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

Abstract

Polyclonal antibodies for domoic acid were generated from rabbits after the animals had been immunized with either domoic acid-keyhole limpet hemocyanin (KLH) or domoic acid-bovine serum albumin (BSA). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of domoic acid in blue mussels and clams. The antibody titers in the serum of rabbits immunized with domoic acid-KLH were considerably higher than those in rabbits immunized with domoic acid-BSA. The antibodies from the rabbits immunized with domoic acid-KLH were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of domoic acid-horseradish peroxidase to the antibodies by domoic acid and a domoic acid analogue, kainic acid, were found to be 0.75 and 200 ng/mL, respectively. In the presence of blue mussel matrix, the detection limit of domoic acid was <25 ng/g. The overall analytical recovery of domoic acid (25-500 ng/g) added to the blue mussels and then extracted with 50% aqueous methanol in the cdELISA was found to be 81.1%. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method. Analysis of domoic acid in shellfish samples showed that 10 of the 15 shellfish examined were contaminated with domoic acid at levels of <50 ng/g.

Development of a Monoclonal Antibody against Domoic Acid and Its Application in Enzyme-Linked Immunosorbent Assay and Colloidal Gold Immunostrip

“…On the basis of 10% inhibition of binding of domoic acid−HRP conjugate, the detection limit of domoic acid in buffer solution of the cdELISA was around 0.01 ng/mL ( Figure 2A ). The mAb-based cdELISA developed here showed a better sensitivity than that developed by Osada et al, showing a detection limit of 0.1 ng/mL (), but similar to the polyclonal-based cdELISA systems established by us or Garthwaite et al ( , ). Both cdELISA and ciELISA showed a very weak cross-reactivity with kainic acid ( Figure A , B ); kainic acid is an excitatory amino acid and a potent agonist of glutamate receptors in the dorsal hippocampus of brain, with a chemical structure similar to domoic acid ().…”

“…To render it immunogenic, it is necessary to conjugate domoic acid to a protein carrier as an immunogen. Using domoic acid-KLH conjugate as an antigen, polyclonal antibodies were produced in our laboratory, and a sensitive ELISA for domoic acid was also established (31). In the present study, a stable hybridoma cell line generating mAb specifically against domoic acid was obtained.…”

Development of a Monoclonal Antibody against Domoic Acid and Its Application in Enzyme-Linked Immunosorbent Assay and Colloidal Gold Immunostrip

“…On the basis of 10% inhibition of binding of domoic acid−HRP conjugate, the detection limit of domoic acid in buffer solution of the cdELISA was around 0.01 ng/mL ( Figure 2A ). The mAb-based cdELISA developed here showed a better sensitivity than that developed by Osada et al, showing a detection limit of 0.1 ng/mL (), but similar to the polyclonal-based cdELISA systems established by us or Garthwaite et al ( , ). Both cdELISA and ciELISA showed a very weak cross-reactivity with kainic acid ( Figure A , B ); kainic acid is an excitatory amino acid and a potent agonist of glutamate receptors in the dorsal hippocampus of brain, with a chemical structure similar to domoic acid ().…”

“…To render it immunogenic, it is necessary to conjugate domoic acid to a protein carrier as an immunogen. Using domoic acid-KLH conjugate as an antigen, polyclonal antibodies were produced in our laboratory, and a sensitive ELISA for domoic acid was also established (31). In the present study, a stable hybridoma cell line generating mAb specifically against domoic acid was obtained.…”

Adsorption and Activity of a Domoic Acid Binding Antibody Fragment on Mesoporous Silicates

“…The antibody fragment adsorbed on SBA-15 and MCM-41/ 80 successfully detected 1 µg/mL domoic acid in solution (Figure 4), which, based on published protocols for preparation of blue mussel tissue for domoic acid immunodetection, 43 corresponds to 20 µg/g domoic acid in shellfish samples. Recovery was low in this study, with only 6-7% of domoic acid bound at adsorbed scFv concentrations from 0.5 to 2 µmol/ g, and 10-12% with 4 µmol/g scFv (Figure 4).…”

“…Nevertheless, the analysis indicates the potential use of mesoporous silicates in the development of a rapid, in situ detection method for domoic acid above safe levels. This preliminary in situ screening could then be followed by laboratory-based quantification of toxin levels using more accurate but labor-intensive methods such as immunoassays (limit of detection 0.025-0.04 µg/g) 28,43 or HPLC-based methods such as LC/UV (0.6 µg/g), 26 LC with fluorimetric detection (6 ng/g), 44 LC/MS (0.1-0.2 µg/g), 24,26 or LC/MS/MS (0.02 µg/g). 45…”

Development of a Sensitive Enzyme-Linked Immunosorbent Assay for the Determination of Ochratoxin A

“…Therefore, in this study the combination of carbodiimide and NHS greatly enhanced the coupling efficiency between OTA and γ-globulin, which could then help generate high-affinity antibodies for OTA. Because the basic structure of OTA contains one reactive carboxylic group, the carbodiimide coupling process includes the formation of a stable intermediate active ester by condensation of the carboxylic group at the OTA with the assistance of NHS, and this intermediate then reacts with a primary amine on γ-globulin to form a stable amide bond (27,29,34).…”

Development of a Monoclonal Antibody against Domoic Acid and Its Application in Enzyme-Linked Immunosorbent Assay and Colloidal Gold Immunostrip

“…On the basis of 10% inhibition of binding of domoic acid−HRP conjugate, the detection limit of domoic acid in buffer solution of the cdELISA was around 0.01 ng/mL ( Figure 2A ). The mAb-based cdELISA developed here showed a better sensitivity than that developed by Osada et al, showing a detection limit of 0.1 ng/mL (), but similar to the polyclonal-based cdELISA systems established by us or Garthwaite et al ( , ). Both cdELISA and ciELISA showed a very weak cross-reactivity with kainic acid ( Figure A , B ); kainic acid is an excitatory amino acid and a potent agonist of glutamate receptors in the dorsal hippocampus of brain, with a chemical structure similar to domoic acid ().…”

“…To render it immunogenic, it is necessary to conjugate domoic acid to a protein carrier as an immunogen. Using domoic acid-KLH conjugate as an antigen, polyclonal antibodies were produced in our laboratory, and a sensitive ELISA for domoic acid was also established (31). In the present study, a stable hybridoma cell line generating mAb specifically against domoic acid was obtained.…”

Adsorption and Activity of a Domoic Acid Binding Antibody Fragment on Mesoporous Silicates

“…The antibody fragment adsorbed on SBA-15 and MCM-41/ 80 successfully detected 1 µg/mL domoic acid in solution (Figure 4), which, based on published protocols for preparation of blue mussel tissue for domoic acid immunodetection, 43 corresponds to 20 µg/g domoic acid in shellfish samples. Recovery was low in this study, with only 6-7% of domoic acid bound at adsorbed scFv concentrations from 0.5 to 2 µmol/ g, and 10-12% with 4 µmol/g scFv (Figure 4).…”

“…Nevertheless, the analysis indicates the potential use of mesoporous silicates in the development of a rapid, in situ detection method for domoic acid above safe levels. This preliminary in situ screening could then be followed by laboratory-based quantification of toxin levels using more accurate but labor-intensive methods such as immunoassays (limit of detection 0.025-0.04 µg/g) 28,43 or HPLC-based methods such as LC/UV (0.6 µg/g), 26 LC with fluorimetric detection (6 ng/g), 44 LC/MS (0.1-0.2 µg/g), 24,26 or LC/MS/MS (0.02 µg/g). 45…”

Development of a Sensitive Enzyme-Linked Immunosorbent Assay for the Determination of Ochratoxin A

“…Therefore, in this study the combination of carbodiimide and NHS greatly enhanced the coupling efficiency between OTA and γ-globulin, which could then help generate high-affinity antibodies for OTA. Because the basic structure of OTA contains one reactive carboxylic group, the carbodiimide coupling process includes the formation of a stable intermediate active ester by condensation of the carboxylic group at the OTA with the assistance of NHS, and this intermediate then reacts with a primary amine on γ-globulin to form a stable amide bond (27,29,34).…”

Development of a Monoclonal Antibody against Domoic Acid and Its Application in Enzyme-Linked Immunosorbent Assay and Colloidal Gold Immunostrip

“…On the basis of 10% inhibition of binding of domoic acid−HRP conjugate, the detection limit of domoic acid in buffer solution of the cdELISA was around 0.01 ng/mL ( Figure 2A ). The mAb-based cdELISA developed here showed a better sensitivity than that developed by Osada et al, showing a detection limit of 0.1 ng/mL (), but similar to the polyclonal-based cdELISA systems established by us or Garthwaite et al ( , ). Both cdELISA and ciELISA showed a very weak cross-reactivity with kainic acid ( Figure A , B ); kainic acid is an excitatory amino acid and a potent agonist of glutamate receptors in the dorsal hippocampus of brain, with a chemical structure similar to domoic acid ().…”

“…To render it immunogenic, it is necessary to conjugate domoic acid to a protein carrier as an immunogen. Using domoic acid-KLH conjugate as an antigen, polyclonal antibodies were produced in our laboratory, and a sensitive ELISA for domoic acid was also established (31). In the present study, a stable hybridoma cell line generating mAb specifically against domoic acid was obtained.…”

Adsorption and Activity of a Domoic Acid Binding Antibody Fragment on Mesoporous Silicates

“…The antibody fragment adsorbed on SBA-15 and MCM-41/ 80 successfully detected 1 µg/mL domoic acid in solution (Figure 4), which, based on published protocols for preparation of blue mussel tissue for domoic acid immunodetection, 43 corresponds to 20 µg/g domoic acid in shellfish samples. Recovery was low in this study, with only 6-7% of domoic acid bound at adsorbed scFv concentrations from 0.5 to 2 µmol/ g, and 10-12% with 4 µmol/g scFv (Figure 4).…”

“…Nevertheless, the analysis indicates the potential use of mesoporous silicates in the development of a rapid, in situ detection method for domoic acid above safe levels. This preliminary in situ screening could then be followed by laboratory-based quantification of toxin levels using more accurate but labor-intensive methods such as immunoassays (limit of detection 0.025-0.04 µg/g) 28,43 or HPLC-based methods such as LC/UV (0.6 µg/g), 26 LC with fluorimetric detection (6 ng/g), 44 LC/MS (0.1-0.2 µg/g), 24,26 or LC/MS/MS (0.02 µg/g). 45…”

Development of a Sensitive Enzyme-Linked Immunosorbent Assay for the Determination of Ochratoxin A

“…Therefore, in this study the combination of carbodiimide and NHS greatly enhanced the coupling efficiency between OTA and γ-globulin, which could then help generate high-affinity antibodies for OTA. Because the basic structure of OTA contains one reactive carboxylic group, the carbodiimide coupling process includes the formation of a stable intermediate active ester by condensation of the carboxylic group at the OTA with the assistance of NHS, and this intermediate then reacts with a primary amine on γ-globulin to form a stable amide bond (27,29,34).…”