ovalbumin and domoic-acid

The potential of immunoassays as high-throughput screening tools for the detection of harmful substances in foods will only be realized when convenient methods are available for production of the high affinity antibodies needed for sensitive assay development. Recombinant antibodies offer advantages over traditional monoclonal antibodies in terms of ease of production, much greater antibody repertoire for selection, and versatility. We describe here the development of recombinant antibodies against the common shellfish toxin, domoic acid (DA), utilizing the sheep immunoglobulin system as an effective method for generating high affinity anti-hapten recombinant antibody fragments. A single-chain antibody fragment (scFv) library was generated from a sheep immunized with DA-bovine serum albumin conjugate, and anti-DA scFvs were isolated by phage-display. Three selected scFvs gave I50s of 2.6 to 58 ng/mL (8.3-186 nM) in competitive enzyme-linked immunosorbent assay (ELISA). Assay optimization with one of these scFvs gave a very reproducible standard curve with a range of 0.3 to 5.6 ng/mL (1.0 to 17.9 nM), a mean limit of quantification (LOQ, defined as the I20) of 0.5 ng/mL (1.6 nM), and a mean I50 of 1.2 ng/mL (3.9 nM). When the assay was used for the analysis of crude methanolic extracts of scallop tissues, results obtained correlated well with standard HPLC assay results (R2, 0.90, n = 40; R2, 0.81, n = 34), although ELISA results were lower than HPLC results. Adjusting the cutoff point for DA concentration accordingly from the regulatory 20 mg/kg, the potential of the sheep scFv-based ELISA for use as a screening assay for DA in shellfish extracts was demonstrated.

Topics: Animals; Chromatography, High Pressure Liquid; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Female; Immunoglobulin Fragments; Kainic Acid; Marine Toxins; Ovalbumin; Peptide Library; Recombinant Proteins; RNA; Serum Albumin, Bovine; Sheep; Shellfish

For production of monoclonal antibodies against domoic acid, a causative agent of amnesic shellfish poisoning, three immunogens, domoic acid conjugated with bovine serum albumin (BSA), ovalbumin (OVA) and human gamma globulin (HGG), were prepared. The antiserum obtained from BALB/c mice immunized with domoic acid-BSA showed the highest affinity for domoic acid. The monoclonal antibody, DA-3, obtained from the mice was highly specific for domoic acid and showed a minor cross-reactivity with the isomers of domoic acid (isodomoic acids B, E, F, G and H), except for isodomoic acid A. Using DA-3 antibody, an indirect competitive enzyme immunoassay (idc-EIA) was developed for measurement of domoic acid. The working range for quantitative measurement of domoic acid and the quantification limit for domoic acid in shellfish were estimated to be 0.15-10 ng/ml and less than 0.04 microg/g, respectively. The mean recovery of domoic acid added to extracts of shellfish at toxin levels of 0.02 to 0.2 microg/ml was 103% with a coefficient of variation of 4.5%. The newly developed idc-EIA seems to be a useful method for monitoring domoic acid in shellfish.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Bivalvia; Brachyura; Cattle; Cross Reactions; Foodborne Diseases; gamma-Globulins; Humans; Immunoenzyme Techniques; Isomerism; Kainic Acid; Marine Toxins; Mice; Mice, Inbred BALB C; Ostreidae; Ovalbumin; Serum Albumin, Bovine; Shellfish

A polyclonal antiserum was raised in mice against domoic acid. Two of three immunogens consisted of domoic acid coupled to ovalbumin (OVA) and keyhole limpet haemocyanin at molar ratios of 47:1 and 44:1, respectively using a carbodiimide reaction. Titres of both antisera exceeded 1/35,000 against domoic acid coupled to the non-relevant carrier. Domoic acid was also conjugated to bovine serum albumin at a molar ratio of 30:1 using N-hydroxysuccinimidyl-4-azidobenzoate, a photoreactive compound. This immunogen, however, produced no measurable serum titres against domoic acid. The antiserum produced against the OVA conjugate displayed the highest affinity for free domoic acid in competitive enzyme-linked immunosorbent assay (ELISA). Furthermore, this antiserum preparation did not significantly cross-react with glutamic acid, aspartic acid, the structural analogue kainic acid, or the paralytic shellfish toxin, saxitoxin. The competitive ELISA was used to quantify domoic acid concentrations in human body fluids spiked with pure domoate. The lower limits of accurate domoic acid determinations in competitive ELISA were 0.2 micrograms/ml in urine, 0.25 micrograms/ml in plasma and 10 micrograms/ml in milk. It was concluded that the competitive ELISA described herein could be used to quantitate directly the concentration of domoic acid in the body fluids of individuals with amnesic shellfish poisoning.

Topics: Animals; Binding, Competitive; Enzyme-Linked Immunosorbent Assay; Female; Foodborne Diseases; Hemocyanins; Humans; Immune Sera; Kainic Acid; Male; Marine Toxins; Mice; Mice, Inbred BALB C; Milk, Human; Neurotoxins; Ovalbumin; Sensitivity and Specificity; Serum Albumin, Bovine; Shellfish Poisoning

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Kainic Acid; Macaca fascicularis; Ovalbumin; Radioimmunoassay; Rats