infections,vol2 Acade Press ,Inc, London.
6 CasMan, EPR,W.Bennett, A.E.Dorsey, JA.Issa,et al. Identification of a fourth staphylococcal enterotoxin, enterotoxinD.J Bacteriol, 1967,94:1875-1882.
7 Bergdoll,MS,CR.Borja,RN Robbins, KFWeiss,et al. Identification of enterotoxin E. Infect .Immun. 1971,4:593-595.
8 Borja, CR,EFanning, IY.Huang, MS.Bergdoll,et al. Purification and some physicocherncial properties of staphylococall enterotoxin F. J Biol.Chen. 1972,247:2456-2463.
9 Munson, SH,and MJ.Betley.1991.Partial characterization of a new staphyloccal enterotoxin gene,abstr. β-36,P-31 In Abstracts of the 91st General Meeting of the AMerican Society for Microhioligy 1991 ,American Society for Microbiology,Washington, D.C.
10 Yi Cheng Su and AMYC,lee Wong,et al Indentification and Environmental. Microbiology.Apr. 1995,P: 1438-1443.
11 Sibyl,H.Munson,MaryT,et al. Indentification and Characterization of staphyloccal Enterotoxin Types I from staphylococcus caureus.J Biol Chen, 1998,27:1234-1242.
12 俞红，钱利生，熊思东等．葡萄球菌肠毒素β突复所的纯化及其活性，
复旦学报，2001，5，28(3)：187-190．
13 Wooster R, Bignell G,Lancaster J,et al. Nature, 1995,278:789.
14 Robbins RMet Detecting the enterotoxigenicity of staphylococcus aureus strains,Appl Microbiol, 1974,28:946.
15 Miller BA.Detection of staphylococcal cells containing protein A as immunoadsorbent. Appl Environ Microbiol, 1978,36:421.
16 Saunders GC.Double-antibody solid-phase enzyme immunoassay for detection of staphylococcal enterotoxinB. Appl Environ Microbiol., 1977,34:518.
17 Freed RC.Enzgme-Linked immunosorbent assay for detection of staphylococcal enterotoxins in foods, Appl Environ Microbiol, 1982,44:1349.
18 Fey.H, Comparative evaluation of different enzyme linked immunosorbont assay systems for the detection of staphylococcal enterotoxins A、B、C and D.J Clin Microbiol ,1984,19:34.
19 Hall JM,Lee MK,NewMan B,et al.Science, 1990,250:1684-1689.
20 Bxown MA,Xu CF,Nicolai H,et al.Oncogen ,1996,12:2507.
21 Ewalds. Tweten,Noskova.J Food Microbiol, 1987,4:207.
22 董邦全、李恩善、刘苏燕等．McAb-ELISA夹心法检测葡萄球菌B型肠毒素第四军医大学学报，1991，12(4)：285-287．
23 李红云、姚咏明、施志国等．双单抗夹心法检测血浆及组织中金葡菌肠毒素B．中华医院感染学杂志，2002，12(9)：651-657．
24 NoterMans S,D.Carpenter, and G.Silverman.J bio, 1988,54:531.
25 MaxaM AM,Davis,J.Angel.J immtmol methods, 1980,65:499.
26 Neill R,Johnson, H.P.Kauffman.J Clin Microbiol, 1990,28:1514.
27 SchMitz FJ,Steierth,Hofmann B,et al.J Med Micobiol,1988,47:335-340.
28 MANISHA MEHROTRA,EEHVA WANG and WENDYM,Johnson,et al.Multiplex PCR for detection of Genes for staphylococcus auteus, J Clinical Microbiology,2000,3:1032-1035.
29 郭胜清、杨宏、李云等．卫生研究．生物传感器技术的进展．卫生研究,27(增刊)：110-113．
30 HarteveldJCN,NiensinhuizenMS,WilsREJ,etal.Biosens Bioelection,1997,12:661-667.
31 Chance TD.Br J BioMed Su,1996,53:284-289.
32 ValRirs GE, Barton R. Immuno concentration-a new format for solid phase immunoassays.ClinChem, 1985,31:1427-1431.
33 Spielberg F,Kabeya CM,Ryder.Field testing and lomparative evaluation of rapid,visuany read sereening assay for antibody to human immunodeficiency vinis,lsncet, 1989,1:590-594.
34 宋农、李君文、王新为，等．胶体金免疫层析法用于产肠毒素葡萄球菌快速检测的研究．中华检验医学杂志，2003，26(7)：443-444．
35 金伯泉主编．细胞和分子免疫学实验技术，第四军医大学出版社，2002，15-16．
36 Beatty JD,et al,J Immunol Methods 1987,100:173.
37 王越剑、王建武、刘庆良，等．McAb亲和常数的简易测定，上海免疫学杂志，1991，11(2)：110．
38 Alsenz J,Loos M.A rapid and simple ELISA for the determination of duplicate McAbs and its application to the study of Ci-INH: J Immunol Methods ,1988,109(1):75-84.
39 梁国栋主编，最新分子生物学实验技术．北京：科学出版社，2001．
40 SaMbrook J,Fritsch EF,Maniatis T，著，分子克隆实验指南．金冬雁、黎孟枫译，第二版，北京：科学出版社，1992．
41 Kohler G, Milstein C. Contionus cultures of fusedcells secreting antibody of predefined specifity.Nature, 1975,256:495.
42 杨唐斌、曲丽娜McAb的研究进展．J航天医学与医学工程，2002，12，15(6)：460-464．
43 李影林主编，中华医学检验全书北京：人民卫生出版社，1996，1152．
44 Hahn IF,Pickenhahn P,lenz W,et al. A BA-ELISA for detection of staphylococcal enterotoxin A and B.J Immunol Methods 1986;92(1):25-29.
45 BoneRC gram positive organisms,Arch Intern Med, 1994,154:26-34.
46 姚咏明、盛志勇、李红云，等．重视金葡菌脓毒症的研究．中华烧伤杂志，2000；16：75-77．
47 许伟石、孙珍、王宏，等．烧伤中心细菌耐药性分析．中华整形烧伤外科杂志，1998，14：199-202．
48 李红去，姚咏明，施志国，等．金葡菌肠毒素BMcAb对烫伤脓毒症大鼠急性肺损伤的影响．中华实验外科杂志，2001，18(3)：228-230．
49 雷祚荣主编，葡萄球菌毒素和葡萄球菌毒素病．北京：中国科学技术出版社．1992，27．
50 雷祚荣主编，葡萄球菌毒素和葡萄球菌毒素病．北京：中国科学技术出版社．1992，20-21．
51 施志国，张延霞，刘伟等金葡菌肠毒素和中毒性休克毒素的分析．中华医院感染学杂志，1994,4：69-71．
2 Casman ,EP Further serological studies of staphylococcal enterotoxin. J Bacteriol, 1960,79:849-856.
3 Bergdoll, M.S, CR, Borja, R.M.Arena, et al. Identification of a enterotoxin as enterotoxin C. J Bacteriol, 1967,90:1481-1485.
4 Avena,RM and MS.Bergdoll. Parification and some physicoclical properties of enterotoxin C. J Biochistry, 1967,6:1474-1480.
5 Bergdoll, MS. Enterotoxins P.559-598 InC. Adlam and C.S EasMon(ed.),staphylococcal infections,vol2 Acade Press ,Inc, London.
6 CasMan, EPR,W.Bennett, A.E.Dorsey, JA.Issa,et al. Identification of a fourth staphylococcal enterotoxin, enterotoxinD.J Bacteriol, 1967,94:1875-1882.
7 Bergdoll,MS,CR.Borja,RN Robbins, KFWeiss,et al. Identification of enterotoxin E. Infect .Immun. 1971,4:593-595.
8 Borja, CR,EFanning, IY.Huang, MS.Bergdoll,et al. Purification and some physicocherncial properties of staphylococall enterotoxin F. J Biol.Chen. 1972,247:2456-2463.
9 Munson, SH,and MJ.Betley.1991.Partial characterization of a new staphyloccal enterotoxin gene,abstr. β-36,P-31 In Abstracts of the 91st General Meeting of the AMerican Society for Microhioligy 1991 ,American Society for Microbiology,Washington, D.C.
10 Yi Cheng Su and AMYC,lee Wong,et al Indentification and Environmental. Microbiology.Apr. 1995,P: 1438-1443.
11 Sibyl,H.Munson,MaryT,et al. Indentification and Characterization of staphyloccal Enterotoxin Types I from staphylococcus caureus.J Biol Chen, 1998,27:1234-1242.
12 俞红，钱利生，熊思东等．葡萄球菌肠毒素β突复所的纯化及其活性，
复旦学报，2001，5，28(3)：187-190．
13 Wooster R, Bignell G,Lancaster J,et al. Nature, 1995,278:789.
14 Robbins RMet Detecting the enterotoxigenicity of staphylococcus aureus strains,Appl Microbiol, 1974,28:946.
15 Miller BA.Detection of staphylococcal cells containing protein A as immunoadsorbent. Appl Environ Microbiol, 1978,36:421.
16 Saunders GC.Double-antibody solid-phase enzyme immunoassay for detection of staphylococcal enterotoxinB. Appl Environ Microbiol., 1977,34:518.
17 Freed RC.Enzgme-Linked immunosorbent assay for detection of staphylococcal enterotoxins in foods, Appl Environ Microbiol, 1982,44:1349.
18 Fey.H, Comparative evaluation of different enzyme linked immunosorbont assay systems for the detection of staphylococcal enterotoxins A、B、C and D.J Clin Microbiol ,1984,19:34.
19 Hall JM,Lee MK,NewMan B,et al.Science, 1990,250:1684-1689.
20 Bxown MA,Xu CF,Nicolai H,et al.Oncogen ,1996,12:2507.
21 Ewalds. Tweten,Noskova.J Food Microbiol, 1987,4:207.
22 董邦全、李恩善、刘苏燕等．McAb-ELISA夹心法检测葡萄球菌B型肠毒素第四军医大学学报，1991，12(4)：285-287．
23 李红云、姚咏明、施志国等．双单抗夹心法检测血浆及组织中金葡菌肠毒素B．中华医院感染学杂志，2002，12(9)：651-657．
24 NoterMans S,D.Carpenter, and G.Silverman.J bio, 1988,54:531.
25 MaxaM AM,Davis,J.Angel.J immtmol methods, 1980,65:499.
26 Neill R,Johnson, H.P.Kauffman.J Clin Microbiol, 1990,28:1514.
27 SchMitz FJ,Steierth,Hofmann B,et al.J Med Micobiol,1988,47:335-340.
28 MANISHA MEHROTRA,EEHVA WANG and WENDYM,Johnson,et al.Multiplex PCR for detection of Genes for staphylococcus auteus, J Clinical Microbiology,2000,3:1032-1035.
29 郭胜清、杨宏、李云等．卫生研究．生物传感器技术的进展．卫生研究,27(增刊)：110-113．
30 HarteveldJCN,NiensinhuizenMS,WilsREJ,etal.Biosens Bioelection,1997,12:661-667.
31 Chance TD.Br J BioMed Su,1996,53:284-289.
32 ValRirs GE, Barton R. Immuno concentration-a new format for solid phase immunoassays.ClinChem, 1985,31:1427-1431.
33 Spielberg F,Kabeya CM,Ryder.Field testing and lomparative evaluation of rapid,visuany read sereening assay for antibody to human immunodeficiency vinis,lsncet, 1989,1:590-594.
34 宋农、李君文、王新为，等．胶体金免疫层析法用于产肠毒素葡萄球菌快速检测的研究．中华检验医学杂志，2003，26(7)：443-444．
35 金伯泉主编．细胞和分子免疫学实验技术，第四军医大学出版社，2002，15-16．
36 Beatty JD,et al,J Immunol Methods 1987,100:173.
37 王越剑、王建武、刘庆良，等．McAb亲和常数的简易测定，上海免疫学杂志，1991，11(2)：110．
38 Alsenz J,Loos M.A rapid and simple ELISA for the determination of duplicate McAbs and its application to the study of Ci-INH: J Immunol Methods ,1988,109(1):75-84.
39 梁国栋主编，最新分子生物学实验技术．北京：科学出版社，2001．
40 SaMbrook J,Fritsch EF,Maniatis T，著，分子克隆实验指南．金冬雁、黎孟枫译，第二版，北京：科学出版社，1992．
41 Kohler G, Milstein C. Contionus cultures of fusedcells secreting antibody of predefined specifity.Nature, 1975,256:495.
42 杨唐斌、曲丽娜McAb的研究进展．J航天医学与医学工程，2002，12，15(6)：460-464．
43 李影林主编，中华医学检验全书北京：人民卫生出版社，1996，1152．
44 Hahn IF,Pickenhahn P,lenz W,et al. A BA-ELISA for detection of staphylococcal enterotoxin A and B.J Immunol Methods 1986;92(1):25-29.
45 BoneRC gram positive organisms,Arch Intern Med, 1994,154:26-34.
46 姚咏明、盛志勇、李红云，等．重视金葡菌脓毒症的研究．中华烧伤杂志，2000；16：75-77．
47 许伟石、孙珍、王宏，等．烧伤中心细菌耐药性分析．中华整形烧伤外科杂志，1998，14：199-202．
48 李红去，姚咏明，施志国，等．金葡菌肠毒素BMcAb对烫伤脓毒症大鼠急性肺损伤的影响．中华实验外科杂志，2001，18(3)：228-230．
49 雷祚荣主编，葡萄球菌毒素和葡萄球菌毒素病．北京：中国科学技术出版社．1992，27．
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