Journal of Clinical Laboratory Analysis 6:65-72 (1992)
A Monoclonal Antibody-Based lmmunoassay for Detecting Tetrodotoxin in Biological Samples* T.J.G. Raybould, G.S. Bignami, L.K. Inouye, Samantha B. Simpson, Jilanne B. Byrnes, RG. Grothaus, and D.C. Vann Hawaii Biotechnology Group, Inc., Aiea, Hawaii Spleen cells from mice hyperimmunized with a keyhole limpet hemocyanin-tetrodotoxin-formaldehyde conjugate were fused with murine P3X63Ag8.653 myeloma cells. A single hybridoma clone was identified that secretes an IgG,,k monoclonal antibody (MAb), designated T20G10, against tetrodotoxin ( T X ) , with an estimated affinityof 1.2 x lo* UM. Competitive inhibition enzyme immunoassays (CIEIAs) for detecting l T X were developed using this MAb. A direct ClElA using alkaline phosphatase-labeled MAb Key words:
marine toxin, fugu, tetrodotoxin, monoclonal antibody, immunoassay
INTRODUCTION Tetrodotoxin (TTX) is an extremely potent low-molecularweight neurotoxin found in widely divergent animal species including puffer fish, gobies, salamanders, frogs, octopus, shellfish and starfish (1,2). TTX intoxication in humans most often results from ingestion of the flesh of certain species of puffer fish, in which elevated levels of TTX are found in the liver, ovaries and eggs. Raw puffer fish flesh, commonly referred to asfugu, is regarded as a delicacy in Japan and several other countries. Those who intentionallyconsume fugu seek a state of mild TTX intoxication, which is said to produce a pleasant peripheral “tingling” sensation. The raw flesh, however, may also contain TTX in sufficient concentration to produce severe intoxication, potentially leading to respiratory paralysis and death. The Japanese and U.S. governments currently require every puffer fish intended for human consumption to be tested. The standard method to assay for TTX is the mouse bioassay (3). This is an expensive and labor-intensive method that suffers from low sensitivity and the inability to discriminate among toxins. In spite of its greater sensitivity, an alternative bioassay employing the house fly (4) has not found wide acceptance, because of the tediousness of such a test. An in vitro cell culture toxin neutralization assay system has recently been reported that employs a rabbit antiserum that cross reacts with TTX and saxitoxin (5). The sensitivity of this system for detecting TTX and saxitoxin was good (15 ng/ml TTX), but the assay was time consuming and did not discriminate between the two toxins. A number of HPLC methods have 0 1992 Wiley-Liss, Inc.
detected mC with sensitivities at ICmand C I, of 6-7 ng/ml and 2-3 ng/ml, respectively. The accuracy of the direct ClElA was comparable with the high-performanceliquid chromatography (HPLC) and the mouse bioassay systems, but the direct ClElA exhibited greater sensitivity. The direct ClElA was also more cost effective, as it required less sample preparation, a shorter assay time, and reduced investment in equipment than either of the other assay systems.
appeared in recent years (6-10). Each of these HPLC methods requires sophisticated equipment and time-consuming sample preparation procedures. In addition, the most sensitive of these HPLC methods was reported to have a detection limit of only 440 ng/ml of TTX (9). Bioreceptor assays using crude brain membranes can detect
A Monoclonal Antibody-Based lmmunoassay for Detecting Tetrodotoxin in Biological Samples* T.J.G. Raybould, G.S. Bignami, L.K. Inouye, Samantha B. Simpson, Jilanne B. Byrnes, RG. Grothaus, and D.C. Vann Hawaii Biotechnology Group, Inc., Aiea, Hawaii Spleen cells from mice hyperimmunized with a keyhole limpet hemocyanin-tetrodotoxin-formaldehyde conjugate were fused with murine P3X63Ag8.653 myeloma cells. A single hybridoma clone was identified that secretes an IgG,,k monoclonal antibody (MAb), designated T20G10, against tetrodotoxin ( T X ) , with an estimated affinityof 1.2 x lo* UM. Competitive inhibition enzyme immunoassays (CIEIAs) for detecting l T X were developed using this MAb. A direct ClElA using alkaline phosphatase-labeled MAb Key words:
marine toxin, fugu, tetrodotoxin, monoclonal antibody, immunoassay
INTRODUCTION Tetrodotoxin (TTX) is an extremely potent low-molecularweight neurotoxin found in widely divergent animal species including puffer fish, gobies, salamanders, frogs, octopus, shellfish and starfish (1,2). TTX intoxication in humans most often results from ingestion of the flesh of certain species of puffer fish, in which elevated levels of TTX are found in the liver, ovaries and eggs. Raw puffer fish flesh, commonly referred to asfugu, is regarded as a delicacy in Japan and several other countries. Those who intentionallyconsume fugu seek a state of mild TTX intoxication, which is said to produce a pleasant peripheral “tingling” sensation. The raw flesh, however, may also contain TTX in sufficient concentration to produce severe intoxication, potentially leading to respiratory paralysis and death. The Japanese and U.S. governments currently require every puffer fish intended for human consumption to be tested. The standard method to assay for TTX is the mouse bioassay (3). This is an expensive and labor-intensive method that suffers from low sensitivity and the inability to discriminate among toxins. In spite of its greater sensitivity, an alternative bioassay employing the house fly (4) has not found wide acceptance, because of the tediousness of such a test. An in vitro cell culture toxin neutralization assay system has recently been reported that employs a rabbit antiserum that cross reacts with TTX and saxitoxin (5). The sensitivity of this system for detecting TTX and saxitoxin was good (15 ng/ml TTX), but the assay was time consuming and did not discriminate between the two toxins. A number of HPLC methods have 0 1992 Wiley-Liss, Inc.
detected mC with sensitivities at ICmand C I, of 6-7 ng/ml and 2-3 ng/ml, respectively. The accuracy of the direct ClElA was comparable with the high-performanceliquid chromatography (HPLC) and the mouse bioassay systems, but the direct ClElA exhibited greater sensitivity. The direct ClElA was also more cost effective, as it required less sample preparation, a shorter assay time, and reduced investment in equipment than either of the other assay systems.
appeared in recent years (6-10). Each of these HPLC methods requires sophisticated equipment and time-consuming sample preparation procedures. In addition, the most sensitive of these HPLC methods was reported to have a detection limit of only 440 ng/ml of TTX (9). Bioreceptor assays using crude brain membranes can detect
