Advertisement

Planta

, Volume 136, Issue 2, pp 173–180 | Cite as

A specific radioimmunoassay for nanogram quantities of the auxin, indole-3-acetic acid

  • William Pengelly
  • Frederick MeinsJr.
Article

Abstract

We have developed a specific radioimmunoassay [RIA] for indole-3-acetic acid (IAA) in the 0.2 ng to 12 ng range which, in principle, can be extended to other indole auxins as well. Methods are presented for obtaining suitable antibody, for the RIA procedure, and for measuring IAA in methanolic extracts of plant tissues. Antibody specific for IAA was obtained from rabbits immunized with IAA bound to bovine serum albumin by formaldehyde treatment. In assays with this antibody, 2,4-dichlorophenoxyacetic acid and indoles structurally related to IAA reacted from 300- to 3000-fold less than did IAA itself. However, α-and β-naphthaleneacetic acid reacted significantly and hence interfered with the assay. Extracts of tobacco (Nicotiana tabacum L.) tissue were immunoassayed after partial purification by buffer-ether partition. Crown-gall tumor tissue, which is auxin-autotrophic, and pith tissue depleted of auxin by the diffusion method contained, respectively, 26.7 ng and <0.5 ng extractable IAA per gram fresh weight.

Key words

Auxin Immunoassay Nicotiana Radioimmunoassay 

Abbreviations

BSA

bovine serum albumin

2,4-D

2,4-dichlorophenoxyacetic acid

IAA

indole-3-acetic acid

α-NAA

α-naphthalenacetic acid

PBS

phosphate-buffered saline

RIA

radioimmunoassay

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Auletta, F.J., Caldwell, B.V., Hamilton, G.L.: Androgens: Testosterone and dihydrotestosterone. In: Methods of hormone radioimmunoassay, pp. 359–370, Jaffe, B.M., Behrman, H.R. eds. New York: Acad. Press 1974Google Scholar
  2. Braun, A.C.: The activation of two growth-substance systems accompanying the conversion of normal to tumor cells in crown gall. Cancer Res. 16, 53–56 (1956)Google Scholar
  3. Braun, A.C.: A demonstration of the recovery of the crown-gall tumor cell with the use of complex tumors of single-cell origin. Proc. Nat. Acad. Sci. USA 45, 932–938 (1959)Google Scholar
  4. Campbell, D.H., Garvey, J.S., Cremer, N.E., Sussdorf, D.H.: Methods in immunology A laboratory text for instruction and research. New York: Benjamin 1970Google Scholar
  5. Chase, M.W.: Coflection and handling of serum. In: Methods in immunology and immunochemistry, pp. 237–241, Williams, C.A., Chase, M.W. eds. New York: Acad. Press 1967Google Scholar
  6. Davis, B.D., Dulbecco, R., Eisen, H.N., Ginsberg, H.S., Wood, B.W., Jr.: Principles of microbiology and immunology. New York-Evanston-London: Harper & Row 1968Google Scholar
  7. Fahey, J.L.: Chromatographic separation of immunoglobulins. In: Methods in immunology and immunochemistry, pp. 321–325, Williams, C.A., Chase, M.W., eds. New York: Acad. Press 1967Google Scholar
  8. Fuchs, S., Fuchs, Y.: Immunological assay for plant hormones using specific antibodies to indoleaceric acid and gibberellic acid. Biochim. Biophys. Acta (Amst.) 192, 528–530 (1969)Google Scholar
  9. Fuchs, S., Haimovich, J., Fuchs, Y.: Immunological studies of plant hormones. Detection and estimation by immunological assays. Europ. J. Biochem. 18, 384–390 (1971)Google Scholar
  10. Goldsmith, M.H.M., Cataldo, C.A., Karn, J., Brenneman, T., Trip, P.: The rapid non-polar transport of auxin in the phloem of intact Coleus plants. Planta [Berl.] 116, 301–317 (1974)Google Scholar
  11. Good, N.E., Andreae, W.A., Van Ysselstein, M.W.H.: Studies on 3-indoleacetic acid metabolism. II. Some products of the metabolism of exogenous indoleacetic acid in plant tissues. Plant Physiol. 31, 231–235 (1956)Google Scholar
  12. Jablonski, J.R., Skoog, F.: Cell enlargement and cell division in excised tobacco pith tissue. Physiol. Plantarum 7, 16–24 (1954)Google Scholar
  13. Jaffe, B.M., Behrman, H.R.: Methods of hormone radioimmunoassay. New York: Acad. Press 1974Google Scholar
  14. Kaldewey, H.: “Simple” indole derivatives and plant growth regulators. Urine metabolites, auxins, gibberellin, and cytokinins. In: Thin-layer chromatogrphy, pp. 471–493, Stahl, E. ed. Rerlin-Heidelberg-New York: Springer 1969Google Scholar
  15. Knegt, E., Bruinsma, J.: A rapid sensitive and accurate determination of indolyl-3-acetic acid. Phytochem. 12, 753–756 (1973)Google Scholar
  16. Kulescha, Z., Gautheret, R.: Sur l'élaboration de substances de croissance par 3 types de cultures de tissue de Scorsonère. Cultures normales, cultures de crown-gall, et cultures accoutumées à l'hėtéro-auxine. C.R. Acad. Sci (Paris) 227, 292–294 (1948)Google Scholar
  17. Landsteiner, K.: The specificity of serological reactions. Cambridge, Mass., USA: Harvard Univ. Press 1945Google Scholar
  18. Linsmaier, E.M., Skoog, F.: Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant 18, 100–127 (1965)Google Scholar
  19. Mann, J.D., Jaworski, E.G.: Minimizing loss of indoleacetic acid during purification of plant extracts. Planta (Berl.) 92, 285–291 (1970)Google Scholar
  20. Pengelly, W.L., Meins, F., Jr.: A radioimmunoassay for indole-3-acetic acid. (Abstr.) Plant Physiol. 57, Suppl., 30 (1976)Google Scholar
  21. Ranadive, N.S., Sehon, A.H.: Antigenicity of 5-hydroxyindole-3-acetic acid, a derivative of serotonin. I. Preparation of protein conjugates of 5-hydroxyindole-3-acetic acid. Canad. J. Biochem. 45, 1681–1688 (1967a)Google Scholar
  22. Ranadive, N.S., Sehon, A.H.: Antibodies to serotonin. Canad. J. Biochem. 45, 1701–1710 (1967b)Google Scholar
  23. Stoessel, A., Venis, M.A.: Determination of submicrogram levels of indole-3-acetic acid: A new, highly specific method. Analyt. Biochem. 34, 344–351 (1970)Google Scholar
  24. Went, F.W.: Wuchsstoff und Wachstum. Rec. Trav. bot néerl. 25, 1–116 (1928)Google Scholar

Copyright information

© Springer-Verlag 1977

Authors and Affiliations

  • William Pengelly
    • 1
  • Frederick MeinsJr.
    • 2
  1. 1.Department of BiologyPrinceton UniversityPrincetonUSA
  2. 2.Department of BotanyUniversity of IllinoisUrbanaUSA

Personalised recommendations