Enzyme Immunoassay for the Determination of Domoic Acid in Mussel Extracts

Development of a Sensitive Enzyme-Linked Immunosorbent Assay for the Determination of Domoic Acid in Shellfish

“…The cdELISA developed here showed a better sensitivity than that of Osada et al (26), with a detection limit of 0.1 ng/mL, but similar to those of Kawatsu et al (21) and Garthwaite et al (14). The group of Smith and Kitts (13) was the first to demonstrate the utility of ciELISA to detect domoic acid levels in shellfish extracts. However, their assay relied upon the limited resource of serum from mice, and the detection limit of the assay is in the range of 500 ng/g.…”

“…Smith and Kitts (12) first reported the generation of polyclonal antibodies against domoic acid in mice; domoic acid was conjugated to either OVA or KLH via the carbodiimide method by linking the carboxyl groups of domoic acid to the amino groups of carrier proteins. Their ELISA method based on mouse polyclonal antibodies had a low sensitivity for domoic acid with a detection limit at 500 ppb (13). The carbodiimide method was initially used by us to conjugate domoic acid to KLH for antibody production in rabbit, but this approach was not successful.…”

Development of a Sensitive Enzyme-Linked Immunosorbent Assay for the Determination of Domoic Acid in Shellfish

“…The cdELISA developed here showed a better sensitivity than that of Osada et al (26), with a detection limit of 0.1 ng/mL, but similar to those of Kawatsu et al (21) and Garthwaite et al (14). The group of Smith and Kitts (13) was the first to demonstrate the utility of ciELISA to detect domoic acid levels in shellfish extracts. However, their assay relied upon the limited resource of serum from mice, and the detection limit of the assay is in the range of 500 ng/g.…”

“…Smith and Kitts (12) first reported the generation of polyclonal antibodies against domoic acid in mice; domoic acid was conjugated to either OVA or KLH via the carbodiimide method by linking the carboxyl groups of domoic acid to the amino groups of carrier proteins. Their ELISA method based on mouse polyclonal antibodies had a low sensitivity for domoic acid with a detection limit at 500 ppb (13). The carbodiimide method was initially used by us to conjugate domoic acid to KLH for antibody production in rabbit, but this approach was not successful.…”

Synthesis of Protein Conjugates and Development of Immunoassays for AAL Toxins

“…A second objective was to develop a simple method for preparing conjugates with a significant fraction of coupling through the carboxyl group(s) of the toxin molecule (C, Figure 7). Water-soluble carbodiimides, like EDC, are employed often for the conjugation of ligands with carboxyl and/or amino functional groups (e.g., peptides, toxins) to proteins and other carrier macromolecules through the formation of amide bonds (Briand et al, 1985;Smith and Kitts, 1995). This approach involves a simple coupling procedure; however, it has several disadvantages including extensive intramolecular cross-linking of the carrier protein, formation of polymers containing protein and ligand monomers, and loss of reagents due to precipitation of side products with high molecular weight.…”

Probing the physiological state of phytoplankton at the single-cell level

“…For example, by binding to bovine serum albumin (BSA), antibodies against brevetoxin, the toxic multi-ring polyether produced by the harmful dinoflagellate Ptychodiscus brevis (Baden et al, 1984(Baden et al, , 1988, and tetrahydropurine-based dinoflagellate toxins causing paralytic shellfish poisoning (Guire et al, 1988) were raised. Antibodies against the amnesic shellfish poisoning toxin domoic acid were produced by ovalbumin binding (Smith and Kitts, 1995). None of those antibodies has been applied to flow cytometry yet but potentially provide tools to assess the cellular toxin concentration in dinoflagellates and its variation with environmental factors.…”

Development of a Sensitive Enzyme-Linked Immunosorbent Assay for the Determination of Domoic Acid in Shellfish

“…The cdELISA developed here showed a better sensitivity than that of Osada et al (26), with a detection limit of 0.1 ng/mL, but similar to those of Kawatsu et al (21) and Garthwaite et al (14). The group of Smith and Kitts (13) was the first to demonstrate the utility of ciELISA to detect domoic acid levels in shellfish extracts. However, their assay relied upon the limited resource of serum from mice, and the detection limit of the assay is in the range of 500 ng/g.…”

“…Smith and Kitts (12) first reported the generation of polyclonal antibodies against domoic acid in mice; domoic acid was conjugated to either OVA or KLH via the carbodiimide method by linking the carboxyl groups of domoic acid to the amino groups of carrier proteins. Their ELISA method based on mouse polyclonal antibodies had a low sensitivity for domoic acid with a detection limit at 500 ppb (13). The carbodiimide method was initially used by us to conjugate domoic acid to KLH for antibody production in rabbit, but this approach was not successful.…”

Synthesis of Protein Conjugates and Development of Immunoassays for AAL Toxins

“…A second objective was to develop a simple method for preparing conjugates with a significant fraction of coupling through the carboxyl group(s) of the toxin molecule (C, Figure 7). Water-soluble carbodiimides, like EDC, are employed often for the conjugation of ligands with carboxyl and/or amino functional groups (e.g., peptides, toxins) to proteins and other carrier macromolecules through the formation of amide bonds (Briand et al, 1985;Smith and Kitts, 1995). This approach involves a simple coupling procedure; however, it has several disadvantages including extensive intramolecular cross-linking of the carrier protein, formation of polymers containing protein and ligand monomers, and loss of reagents due to precipitation of side products with high molecular weight.…”

Probing the physiological state of phytoplankton at the single-cell level

“…For example, by binding to bovine serum albumin (BSA), antibodies against brevetoxin, the toxic multi-ring polyether produced by the harmful dinoflagellate Ptychodiscus brevis (Baden et al, 1984(Baden et al, , 1988, and tetrahydropurine-based dinoflagellate toxins causing paralytic shellfish poisoning (Guire et al, 1988) were raised. Antibodies against the amnesic shellfish poisoning toxin domoic acid were produced by ovalbumin binding (Smith and Kitts, 1995). None of those antibodies has been applied to flow cytometry yet but potentially provide tools to assess the cellular toxin concentration in dinoflagellates and its variation with environmental factors.…”

Development of a Sensitive Enzyme-Linked Immunosorbent Assay for the Determination of Domoic Acid in Shellfish

“…The cdELISA developed here showed a better sensitivity than that of Osada et al (26), with a detection limit of 0.1 ng/mL, but similar to those of Kawatsu et al (21) and Garthwaite et al (14). The group of Smith and Kitts (13) was the first to demonstrate the utility of ciELISA to detect domoic acid levels in shellfish extracts. However, their assay relied upon the limited resource of serum from mice, and the detection limit of the assay is in the range of 500 ng/g.…”

“…Smith and Kitts (12) first reported the generation of polyclonal antibodies against domoic acid in mice; domoic acid was conjugated to either OVA or KLH via the carbodiimide method by linking the carboxyl groups of domoic acid to the amino groups of carrier proteins. Their ELISA method based on mouse polyclonal antibodies had a low sensitivity for domoic acid with a detection limit at 500 ppb (13). The carbodiimide method was initially used by us to conjugate domoic acid to KLH for antibody production in rabbit, but this approach was not successful.…”

Synthesis of Protein Conjugates and Development of Immunoassays for AAL Toxins

“…A second objective was to develop a simple method for preparing conjugates with a significant fraction of coupling through the carboxyl group(s) of the toxin molecule (C, Figure 7). Water-soluble carbodiimides, like EDC, are employed often for the conjugation of ligands with carboxyl and/or amino functional groups (e.g., peptides, toxins) to proteins and other carrier macromolecules through the formation of amide bonds (Briand et al, 1985;Smith and Kitts, 1995). This approach involves a simple coupling procedure; however, it has several disadvantages including extensive intramolecular cross-linking of the carrier protein, formation of polymers containing protein and ligand monomers, and loss of reagents due to precipitation of side products with high molecular weight.…”

Probing the physiological state of phytoplankton at the single-cell level

“…For example, by binding to bovine serum albumin (BSA), antibodies against brevetoxin, the toxic multi-ring polyether produced by the harmful dinoflagellate Ptychodiscus brevis (Baden et al, 1984(Baden et al, , 1988, and tetrahydropurine-based dinoflagellate toxins causing paralytic shellfish poisoning (Guire et al, 1988) were raised. Antibodies against the amnesic shellfish poisoning toxin domoic acid were produced by ovalbumin binding (Smith and Kitts, 1995). None of those antibodies has been applied to flow cytometry yet but potentially provide tools to assess the cellular toxin concentration in dinoflagellates and its variation with environmental factors.…”