Development of a New Monoclonal Antibody against Brevetoxins in Oyster Samples Based on the Indirect Competitive Enzyme-Linked Immunosorbent Assay

Abstract

The consumption of shellfish contaminated with brevetoxins, a family of ladder-frame polyether toxins formed during blooms of the marine dinoflagellate Karenia brevis, can cause neurotoxic poisoning, leading to gastroenteritis and neurotoxic effects. To rapidly monitor brevetoxin levels in oysters, we generated a broad-spectrum antibody against brevetoxin 2 (PbTx-2), 1 (PbTx-1), and 3 (PbTx-3) and developed a rapid indirect competitive enzyme-linked immunosorbent assay (icELISA). PbTx-2 was reacted with carboxymethoxylamine hemihydrochloride (CMO) to generate a PbTx-2-CMO hapten and reacted with succinic anhydride (HS) to generate the PbTx-2-HS hapten. These haptens were conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare immunogen and coating antigen reagents, respectively, using the active ester method. After immunization and cell fusion, a broad-spectrum monoclonal antibody (mAb) termed mAb 1D3 was prepared. The 50% inhibitory concentration (IC50) values of the icELISA for PbTx-2, PbTx-1, and PbTx-3 were 60.71, 52.61, and 51.83 μg/kg, respectively. Based on the broad-spectrum mAb 1D3, an icELISA was developed to determine brevetoxin levels. Using this approach, the limit of detection (LOD) for brevetoxin was 124.22 μg/kg and recoveries ranged between 89.08% and 115.00%, with a coefficient of variation below 4.25% in oyster samples. These results suggest that our icELISA is a useful tool for the rapid monitoring of brevetoxins in oyster samples.

An immunochromatographic strip sensor for Marbofloxacin residues

“…Preparation of immunogen, coating antigen and anti-MBF mAbs. The EDC/NHS method for the preparation of MBF-BSA and MBF-OVA conjugates was modi ed from a previous report 28, 29 . Brie y, 18.12 mg BMF were dissolved in 3 mL DMF, and mixed with 5.75 mg NHS and 9.59 mg EDC at room temperature (RT) for 12 h. The mixture was centrifuged at 3950 rpm for 5 min to collect the supernatant.…”

Development of a Lateral Flow Immunoassay Based on a Highly Specific Monoclonal Antibody To Detect 4-Methylaminoantipyrine

“…Hapten conjugation of proteins was performed as previously described. − Briefly, 20 mg of AA-BA was redissolved in 1.0 mL N , N -dimethylformamide containing 30 mg N , N ′-dicyclohexylcarbodiimide and 20 mg N -hydroxysuccinimide and reacted at room temperature overnight. After centrifugation at 8000 g for 15 min, the resulting supernatant was an active hapten solution.…”

“…Booster immunizations were given every 21 days after initial immunization, and when serum was collected from mice 7–10 days after the final immunization, indirect ELISA was used to determine antibody titers, and icELISA was used to determine the specificity of the competing MAA. Eventually, mice with the highest affinity and inhibition ratios were sacrificed by cervical dislocation prior to the cell-fusion procedure. − The formula for calculating the inhibition rate was as follows: Inhibition ratio 0.25em ( % ) = ( 1 − B / B 0 ) × 100 % …”

“…The performance of mAb was evaluated by indirect ELISA and icELISA, for which protocols were almost the same as those previously described. , The sensitivity of mAb was assessed using the IC 50 value, defined as the value of the half-maximal inhibitory concentration of the analyte. The specificity of mAb was evaluated using cross-reactivity (CR) values calculated using the following formula: CR 0.25em ( % ) = ( IC 50 of MAA / IC 50 of analyte false[ ng / mL false] ) × 100 …”

Application of Antibody and Immunoassay for Food Safety

“…Furthermore, the practicability of the developed ELISA was verified by detecting ABZs in spiked milk, beef, and liver samples with recoveries. Zhang et al [ 10 ] explored and developed novel haptens using molecular modeling to prepare broad-spectrum mAbs against brevetoxin 2 (PbTx-2), 1 (PbTx-1), and 3 (PbTx-3), followed by an ELISA method to detect brevetoxins in oyster samples was developed. In particular, the differences between the haptens of PbTx-2-CMO and PbTx-2-HS were evaluated using molecule alignment and electrostatic potential analysis.…”