The Protein Journal

, Volume 29, Issue 7, pp 475–480 | Cite as

Preparation of Immunoglobulin Y (IgY) Against Lipopolysaccharide using Gel Chromatography from the Yolks of Eggs Laid by Immunized Hens

  • Siyuan Ma
  • Yaping Zhang


The objective is to prevent and treat injuries caused by lipopolysaccharide (LPS) from gram negative bacteria in animals and humans, we produced antibodies against LPS from egg yolk. LPS from E. coli (O111:B4) mixed with Freund’s Adjuvant was used as the immunogen to immunize Roman hens. Immunized eggs were collected, and immunoglobulin Y (IgY) was purified using a water solution, salt precipitation and gel chromatography. The molecular weight and purity were determined by SDS–PAGE, the antibody titer by noncompetitive enzyme-linked immunosorbent assay (ELISA) and antibody activity against LPS by the mortality of mice intraperitoneally injected with LPS or LPS-IgY solutions. IgY against LPS showed two protein bands at 68 and 26 kDa on the gel; the antibody titer was almost 1:25,600. After incubation with LPS, IgY decreased the mortality of mice challenged with LPS. This study provided an efficient way to produce high-titer egg yolk antibodies, which could attenuate lethal effects of LPS, by immunizing hens. Furthermore, the LPS antibody was purified well using a water solution, salting-out and gel chromatography.


Purification Lipopolysaccharide Immunoglobulins Egg yolk Antibody 



Immunoglobulin Y; egg yolk antibody



E. coli

Escherichia coli


Hydrogen chloride


Sodium sulfate


Optical density


Phosphate-buffered saline



The authors thank the staff of the State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, for their assistance in caring for the animals, collecting eggs and providing technical services used in this study.


  1. 1.
    Akita EM, Nakai S (1993) J Immunol Methods 160:207–214CrossRefGoogle Scholar
  2. 2.
    Cross AS, Opal SM, Palardy JE, Drabick JJ, Warren HS, Huber C, Cook P, Bhattacharjee AK (2003) Vaccine 21:4576–4587CrossRefGoogle Scholar
  3. 3.
    Amaral JA, De Franco MT, Zapata-Quintanilla L, Carbonare SB (2008) Vet Res Commun 32:281–290CrossRefGoogle Scholar
  4. 4.
    Bhattacharjee AK, Opal SM, Palardy JE, Drabick JJ, Collins H, Taylor R, Cotton A, Cross AS (1994) J Infect Dis 70:622–629Google Scholar
  5. 5.
    Chalghoumi R, Thewis A, Beckers Y, Marcq C, Portetelle D, Schneider YJ (2009) Foodborne Pathog Dis 6:593–604CrossRefGoogle Scholar
  6. 6.
    Raetz C, Whitfield C (2002) Annu Rev Biochem 71:635–700CrossRefGoogle Scholar
  7. 7.
    de Almeida CMC, Da Silva CL, Couto HP, de Escocard RCM, da Rocha DG, de Paula Sentinelli L, Kipnis TL, da Silva WD (2008) Toxicon 52:293–301CrossRefGoogle Scholar
  8. 8.
    Derkx B, Witter J, Mccloskey R (1999) Clin Infect Dis 28:770–777CrossRefGoogle Scholar
  9. 9.
    Di Padova FE, Brade H, Barclay GR, Poxton IR, Liehl E, Schuetze E, Kocher HP, Ramsay G, Schreier MH, McClelland DB (1993) Infect Immun 61:3863–3872Google Scholar
  10. 10.
    Fomsgaard A, Baek L, Fomsgaard JS, Engquist A (1989) Scand J Infect Dis 21:697–708CrossRefGoogle Scholar
  11. 11.
    Yi-bin G, Hong-wei C, Liping C, Yong-ling L, Jiang Z, Guang-xia X (2007) Chin Jl Burns 23:104–107Google Scholar
  12. 12.
    Harkin DW, Barros D’Sa AA, Yassin MM, Hoper M, Halliday MI, Parks TG, Campbell FC (2001) J Vas Surg 33:840–846CrossRefGoogle Scholar
  13. 13.
    Amaral JA, De Franco MT, Zapata-Quintanilla L, Carbonare SB (2008) Vet Res Commun 32:281–290CrossRefGoogle Scholar
  14. 14.
    Krief A, Letesson JJ, Billen D (2002) Tetrahedron Lett 43:1843–1846CrossRefGoogle Scholar
  15. 15.
    Lachman E, Pitsoe SB, Gaffin SL (1984) Lancet 1:981–983CrossRefGoogle Scholar
  16. 16.
    Le Roy D, Di Padova F, Tees R, Lengacher S, Landmann R, Glauser MP, Calandra T, Heumann D (1999) J Immunol 162:7454–7460Google Scholar
  17. 17.
    Mayumi T, Takezawa J, Takahashi H, Kuwayama N, Fukuoka T, Shimizu K, Yamada K, Kondo S, Aono K (1999) Shock 11:82–86CrossRefGoogle Scholar
  18. 18.
    MT Madigan, JM Martinko, J Parker (2003) In: Brock biology of microorganisms. Pearson-Prentice Hall, Madrid, 1012 pp. ISBN 84-205-3679-2Google Scholar
  19. 19.
    Nomura S, Suzuki H, Masaoka T, Kurabayashi K, Ishii H, Kitajima M, Nomoto K, Hibi T (2005) Helicobacter 10:43–52CrossRefGoogle Scholar
  20. 20.
    van Langevelde P, Kwappenberg KMC, Groeneveld PHP, Mattie H, van Dissel JT (1998) Antimicrob Agents Chemother 42:739–743Google Scholar
  21. 21.
    Shi z, Zhang Y, Ma S (2009) Immunol J 25:337–340Google Scholar
  22. 22.
    Steven AM, James PT (2001) Biochemisty 40:5777–5785CrossRefGoogle Scholar
  23. 23.
    Suzuki H, Nomura S, Masaoka T, Goshima H, Kamata N, Kodama Y, Ishii H, Kitajima M, Nomoto K, Hibi T (2004) Aliment Pharmacol Ther 20(Suppl 1):185–192CrossRefGoogle Scholar
  24. 24.
    Tsuji M, Matsuda H, Miwa H, Miyazaki S (2003) J Antimicrob Chemother 51:353–359CrossRefGoogle Scholar
  25. 25.
    Lu Y, Liu J, Jin L, Li X, Zhen YH, Xue H, You J, Xu Y (2008) Fish Shellfish Immunol 25:604–610CrossRefGoogle Scholar
  26. 26.
    Zhen YH, Jin LJ, Li XY, Guo J, Li Z, Zhang BJ, Fang R, Xu YP (2009) Vet Microbiol 133:317–322CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined InjuryThe Third Military Medical UniversityShapingba District, ChongqingPeople’s Republic of China

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