Table 1.
Haptens, Antigens, and Immunochemical Specifications of anti-(+)-METH Monoclonal Antibodies.
| (+)-METH-like hapten and antigen | Monoclonal antibody | IgG isotype, light chain | (+)-METH KD (nM) | (+)-AMP KD (nM) or KI (nM)a | (+)-MDMA KI (nM)b |
|---|---|---|---|---|---|
| MO6 bound to c-BSA | mAb9B11 | IgG1, λ | 110 | >1,000 | 24 |
| mAb10E1 | IgG1, λ | 91 | >5,000 | 24 | |
| mAb7H11 | IgG1, κ | 52 | >5,000 | 30 | |
| mAb5C2 | IgG1, κ | 39 | >5,000 | 28 | |
| mAb9C4 | IgG2a, κ | 77 | >5,000 | 45 | |
| mAb1C1 | IgG1, κ | 58 | >5,000 | 22 | |
| MO10 bound to OVA | mAb4G9 | IgG2b, κ | 16 | 50 | 68 |
| mAb2F11 | IgG2a, κ | 13 | 49 | 65 | |
| mAb1A12 | IgG2a, κ | 13 | >1,000 | 5 | |
| mAb6C1 | IgG1, κ | 47 | 47 | 52 | |
| mAb10D1 | IgG1, κ | 34 | 51 | 69 | |
| MO10 bound to BSA | mAb7F9 | IgG1, κ | 9 | >1,000 | 14 |
| mAb9C10 | IgG1, κ | 38 | >1,000 | 27 | |
Only four mAbs (all from immunization with a MO10-OVA antigen) had high affinity for (+)-AMP, and these KD values were determine in a homologous immunoassay with [3H]-AMP as the radioligand and inhibition with unlabeled (+)-AMP. If we determined in screening assays that the mAb would have a very high KD value >1000–5,000 nM, we conducted a heterologous immunoassay with [3H]-METH as the radioligand and inhibition with a high range of unlabeled (+)-AMP doses to obtain an approximate KI value for (+)-AMP binding.
[3H]-(+)-MDMA was unavailable, so we determined a KI value for binding to each mAb using [3H]-METH as the radioligand and inhibition with unlabeled (+)-MDMA.