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. 2002 Feb;110(2):179–185. doi: 10.1289/ehp.02110179

A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shellfish, and mammalian body fluid.

Jerome Naar 1, Andrea Bourdelais 1, Carmelo Tomas 1, Julia Kubanek 1, Philip L Whitney 1, Leanne Flewelling 1, Karen Steidinger 1, Johnny Lancaster 1, Daniel G Baden 1
PMCID: PMC1240733  PMID: 11836147

Abstract

We developed a competitive enzyme-linked immunosorbent assay (ELISA) to analyze brevetoxins, using goat anti-brevetoxin antibodies obtained after immunization with keyhole limpet hemocyanin-brevetoxin conjugates, in combination with a three-step signal amplification process. The procedure, which used secondary biotinylated antibodies, streptavidine-horseradish peroxidase conjugate, and chromogenic enzyme substrate, was useful in reducing nonspecific background signals commonly observed with complex matrices. This competitive ELISA detected brevetoxins in seawater, shellfish extract and homogenate, and mammalian body fluid such as urine and serum without pretreatment, dilution, or purification. We investigated the application of this technique for shellfish monitoring by spiking shellfish meat with brevetoxins and by analyzing oysters from two commercial shellfish beds in Florida that were exposed to a bloom of Karenia brevis (formerly Gymnodinium breve). We performed brevetoxin analysis of shellfish extracts and homogenates by ELISA and compared it with the mouse bioassay and receptor binding assay. The detection limit for brevetoxins in spiked oysters was 2.5 microg/100 g shellfish meat. This assay appears to be a useful tool for neurotoxic shellfish poisoning monitoring in shellfish and seawater, and for mammalian exposure diagnostics, and significantly reduces the time required for analyses.

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Selected References

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  1. Baden D. G., Melinek R., Sechet V., Trainer V. L., Schultz D. R., Rein K. S., Tomas C. R., Delgado J., Hale L. Modified immunoassays for polyether toxins: implications of biological matrixes, metabolic states, and epitope recognition. J AOAC Int. 1995 Mar-Apr;78(2):499–508. [PubMed] [Google Scholar]
  2. Baden D. G., Mende T. J., Szmant A. M., Trainer V. L., Edwards R. A., Roszell L. E. Brevetoxin binding: molecular pharmacology versus immunoassay. Toxicon. 1988;26(1):97–103. doi: 10.1016/0041-0101(88)90141-9. [DOI] [PubMed] [Google Scholar]
  3. Baden D. G., Mende T. J. Toxicity of two toxins from the Florida red tide marine dinoflagellate, Ptychodiscus brevis. Toxicon. 1982;20(2):457–461. doi: 10.1016/0041-0101(82)90009-5. [DOI] [PubMed] [Google Scholar]
  4. Baden D. G., Mende T. J., Walling J., Schultz D. R. Specific antibodies directed against toxins of Ptychodiscus brevis (Florida's red tide dinoflagellate). Toxicon. 1984;22(5):783–789. doi: 10.1016/0041-0101(84)90161-2. [DOI] [PubMed] [Google Scholar]
  5. Benson J. M., Tischler D. L., Baden D. G. Uptake, tissue distribution, and excretion of brevetoxin 3 administered to rats by intratracheal instillation. J Toxicol Environ Health A. 1999 Jul 9;57(5):345–355. doi: 10.1080/009841099157656. [DOI] [PubMed] [Google Scholar]
  6. Bossart G. D., Baden D. G., Ewing R. Y., Roberts B., Wright S. D. Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996 epizootic: gross, histologic, and immunohistochemical features. Toxicol Pathol. 1998 Mar-Apr;26(2):276–282. doi: 10.1177/019262339802600214. [DOI] [PubMed] [Google Scholar]
  7. Dickey R., Jester E., Granade R., Mowdy D., Moncreiff C., Rebarchik D., Robl M., Musser S., Poli M. Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts. Nat Toxins. 1999;7(4):157–165. doi: 10.1002/(sici)1522-7189(199907/08)7:4<157::aid-nt52>3.0.co;2-#. [DOI] [PubMed] [Google Scholar]
  8. Levine L., Shimizu Y. Antibodies to brevetoxin B: serologic differentiation of brevetoxin B and brevetoxin A. Toxicon. 1992 Apr;30(4):411–418. doi: 10.1016/0041-0101(92)90537-f. [DOI] [PubMed] [Google Scholar]
  9. Melinek R., Rein K. S., Schultz D. R., Baden D. G. Brevetoxin PbTx-2 immunology: differential epitope recognition by antibodies from two goats. Toxicon. 1994 Aug;32(8):883–890. doi: 10.1016/0041-0101(94)90367-0. [DOI] [PubMed] [Google Scholar]
  10. Music S. I., Howell J. T., Brumback C. L. Red tide. Its public health implications. JFMA. 1973 Nov;60(11):27–29. [PubMed] [Google Scholar]
  11. Naar J., Branaa P., Chinain M., Pauillac S. An improved method for the microscale preparation and characterization of hapten-protein conjugates: the use of cholesterol as a model for nonchromophore hydroxylated haptens. Bioconjug Chem. 1999 Nov-Dec;10(6):1143–1149. doi: 10.1021/bc990042g. [DOI] [PubMed] [Google Scholar]
  12. Okabayashi T., Mihara S., Repke D. B., Moffatt J. G. A radioimmunoassay for 1-beta-D-arabinofuranosylcytosine. Cancer Res. 1977 Feb;37(2):619–624. [PubMed] [Google Scholar]
  13. Pauillac S., Naar J., Branaa P., Chinain M. An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate. J Immunol Methods. 1998 Nov 1;220(1-2):105–114. doi: 10.1016/s0022-1759(98)00148-3. [DOI] [PubMed] [Google Scholar]
  14. Poli M. A., Mende T. J., Baden D. G. Brevetoxins, unique activators of voltage-sensitive sodium channels, bind to specific sites in rat brain synaptosomes. Mol Pharmacol. 1986 Aug;30(2):129–135. [PubMed] [Google Scholar]
  15. Poli M. A., Musser S. M., Dickey R. W., Eilers P. P., Hall S. Neurotoxic shellfish poisoning and brevetoxin metabolites: a case study from Florida. Toxicon. 2000 Jul;38(7):981–993. doi: 10.1016/s0041-0101(99)00191-9. [DOI] [PubMed] [Google Scholar]
  16. Poli M. A., Rein K. S., Baden D. G. Radioimmunoassay for PbTx-2-type brevetoxins: epitope specificity of two anti-PbTx sera. J AOAC Int. 1995 Mar-Apr;78(2):538–542. [PubMed] [Google Scholar]
  17. Poli M. A., Templeton C. B., Thompson W. L., Hewetson J. F. Distribution and elimination of brevetoxin PbTx-3 in rats. Toxicon. 1990;28(8):903–910. doi: 10.1016/0041-0101(90)90020-8. [DOI] [PubMed] [Google Scholar]
  18. STARR T. J. Notes on a toxin from Gymnodinium breve. Tex Rep Biol Med. 1958;16(4):500–507. [PubMed] [Google Scholar]
  19. Smith D. S., Kitts D. D. A competitive enzyme-linked immunoassay for domoic acid determination in human body fluids. Food Chem Toxicol. 1994 Dec;32(12):1147–1154. doi: 10.1016/0278-6915(94)90130-9. [DOI] [PubMed] [Google Scholar]
  20. Trainer V. L., Baden D. G. An enzyme immunoassay for the detection of Florida red tide brevetoxins. Toxicon. 1991;29(11):1387–1394. doi: 10.1016/0041-0101(91)90126-c. [DOI] [PubMed] [Google Scholar]
  21. Van Dolah F. M., Finley E. L., Haynes B. L., Doucette G. J., Moeller P. D., Ramsdell J. S. Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins. Nat Toxins. 1994;2(4):189–196. doi: 10.1002/nt.2620020407. [DOI] [PubMed] [Google Scholar]
  22. van Oss C. J., Naim J. O. Aspecific immunoglobulin binding to hydrophobic surfaces. Curr Top Microbiol Immunol. 1996;210:85–91. doi: 10.1007/978-3-642-85226-8_9. [DOI] [PubMed] [Google Scholar]

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