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ABBREVITIONS 16
ABSTRACT 18
I. 서론 22
II. 재료 및 방법 30
1. 실험 재료 및 기기 30
1) 사용균주 및 배지 30
2) 시약 및 재료 30
3) 사용기기 31
2. 실험방법 32
1) 단클론성 항체의 생산 32
(1) 면역원 조제 32
(2) 면역 33
(3) 면역한 mouse 혈청의 역가 측정 33
(4) 단클론 항체 생성 hybridoma 세포주 개발 34
(5) 단클론 항체의 생산 및 정제 35
(6) 단클론 항체의 특이성 확인 36
2) 계란항체(immunoglobulin yolk, IgY)의 생산 37
(1) 항원의 면역 37
(2) 항혈청의 역가 측정 37
(3) 계란항체의 정제방법 및 역가 측정 37
(4) 계란항체의 특이성 확인 39
3) 효소면역 분석법의 확립 40
(1) 단클론성 항체를 이용한 indirect ELISA법 40
(2) 계란항체(IgY)를 이용한 indirect ELISA법 41
(3) 단클론성 항체 및 계란항체를 이용한 sandwich ELISA법 43
4) Immunochromatography(ICG)법 개발 45
(1) Protein G agarose를 이용한 항체 정제 45
(2) Colloidal gold의 제작 45
(3) Colloidal gold와 항체 합성 45
(4) ICG법의 최적화 46
(5) 교차 반응성 확인 48
(6) L. monocytogenes에 대한 ICG법의 검출한계 48
5) 면역자석입자분리(Immuno-magnetic separation) system의 확립 49
(1) 자석 입자(magnetic bead)와 항체 합성 및 확인 49
(2) 교차반응성 확인 49
(3) Immuno-magnetic bead(IMB) 첨가량 결정 50
(4) 회수율 확인 50
6) 식육제품 시료에의 응용 51
(1) 시료 선정 51
(2) 면역분석법 적용을 위한 시료 전처리 51
(3) 면역분석법에 의한 L. monocytogenes의 screening 52
(4) PCR법에 의한 확인검사 53
III. 결과 및 고찰 55
1. 단클론성 항체의 생산 및 특성 확인 55
1) 면역한 마우스 혈청의 역가 55
2) 단클론성 항체 생성 세포주 57
3) 단클론성 항체의 생산 및 정제 63
4) 단클론성 항체의 특이성 64
2. 계란항체의 생산 및 특성 확인 66
1) 항혈청의 역가 66
2) 계란항체의 정제방법 비교 67
3) 계란항체(IgY)의 역가 68
4) 계란항체의 특이성 70
3. 효소면역분석법의 확립 71
1) 단클론성 항체를 이용한 indirect ELISA법 71
2) 계란항체 를 이용한 indirect ELISA법 77
3) 단클론성 항체 및 계란항체를 이용한 sandwich ELISA법 82
4. Immunochromatography법을 이용한 test strip 개발 91
1) Protein G agarose를 이용한 항체 정제 91
2) Colloidal gold의 제작 92
3) Colloidal gold와 항체 합성 92
4) ICG법의 최적화 94
5) 교차반응성 97
6) ICG법의 검출한계 98
5. 확립된 면역 분석법의 비교분석 99
6. 면역자석입자분리(Immuno-magnetic separation) 시스템 개발 100
1) Immuno-magnetic bead(IMB) 합성 100
2) 교차반응성 100
3) IMB 첨가량 101
4) 회수율 102
7. 시료 적용 103
1) 면역 분석법 적용을 위한 시료 전처리 103
2) L. monocytogenes의 모니터링 107
IV. 적요 111
참고문헌 114
Table 1. Sample sources for monitoring of L. monocytogenes 51
Table 2. Primer sequences for PCR amplification of a hlyA gene fragment 53
Table 3. PCR conditions for amplification of a listeriolysin hlyA gene fragment 54
Table 4. Fusion and frequency of hybrids selected from mice immunized with HKLM 58
Table 5. Fusion and frequency of hybrids selected from mice immunized with FKLM 60
Table 6. Comparision of combinations of gold-antibody conjugates in the conjugate pad and antibodies in the test line 94
Table 7. Comparison of four immunoassay methods for the detection of L. monocytogenes 99
Table 8. Confirmation of the ability of immuno-magnetic bead to recover L. monocytogenes Cells 100
Table 9. Cross-reactivity of the 1MB for Listeria spp. and other bacteria 101
Table 10. Optimum addition volume of 1MB for recovering L. monocytogenes cells 101
Table 11. Recovery ratios for L. monacytogenes cells at different cell counts by IMB 102
Table 12. Comparison of immunoassays with a PCR method to detect L. monocytogenes 109
Fig. 1. The main steps of indirect ELISA determining anti-sera titer 34
Fig. 2. The schematic diagram of ICG designed for the detection of L. monocytogenes 47
Fig. 3. Titration of anti-sera from mice immunized with viable cells of L. monocytogenes 55
Fig. 4. Titration of anti-sera from mice immunized with crude cell surface proteins of L. monocytogenes 56
Fig. 5. Titration of anti-sera from mice immunized with heat killed L. monocytogenes(HKLM) 56
Fig. 6. Titration of anti-sera from mice immunized with formalin killed L. monocytogenes(FKLM) 57
Fig. 7. Titration of culture media from fused cells between myeloma cells and spleen cells isolated from mice immunized with HKLM by indirect ELISA 58
Fig. 8. Western blot results of culture media from fused cells between myeloma cells and spleen cells isolated from mice immunized with HKLM by indirect ELISA 59
Fig. 9. Characterization of antibodies produced from cloned hybridoma cells(HKLM-3H1-2B8 and HKLM-3H1-2E4) by western blot 60
Fig. 10. Titration of culture media from fused cells between myeloma cells and spleen cells isolated from mice immunized with FKLM by indirect ELISA 61
Fig. 11. Western blot Results of culture media of fused cells between myeloma cells and spleen cells isolated from mice immunized with FKLM by indirect ELISA 61
Fig. 12. Characterization of antibodies produced from cloned hybridoma cells(FKLM-3BI2-17, 19, 21 and 41) by western blot 62
Fig. 13. Titration of four different kinds of MAbs against L. monocytogenes by indirect ELISA 63
Fig. 14. Cross-reactivity patterns of four different kinds of MAbs against Listeria spp 64
Fig. 15. Cross-reactivity patterns of four different kinds of MAbs against other bacteria 65
Fig. 16. Titration of anti-sera from hens immunized with FKLM 66
Fig. 17. Comparison of four different purification methods for IgYs by SDS-PAGE 68
Fig. 18. Titration of IgYs purified from hens immunized with FKLM 69
Fig. 19. Titration of IgYs against L. monocytogenes by indirect ELISA 69
Fig. 20. Cross-reactivity patterns of IgY with Listeria spp. and other bacteria 70
Fig. 21. Comparison of PBS with carbonate buffer as a coating buffer on the efficiency of indirect ELISA using MAb 71
Fig. 22. Determination of the optimum coating temperature and time for indirect ELISA using MAb 72
Fig. 23. Selection of an optimum blocking reagent for indirect ELISA using MAb 73
Fig. 24. Determination of the optimum concentration of a blocking reagent for indirect ELISA using MAb 73
Fig. 25. Determination of the optimum blocking temperature and time for indirect ELISA using MAb 73
Fig. 26. Determination of the optimum dilution ratio of MAb for indirect ELISA 74
Fig. 27. Determination of the optimal MAb reaction temperature and time for indirect ELISA 74
Fig. 28. Cross-reactivities with Listeria spp. and other bacteria by indirect ELISA using MAb 75
Fig. 29. A standard curve for detection of L. monocytogenes by indirect ELISA based on MAb 76
Fig. 30. Comparison of PBS with carbonate buffer as a coating buffer for indirect ELISA using IgY 77
Fig. 31. Determination of the optimum coating temperature and time for indirect ELISA using IgY 78
Fig. 32. Selection of an optimum blocking reagent for indirect ELISA using IgY 79
Fig. 33. Determination of the optimum concentration of a blocking reagent for indirect ELISA using IgY 79
Fig. 34. Determination of the optimum blocking temperature and time for indirect ELISA using IgY 79
Fig. 35. Determination of the optimum dilution ratio of IgY for indirect ELISA 80
Fig. 36. Determination of the optimal IgY reaction temperature and time for indirect ELISA 80
Fig. 37. Cross-reactivities with Listeria spp. and other bacteria by indirect ELISA using IgY 81
Fig. 38. A standard curve for detection of L. monocytogenes by indirect ELISA using IgY 82
Fig. 39. Selection of a coating antibody for sandwich ELISA 83
Fig. 40. Selection of the optimal antibody concentration for sandwich ELISA 84
Fig. 41. Comparison of PBS with carbonate buffer as a coating buffer for sandwich ELISA 84
Fig. 42. Determination of the optimum coating temperature and time for sandwich ELISA 85
Fig. 43. Selection of the optimum blocking reagent for sandwich ELISA 86
Fig. 44. Determination of the optimum concentration of a blocking reagent for sandwich ELISA 86
Fig. 45. Determination of the optimum blocking temperature and time for sandwich ELISA 86
Fig. 46. Selection of a MAb for antibody reaction step for sandwich ELISA 87
Fig. 47. Determination of the optimum dilution ratio of MAb for sandwich ELISA 87
Fig. 48. Determination of the optimal reaction temperature and time for sandwich ELISA 88
Fig. 49. Cross-reactivities with Listeria spp. and other bacteria by sandwich ELISA 89
Fig. 50. A standard curve for detection of L. monocytogenes by sandwich ELISA 90
Fig. 51. SDS-PAGE patterns of FKL3B12-41 MAb purified by protein G agarose 91
Fig. 52. Photograph of colloidal gold observed by transmission electron microscope(×110,000) 92
Fig. 53. Determination of the amount of FKLM-3B12-41 MAb to stabilize gold particle 93
Fig. 54. Confirmation of FKLM-3B12-41 MAb-gold conjugate 93
Fig. 55. Determination of the amount of FKLM-3B12-41 MAb applied to test line 95
Fig. 56. Determination of the amount of FKLM-3B12-41 MAb-gold conjugate applied to conjugate pad 95
Fig. 57. Selection of type of membrane for immunochromatography strip 96
Fig. 58. Selection of working solution for sample preparation for ICG strip 97
Fig. 59. Cross-reactivities of a test strip with Listeria spp. and other bacteria 98
Fig. 60. Sensitivities of a immunochromatography strip for the detection of L. monocytogenes. NC: negative control 98
Fig. 61. Determination of the matrix effect and recovery yields for six samples spiked with L. monocytogenes by indirect ELISA 103
Fig. 62. Determination of the optimum incubation time for indirect ELISA 106
Fig. 63. PCR amplification results obtained from L. monocytogenes strains isolated from the pork 108
Fig. 64. PCR amplification results obtained from L. monocytogenes strains isolated from beef and fish 108
Fig. 65. PCR amplification results obtained from L. monocytogenes strains isolated from chicken 109
초록보기 더보기
The aims of this study were production of monoclonal antibodies(MAb) and immunoglobulin yolks(IgY) against L. monocytogenes, and establishment of immunoassays and immunomagnetic separation(IMS) systems for the rapid isolation of L. monocytogenes. Established immunoassays were tested for detection of L. monocytogenes from meat samples. PCR was used to confirm the results. The results obtained were as follows :
1. To obtain MAb against L. monocytogenes, four different immunogens such as viable cell(VC), crude cell surface protein(CCSP), heat killed L. monocytogenes(HKLM), and formalin killed L. monocytogenes(FKLM) were prepared and injected to mice. The mice immunized with HKLM or FKLM showed higher titers, and spleen cells from mice were fused with myeloma cells.
2. Two kinds of fused cells(HKLM-3H1 and FKLM-3B12) were obtained by cell fusion. After cloning step, hybridoma cell lines from HKLM-3H1 showed a low titer to L. monocytogenes. However, hybridoma cell lines from FKLM-3B12 showed a high titer to L. monocytogenes four hybridoma cell lines(FKLM-3B12-17, 19, 21, 41) producing specific antibodies were selected for antibody production. Four different kinds of antibodies were tested by indirect ELISA. FKLM-3B12-41 antibodies showed the high of sensitivity and specificity against L. monocytogenes. Therefore, FKLM-3B12-41 was selected for development of several immunoassays and a IMS system.
3. For the production of IgY, FKLM showing high efficiency as an immunogen was injected to hens. All eggs were collected and stored at 4℃. Egg yolk was purified by the method of water dilution & salt precipitation. Titer of IgY was investigated by indirect ELISA. Titer of IgY increased and preserved until 13 weeks after 2 weeks of initial immunization. In this study, IgY at 6 weeks after initial immunization was selected and used to develop indirect ELISA and sandwich ELISA because this IgY showed the highest titer.
4. The optimized conditions for an indirect ELISA using MAb were as follows : coating step(carbonate buffer, 37℃, 1 hr), blocking step (1% skim milk, 37℃, 1 hr), and antibody reaction step (1/5,000 dilution time, RT, 1hr). The assay showed a little cross-reactivity to Listeria spp. and S. aureus but had no cross-reactivity to other pathogenic bacteria. Detection limit of the indirect ELISA using MAb was 105 CFU/ml and it took 5 hrs. to complete the analysis.
5. The optimized conditions for an indirect ELISA using IgY were as follows : coating step(PBS, 37℃, 1hr), blocking step(0.5% skim milk, 37℃, 1 hr), and antibody reaction step (MAB 1/4,000 dilution, RT, 1 hr). The assay showed a little cross-reactivity to Listeria spp, S. aureus, and B. cereus but had no cross-reactivity to other pathogenic bacteria. Detection limit of the indirect ELISA using IgY was 108 CFU/ml and it took 5hrs. to complete the analysis.
6. The optimized conditions for a sandwich ELISA using MAb and IgY were as follows: coating step(IgY, PBS, 37℃, 1 hr), blocking step(0.5% skim milk, 37℃, 1 hr), and antibody reaction step(MAb 1/1,000 dilution, RT, 1 hr). The assay showed a little cross-reactivity to Listeria spp. and S. aureus but had no cross-reactivity to other pathogenic bacteria. Detection limit of a sandwich ELISA using MAb and IgY was 107 CFU/ml and it took 6hrs. to complete the analysis.
7. A test strip based on immunochromatography principle was developed for rapid detection of L. monocytogenes. For conjugation of colloidal gold and MAb, the minimum amount of antibody necessary for the stabilization of colloidal gold particles was determined. In our experiment, 7g of MAb was confirmed as the minimum amount necessary for the stabilization of colloidal gold. The optimal conditions for the test strip were determined as follows : 0.33㎎/ml MAb was treated in the test line on the membrane and 5μI of colloidal gold-MAb conjugate diluted by 4 times was sprayed on the conjugate pad. Nitrocellulose membrane(SHF 09004) and working solution(PSB) showed a clear red band at the test and the control line. The optimized test strip showed a little cross-reactivity to Listeria spp. but had no cross-reactivity to other pathogenic bacteria. The detection limit of this assay was 105 CFU/ml and it took 15 min. to complete the analysis.
8. A IMS system using a magnetic bead and MAb was developed for the rapid isolation of L. monocytogenes from samples and culture media. The effect on adding volume of immuno-magnetic bead(IMB) was determined. The optimal adding volume was estimated as 30μI for high isolation ratio of L. monocytogenes. The isolation ratio was investigated with different cell counts of L. monocytogenes(107~10¹ cell/ml). The highest isolation ratio, 54%, was observed at 10³ cells. Finally, cross-reactivity of IMS was investigated. IMS also showed a little cross-reactivity to Listeria spp. but did not show cross-reactivity to other pathogenic bacteria.
9. Six different samples were selected for the screening of L. monocytogenes. Before screening, a matrix effect of sample for immunoassays was determined. All samples showed high matrix effect on immunoassay. It was concluded that all samples could not be directly applied to immunoassays and preenrichment was needed. Therefore, 10 cells of L. monocytogenes were spiked to 10g of six different samples and spiked samples were cultured for 48hrs. at 37℃. During the cultivation, culture media at two hour intervals were tested by indirect ELISA. After 24hrs. of cultivation, all samples were shown positive by indirect ELISA.
10. For the screening of L. monocytogenes, 116 meat samples were collected from ]inju, Gyeongnam, Korea. All samples were cultured for 24 hrs. and analyzed by ICG test strip and indirect ELISA. 27 meat samples were shown positive by immunoassays and confirmed by PCR assay. Only 17 samples among 27 positive samples by immunoassays were identified positive for the presence of L. monocytogenes by PCR. The results by immunoassays were in a good agreement with the results by PCR. The immunoassays developed in this study can be applied for the preliminary screening of L. monocytogenes from a large size of samples.
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