ABSTRACT
Monoclonal antibodies(MoAbs) very specific to diquat (DQ) were produced. An immunogen was synthesized by binding DQ to bovine serum albumin via a diazo-coupled derivative. BALB/c mice were immunized i.p. monthly with 0.25mg of the immunogen for five months. Their spleen cells were fused with P3U1 myeloma cells and hybridoma clones secreting MoAbs were obtained. Two MoAbs were selected and subtyped to be IgM and IgG3. The MoAbs recognized DQ but did not bind to paraquat and other analogues at all. A datum obtained from a clinical sample demonstrates that an enzyme-linked immunosorbent assay system using one of the MoAbs is useful in the practice of toxicological analysis.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Diquat/immunology , Enzyme-Linked Immunosorbent Assay , Adult , Animals , Cross Reactions , Diquat/analysis , Diquat/poisoning , Humans , Male , Mice , Mice, Inbred BALB C , Poisoning/diagnosisABSTRACT
Four kinds of monoclonal antibodies (moAbs) specific to paraquat, diquat, methamphetamine (MA) and melatonin (MT) were produced. 1-Methyl,1'-hexanoic acid-4,4'-bi-pyridinium, 4-amino-D,L-phenylalanine-diazo-coupled diquat, p-amino-methamphetamine as haptens were synthesized and coupled to bovine serum albumin (BSA) directly or to BSA via glutaraldehyde, and an antigen of MT was prepared by binding MT to BSA with the Mannich reaction. These immunogens of BSA conjugates were immunized i.p. to BALB/C mice. Usable moAbs were established 4 to 6 months after the first immunization. Amounts of paraquat, diquat, MA and MT causing a 50% of inhibition by enzyme-linked immunosorbent assay or radioimmunoassay with one of the most specific moAbs from each hapten were 1.1 ng, 0.67 nmol, 10.0 ng and 1.4 ng, respectively.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Haptens/immunology , Animals , Antibody Specificity , Diquat/immunology , Enzyme-Linked Immunosorbent Assay , Melatonin/immunology , Methamphetamine/immunology , Mice , Mice, Inbred BALB C , Paraquat/immunology , RadioimmunoassayABSTRACT
The anti-diquat (DQ) monoclonal antibodies with high specificity were produced. An immunogen was synthesized by binding DQ to bovine serum albumin via a diazo-coupled intermediate. BALB/c mice were injected intraperitoneally once a month with 0.25 mg of the immunogen for 5 months. Their spleen cells were fused with P3U1 myeloma cells to get hybridoma clones secreting anti-DQ antibodies. Two anti-DQ monoclonal antibodies (ADM-1, ADM-2) were subtyped to be IgM and IgG3, respectively. A competitive ELISA was developed with ADM-2. More than 0.05 micrograms of DQ was measured without any interference from human serum. The ADM-2 showed high affinity for DQ and no cross-reactivities with paraquat and other analogues. DQ in sera of poisoning patients were successfully determined by the ELISA. On the other hand, the ADM-2 was applicable to the immunohistochemical demonstration of DQ distribution in experimental animals. An avidin-biotin-peroxidase complex method was used in this immunohistochemical study. DQ-intoxicated rats were killed at 3 h, 12 h, 24 h, 3 days and 7 days after intravenous administration of DQ (30 mg/kg). The macrophages containing DQ in the lung started to be observed at 12 h after injection and the number increased till 7 days. From 3 hours after injection, DQ was localized in the epithelial cells of the distal tubules and collection tubules, but not in the glomeruli in the kidney. In the heart, at every time from 3 h to 7 days after DQ administration, a few myocardial cells were positive with the immunohistochemical staining. The ADM-2 was expected to be available in practice of forensic and analytical toxicology.
