The Reporter Antigen Popliteal Lymph Node Assay

Stefan Nierkens1, Raymond Pieters1

1 Institute for Risk Assessment Sciences, Utrecht University, Utrecht
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.12
DOI:  10.1002/0471140856.tx1812s29
Online Posting Date:  September, 2006
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Many chemicals, including drugs and environmental pollutants, may have the intrinsic capacity to stimulate or dysregulate immune responses. These responses may create considerable problems for exposed subjects in terms of development of autoimmunity or hypersensitivity reactions. The popliteal lymph node assay (PLNA) provides a suitable tool to assess the immunostimulating potential of chemicals and might be a potential candidate as a screening tool in immunotoxicological hazard identification. The use of so‐called reporter antigens (RA) in this assay additionally enables differentiation between immunosensitizing (sensitizers), immunostimulating (irritants), and innocent chemicals. In the RA‐PLNA, the compound of interest is injected into the hind footpad together with a nonsensitizing dose of the RA. After 6 to 8 days, RA‐specific responses are monitored in the draining PLN by measuring RA‐specific antibody formation, cytokine secretion, and shifts in immune cell numbers. Hence, this simple and straightforward assay provides immunologically relevant information about the immunomodulating properties of a chemical.

Keywords: popliteal lymph node assay; immunostimulation; sensitization; hypersensitivity; allergy; autoimmunity; predictive screening assay

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Female mice (weighing ∼20 g), 6 to 8 weeks old at time of arrival
  • Standard laboratory mouse food pellets
  • Test compound(s) and vehicle(s)
  • T‐dependent reporter antigen (e.g., TNP‐OVA) and/or T‐independent reporter antigen [e.g. TNP‐Ficoll: available from Biosearch Technologies (; prepare solutions according to manufacturer's instructions, test for immunogenicity in PLNA against positive control; store in aliquots; Inman, ]
  • 70% ethanol
  • Solid‐bottom mouse cages with wood‐chip bedding
  • 1‐ml syringes
  • 25‐G, 5/8‐in. or 26‐G, 1/2‐in. needles
  • Dissecting equipment: forceps and dissecting scissors
  • Additional reagents and equipment for footpad injection of the mouse (Donovan and Brown, ) and euthanasia of the mouse (Donovan and Brown, )
NOTE: All reagents should be screened for endotoxin.
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Literature Cited

Literature Cited
   Albers, R., Broeders, A., van der Pijl, A., Seinen, W., and Pieters, R. 1997. The use of reporter antigens in the popliteal lymph node assay to assess immunomodulation by chemicals. Toxicol. Appl. Pharmacol. 143:102‐109.
   Bloksma, N., Kubicka‐Muranyi, M., Schuppe, H.C., Gleichmann, E., and Gleichmann, H. 1995. Predictive immunotoxicological test systems: Suitability of the popliteal lymph node assay in mice and rats. Crit. Rev. Toxicol. 25:369‐396.
   Donovan, J. and Brown, P. 2006a. Parenteral injections. In Current Protocols in Immunology (J.E. Coligan, B.E. Bierer, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.6.1‐1.6.9. John Wiley & Sons, Hoboken, N.J.
   Donovan, J. and Brown, P. 2006b. Euthanasia. In Current Protocols in Immunology (J.E. Coligan, B.E. Bierer, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.8.1‐1.8.4. John Wiley & Sons, Hoboken, N.J.
   Gutting, B.W., Schomaker, S.J., Kaplan, A.H., and Amacher, D.E. 1999. A comparison of the direct and reporter antigen popliteal lymph node assay for the detection of immunomodulation by low molecular weight compounds. Toxicol. Sci. 51:71‐79.
   Gutting, B.W., Updyke, L.W., and Amacher, D.E. 2002. Investigating the TNP‐OVA and direct popliteal lymph node assays for the detection of immunostimulation by drugs associated with anaphylaxis in humans. J. Appl. Toxicol. 22:177‐183.
   Inman, J.K. 1975. Thymus‐independent antigens: The preparation of covalent, hapten‐Ficoll conjugates. J. Immunol. 114:704‐709.
   Nierkens, S., van Helden, P., Bol, M., Bleumink, R., van Kooten, P., Ramdien‐Murli, S., Boon, L., and Pieters, R. 2002. Selective requirement for CD40‐CD154 in drug‐induced type 1 versus type 2 responses to trinitrophenyl‐ovalbumin. J. Immunol. 168:3747‐3754.
   Schielen, P., van Rodijnen, W., Tekstra, J., Albers, R., and Seinen, W. 1995. Quantification of natural antibody producing B cells in rats by an improved ELISPOT technique using the polyvinylidene difluoride membrane as the solid support. J. Immunol. Methods 188:33‐41.
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