Production and Characterization of High Affinity Rabbit Monoclonal Antibodies (RmAbs) to Amyloid  38 (A 38) Peptide (P02.060)

Objective: To generate high affinity RmAbs to human Aβ38, and develop an ELISA to measure concentrations in CSF. Background The three best known C-terminally truncated Aβ peptides are Aβ38, Aβ40 and Aβ42. Studies indicated that Aβ38 is the second most prominent Aβ form next to Aβ40, and CSF Aβ38/42 ratio was higher in Alzheimer disease (AD) patients than controls. We had generated high affinity rabbit polyclonal antibodies to Aβ38; however, their main drawback was limited supply. Design/Methods: Rabbits were immunized with C-terminal part of Aβ38 (nh2-AIIGLMVGG-oh) (Miller et al., 2010). Spleens were sent to Epitomics Inc., which generated hybridomas. The clones were examined for specificity, sensitivity, and affinity using an ELISA and immunoblotting. Results: RmAb 7-14-4 clone specific for Aβ38 was characterized. Inhibition ELISA showed that 1nM of Aβ38 inhibited 50% of binding between 7-14-4 and Aβ38, whereas 100nM of Aβ37, Aβ40 or Aβ42 showed no inhibition. The Kd of RmAb was 4nM. Immunoblots detected 1fmole of Aβ38 but 200fmole of Aβ40 showed no reactivity. In ELISA RmAb detected Aβ38 at 4fmole/ml; whereas 2.4pmole/ml of Aβ40 or Aβ42 showed no reactivity. Using RmAb and polyclonal antibodies to Aβ38 we quantitated Aβ38 levels in CSF of 25 AD or controls by ELISA. There was a strong correlation between RmAb and polyclonal antibodies to Aβ38 (r = .889, p Conclusions: The RmAb possessed high affinity, sensitivity and specificity. Immunohistological data indicate that Aβ38, like Aβ40 is mainly deposited as vascular amyloid. Further studies will determine whether CSF Aβ38/42 ratio is more useful than CSF Aβ40/42 ratio to support the diagnosis of AD or monitor disease activity. Supported by: NY State OPWDD, and grants from the NIH. Disclosure: Dr. Mehta has nothing to disclose. Dr. Barshatzky has nothing to disclose. Dr. Potempska has nothing to disclose. Dr. Patrick has nothing to disclose. Dr. Mehta has nothing to disclose. Dr. Wegiel has nothing to disclose. Dr. Miller has nothing to disclose.