A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shellfish, and mammalian body fluid.
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.
Naar
Jerome
J
Center for Marine Science, University of North Carolina at Wilmington, 5600 Marvin K. Moss Lane, Wilmington, NC 28409, USA.
Bourdelais
Andrea
A
Tomas
Carmelo
C
Kubanek
Julia
J
Whitney
Philip L
PL
Flewelling
Leanne
L
Steidinger
Karen
K
Lancaster
Johnny
J
Baden
Daniel G
DG
D000818:Animals; D000906:Antibodies; D001681:Biological Assay; D004784:Environmental Monitoring; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006041:Goats; D006433:Hemocyanins / Q000276:immunology; D008387:Marine Toxins / Q000032:analysis*; D051379:Mice; D010125:Ostreidae / Q000737:chemistry*; D035964:Oxocins; D012680:Sensitivity and Specificity; D012758:Shellfish; D014867:Water / Q000737:chemistry; D000095702:Polyether Toxins
D000906:Antibodies; D006433:Hemocyanins; D008387:Marine Toxins; D035964:Oxocins; D014867:Water; C041017:brevetoxin T17; D000095702:Polyether Toxins; C032808:keyhole-limpet hemocyanin