The Korean Journal of Nuclear Medicine (대한핵의학회지)

The Korean Society of Nuclear Medicine (대한핵의학회)
권호 표지

Radioimmunoassay for Determination of Serum Macrophage Migration Inhibitory Factor

혈중 대식세포 유주 저지 인자 측정을 위한 방사면역측정법

  • Lee, Tae-Sup (Laboratory of Cyclotron Application, Korea Institute of Radiological and Medical Science (KIRAMS)) ;
  • Shin, Seok-Hwan (Department of Surgery, College of Medicine, Inha University) ;
  • Song, Jee-In (Department of Surgery, College of Medicine, Inha University) ;
  • Woo, Kwang-Sun (Laboratory of Cyclotron Application, Korea Institute of Radiological and Medical Science (KIRAMS)) ;
  • Chung, Wee-Sup (Department of Nuclear Medicine, Korea Institute of Radiological and Medical Science (KIRAMS)) ;
  • Choi, Chang-Woon (Department of Nuclear Medicine, Korea Institute of Radiological and Medical Science (KIRAMS)) ;
  • Lim, Sang-Moo (Laboratory of Cyclotron Application, Korea Institute of Radiological and Medical Science (KIRAMS))
  • 이태섭 (원자력의학원 싸이클로트론응용연구실) ;
  • 신석환 (인하대학교 외과학교실) ;
  • 송지인 (인하대학교 외과학교실) ;
  • 우광선 (원자력의학원 싸이클로트론응용연구실) ;
  • 정위섭 (원자력의학원 싸이클로트론응용연구실) ;
  • 최창운 (원자력의학원 핵의학과) ;
  • 임상무 (원자력의학원 핵의학과)
  • Published : 2004.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose: There has been a renewal of interest in Macrophage migration inhibitory factor (MIF), especially correlation in pathogenesis of sepsis by many infectious diseases and in regulation of host inflammatory and immune response. We developed immunoradiometric assay (IRMA) to determine serum human MIF concentration. Materials and Methods: The IRMA system utilizes solid phase bound monoclonal anti-recombinant human MIF (rhMIF) antibody as a capture antibody, biotinylated polyclonal anti-rhMIF antibody as a detector antibody. We applied with rhMIF that concentration of standard solutions increased from 0 ng/ml to 100 ng/ml. We used $^{125}I$-streptavidin (SA) as radiotracer to determination of rhMIF concentration. Streptavidin was labeled with $^{125}I$ by Chloramine-T method and $^{125}I$-SA was purified by ultracentrifugation. $^{125}I$-SA stability was evaluated by ITLC analysis at $4^{\circ}C$ and room temperatures until 60days. To validate IRMA system for MIF, we experimented intra-assay and inter-assay coefficients of variation, recovery test and dilution test. Results: Radiolabeling yield of $^{125}I$-SA was 87% and purified $^{125}I$-SA retained above 99% radiochemical purity. $^{125}I$-SA showed above 93% stability in $4^{\circ}C$ until 60days that it is good for immunoradiometric assay as radiotracer. Plotted standard dose response curve showed that increased concentration of rhMIF linearly correlated (R2=0.99) with bound radioactivity of $^{125}I$-SA. The highest intra- and inter-assay coefficients of variation were 5.5% and 7.6%, respectively. The average of recovery of MIF in samples was 102%. In dilution test, linear response curves were obtained (R2=0.97). Conclusion: Radioimmunoassay using $^{125}I$-SA as radiotracer thought to be useful for the determination of serum MIF concentration, and further, its data will be used to evaluate the correlation between clinical significance and serum MIF concentration in patients with various inflammatory diseases.

목적: 사람 대식세포 유주 저지 인자는 많은 감염성 질환에 의한 패혈증의 병인론과 숙주의 염증 및 면역 반응의 조절에 중요한 역할은 하는 것으로 알려져 있다. 본 연구에서는 사람의 혈중에서 대식세포 유주 저지 인자를 측정할 수 있는 방사면역측정법을 개발하고자 하였다. 대상 및 방법 : 사람 대식세포 유주저지 인자에 대한 단클론 항체를 포획항체로, 비오틴화된 다클론항체를 검출 항체로 사용하였다. 사람 대식세포 유주 저지인자를 검출하기 위하여 스트렙타비딘에 $^{125}I$를 방사성추적자로 사용하고 재조합 사람 대식세포 유주저지인자를 이용하여 표준투여 응답곡선을 작성하였다. 스트렙타비딘에 $^{125}I$의 표지는 Chloramine-T법을 사용하고, 분리정제는 한외여과법을 사용하였다. $^{125}I$-스트렙타비딘의 안정성을 60일까지 평가하였다. 표지수율과 안정성은 ITLC법을 사용하였다. 방사면역측정법의 유용성은 intra- 와 inter-assay의 변이계수 측정, 재현도 및 희석 실험 등을 시행하였다. 결과: $^{125}I$-스트렙타비딘의 표지수율은 88%이었으며, 분리 정제된 $^{125}I$-SA의 방사화학적 수율은 99%였다. $^{125}I$-스트렙타비딘는 60일까지 93%의 안정성을 나타내어 방사면역측정의 방사성추적자로 사용하는데 적합하였다. 작성된 표준투여 응답곡선에서 재조합 사람 대식세포 유주 저지 인자의 농도와 결합된 $^{125}I$-스트렙타비딘의 방사능 값은 높은 상관관계를 나타내었다($R^2=0.99$). 가장 높은 intra-와 inter-assay의 변이계수 간이 각각 5.5%와 7.6%로 나타났다. 시료 내에서 평균 recovery 측정값은 102%였다. 시료의 농도 희석에 따른 방사능의 측정치는 직선적인 상관관계를 나타내었다($R^2=0.97$). 결론: 대식세포 유주 저지 인자의 농도 측정을 위하여 방사성추적자로 $^{125}I$-스트렙타비딘를 이용한 방사면역측정법을 확립하였으며, 이 방법을 이용하여 다양한 염증성 질환을 가진 임상환자에서 대식세포 유주 저지 인자의 혈중 농도와 임상적 의의와의 상관관계를 규명하는데 이용될 수 있을 것으로 기대된다.

Keywords

References

  1. Bloom BR, Bennett B. Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science 1966;153:80-2 https://doi.org/10.1126/science.153.3731.80
  2. David JR. Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction. Proc Natl Acad Sci U S A 1966;56:72-7 https://doi.org/10.1073/pnas.56.1.72
  3. Weiser WY, Temple PA, Witek-Giannotti JS, Remold HG, Clark SC, David JR. Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor. Proc Natl Acad Sci U S A 1989;86:7522-6 https://doi.org/10.1073/pnas.86.19.7522
  4. Calandra T, Bernhagen J, Mitchell RA, Bucala R. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med 1994;179:1895-902 https://doi.org/10.1084/jem.179.6.1895
  5. Onodera S, Suzuki K, Matsuno T, Kaneda K, Takagi M, Nishihira J. Macrophage migration inhibitory factor induces phagocytosis of foreign particles by macrophages in autocrine and paracrine fashion. Immunology 1997;92:131-7 https://doi.org/10.1046/j.1365-2567.1997.00311.x
  6. Bacher M, Metz CN, Calandra T, Mayer K, Chesney J, Lohoff M, et al. An essential regulatory role for macrophage migration inhibitory factor in T-cell activation. Proc Natl Acad Sci U S A 1996;93:7849-54. https://doi.org/10.1073/pnas.93.15.7849
  7. Abe R, Peng T, Sailors J, Bucala R, Metz CN. Regulation of the CTL response by macrophage migration inhibitory factor. J Immunol 2001;166:747-53 https://doi.org/10.4049/jimmunol.166.2.747
  8. Calandra T, Bernhagen J, Metz CN, Spiegel LA, Bacher M, Donnelly T, et al. MIF as a glucocorticoid-induced modulator of cytokine production. Nature 1995;377:68-71 https://doi.org/10.1038/377068a0
  9. Meinhardt A, Bacher M, McFarlane JR, Metz CN, Seitz J, Hedger MP, et al. Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function. Endocrinology 1996;137:5090-5 https://doi.org/10.1210/en.137.11.5090
  10. Waeber G, Calandra T, Roduit R, Haefliger JA, Bonny C, Thompson N, et al. Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor. Proc Natl Acad Sci U S A 1997;94:4782-7 https://doi.org/10.1073/pnas.94.9.4782
  11. Santos L, Hall P, Metz C, Bucala R, Morand EF. Role of macrophage migration inhibitory factor (MIF) in murine antigen-induced arthritis: interaction with glucocorticoids. Clin Exp Immunol 2001;123:309-14 https://doi.org/10.1046/j.1365-2249.2001.01423.x
  12. Bernhagen J, Calandra T, Mitchell RA, Martin SB, Tracey KJ, Voelter W, et al. MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature 1993;365:756-9 https://doi.org/10.1038/365756a0
  13. Calandra T, Spiegel LA, Metz CN, Bucala R. Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of Gram-positive bacteria. Proc Natl Acad Sci U S A 1998;95:11383-8 https://doi.org/10.1073/pnas.95.19.11383
  14. Bozza M, Satoskar AR, Lin G, Lu B, Humbles AA, Gerard C, et al. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis. J Exp Med 1999;189:341-6 https://doi.org/10.1084/jem.189.2.341
  15. Mikulowska A, Metz CN, Bucala R, Holmdahl R. Macrophage migration inhibitory factor is involved in the pathogenesis of collagen type II-induced arthritis in mice. J Immunol 1997;158:5514-7
  16. Leech M, Metz C, Hall P, Hutchinson P, Gianis K, Smith M, et al. Macrophage migration inhibitory factor in rheumatoid arthritis: evidence of proinflammatory function and regulation by glucocorticoids. Arthritis Rheum 1999;42:1601-8 https://doi.org/10.1002/1529-0131(199908)42:8<1601::AID-ANR6>3.0.CO;2-B
  17. Lan HY, Yang N, Brown FG, Isbel NM, Nikolic-Paterson DJ, Mu W, et al. Macrophage migration inhibitory factor expression in human renal allograft rejection. Transplantation 1998;66:1465-71 https://doi.org/10.1097/00007890-199812150-00009
  18. Lan HY, Bacher M, Yang N, Mu W, Nikolic-Paterson DJ, Metz C, et al. The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat. J Exp Med 1997;185:1455-65 https://doi.org/10.1084/jem.185.8.1455
  19. Donnelly SC, Haslett C, Reid PT, Grant IS, Wallace WA, Metz CN, et al. Regulatory role for macrophage migration inhibitory factor in acute respiratory distress syndrome. Nat Med 1997;3:320-3. https://doi.org/10.1038/nm0397-320
  20. Takahashi N, Nishihira J, Sato Y, Kondo M, Ogawa H, Ohshima T, et al. Involvement of macrophage migration inhibitory factor (MIF) in the mechanism of tumor cell growth. Mol Med 1998;4:707-14
  21. Chesney J, Metz C, Bacher M, Peng T, Meinhardt A, Bucala R. An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of a murine lymphoma. Mol Med 1999; 5:181-91
  22. Hudson JD, Shoaibi MA, Maestro R, Carnero A, Hannon GJ, Beach DH. A proinflammatory cytokine inhibits p53 tumor suppressor activity. J Exp Med 1999;190:1375-82 https://doi.org/10.1084/jem.190.10.1375
  23. Ogawa H, Nishihira J, Sato Y, Kondo M, Takahashi N, Oshima T, et al. An antibody for macrophage migration inhibitory factor suppresses tumour growth and inhibits tumour-associated angiogenesis. Cytokine 2000;12:309-14 https://doi.org/10.1006/cyto.1999.0562
  24. Calandra T, Echtenacher B, Roy DL, Pugin J, Metz CN, Hultner L, et al, Mannel D, Bucala R, Glauser MP. Protection from septic shock by neutralization of macrophage migration inhibitory factor. Nat Med 2000;6:164-70 https://doi.org/10.1038/72262
  25. Mizue Y, Nishihira J, Miyazaki T, Fujiwara S, Chida M, Nakamura K, et al. Quantitation of macrophage migration inhibitory factor (MIF) using the one-step sandwich enzyme immunosorbent assay: elevated serum MIF concentrations in patients with autoimmune diseases and identification of MIF in erythrocytes. Int J Mol Med 2000;5:397-403